Screening Assessment - Part 2

Aromatic Azo and Benzidine-based Substance Grouping
Certain Benzidine-based Dyes and Related Substances

Environment Canada
Health Canada
November 2014

Table of Contents

• List of Tables
• 7.  Potential to Cause Harm to Human Health
  • 7.1  Exposure Assessment
  • 7.2  Health Effects Assessment
  • 7.3  Characterization of Risk to Human Health
• 8.  Conclusion
• References
• Go to the Screening Assessment - Appendices
• Back to the Screening Assessment - Synopsis
• Back to the Screening Assessment - Part 1

List of Tables

• Table 7-1. Consumer products containing 3,3′-DMB and 3,3′-DMOBnotified in RAPEX database from 2010 to September 2012 (RAPEX 2012)
• Table 7-2. Results of the analysis of 117 samples of 86 commercial textile products purchased in Japan in 2009 for 3,3′-DMB and 3,3′-DMOB (Kawakami et al. 2010)
• Table 7-3. Twenty-six Benzidine-based Substances based on EU22 aromatic amines
• Table 7-4. Eight Benzidine-based Substances based on non-EU22 benzidine derivatives
• Table 7-5. Information considered in the determination of azo bond reductive cleavage
• Table 7-6. Available information for genotoxicity and carcinogenicity of Benzidine-based Substances that may release benzidine, 3,3′-DMOB, 3,3′-DMB or 3,3′-DCB
• Table 7-7. Benzidine-based Substances that may release 2,2′-DMB, 2,2′-DCB, 2,2′-DSB, 3,3′-DCAB or 3,3′-DCMB

7. Potential to Cause Harm to Human Health

The human health assessment for Benzidine-based Dyes and Related Substances focuses on substances that are in commerce (based on information received in response to the section 71 survey) and/or for which available information indicates potential exposure to the general population of Canada: 3,3’-DMB and Acid Red 97. Direct Blue 14 is also in commerce based on available information (Environment Canada 2009; DPD 2012), but reported uses of this substance do not result in exposure for the general population in Canada.

7.1 Exposure Assessment

7.1.1 Environmental Media

As described in the section on Sources, limited quantities of Acid Red 97 and Direct Blue 14 were reported to be in commerce.  Based on the uses of these substances and the overall low volumes of Benzidine-based Substances or Benzidine Derivatives in commerce, exposure from environmental media is not expected.

As none of the substances in this assessment were identified to be used in food packaging or food-related applications, these sources are not considered to contribute to exposure to Benzidine-based Substances or Benzidine Derivatives.

7.1.2 Consumer Products

In general, Benzidine-based Dyes are used predominantly for dyeing textiles, Benzidine-based Cationic Indicators as laboratory reagents and Benzidine-based Precursors and Benzidine Derivatives as intermediates in the manufacturing of dyes, pigments, colouring agents and chemicals.

To characterize potential general population exposure to Benzidine-based Substances and Benzidine Derivatives from contact with products, the following information was taken into consideration: information received from section 71 surveys; information on the use of these substances and their presence in products based upon publicly available sources; information submitted to Health Canada (e.g., pursuant to the Food and Drugs Act); recent data from testing of products on the Canadian market; and market surveillance/monitoring data from Europe and Japan.

Leaching of 3,3’DMB from black polyamide cooking utensils was reported in an Irish study (McCall et al. 2012). Acid Red 97 is identified to be used primarily for dyeing textiles and leather (CII 2011). Direct Blue 14 was reported to be used in medical devices and research and development (Environment Canada 2008); this substance is not present in consumer products and therefore exposure of the general population to this substance is not expected.  The presence of other Benzidine-based Substances or Benzidine Derivatives in products, in particular in textiles, in Canada has not been identified. 

Benzidine Derivatives

In the study conducted in Ireland (McCall et al. 2012), 84 black polyamide cooking utensils (e.g., spatulas, slotted spoons) purchased from various retail locations were analyzed for release of primary aromatic amines, including 3,3′-DMB, during a simulated use scenario. The contact area of the utensil was immersed in 3% acetic acid simulant solutions and left for 2 hours at 100°C. This was repeated two additional times to simulate repeated use. The results of the study showed leaching of 3,3′-DMB from 16 cooking utensils; the median, average and maximum concentrations from the third extraction were 1.4 µg/kg food, 3.9 µg/kg food and 30 µg/kg food, respectively. The authors indicated that primary aromatic amines leached from these utensils, under these test conditions, primarily due to incomplete polymerization. Significant variation in leaching levels was observed from identical utensils and among different types of cooking utensils.

Since the frequency of detection of 3,3′-DMB was less than 20%, the use of the median concentration was considered an appropriate metric for use in characterizing potential exposure of the general population. Using the median leaching level of 3,3′-DMB (1.4 µg/kg food) under acidic and elevated temperature, conservative estimates of potential exposure from the use of polyamide cooking utensils were derived to range from 0.002 µg/kg-bw per day (12 years of age and older) to 0.0065 µg/kg-bw per day (toddlers 0.5–4 years of age). See Appendix D for derivation of the exposure estimates.

Testing of products on the Canadian market conducted by Health Canada (2013) did not identify any detectable levels of 3,3′-DMB or 3,3′-DMOB in imported and domestic textile and leather products in Canada (limits of detections of 1.9 ppm for 3,3′-DMB and 1.5 ppm for 3,3′-DMOB). The investigation tested 66 samples of imported and domestic textile and leather products for EU22 aromatic amines^Footnote[6] which are regulated under the EU22 regulation (EU 2006; Environment Canada and Health Canada 2013). The testing focused on children’s toys, leather slippers, children’s clothing and woollen items purchased in retail stores in Ottawa, Ontario, in August 2012 (Health Canada 2013). The testing procedure followed that of EU Standard BS EN 14362-1:2012 for testing EU22 aromatic amines (ECS 2012) and had a limit of quantification (LOQ) of 5.6 parts per million (ppm) for 3,3′-DMB and 4.4 ppm for 3,3′-DMOB. Results are consistent with the global phaseout of EU22 aromatic amines and the corresponding azo dyes due to restrictions in other countries (Environment Canada and Health Canada 2013). Combined with no manufacture and import activities above the reporting threshold of 100 kg for these substances reported in response to section 71 surveys, direct and prolonged exposure to 3,3′-DMB and 3,3′-DMOB for the general population of Canada from contact with textile and leather is not expected.

Notwithstanding the restrictions in other countries, the presence of some EU22 aromatic amines has been reported in other jurisdictions. Compliance reports in Europe, the RAPEX alert system (RAPEX 2012) and the EurAzos project (EurAzos 2007), as well as a recent Japanese market survey (Kawakami et al. 2010), show the presence of 3,3′-DMOB and 3,3′-DMB in some textile, clothing and leather products, some of which were reported to be imported from other countries.

RAPEX is the EU rapid alert system shared by EU member states that facilitates the rapid exchange of information on products posing a serious risk to the health and safety of consumers. The operational procedures for RAPEX are described within the EU Product Safety Directive 2001/95/EC (EU 2001), which imposes a general safety requirement on any product put on the market for consumers. The EU22 aromatic amines listed in Appendix 8 of Regulation (EC) No 1907/2006 (EU 2006) are monitored by the RAPEX alert system (RAPEX 2012). A search of the RAPEX database for alert notifications made from 2010 to 2012 identified notifications for 5 and16 textile articles containing 3,3′-DMB and 3,3′-DMOB, respectively; concentrations ranged from 5.16 to 640 mg/kg for 3,3′-DMB and from 8.7 to 615 mg/kg for 3,3′-DMOB based on standardized test methods (see Table 7-1).

The total number of products tested is unknown. These Benzidine Derivatives are either breakdown products of benzidine-based substances used as dyes or residuals from use as chemical intermediates in the dyeing process. Notified products include clothing, shoes and textile accessories that may be used by the general population, including products intended for infants, and were originally manufactured in India, China, Turkey and the Philippines (RAPEX 2012).

The EurAzos project (EurAzos 2007) is a European enforcement project conducted in 2007, similar to the RAPEX alert system, which aimed to assess the compliance of textile and leather products in the European market with the provisions regarding the EU22 aromatic amines (EU 2006). There were nine violations reported among 361 textile and leather products analyzed, in which the concentrations of EU22 aromatic amines were found to be above the regulated concentration of 30 mg/kg; two of the nine products contained 3,3′-DMOB (31 mg/kg in a hat and 590 mg/kg in an unidentified product). The countries of origin for these two products were not reported.

A Japanese study surveyed 86 textile products purchased at retail stores in Japan between January and March 2009 for 26 primary aromatic amines released from azo dyes (Kawakami et al. 2010). In addition to the EU22 aromatic amines, four additional aromatic amines (2,4-xylidine, 2,6-xylidine, aniline and 1,4-phenylenediamine) were tested. In total, 117 samples of 86 textile products were analyzed for their content of aromatic amines released from azo dyes when extracted under reductive conditions. Two separate standard European sample processing methods, with slight modifications, were used, depending on the type of material. The EN 14362-1 “without solvent extraction” method (ECS 2003a) was used for 77 samples composed of natural fibres (e.g., cellulosic and protein-based fibres). The EN 14362-2 “with solvent extraction” method (ECS 2003b) was used for 40 samples composed of synthetic fibres (e.g., polyester). Both sample processing methods were conducted for “mixed-fibre samples.” Table 7-4 summarizes the results of the analysis for 3,3′-DMB and 3,3′-DMOB. Based on the results of the analysis, almost all concentrations of the investigated aromatic amines were measured below the 30 mg/kg limit set by the EU. However, 3,3′-DMOB concentrations were found to be particularly high (up to 390 mg/kg) in several cotton placemat samples manufactured in India. However, direct and prolonged exposure from the intended use of placemats is not expected.

