Appendices of the Final Screening Assessment Petroleum Sector Stream Approach Gas Oils [Industry-Restricted] Chemical Abstracts Service Registry Numbers 64741-59-9 64741-82-8 Environment Canada Health Canada July 2013

Lowest dermal LOAEL: 50 mg/kg-bw per day was identified based on decreased maternal rat body weight gain and body weight (likely due to reduced feed consumption), as well as skin irritation. Pregnant Sprague-Dawley rats were exposed to 0, 25, 50, 125, 250 and 500 mg/kg-bw per day of Mobil LCO on GDs 0–19 and to 1000 mg/kg-bw per day on GDs 6–15. Increased cholesterol and triglycerides were observed at doses greater than or equal to  250 mg/kg-bw per day, and severe sensory irritation was noted at doses greater than or equal to  500 mg/kg-bw per day (Mobil 1988a).

Other dermal studies:
API 83-07 (0, 250, 500 and 1000 mg/kg-bw per day) and API 83-08 (0, 200, 1000 and 2000 mg/kg-bw per day) were applied to the clipped dorsal skin of New Zealand White rabbits (five of each sex per group) 3 times per week for 4 weeks. Dose-dependent skin irritation was observed, from no irritation in the control groups to moderate and severe irritation in the test groups. Histological examination revealed moderate to severe proliferation and inflammation in the high-dose groups. Other findings were not considered treatment related and included decreased body weight gain and body weight, reduced ovary weight, hypoplasia of the seminiferous tubules and mortality (API 1985c,d).

Dermal NOAEL: 25 mg/kg-bw per day. Male Sprague-Dawley rats were exposed to Mobil LCO at 0, 8, 25, 125, 500 or 1250 mg/kg-bw per day, 5 times per week for 13 weeks (the highest dose was applied for only 2 weeks). Test substance was applied unoccluded to the clipped back skin of 10 animals of each sex per group. Dose-dependent, slightly reduced thymus weights (likely due to lymphocyte depletion) were observed at 125 mg/kg-bw per day. Severe erythema and edema with visibly thick, stiffened skin were observed in the 500 mg/kg-bw per day group; histological examination confirmed moderate chronic inflammatory changes in the skin and hair follicles. Systemic toxicity was noted at 500 and 1250 mg/kg-bw per day (Mobil 1985).

Other dermal study: In a similar study, a statistically significant increase in relative liver weights occurred in male and female Sprague-Dawley rats (TAC:N(SD)fBR MPF) dermally exposed to LCO at 500 mg/kg-bw per day for 13 weeks (Feuston et al. 1994). Liver weights of animals exposed to 1250 mg/kg-bw per day were not reported.

Lowest dermal LOAEL: 30 mg/kg-bw per day was identified based on increased lymphocytes in female rats and a 10% decrease in thymus weight in male rats. Sprague-Dawley rats (10 animals of each sex per group) were exposed 5 days/week for 13 weeks to 30, 125, 500 or 2000 mg/kg-bw via application of the substance to shaved skin. At doses greater than or equal to  125 mg/kg-bw, changes in megakaryocytes, increased lymphocytes and decreased body weight in male rats were observed. Additional effects were observed at doses greater than or equal to  500 mg/kg-bw, including severe skin irritation and decreased body weight in females. Daily exposure to the highest dose, 2000 mg/kg-bw, resulted in increased leukocytes and segmented neutrophils, as well as a reduction in erythropoietic cells and megakaryocytes. Basophilia in the renal tubular cortex was also observed in male rats (Mobil 1991).

Other dermal study: Sprague-Dawley rats were exposed to 30, 125, 500 or 2000 mg/kg-bw per day of test substance 5 days/week for 13 weeks. Increased relative liver weights in male and female rats were noted at 125 mg/kg-bw per day. Other possible effects, noted at unspecified doses, included decreased body and thymus weights, skin irritation and altered serum chemistry and hematology. However, the study examined several different substances, and the authors did not explicitly state whether any or all of the latter aforementioned effects were due to CAS RN 64741-82-8 (Feuston et al. 1994).