These three product surveys in Europe and Japan (i.e., EurAzos 2007; Kawakami et al. 2010; RAPEX 2012) indicate that 3,3′-DMB and 3,3′-DMOB can be  present in imported products in these foreign markets. Given that the Canadian textile market is composed predominantly of imported products (Industry Canada 2012; Environment Canada and Health Canada 2013b), the potential for these substances to be present in a limited number of imported products in Canada is recognized.

The remaining three Benzidine Derivatives, 3,3′-DMB·2HCl, TODI and 4N-TMB, are not part of the EU22 aromatic amines and therefore were not targeted for analysis in the product surveys mentioned above. These substances are used as intermediates in chemical synthesis, and no consumer products were identified as containing these substances in Canada. The presence of these substances in imported products is not expected except potentially as residuals at low levels; as such, direct exposure of the general population to 3,3′-DMB·2HCl, TODI or 4N-TMB is not expected.

Benzidine-based Substances

Within the Benzidine-based Substances, 26 substances (Table 7-3) are capable of breaking down to one of four EU22 aromatic amines; benzidine, 3,3′-DCB, 3,3′-DMOB or 3,3′-DMB. In the dye industry, there are approximately 250 azo dyes that are based on benzidine (CAS RN 92-87-5), 6 azo dyes that are based on 3,3′-DCB (CAS RN 91-94-1), 95 azo dyes that are based on 3,3′-DMB and 89 azo dyes that are based on 3,3′-DMOB (SCCNFP 2002). These four aromatic amines (benzidine, 3,3′-DCB, 3,3′-DMOB or 3,3′-DMB) were not detected in testing of domestic and imported textile and leather products on the Canadian market (Health Canada 2013) (limits of detection ranged from 1.4 to 1.9 mg/kg; study described above).  Notwithstanding the restrictions in other jurisdictions, the presence of some of the EU22 aromatic amines in textile and leather products has been reported in product compliance surveys in other countries (e.g., the RAPEX alert system (RAPEX 2012), the EurAzos project (EurAzos 2007) and the recent Japanese survey (Kawakami et al. 2010)). In these surveys, the detection of an EU22 aromatic amine may suggest the presence of a dye based on the detected aromatic amine; however, the specific dye is not identified. Therefore, while some of these EU22 aromatic amines (benzidine, 3,3′-DCB, 3,3′-DMOB or 3,3′-DMB) may be detected in imported products in compliance and market product surveys in other countries, the available information does not indicate direct and prolonged exposure of the general population of Canada to the 26 Benzidine-based Substances.

Eight of the Benzidine-based Substances are based on benzidine derivatives that are not EU22 aromatic amines (Table 7-4). No information regarding consumer product use was identified for these Benzidine-based Substances in Canada, except for Acid Red 97. Dermal exposure to Acid Red 97 from dermal contact with textile clothing and leather articles is estimated to range from 2.6 × 10⁻³ to 4.0 × 10⁻³ mg/kg body weight (kg-bw) per day for textile clothing and from 2.1 × 10⁻³ to 7.7 × 10⁻² mg/kg-bw for leather articles (refer to Appendix E). Oral exposure due to mouthing of textiles by infants is estimated to be 2.7 × 10⁻⁵ mg/kg-bw per day as a conservative estimate. The dermal exposure estimates from textiles are based on conservative assumptions (e.g., full body coverage and direct skin contact). Benzidine-based dyes are relatively water soluble, therefore the effect of laundering is expected to significantly reduce any dye that is not fixed to the textile fibre, thereby reducing exposures over time. It is not expected that Acid Red 97 would be present in all consumer products made of textiles in Canada. Therefore, exposures were derived assuming, based on professional judgement, that there is a 10% probability that this substance is used in dyeing products made of textile in Canada. From the limited data available (Danish EPA 1998; Brüschweiler et al. 2014), the detection of most non-EU22 amines in textiles is usually less than 10%. Accordingly, the presence of associated dyes in textiles would be the same or lower. This adjustment factor of 10% used in this assessment is similar to the 8% value used in the Danish assessment in estimating exposures to aromatic amines and azo dyes from textile garments in the Dutch market (Zeilmaker et al. 1999). See Appendix E for further explanation on the estimated exposures to Acid Red 97.

As the non-EU22 benzidine derivatives were not within the scope of the three product surveys described above (i.e., EurAzos 2007; Kawakami et al. 2010; RAPEX 2012), no information is available from these surveys on the presence of these benzidine derivatives in products in Europe or Japan. From the limited data available (Danish EPA 1998; Brüschweiler et al. 2014), the detection of most non-EU22 amines in textiles is usually less than 10%. Accordingly, the presence of associated dyes in textiles would be the same or lower. Testing of products on the Canadian market included the analysis of 2,2′-DCB and 2,2′-DMB in imported and domestic products, and did not detect these two benzidine derivatives (limits of detections of 3.1 ppm for 2,2′-DMB and 1.0 ppm for 2,2′-DCB; Health Canada 2013). Overall, direct and prolonged exposure to the 7 remaining Benzidine-based Substances from contact with textiles and leather is not expected.

The two Benzidine-based Cationic Indicators (TDBD and TDBPD) were identified for use primarily as laboratory reagents (Merck Index 2001; Sigma-Aldrich Canada 2010; Ullmann’s Encyclopedia 2010), and the Benzidine-based Precursor Naphthol AS-BR was identified as a dye precursor (Freeman 2011). Use of these three substances by the general population is not expected; therefore, exposure to these three substances is not expected.

7.2 Health Effects Assessment

Carcinogenicity and genotoxicity are the critical health effects of potential concern for Aromatic Azo and Benzidine-based Substances (Environment Canada and Health Canada 2013). The mechanism by which Benzidine-based Substances exert their toxicity involves the reductive cleavage of the azo bonds and the subsequent release of the free aromatic amines. These aromatic amines are, in turn, converted to reactive electrophilic intermediates through metabolic oxidation (Environment Canada and Health Canada 2013).

The health effects of the Benzidine-based Substances are evaluated in this assessment by examining their ability to undergo reductive cleavage and their hazard potential. This analysis is based on consideration of the available information and presented in the next two sections. Similarly, the hazard potential of the five Benzidine Derivatives is evaluated and discussed. Health effects data on two substances previously assessed in the Challenge Initiative (Acid Red 111 and Direct Black 38) were included to inform the health effects assessment of the Benzidine-based Substances.  

The focus of the health effects assessment was on those Benzidine-based Substances and Benzidine Derivatives for which exposure to the general population is expected (see section Exposure Assessment).

7.2.1 Azo Bond Cleavage Potential

The azo bond cleavage potential of the substances considered in this assessment was determined based on several lines of evidence that have been previously discussed (Environment Canada and Health Canada 2013). The types of information considered in this assessment range from in vivo assays to read-across approaches.

In vivo metabolism studies provide the most direct evidence for reductive cleavage, and this type of study was found for 10 Benzidine-based Dyes (Table 7-5). In all instances, one or more of the released aromatic amines were identified in the urine and feces of one or more mammalian species that were orally exposed to the dye. The amounts of the aromatic amines present in the urine and feces were greater than those present as impurities in the testing material, indicating in vivo cleavage of the azo bond (Rinde 1974; Rinde and Troll 1975; Lynn et al. 1980; Nony and Bowman 1980; Robens et al. 1980; Bowman et al. 1982, 1983; Kennelly et al. 1982; Levine et al. 1982; Nony et al. 1983; NTP 1983). Reactive aromatic amine metabolites were also found when hemoglobin adducts were used to monitor the bioavailability of Direct Red 28 and Direct Red 46 (Birner et al. 1990; Sagelsdorff et al. 1996).

In vitro metabolism studies were found for six Benzidine-based Dyes (Table 7-5). The aromatic amines generated from the reductive cleavage of the azo bond were identified following incubation of the dye with either intestinal contents from various species or human skin cultures. In all these studies, the potential for reductive cleavage was demonstrated (Hartman et al. 1978; Cerniglia et al. 1982a, b, 1986; Bos et al. 1986a; Chung et al. 1992; Platzek et al. 1999). The results of studies investigating liver metabolism, in contrast, were mixed (Martin and Kennelly 1981; Bos et al. 1984). Only Direct Black 38 and Direct Brown 95 released their aromatic amine or its acetylated form following incubation with rat liver supernatant or rat hepatocytes. In general, liver metabolism plays a minor or negligible role in the azo reduction of dyes derived from benzidine or its derivatives (Martin and Kennelly 1981).

Another line of evidence that was considered in the assessment of reductive cleavage potential is the results obtained from the Ames assay under reductive conditions. These reductive conditions include incubation with intestinal contents, incorporation of the Prival modifications or presence of sodium dithionite. If the Ames test yielded positive results only after such conditions were employed, then the potential for the substance to cleave into genotoxic metabolites in vivo was inferred (Environment Canada and Health Canada 2013). Eleven Benzidine-based Dyes were evaluated in this type of assay, and all had data to support azo bond cleavage (Table 7-5).

In the absence of empirical data, the reductive cleavage potential of a Benzidine-based Substance can be inferred based on read-across among closely related analogues (Environment Canada and Health Canada 2013). These closely related analogues were determined based on a number of factors, including the number of azo bonds, the number of rings, the types of rings and water solubility. Substances with similar properties were assumed to be similarly likely to undergo azo reductive cleavage. The division of Benzidine-based Substances into structurally related groups (Table 7-5) allowed for read-across from substances with empirical data for azo bond reductive cleavage to those with no data. For two structurally related groups and a number of stand-alone substances, however, there were no empirical data that could be used to infer azo bond reductive cleavage. In such instances, a general read-across was done for Benzidine-based Substances, based on the fact that for this class of substances, all available empirical data indicate that reductive cleavage occurs. Therefore, the genotoxicity and carcinogenicity of the released aromatic amines are also considered in the assessment of the Benzidine-based Substances.

The information available to determine azo bond reductive cleavage for this class of substances is generally strong. The reductive cleavage potential for many substances and structurally related groups is based on several types of evidence. All the Benzidine-based Acid and Direct Dyes evaluated in this assessment as well as the Benzidine-based Precursor TCDB are expected to release aromatic amines. Confidence in the read-across approach for substances without empirical data is high, as they have been grouped together based on their similar uses, properties and structures. The carcinogenicity and genotoxicity of the released aromatic amines are therefore considered when determining the hazard potential of Benzidine-based Acid and Direct Dyes as well as the Benzidine-based Precursor TCDB.

In contrast, the two Benzidine-based Cationic Indicators, TDBPD and TDBD, and the Benzidine-based Precursor Naphthol AS-BR do not have the typical azo bond. Therefore, only the hazard potential of these substances is considered in the following section.

7.2.2 Health Effects

Carcinogenicity and genotoxicity are the critical health effects of potential concern for Aromatic Azo and Benzidine-based Substances and, in particular, Benzidine-based Dyes. Studies for these endpoints, especially for carcinogenicity, are not available for many of these dyes. To address the lack of data for benzidine- and benzidine congener–derived dyes, the Benzidine Dye Initiative was established by the US National Toxicology Program (NTP). This research program generated data for two benzidine derivatives and a select group of prototypical dyes derived from those amines and applied the basic information generated from those studies to the toxicity and carcinogenicity associated with other benzidine- and benzidine derivative–based dyes after conducting only a small number of experiments (Morgan et al. 1994). This potential for read-across is also considered, as applicable.

When available, empirical data from carcinogenicity and genotoxicity studies for the Benzidine-based Substances that are being assessed, as well as for relevant aromatic amine metabolites, were considered (see Tables 18 and 19). When required, read-across from substances that release similar aromatic amine metabolites, SAR analyses and (Q)SAR models were also used.

The structures of the potential metabolites released for each Benzidine-based Substance were proposed based on theoretical cleavage of the azo bond. The resulting structures were used to identify CAS RNs associated with each metabolite, when possible.

Benzidine-based Substances That May Release Benzidine, 3,3′-DMOB, 3,3′-DMB or 3,3′-DCB

Of the Benzidine-based Substances included in this assessment, 26 may release benzidine or one of three benzidine derivatives that have been previously assessed and classified by international agencies. Benzidine, 3,3′-DMOB, 3,3′-DMB and 3,3′-DCB are groups 1 and 2B International Agency for Research on Cancer (IARC) carcinogens (known and possible human carcinogens, respectively). They are also regulated in Europe as part of the EU22 aromatic amines. Benzidine and these benzidine derivatives are responsible for the high hazard potential of the Benzidine-based Substances. Studies with reproductive and developmental endpoints are available for seven of these dyes (Table 7-6).

Benzidine-based Substances That May Release Benzidine

Direct Red 28 and Direct Brown 95 may release benzidine following azo bond reductive cleavage (refer to Table 7-6). In addition, data on Direct Black 38 were included to inform the health effects assessment. Both in vitro and in vivo genotoxicity assays are essentially positive for all three dyes. In vitro mutagenicity studies indicate that reductive metabolism and metabolic activation are required for Direct Red 28 and Direct Brown 95 to exert mutagenicity, while Direct Black 38 requires only metabolic activation (Gregory et al. 1981; Martin and Kennelly 1981; Prival and Mitchell 1982; Robertson et al. 1982; Brown and Dietrich 1983; Reid et al. 1983, 1984; Joachim and Decad 1984; Prival et al. 1984; Joachim et al. 1985; Cerniglia et al. 1986; De France et al. 1986; Krishna et al. 1986; Mortelmans et al. 1986; Chung et al. 2006; ILS 2011a). Direct Red 28 was also weakly positive for DNA repair induction in hamster hepatocytes and positive for DNA damage in mammalian cells (Kornbrust and Barfknecht 1984; Bafana et al. 2009b). Direct Brown 95, however, was negative for chromosomal aberrations, unscheduled DNA synthesis and sister chromatid exchange in mammalian cells (Joachim and Decad 1984; Galloway et al. 1987); this was presumably due to the absence of reductive conditions. In vivogenotoxicity assays were positive for DNA damage for Direct Red 28 (Kennelly et al. 1984a; Yi et al. 1993), for mutagenicity and unscheduled DNA synthesis for Direct Black 38 and Direct Brown 95 (Nony 1979; Bos et al. 1984, 1986a, b; Joachim and Decad 1984; Joachim et al. 1985; Beije 1987; Ashby and Mohammed 1988), as well as for chromosome damage and DNA damage for Direct Black 38 (Beije 1987; Tsuda et al. 2000).

A number of studies showing a carcinogenic effect in laboratory animals were identified for Direct Black 38 and Direct Brown 95. Hepatocellular carcinomas and mammary carcinomas were found in mice orally exposed to Direct Black 38 in their drinking water for 55–60 weeks (US EPA 1987a). In rats, Direct Black 38 produced hepatocellular carcinomas within 13 weeks after it was administered in the diet and carcinomas in the urinary bladder, liver and colon after it was administered in drinking water (Okajima et al. 1975; Robens et al. 1980). In a well-conducted 13-week study, Direct Brown 95 produced neoplastic nodules in the livers of four of eight female rats, one of which showed a hepatocellular carcinoma (US EPA 1987b). Due to the increased mortality in male rats, neoplastic lesions were not seen, but significant increases in basophilic foci were observed in these rats.

IARC has evaluated both Direct Black 38 and Direct Brown 95 and previously classified both as group 2A (probable human carcinogens). The working group found that while there is inadequate evidence in humans for the carcinogenicity of dyes metabolized to benzidine, there is sufficient evidence in experimental animals for the carcinogenicity of both dyes (IARC 2010a). More recently, IARC has classified dyes that are metabolized to benzidine as group 1 (carcinogenic to humans) (IARC 2012a). The conclusion was based on a) sufficient evidence in experimental animals for the carcinogenicity of dyes metabolized to benzidine; b) strong mechanistic evidence indicating that benzidine-based dyes are converted by azo reduction to benzidine in humans and in experimental animals and, consequently, produce DNA adducts and genotoxic effects similar to those of benzidine; c) sufficient evidence in humans and in experimental animals for the carcinogenicity of benzidine; and d) evidence that benzidine-based dyes induce chromosomal aberrations in humans and in all experimental animal species studied. In the EU, benzidine-based azo dyes are classified as carcinogenicity category 1B (known to be carcinogenic to humans) with hazard code H350 (“may cause cancer”) (ESIS ©1995–2012). Dyes metabolized to benzidine are listed as “known to be human carcinogens” in the US NTP Report on Carcinogens (NTP 2011).

The Government of Canada has also evaluated Direct Black 38 and has recognized that it may be harmful to human health (Environment Canada and Health Canada 2009).

The Government of Canada has evaluated benzidine and concluded that it was harmful to human health, based on genotoxicity and carcinogenicity (Canada 1993a). More recently, IARC concluded that studies on occupational exposure to benzidine and incidences of bladder cancer showed “consistent positive associations with some indication of dose–response relationships.” In addition, oral, subcutaneous and intraperitoneal administration of benzidine to mice, rats, dogs and hamsters resulted in tumours at a variety of sites. On this basis, benzidine was classified as a group 1 carcinogen (carcinogenic to humans) by IARC (2010a, 2012b).

The health effects of Direct Red 28 and Direct Brown 95 are expected to be similar to those of benzidine, due to the release of benzidine following azo bond reductive cleavage.

3,3′-DMOB and Benzidine-based Substances That May Release 3,3′-DMOB

Fifteen of the Benzidine-based Substances may release 3,3′-DMOB upon azo bond reductive cleavage (refer to Table 18).

In vitro genotoxicity studies were found for Direct Blue 8, Direct Blue 15, Direct Blue 151 and BABHS. All four dyes were mutagenic in the presence of metabolic activation and reductive conditions in bacterial mutagenicity assays (EI DuPont de Nemours and Co. Inc. 1978; Gregory et al. 1981; Brown and Dietrich 1983; Prival et al. 1984; Reid et al. 1984; Krishna et al. 1986; Mortelmans et al. 1986; Zhou et al. 1987; ILS 2011b). While Direct Blue 15 and BABHS also showed some equivocal or positive results under metabolic activation only, incorporation of reductive conditions resulted in an increase in mutagenic potency for Direct Blue 15 (Prival et al. 1984; Reid et al. 1984). Incubation of Direct Blue 15 with mammalian cells did not produce increases in sister chromatid exchanges or chromosomal aberrations with or without metabolic activation, but the assays were not carried out under reductive conditions (Galloway et al. 1987).