Chronic dermal studies
Lowest dermal effect level: 343 mg/kg-bw per day. A statistically significant (p less than 0.05) increase in the number of mice with squamous cell carcinomas or papillomas occurred after long-term dermal exposure to 343 mg/kg-bw per day of MD-7 LCO. Groups of male mice (C3H/HeNCrlBR; 50 per group) were exposed daily for 104 weeks to 35 µL of highly refined mineral oil (negative control), 50 µL of 5% heavy clarified oil (positive control), 28.5% MD-7 LCO (7 times per week = 343 mg/kg-bw per day^{[c] [d] [e] [f]}), 50% MD-7 LCO (4 times per week = 601 mg/kg-bw per application^{[c] [d] [e] [f]}) or 100% MD-7 LCO (2 times per week = 1203 mg/kg-bw per application^{[c] [d] [e]}). Of mice exposed to 28.5% (343 mg/kg-bw) MD-7 LCO, 7/50 developed skin tumours (compared with 0/50 in the negative control and 47/50 in the positive control), with the first tumour visible by day 301. Mice exposed to 50% (601 mg/kg-bw) MD-7 LCO also developed skin tumours (17/50, p less than 0.01), and these were confirmed histologically (the first tumour was visible by day 266). In addition to the 17 mice with confirmed tumours in this group, other mice had unconfirmed tumours (1/50) or developed only fibrosarcomas (4/50) or melanomas (1/50). An insignificant increase in skin tumours occurred in the group exposed to 100% (1203 mg/kg-bw) MD-7 LCO (1/50, tumour visible by day 651). The lack of tumours in this group was thought to be due to substantial cellular necrosis of skin cells due to the high concentration of the substance (Nessel et al. 1998).

Other chronic dermal studies:
Application twice weekly of 50 µL (1203 mg/kg-bw per application^{[c] [d] [e]}) LCCD to the shaved interscapular region of the backs of 50 C3H/HeJ male mice for 104 weeks resulted in a statistically significant (p less than 0.05) increased incidence of skin tumours, with 63% of mice developing at least one skin tumour (mean latency of onset = 79 weeks). Squamous cell carcinomas (13/50) and fibrosarcomas (12/50) were predominantly formed (compared with a zero tumour incidence in controls). An ~50% reduction in survival (relative to the control group) at 78 weeks was noted for mice exposed to LCCD, and only 6% survived to 104 weeks (compared with 52% of control mice) (Broddle et al. 1996).

Application twice weekly of 50 µL (1203 mg/kg-bw per application^{[c] [d] [e]}) LCCD to the shaved intrascapular region of the backs of 50 C3H/HeJ male mice for 104 weeks resulted in the formation of squamous cell carcinomas (54% of test mice) and papillomas (14% of test mice), as well as fibrosarcomas (24% of test mice) (39/50 test mice developed skin tumours, mean latency of onset = 40 weeks) (Skisak et al. 1994).

Initiation/promotion dermal studies
Initiation: 30 male CD-1 mice received five consecutive daily applications of 50 µL (1203 mg/kg-bw^{[c] [d] [e]}) LCCD and then 50 µL of tumour promoter (phorbol-12-myristate-13-acetate) twice weekly for 25 weeks. Squamous cell papillomas and keratoacanthomas developed in 9/30 mice, but this was not considered statistically significant (3/30 tumours in the negative versus 30/30 in the positive control groups).
Promotion: 30 mice were initiated with 50 µL 7,12-dimethylbenzanthracene and then received 50 µL (1203 mg/kg-bw^{[c] [d] [e]}) LCCD twice weekly for 25 weeks. Mice treated with LCCD exhibited a statistically significant increased incidence of skin tumours (28/30 versus zero incidence in the negative control group), with a 90% incidence of squamous cell papillomas and a 33% incidence of keratoacanthomas. Two malignant tumours were noted in the LCCD-exposed group (Skisak et al. 1994).

Other dermal studies with similar results have been described in API (1989b).

No oral or inhalation studies were identified.

Test substance was a blend of the CAS RNs listed. Two different formulations (ARCO Base LB-7979 and Provalent 4A) were used.