An in vivo genotoxicity study and a carcinogenicity study were identified for Direct Blue 15. Direct Blue 15 produced DNA damage in the liver of male mice 3 hours after treatment (Tsuda et al. 2000). When rats were exposed to Direct Blue 15 (purity 50%; 35 impurities, including 3,3′-DMOB·2HCl [836–1310 ppm] and benzidine [ less than 1 ppm]) in their drinking water for 22 months (early termination due to extensive mortality associated with chemical-related neoplasia), neoplasms were observed as early as 9 months in the Zymbal gland and clitoral gland. At 22 months, neoplasms were found at multiple sites, including Zymbal gland, skin, oral cavity and preputial or clitoral gland, in a dose-dependent manner. Neoplasms were also observed in the small and large intestine, liver, uterus and brain. The incidence of mononuclear cell leukemia was also increased in female rats (NTP 1992). Given the dye’s low purity, there is uncertainty as to whether the effects observed in this otherwise well-conducted NTP study can be solely attributed to Direct Blue 15. It was, however, the only study identified for dyes based on 3,3′-DMOB.

IARC has evaluated Direct Blue 15 and has classified it as a group 2B carcinogen (possibly carcinogenic to humans). The working group has found that while there is inadequate evidence in humans, there is sufficient evidence in experimental animals for the carcinogenicity for Direct Blue 15 (IARC 1993a).

3,3′-DMOB was classified as a group 2B carcinogen (possibly carcinogenic to humans) by IARC; the working group concluded that “there is sufficient evidence in experimental animals for the carcinogenicity of 3,3′-dimethoxybenzidine” (IARC 1974a, 1987a, 2010b). In the EU, 3,3′-DMOB and 3,3′-DMOB-based azo dyes are classified as carcinogenicity category 1B (known to be carcinogenic to humans) with hazard code H350 (“may cause cancer”) (ESIS ©1995–2012). 3,3′-DMOB and dyes metabolized to 3,3′-DMOB are listed as “reasonably anticipated to be human carcinogens” in the US NTP Report on Carcinogens (NTP 1998, 2011). Data on the hydrochloride salts of 3,3′-DMOB were considered relevant, as these salts are expected to dissociate in physiological media to generate 3,3′-DMOB and are therefore considered to be toxicologically equivalent.

Following administration of 3,3′-DMOB·2HCl in the drinking water for up to 21 months, tumours were induced in rats at multiple sites, including the skin, Zymbal gland and intestines in both sexes, as well as in the liver, preputial gland and oral cavity in males and in the clitoral and mammary glands in females (Morgan et al. 1990; NTP 1990). The study was originally intended to have a 2-year treatment period, but it was terminated at 21 months because of reduced animal survival in all dose groups, primarily due to neoplasm-related deaths. At an interim (9 months) sacrifice of some high-dose animals, malignant and benign tumours were observed at multiple sites, indicating an early onset of some treatment-related tumours. At the 21-month time point, tumour incidences were clearly increased and showed a dose–response relationship.

Rats given 3,3′-DMOB by stomach intubation for 12–13 months developed tumours in the bladder, intestine, skin, ovaries, mammary gland and Zymbal gland (Pliss 1963, 1965; Hadidian et al. 1968). In a study in which male and female hamsters were given 3,3′-DMOB in the diet for 144 weeks (approximately 2.7 years), there was a significant increase in the incidence of forestomach papillomas, and one treated animal developed a rare transitional cell carcinoma in the bladder (Saffiotti et al. 1967; Sellakumar et al. 1969). No evidence of carcinogenicity was observed in a 2-year drinking water study in mice (Schieferstein et al. 1990).

No studies on the relationship between exposure to 3,3′-DMOB alone and cancer in humans were identified. In the epidemiological studies identified, subjects were exposed to benzidine or other carcinogenic aromatic amines in addition to 3,3′-DMOB, and therefore the studies cannot be used to evaluate the effects of 3,3′-DMOB exposure specifically. IARC has concluded that the evidence for carcinogenicity in humans is insufficient (IARC 2010b).

Benchmark doses (BMD) associated with a 10% increase in tumour incidence above controls (i.e., the BMD₁₀) and the corresponding lower limit of a one-sided 95% confidence interval (BMDL₁₀) were derived for 3,3′-DMOB·2HCl using the US EPA Benchmark Dose Software (BMDS version 2.3.1) (US EPA 2013). The BMD approach, which includes dose–response modelling, provides a quantitative alternative to the traditional dose–response assessment. BMD₁₀ and BMDL₁₀ values were calculated for nine tumour sites from the 21-month rat drinking water study (NTP 1990) (see Appendix F for details). The lowest calculated BMD₁₀ for 3,3′-DMOB·2HCl is 0.32 mg/kg-bw per day, based on 3,3′-DMOB·2HCl-induced skin basal cell or sebaceous gland neoplasms in male F344/N rats; the lower 95% confidence limit (BMDL₁₀) for this value is 0.22 mg/kg-bw per day. BMD₁₀ and BMDL₁₀ values of 0.91 and 0.66 mg/kg-bw per day, respectively, were obtained for female clitoral gland neoplasms, which supports the high tumour-inducing potency of 3,3′-DMOB in rats. When the lowest BMDL₁₀ for 3,3′-DMOB·2HCl (0.22 mg/kg-bw per day) is adjusted for the molecular weight difference, it is equivalent to a BMDL₁₀ of 0.17 mg/kg-bw per day for 3,3′-DMOB. The derived BMDL₁₀ values are similar in magnitude to the previously published LTD10 (the lowest tumorigenic dose associated with a 10% increase in tumour incidence above controls), which is 0.12 mg/kg-bw per day for 3,3′-DMOB·2HCl (CPDB 2012).

The majority of in vitro and in vivo genetic toxicology test results for 3,3′-DMOB were positive. In vivo, 3,3′-DMOB induced chromosomal aberrations and sister chromatid exchanges in the bone marrow of mice treated by injection (Gorecka-Turska 1983; You et al. 1993). However, a micronucleus assay was negative in the bone marrow of mice treated by intraperitoneal injection and gave mixed results when the mice were dosed by gavage (Morita et al. 1997). DNA damage was detected by the comet assay in tissues of mice following oral gavage (Sasaki et al. 1999; Martelli et al. 2000); tests for sex-linked recessive lethal mutations in Drosophila melanogaster adults and larvae were negative (Yoon et al. 1985; Zimmering et al. 1989). In mammalian cells in vitro, 3,3′-DMOB was mutagenic in the mouse lymphoma assay (Mitchell et al. 1988; Myhr and Caspary 1988) and induced DNA and chromosome damage (Martin et al. 1978; Probst et al. 1981; Galloway et al. 1985; JETOC 2000; Martelli et al. 2000; Chen et al. 2003).In reverse mutation assays in bacteria (Ames test), 3,3′-DMOB was generally positive in Salmonella typhimurium strains TA98, TA100, TA102 and TA1538 with metabolic activation, but negative without metabolic activation. Negative results were obtained in TA1535 and TA1537 both with and without metabolic activation (Probst et al. 1981; Haworth et al. 1983; Krishna et al. 1986; You et al. 1993; Chung et al. 2000; Makena and Chung 2007). Other bacterial tests (SOS/umu, forward mutation) were negative both with and without metabolic activation (Nakamura et al. 1987; Von der Hude et al. 1988; Shimada et al. 1989).

The health effects of the 15 3,3′-DMOB-based substances are expected to be similar to those of 3,3′-DMOB, due to the release of 3,3′-DMOB following azo bond reductive cleavage.

3,3′-DMB and Benzidine-based Substances That May Release 3,3′-DMB

Eight of the Benzidine-based Substances may release 3,3′-DMB following azo bond reductive cleavage (refer to Table 18).

In vitro genotoxicity studies were found for Direct Blue 14 (also referred to as trypan blue), Direct Blue 25, Direct Red 2 and Acid Red 114. All four dyes were mutagenic in the presence of reductive metabolism followed by oxidative metabolism with S9 liver enzymes (Brown et al. 1978; Hartman et al. 1978; Elliott and Gregory 1980; Gregory et al. 1981; Prival and Mitchell 1982; Brown and Dietrich 1983; Prival et al. 1984; Reid et al. 1984; Joachim et al. 1985; De France et al. 1986; Krishna et al. 1986; Mortelmans et al. 1986; Cameron et al. 1987; Zeiger et al. 1987; Dellarco and Prival 1989; NTP 1991a; Chung et al. 2006). Acid Red 114 was also mutagenic without prior reduction; positive results were observed in the presence of metabolic activation for Salmonella typhimurium strains TA98 and TA1538 (ETAD 1983a, 1985, 1986; Mortelmans et al. 1986; NTP 1991a). Direct Red 2 exhibited mutagenicity in the bacterial forward mutation assay in the presence of metabolic activation; this is in contrast to its results in the Ames assay (Prival and Mitchell 1982). In mammalian cells, Direct Red 2 did not induce any DNA repair (Von der Hude et al. 1988), nor did Acid Red 114 induce sister chromatid exchange or chromosomal aberrations in Chinese hamster ovary cells with or without S9 activation (NTP 1991a). Reductive metabolism was, however, not used in these cytogenetic tests. Direct Blue 14 was positive for chromosomal aberrations, but negative for mouse lymphoma, and mixed results were obtained for induction of DNA repair and morphological transformation (Joneja and Ungthavorn 1968; Amacher and Zelljadt 1983; Joachim and Decad 1984; Kornbrust and Barfknecht 1984, 1985; Longstaff et al. 1984; Cameron et al. 1987; Von der Hude et al. 1988). Addition of 2 mM flavin mononucleotide (FMN) had no effect on induction of DNA repair, and no other assays conducted in mammalian cells were performed under reductive conditions.

Among the in vivo genotoxicity assays identified for these Benzidine-based Substances, a dose-dependent increase in chromosomal damage was observed in male mice exposed to Direct Red 2 by oral gavage. When mice were treated with both Direct Red 2 and acidified water, which reduces indigenous intestinal microflora, a significant decrease in micronucleus formation was observed (Rajaguru et al. 1999). Hepatocytes of rats orally exposed to purified Direct Blue 14 displayed unscheduled DNA synthesis (Joachim and Decad 1984). In addition, urine extracts of rats treated with Direct Blue 14 at 500 mg/kg-bw were weakly mutagenic in TA1538 with metabolic activation (Joachim et al. 1985). Chromosomal aberrations were also found in normal-looking embryos from pregnant mice treated on gestational day 9 with Direct Blue 14 at 500 mg/kg-bw (Joneja and Ungthavorn 1968). DNA repair and DNA damage assays, however, were negative for Direct Blue 14 (Kornbrust and Barfknecht 1985; Tsuda et al. 2000). Similarly, no increase in sex-linked recessive lethal mutations in Drosophilia melanogaster and no DNA damage in mouse tissues were observed following exposure to Acid Red 114 (Woodruff et al. 1985; Tsuda et al. 2000).

Carcinogenicity studies were identified for Direct Blue 14 and Acid Red 114. Numerous studies using various suppliers of Direct Blue 14 and different routes of exposure have investigated the carcinogenicity of Direct Blue 14. Many of these studies were reviewed by IARC (1975). Direct Blue 14 produced reticulum cell sarcomas, mainly of the liver, as well as fibrosarcomas at the site of injection in rats following subcutaneous or intraperitoneal injection (Gillman and Gillman 1952; Simpson 1952; Brown and Thorson 1956; Brown 1963a, b; Papacharalampous 1966; Gillman et al. 1973; Field et al. 1977; Ford and Becker 1982). Tumours were found in other organs only when the liver was involved (Brown and Thorson 1956), indicating that metabolism of the dye was required for carcinogenicity. Liver spindle cell sarcomas were also induced in rats by single intraperitoneal injections (Papacharalampous 1957). In a 40-week study in which rats were exposed to four forms of Direct Blue 14 (Grubler, crude synthesized, dialysed and pure), a decrease in potency was observed as more purified forms of the dye were used. Only stage 1 or at most stage 2 changes in the liver were observed when the rats were treated with the purified dye (Field et al. 1977). Experiments conducted in rats that were orally exposed to Direct Blue 14 or in mice exposed by subcutaneous injection were not evaluated by IARC because of the small number of animals used or because the adequacy of the dose used could not be assessed. In a more recent study, neoplasms were observed in the liver and occasionally in the portahepatic lymph nodes of female mice subcutaneously exposed to Direct Blue 14 for 52 weeks. Similar types of tumours were also seen following a 6-week treatment and a latency period of 20 months (Ford and Becker 1982).

Acid Red 114 was tested for carcinogenicity in a 2-year drinking-water study. A clear carcinogenic response was produced in the skin, Zymbal gland and liver of male and female rats and in the clitoral gland, oral cavity epithelium, small and large intestine, and lung in female rats after 2 years. Treatment-related increases in the incidences of neoplasms were also seen in the oral cavity epithelium, adrenal gland and lungs of male rats and in mammary glands and adrenal glands in female rats. An increase in mononuclear cell leukemia was observed in female rats. Neoplasms were found at various sites at the interim time points of 9 and 15 months, and the number of neoplasms at these sites increased with time (NTP 1991a). The malignancy of moderately well and well differentiated preputial gland carcinomas, malignant epidermal basal cell tumours and epidermal squamous cell carcinomas from this 2-year study was confirmed in transplantation studies (Ulland et al. 1989).

IARC has reviewed Direct Blue 14 and Acid Red 114 and has classified each of them as a group 2B carcinogen (possibly carcinogenic to humans) (IARC 1987b, 1993b, 1997). 3,3′-DMB is also classified by IARC as a group 2B carcinogen (possibly carcinogenic to humans) (IARC 1972, 1987c, 2010d). 3,3′-DMB and 3,3’-DMB-based dyes are also classified as carcinogenicity category 1B in the EU, with hazard code H350 (“may cause cancer”) (ESIS ©1995–2012). 3,3′-DMB and dyes known to metabolize to 3,3′-DMB are listed as “reasonably anticipated to be human carcinogens” by the US NTP (NTP 2011). Data on the hydrochloride salts of 3,3′-DMB were considered relevant, as these salts are expected to dissociate in physiological media to generate 3,3′-DMB and are therefore considered to be toxicologically equivalent. The dihydrochloride salt of 3,3′-DMB  is also specifically addressed in this assessment.

The oral administration of 3,3′-DMB·2HCl in drinking water to rats for 14 months induced tumours at multiple sites in both sexes, including the skin, Zymbal gland, liver, oral cavity and gastrointestinal tract, as well as in the lung and preputial gland in males and in the mammary and clitoral glands in females (Morgan et al. 1991; NTP 1991b). This study was originally intended to have a 2-year treatment period, but it was terminated at 14 months because of reduced animal survival in all dose groups, due primarily to neoplasm-related deaths. At an interim (9 months) sacrifice of some high-dose animals, malignant and benign tumours were observed at multiple sites, indicating an early onset of some treatment-related tumours. At the 14-month time point, tumour incidences were clearly increased and showed a dose–response relationship. In an earlier study, 3,3′-DMB given to female rats by gavage induced mammary tumours (Griswold et al. 1968).

In mice given 3,3′-DMB·2HCl in drinking water for up to 116 weeks, there was a dose-related increase in the incidence of lung neoplasms, but only in males that died or were sacrificed moribund before study termination (Schieferstein et al. 1989). Zymbal gland tumours were observed in two studies in which rats were administered 3,3′-DMB by subcutaneous injection (Spitz et al. 1950; Pliss and Zabezhinsky 1970). A carcinogenic effect was not observed in hamsters following oral administration of 3,3′-DMB (Saffiotti et al. 1967). No case reports or epidemiological studies on 3,3′-DMB were identified.

BMD₁₀ and the corresponding BMDL₁₀ values were derived for 3,3′-DMB·2HCl using the US EPA Benchmark Dose Software (BMDS version 2.3.1) (US EPA 2013). BMD₁₀ and BMDL₁₀ values were calculated for 11 tumour sites from the 14-month rat drinking water study (NTP 1991b) (see Appendix G for details). The lowest calculated BMDL₁₀ for 3,3′-DMB·2HCl is 0.51 mg/kg-bw per day, derived from one of the lowest calculated BMD₁₀ values of 1.07 mg/kg-bw per day, based on 3,3′-DMB·2HCl-induced skin basal cell neoplasms in male F344/N rats. In addition, BMDL₁₀ and BMD₁₀values of 0.59 and 0.76 mg/kg-bw per day, respectively, were obtained for female clitoral gland neoplasms, which supports the high tumour-inducing potency of 3,3′-DMB in rats. When the lowest BMDL₁₀ for 3,3′-DMB·2HCl (0.51 mg/kg-bw per day) is adjusted for the molecular weight difference, it is equivalent to a BMDL₁₀ of 0.38 mg/kg-bw per day for 3,3′-DMB. The derived BMDL₁₀ values are similar in magnitude to the previously published LTD10 (the lowest tumorigenic dose associated with a 10% increase in tumour incidence above controls), which is 0.08 mg/kg-bw per day for 3,3′-DMB·2HCl (CPDB 2012).

The majority of in vitro and in vivo genetic toxicology test results for 3,3′-DMB were positive. In vivo, 3,3′-DMB induced micronuclei in rats and mice treated orally by gavage (Cihak 1979; Rajaguru et al. 1999), but not in mice administered 3,3′-DMB by intraperitoneal injection (Morita et al. 1997). Chromosomal aberrations were induced in bone marrow when mice were given 3,3′-DMB by intraperitoneal injection (You et al. 1993), and DNA damage was observed (by comet assay) in all tissues examined from mice following a single oral (gavage) dose (Sasaki et al. 1999). 3,3′-DMB also induced sex-linked recessive mutations in Drosophila when given in feed or by injection (NTP 1991b). In mammalian cells in vitro, 3,3′-DMB was mutagenic both with and without metabolic activation in mouse lymphoma assays (Mitchell et al. 1988; Myhr and Caspary 1988). DNA and chromosome damage were induced without activation, but mixed results were obtained in the presence of metabolic activation (Martin et al. 1978; Waalkens et al. 1981; Kornbrust and Barfknecht 1984; Barfknecht et al. 1987; Galloway et al. 1987; NTP 1991b). In mutagenicity tests in bacteria (Ames assay), 3,3′-DMB was positive in the frameshift mutation-detecting strains TA98 and 1538 only in the presence of metabolic activation. In other strains (TA97, TA100, TA102, TA1535 and TA1537), negative results were generally observed both with and without activation (Tanaka et al. 1980; Waalkens et al. 1981; Tanaka et al. 1982; Omar 1983; Kennelly et al. 1984b; Prival et al. 1984; Reid et al. 1984; De France et al. 1986; Krishna et al. 1986; Zeiger et al. 1988; NTP 1991b; You et al. 1993; Makena and Chung 2007). Mixed results were obtained in bacterial DNA damage tests (SOS/umu) (Oda et al. 1995; Oda 2004).

The health effects of the 3,3′-DMB-based substances are expected to be similar to those of 3,3′-DMB, due to the release of 3,3′-DMB following azo bond reductive cleavage.

Benzidine-based Dye That May Release 3,3′-DCB

Direct Red 46 releases 3,3′-DCB upon azo bond reductive cleavage (refer to Table 18).

Direct Red 46 was not mutagenic in the Ames assay in the presence or absence of metabolic activation (Joachim and Decad 1984; Reid et al. 1984). Mutagenicity was observed in Salmonella typhimurium strains TA98 and TA1538 in the presence of metabolic activation only after reductive conditions, such as Prival modifications, incubations with sodium dithionite or incubations with rat cecal bacteria were incorporated into the assay (Gregory et al. 1981; Reid et al. 1984). One study that used the Prival modifications, however, observed no mutagenicity in TA98 following incubation with the purified dye (ILS 2011b). This may be attributed to concentrations that were not sufficiently high to achieve mutagenicity. Equivocal results were obtained for unscheduled DNA synthesis when the purified dye was either incubated with primary rat hepatocytes or orally administered to rats (Joachim and Decad 1984). DNA damage, however, was clearly detected in the liver of female rats exposed to Direct Red 46 for 4 weeks in their drinking water (Sagelsdorff et al. 1996). No carcinogenicity study was identified for Direct Red 46.

The Government of Canada has evaluated 3,3′-DCB  and concluded that it was harmful to human health, based on carcinogenicity and genotoxicity (Canada 1993b). It is also classified as a group 2B carcinogen (possibly carcinogenic to humans). The IARC working group concluded that “there is sufficient evidence in experimental animals for the carcinogenicity of 3,3′-dichlorobenzidine” (IARC 1974b, 1982b, 1987b, 2010c). The health effects of Direct Red 46 are expected to be similar to those of 3,3′-DCB, due to the release of 3,3′-DCB following azo bond reductive cleavage.

Benzidine-based Substances That May Release 2,2′-Dimethylbenzidine (2,2′-DMB), 2,2′-Dichlorobenzidine (2,2′-DCB), 2,2′-Disulfobenzidine (2,2′-DSB), 3,3′-Dicarboxybenzidine (3,3′-DCAB) or 3,3′-Di(carboxymethoxy)benzidine (3,3′-DCMB)

Eight of the Benzidine-based Substances release benzidine derivatives that have not been assessed or classified by any national or international agencies (2,2’-DMB; 2,2’-DCB; 2,2’-DSB; 3,3’-DCAB; 3,3’-DCMB). Limited data were identified on these eight Benzidine-based Dyes or the benzidine derivatives that may be released.Therefore, SAR analysis was also considered. The hazard potential for non-benzidine aromatic amines released following azo bond cleavage was considered for one substance for which exposure

Benzidine-based Substances That May Release 2,2′-DMB or 2,2′-DCB

Acid Red 99 and BADB release 2,2′-dimethylbenzidine (2,2’-DMB; CAS RN 84-67-3) following azo bond reductive cleavage. In addition, data on Acid Red 111 (which also releases 2,2’-DMB) were included to inform the health effects assessment. BAHSD releases 2,2′-dichlorobenzidine (2,2’-DCB; CAS RN 84-68-4) following azo bond reductive cleavage.

In vitro genotoxicity assays indicate that Acid Red 111 and Acid Red 99 are mutagenic only in the presence of reductive conditions and metabolic activation in the Salmonellamutagenicity assay (Venturini and Tamaro 1979; Gregory et al. 1981; Zhou et al. 1987; ILS 2011a). Acid Red 111 was negative for bacterial DNA damage, but no reductive conditions were used in the assay (Kosaka and Nakamura 1990). BADB, however, was not mutagenic following reductive metabolism and metabolic activation in both TA98 and TA100 in the Salmonella mutagenicity assay for doses up to 1750 µg/plate (ILS 2011b). The top dose was considerably less than the 5 mg/plate recommended by OECD guidelines (OECD 1997), and it is therefore possible that the dye may be mutagenic at higher doses. No data were identified for BAHSD.

2,2′-DMB was mutagenic in TA98 and TA100 with metabolic activation, and 2,2′-DCB was positive for unscheduled DNA synthesis in HeLa cells (Martin et al. 1978; Hinks et al. 2000). Similar results were obtained in genotoxicity assays conducted for 3,3′-DMB and 3,3′-DCB, and there is no evidence to suggest that changing the position of the methyl group or the chlorine atom from the ortho to the meta position would eliminate mutagenicity.

The health effects of these four Benzidine-based Dyes are expected to be similar to those of 2,2-DMB and 2,2’-DCB, due to the release of 2,2-DMB or 2,2’-DCB following azo bond reductive cleavage.

Benzidine-based Substances That May Release 2,2′-DSB

Acid Red 97, Acid Orange 56 and NAADD release 2,2′-disulfobenzidine (2,2′-DSB; CAS RN117-61-3) following reductive cleavage of the azo bond.

Mixed results were obtained in in vitro mutagenicity assays for Acid Red 97: in one study, Acid Red 97 was mutagenic in the presence of reductive conditions and metabolic activation in TA98 and TA100, while in another, it was not (Gregory et al. 1981; ILS 2011a). An in vitro mutagenicity study conducted under reductive conditions was also identified for Acid Orange 56, but it was disregarded due to the low purity of the dye (41.8%) (ILS 2011a).

In vitro genotoxicity studies indicate that 2,2′-DSB is not mutagenic in five strains of Salmonella typhimuriumwith and without metabolic activation as well as with and without reductive conditions (ETAD 1989; NTP 1993; ILS 2011a). Furthermore, available data pertaining to other sulfonated aromatic amines indicate that these substances generally have no or very low genotoxic effects. In a review, Jung et al. (1992) showed that mutagenicity seen in several aromatic amines is absent in their corresponding sulfonated analogues. This was discussed for benzidines specifically in a review by Chung et al. (2006). Several groups have also postulated mechanistic considerations that may be responsible for the mitigating effect of sulfonation on the mutagenic potential of aromatic amines, including increased electronegativity and water solubility (Marchisio et al. 1976; Lin and Solodar 1988; OECD QSAR Toolbox 2011).

The other postulated azo bond cleavage product of Acid Red 97 is 1-amino-2-napthol (CAS RN 2834-92-6). The available data on this aromatic amine indicates that it is not directly mutagenic in vitro. (Garner and Nutman 1977; Chung et al. 1981; Freeman et al. 1987; Dillon et al. 1994)

Data on other endpoints were not available for Acid Red 97 or its metabolite 2,2’-DSB. Limited data were available on the metabolite 1-amino-2-napthol; however, the azo acid dye Orange II also releases this aromatic amine upon azo cleavage. In oral repeated-dose studies on Orange II in rats, spleen and blood effects were consistently observed, characteristic of amine-induced anemia (Hamann et al. 2000; Rofe 1957; Rosner 1999a,b; Singh and Khanna 1979; Singh et al. 1987). Effect levels in these studies ranged from 10 to 250 mg/kg-bw per day. No effects were observed in a skin painting study in which mice received weekly uncovered skin applications of Orange II for 18 months at approximately 5 mg/kg-bw per day (Carson 1984).

Benzidine-based Substance That May Release 3,3′-DCAB

BDAAH may release 3,3′-dicarboxybenzidine (3,3′-DCAB; CAS RN 2130-56-5) following azo bond reductive cleavage. No empirical data were identified for this dye. No empirical data were identified for the benzidine derivative 3,3′-DCAB. According to the Benigni/Bossa rulebase for mutagenicity and carcinogenicity, aromatic amines with a carboxylic acid group ortho to the nitrogen substituent may have diminished mutagenic potential (Benigni et al. 2008). The authors postulated that the carboxylic acid group in this position may hinder the metabolic activation of the nitrogen substituent to its reactive metabolite. This is further supported by studies on other aromatic amines (Takagi et al. 1995; ECJRC 2001).

Benzidine-based Substance That May Release 3,3′-DCMB

Direct Blue 158 releases 3,3′-di(carboxymethoxy)benzidine (3,3′-DCMB; CAS RN 3366-63-0) following azo bond reductive cleavage.

Direct Blue 158 was not mutagenic in Salmonella typhimurium strain TA98 or TA100 in the presence of metabolic activation alone or when reductive conditions and metabolic activation were incorporated into the bacterial mutagenicity assay (ILS 2011a).

No empirical data were identified for 3,3′-DCMB. No existing SAR analysis looking specifically at aromatic amines substituted with a glycolic acid group was identified. The presence of electron-donating groups in the ortho position to the amine (e.g., the divalent oxygen of the glycolic acid group) may promote the activation of the aromatic amine by stabilizing the nitrenium ion through delocalization of charge in the aromatic ring system. However, the glycolic acid group may also hinder N-hydroxylation (a precursor metabolic step in the formation of the nitrenium ion) due to steric hindrance at the adjacent ortho position. The glycolic acid group will also increase the water solubility of benzidine, which would favour the elimination of unchanged chemical and make N-hydroxylation (a precursor metabolic step in the formation of the nitrenium ion) less likely to occur (OECD 2011; DEREK Nexus 2010).

Benzidine-based Substances Without Azo Bond

The Benzidine-based Cationic Indicators TDBPD and TBDB and the Benzidine-based Precursor Naphthol AS-BR do not contain azo bonds. Therefore, the azo bond reduction mechanism of toxicity in which the benzidine derivative may be released as a metabolite through azo bond cleavage is not applicable to these substances.

TDBPD was negative for mutagenicity in all strains of Salmonella and Escherichia coli tested with or without metabolic activation (Venitt and Crofton-Sleigh 1979). It was also negative in the in vitro chromosomal aberration assay in Chinese hamster ovary cells with and without metabolic activation (Au and Hsu 1979). Simulated mammalian liver metabolism on the neutral form (non-chloride) of TDBPD was conducted using the software OASIS TIMES Mix version 2.27.3(Mekenyan et al. 2004). The preferred transformation of TDBPD involved the dealkylation of the methoxy group(s) to a hydroxyl group followed by sulfation or O-glucuronidation. No release of 3,3′-DMOB was predicted. However, since the structure was not in the domain of the model, the confidence in this prediction is considered low.

TDBD was positive in the Ames assay under reductive conditions in the presence of metabolic activation, but showed equivocal results in the presence of metabolic activation alone in Salmonella typhimurium strain TA98. The maximum response in TA98 with and without FMN was seen at the lowest dose tested, and the response decreased with higher doses. TDBD was also negative in TA100 with and without reductive conditions (ILS 2011a). Simulated mammalian liver metabolism on the neutral form (non-chloride) of TDBD was conducted using the software OASIS TIMES Mix version 2.27.3 (Mekenyan et al. 2004). The preferred transformation of TDBD involved the dealkylation of the methoxy group(s) to a hydroxyl group followed by sulfation or O-glucuronidation. No release of 3,3′-DMOB was predicted. Since the structure was not in the domain of the model, the confidence in this prediction is considered low.

No empirical data were identified for Naphthol AS-BR Simulated S9-mediated metabolism of Naphthol AS-BR was conducted using the software OASIS TIMES Mix version 2.27.3(Mekenyan et al. 2004). The metabolic tree created by the TIMES simulator indicates that the preferred metabolic transformation is O-glucuronidation or sulfation at the hydroxyl position on the naphthalene ring. No amide bond hydrolysis takes place, and therefore 3,3′-DMB is not released in the simulation. However, the structure was out of the model’s domain, indicating that the reliability of this prediction should be considered low. The physical and chemical properties of Naphthol AS-BR were also analysed; this substance was predicted not to be bioavailable due to its high log K_ow( greater than 5) and its high molecular weight ( greater than 500 Da), according to the Lipinski Rule of Five (Lipinski et al. 2001). Considering the likely low potential bioavailability of this compound, amide bond hydrolysis in vivo does not seem likely. Therefore, 3,3′-DMB is not likely released due to the metabolism of this compound.

Benzidine Derivatives: TODI and 4N-TMB

TODI

In a mutagenicity study in bacteria, TODI was found to be mutagenic in Salmonella typhimurium strains TA98 and TA1538 in the presence, but not in the absence, of metabolic activation. This Benzidine Derivative was negative in Salmonella typhimurium strains TA100, TA1535 and TA1537 and in Escherichia coli WP2uvrA, with or without metabolic activation. In the same study, TODI induced chromosomal aberrations in Chinese hamster lung cells in the presence, but not in the absence, of metabolic activation (JETOC 1996). In unpublished reports identified as submissions to REACH in the EU, TODI was positive for gene mutation in mammalian cells in the presence, but not in the absence, of metabolic activation; negative for unscheduled DNA synthesis in hepatocytes of rats given a single dose by oral gavage; and negative for micronuclei in the bone marrow of mice treated once by intraperitoneal injection (ECHA 2012). All of the studies identified for this Benzidine Derivative were from secondary sources; therefore, the reliability is uncertain.

The in vitro genotoxicity test results for TODI are consistent with the results for other benzidine derivatives, including 3,3′-DMOB and 3,3′-DMB. In addition, the related benzidine derivative 3,3′-dimethoxybenzidine-4,4′-diisocyanate (CAS RN 91-93-0), which can be hydrolysed to 3,3′-DMOB, was mutagenic in Ames tests in strain TA98 with metabolic activation and was carcinogenic to rats in a 2-year oral bioassay (NTP 1979; IARC 1986). As the only difference between 3,3′-dimethoxybenzidine-4,4′-diisocyanate and TODI is the change from methoxy groups to methyl groups at the 3,3′-positions, this implies that TODI could similarly be hydrolyzed to 3,3′-DMB.

4N-TMB

In vitro, 4N-TMB was mutagenic in Salmonellatyphimurium strain TA98 with metabolic activation in two studies. In the same studies, negative results were obtained in strain TA100, both with and without metabolic activation, and in strain TA98 without activation (Messerly et al. 1987; Chung et al. 2000). In a third bacterial mutagenicity study, negative results were obtained with Salmonella strains TA98, TA100, TA1535 and TA1537, both with and without activation (McCann et al. 1975). McConlogue et al. (1980) reported in an abstract that 4N-TMB was negative in a sister chromatid assay in Chinese hamster ovary cells. However, the authors also reported that the test compound was insoluble at concentrations equivalent to those at which benzidine induced sister chromatid exchanges.One research group has reported the peroxidase-catalyzed oxidative N-demethylation of 4N-TMB (O’Brien and Gregory 1985; McGirr and O’Brien 1987) and observed that the products of this biotransformation can bind DNA.

7.3 Characterization of Risk to Human Health

Exposure of the general population of Canada to Benzidine-based Substances and Benzidine Derivatives from environmental media is not expected due to limited commercial quantities in Canada; therefore risk from these sources is not expected.

7.3.1 Benzidine Derivatives

A review of the available health effects data on 3,3′-DMB and 3,3′-DMOB indicates strong evidence for the mutagenicity and carcinogenicity of these substances. In rat studies, tumours were observed at less than lifetime exposure durations and in both sexes at multiple sites. The mode of action of carcinogenicity is relatively well understood and likely occurs through the direct interaction of reactive intermediates with DNA. 3,3′-DMB·2HCl is expected to dissociate in physiological media and is therefore considered to be toxicologically equivalent to 3,3′-DMB. As such, the health effects information regarding 3,3′-DMB is considered applicable for 3,3′-DMB·2HCl.

In Europe and Japan, 3,3′-DMB and 3,3′-DMOB were detected in some textiles and leather products, some of which were reported to be imported from other countries, and may therefore be present in imported products in Canada, as the Canadian textile market is predominantly composed of imported products. Testing of products on the Canadian market, however, did not identify these two Benzidine Derivatives in imported and domestic textile products (Health Canada 2013). Overall, exposure to 3,3′-DMB and 3,3′-DMOB from textiles and leather is considered to be limited; direct and prolonged skin contact is not expected. Therefore, risk to human health for the general population from use of textile or leather products is not expected.

Potential daily oral exposure to3,3′-DMB from use of polyamide cooking utensils was conservatively estimated to range from 2 × 10⁻⁶ mg/kg-bw per day (12 years of age and older) to 6.5 × 10⁻⁶ mg/kg-bw per day (toddlers 0.5–4 years of age). The critical effect level for 3,3′-DMB is a BMDL₁₀ of 0.38 mg/kg-bw per day, based on skin basal cell neoplasms in male F344/N rats in a chronic oral study. Comparison of the upper-bounding estimate of oral exposure to3,3′-DMB with the critical effect level results in an MOE greater than 58,000, which is considered adequate to address uncertainties in the health effects and exposure databases.

Since 3,3′-DMB·2HCl, TODI and 4N-TMB are used as intermediates in chemical synthesis, the presence of these substances in Canadian or imported products is not expected, except potentially as residuals at very low levels. Exposure of the general population to these substances is not expected; therefore the risk to human health is not expected.

7.3.2 Benzidine-based Substances

The genotoxic and carcinogenic properties of benzidine-based dyes are due to the release of free benzidine, benzidine derivatives and other aromatic amines via reductive cleavage of the azo bond and subsequent conversion of these substances to reactive electrophilic intermediates (Environment Canada and Health Canada 2013). The human health effects of these substances are therefore assessed based on their capacity to undergo reductive azo bond cleavage and on the genotoxic and carcinogenic properties of the aromatic amines released. All of the available in vivo and in vitro metabolism data as well as reductive Ames test results for the Benzidine-based Substances with azo bonds support the potential for reductive cleavage. Two Benzidine-based Cationic Indicators and one Benzidine-based Precursor do not have azo bonds.

Benzidine-based Substances That May Release Benzidine, 3,3′-DCB, 3,3′-DMOB or 3,3′-DMB

Following reductive azo bond cleavage, 26 Benzidine-based Substances are considered to have the potential to release benzidine or one of three other EU22 benzidine derivatives (3,3′-DCB, 3,3′-DMOB and 3,3′-DMB) (Table 7-3). Benzidine and 3,3′-DCB have been previously assessed by the Government of Canada, while 3,3′-DMOB and 3,3′-DMB are substances currently being evaluated in this assessment. A review of the available data on these four benzidine derivatives clearly shows that they are carcinogenic and mutagenic. In addition, empirical data for genotoxicity are available for approximately half of the Benzidine-based Substances being evaluated in this assessment, and carcinogenicity data are available for five of these substances. Where data are available, the genotoxicity and carcinogenicity databases of the Benzidine-based Substances are consistent with those of the corresponding benzidine derivative. The potential health effects of these 26 Benzidine-based Substances can be attributed to the toxicity of the benzidine derivative released.

Based on the available information, no manufacture or import activities above the reporting threshold in Canada was confirmed for any of these Benzidine-based Substances. Direct Blue 14 was reported to be imported in Canada in the DSL Inventory Update survey for use as a laboratory substance in quantities below or equal to the reporting threshold. The four EU22 aromatic amines were occasionally detected in imported textiles and leather products in Europe and Japan and may therefore be present in imported products in Canada, as the Canadian textile market is predominantly composed of imported products. Testing of products on the Canadian market, however, did not identify these four aromatic amines in imported and domestic textile and leather products (Health Canada 2013). The detection of an EU22 aromatic amine does not indicate the presence or absence of an individual dye, as specific dyes are not identified by the testing method.

Exposure of the general population to any of these Benzidine-based Substances from textiles and leather is considered to be limited; direct and prolonged skin contact is not expected. Therefore, risk to human health is not expected.

Benzidine-based Substances that May Release 2,2′-DMB, 2,2′-DCB, 2,2′-DSB, 3,3′-DCAB or 3,3′-DCMB

There are three Benzidine-based Substances that may release 2,2′-DMB or 2,2′-DCB, following reductive cleavage of the azo bond (Table 7-4). It is considered that the benzidine derivatives 2,2′-DMB and 2,2′-DCB may be oxidized to active intermediates through the same pathway as for benzidine, 3,3′-DMB and 3,3′-DCB. The potential health effects of these three Benzidine-based Substances can be attributed to the toxicity of the benzidine derivative released. There are five Benzidine-based Substances that may release 2,2′-DSB, 3,3′-DCAB or 3,3′-DCMB, following reductive cleavage of the azo bond (Table 7-4). Limited international data is available on the presence of non-EU22 aromatic amines in textiles. Testing of products on the Canadian market did not identify the aromatic amines 2,2′-DMB or 2,2′-DCB in imported and domestic textile and leather products (Health Canada 2013).

Only one substance (Acid Red 97) had confirmed commercial activity in Canada based on information from industry (2010 email from ETAD to Program Development and Engagement Division, Environment Canada; unreferenced). The major use of Acid Red 97 is to dye leather and textile materials, which may potentially be used for consumer products that lead to direct exposure. Conservative estimates of dermal exposure to Acid Red 97 due to skin contact with textile clothing and leather articles range from 0.0026 to 0.0040 mg/kg-bw per day and from 2.1 × 10⁻³ to 7.7 × 10⁻² mg/kg-bw, respectively (see Appendix E).  Based on the available data, carcinogenicity and genotoxicity are not expected to be endpoints of concern for Acid Red 97, and the hazard potential is low. Therefore, risk to human health for the general population from Acid Red 97 in textiles and leather is considered to be low.

Exposure of the general population to any of the remaining Benzidine-based Substances from textiles and leather is considered to be limited; direct and prolonged skin contact is not expected. Therefore, risk to human health is not expected.

Benzidine-based Substances Without Azo Bond

The three Benzidine-based Substances without an azo bond are not expected to result in direct exposure of the general population due to their general use as laboratory reagents and chemical precursors. In addition, none of these three Benzidine-based Substances are expected to release aromatic amines by azo bond reductive cleavage. Therefore, risk to human health is not expected.

7.3.3 Uncertainties in Evaluation of Risk to Human Health

Acid Red 97 was identified in a list of dyes known by ETAD as being sold or used in Canada (personal communication, email from ETAD to Environment Canada, dated 2010; unreferenced); however, the specific use of Acid Red 97 in Canada was not indicated. It is assumed, based on the information available elsewhere (CII 2013), that Acid Red 97 is used as a dye for textiles and leather that may come in contact with consumers in Canada.

The detection of the Benzidine Derivatives 3,3′-DMB and 3,3′-DMOB in the product surveys (EurAzos 2007; Kawakami et al. 2010; RAPEX 2012) is specific primarily to imported textile and leather products in the European and Japanese markets, and uncertainty exists in the potential exposure of the general population of Canada to these textile and leather products based on these data. Uncertainty is also recognized in the results of testing of products on the Canadian market (Health Canada 2013), which involved a limited number of samples. Despite the uncertainties, the results are consistent with the global phase out of dyes based on EU22 aromatic amines, which include 3,3′-DMB and 3,3′-DMOB.

Exposure estimates presented in this assessment assume conditions of intended or reasonably foreseeable use behaviours resulting in direct and/or prolonged exposure. They are developed for use of polyamide cooking utensils, dermal contact with textile apparel, mouthing of textile objects (e.g., toys and blankets) by infants and dermal contact with leather articles (e.g., apparel, furniture, and children’s toys). These exposure scenarios are considered to represent the predominant sources of exposure based on use patterns. There exists uncertainty in some of the parameter inputs used in estimating exposures. However, conservative assumptions were used to yield upper-bounding exposure estimates, such as considering full adult body coverage for dermal exposure via textile apparel.

Since Acid Red 97 is relatively water soluble, the effect of laundering is expected to significantly reduce any dye that is not fixed to the textile fibre, thereby reducing the exposure after the initial washes. This effect was not factored in the derivation of exposure; therefore the estimated exposures to Acid Red 97 from textiles are conservative.

Overall confidence in the health effects database ranges from low to high for the Benzidine-based Substances and Benzidine Derivatives in this assessment. For the Benzidine-based Substances that were evaluated for azo bond cleavage, confidence is high that aromatic amines are released. Confidence is also high in the approach taken in which the potential hazard of aromatic amine metabolites was used to infer the potential hazard of the Benzidine-based Substances. However, uncertainty is recognized in association with the identity of the metabolites and the relative efficiency of in vivo azo reductive cleavage.

For approximately one-third of these substances, all of which are based on benzidine, 3,3′-DMOB, 3,3′-DMB or 3,3′-DCB, the identity of the metabolites produced in vivo was confirmed. The relative efficiency of azo bond reductive cleavage in vivois considered highly variable, and uncertainty around the degree of cleavage in vivo also results in uncertainty in the amount of aromatic amine metabolites released.

There is more uncertainty in the health effects databases for the Benzidine-based Substances based on benzidine derivatives other than benzidine, 3,3′-DMOB, 3,3′-DMB or 3,3′-DCB. Those substances were generally more data poor; for example, azo bond cleavage was inferred from reductive Ames tests or by general read-across: no in vivo or in vitro metabolism studies were available to confirm the presence or identity of aromatic amine metabolites, including the benzidine derivatives. Empirical hazard data were limited to in vitromutagenicity assays for the substances or metabolites. Further mutagenicity testing of parent Benzidine-based Substances under reductive conditions would increase the confidence in the health effects database for data-poor Benzidine-based Substances.

The carcinogenic potential of benzidine has been demonstrated in humans. As the pathway for tumour induction is suspected to be the same for Benzidine Derivatives, including 3,3′-DMOB and 3,3′-DMB, it is reasonable to assume that 3,3′-DMOB and 3,3′-DMB are also potential human carcinogens. Similarly, it is reasonable to assume that substances capable of releasing benzidine and the Benzidine Derivatives 3,3′-DCB, 3,3′-DMB and 3,3′-DMOB upon reductive cleavage of the azo bond are potential human carcinogens. For the Benzidine-based Substances that release 2,2′-DMB and 2,2′-DCB, it is possible that tumours could be induced in humans by the same pathway as those based on benzidine. However, there is not enough information in the toxicity database to determine the carcinogenic potential of these dyes with confidence.

There is also uncertainty associated with substance purity; in many instances, the purity of the substance used in experimental studies was not reported, or the substance was reported to contain a number of impurities, including their corresponding benzidine and other aromatic amine precursors.

7.3.4 Benzidine Derivatives and Benzidine-based Substances with Human Health Effects of Concern

Overall, human health risk from the substances in this assessment is low based on the current levels of exposure. However, as indicated above, some of the Benzidine Derivatives, Benzidine-based Acid Dyes, Benzidine-based Direct Dyes, and Benzidine-based Precursors in this assessment have human health effects of concern based on potential carcinogenicity. These include the Benzidine Derivatives 3,3′-DMOB, 3,3′-DMB and 3,3′-DMB·2HCl, as well as those Benzidine-based Substances which may release benzidine, 3,3′-dichlorobenzidine (3,3′-DCB), 3,3′-DMB, 3,3′-DMOB, 2,2′-dimethylbenzidine (2,2′-DMB) or 2,2′-dichlorobenzidine (2,2′-DCB). A list of these substances with human health effects of concern based on potential carcinogenicity are shown in Appendix H.

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8. Conclusion

Considering all available lines of evidence presented in this Screening Assessment, there is low risk of harm to organisms and the broader integrity of the environment from the 42 Benzidine-based Dyes and Related Substances evaluated in this assessment. It is concluded that these Benzidine-based Dyes and Related Substances do not meet the criteria under paragraphs 64(a) or 64(b) of CEPA 1999, as they are not entering the environment in a quantity or concentration or under conditions that have or may have an immediate or long-term harmful effect on the environment or its biological diversity or that constitute or may constitute a danger to the environment on which life depends.

Based on the information presented in this Screening Assessment, it is concluded that the Benzidine-based Dyes and Related Substances evaluated in this assessment do not meet the criteria under paragraph 64(c) of CEPA 1999, as they are not entering the environment in a quantity or concentration or under conditions that constitute or may constitute a danger in Canada to human life or health.

It is concluded that the Benzidine-based Dyes and Related Substances evaluated in this assessment do not meet any of the criteria set out in section 64 of CEPA 1999.

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