Chronic dermal studies:
C3H/HeJ mice (50 animals per group) were exposed to 50 mg (1389 mg/kg-bw^[c] [g]) ARCO LB-7979 twice per week for 80 weeks. Substance was applied to shaved interscapular skin. Benign skin tumours developed in 2/50 exposed mice after 17 weeks of observation, with a mean latency period of 14 weeks (the positive control group, exposed to benzo[a]pyrene, developed 3/50 tumours after 10 weeks). Following 37 weeks of observation, 47/50 mice in the test group had skin tumours (and of the mice with tumours, 29 were moribund), with a mean latency period of 24.5 weeks (28/50 mice in the positive control group had tumours, and 9 of the 28 were moribund after 27.8 weeks). After 80 weeks of observation, 46/47 mice in the test group had skin tumours (39 malignant, 7 benign) (in the positive control group, 47/49 mice had tumours, with 32 malignancies) (ARCO 1980a,b, 1981).

Provalent 4A was tested as above. After 17 weeks of observation, 16/50 exposed mice developed benign skin tumours, with a mean latency period of 15.8 weeks (3/50 mice of the positive control group had tumours after 10 weeks). Following 37 weeks of observation, 47/50 exposed mice had tumours, and 41 with tumours were moribund (mean latency period of 20.7 weeks) (27/50 mice in the positive control group had tumours after 27.8 weeks, and 9 with tumours were moribund). After 80 weeks of observation, 46/47 exposed mice had skin tumours (42 malignant, 4 benign) (47/49 in positive control, with 32 malignancies) (ARCO 1980a,b, 1981).

Dermal reproductive LOAEL: 1000 mg/kg-bw per day was identified based on a statistically significant increased incidence of resorptions after dermal application of 0, 25, 50, 125, 250 or 500 mg/kg-bw per day of Mobil LCO to 11-week-old pregnant CD rats (VAF/Plus Crl:CD(SD)BR) on GDs 0–19 and of 1000 mg/kg-bw per day on GDs 6–15 (Feuston et al. 1994).

Dermal developmental LOAEL: 1000 mg/kg-bw per day was identified based on statistically significant decreased fetal body weights after dermal application of 0, 25, 50, 125, 250 or 500 mg/kg-bw per day of Mobil LCO to the shorn dorsal skin of pregnant Sprague-Dawley rats on GDs 0–19 and of 1000 mg/kg-bw per day on GDs 0–6 and 6–15. Fetal body weights were decreased at 500 mg/kg-bw per day, but this was not statistically significant. No developmental malformations or reproductive effects were noted (Mobil 1988a).

Dermal studies:
Doses of 15, 60, 250 or 500 mg/kg-bw were applied to the shaved dorsal skin of pregnant Sprague-Dawley rats (10 animals per group) from GD 0–19. No differences were observed in the number of females who aborted, dams with viable fetuses, dams with resorptions, corpora lutea, implantation sites, percent preimplantation loss, viable fetuses or resorptions. No differences were observed in litter sizes, viable male/female fetuses, dead fetuses, fetal body weight or fetal crown-to-rump length (Mobil 1988b).

Pregnant Sprague-Dawley rats were exposed to 15 or 60 mg/kg-bw per day of test substance from GD 0–19 or to 250 mg/kg-bw per day from GD 0–15. No increased incidence of resorptions was observed (Feuston et al. 1994).

Sprague-Dawley rats (10 animals of each sex per dose) were exposed to test substance at 30, 125, 500 or 2000 mg/kg-bw per day, 5 days/week for 13 weeks. No effects were observed on spermatid and spermatozoa counts or morphology of testes and epididymides. Effects in females were not reported (Mobil 1991).

Cytogenetic assay
Negative: API 83-07 was administered by intraperitoneal injection to Sprague-Dawley rats (15 of each sex per group) at doses of 0, 0.2, 0.67 and 2 g/kg-bw. Bone marrow was obtained at 6, 24 and 48 h after exposure to the test substance. Lethargy and mortality were noted for some rats receiving the highest dose. API 83-07 was found not to affect the mitotic index of bone marrow cells. Testing of API 83-08, using the same protocol, was also observed to be negative (API 1985e, 1986d).

Sister chromatid exchange assay
Positive: API 83-07 was positive for sister chromatid exchange when administered to mice via intraperitoneal injection at doses of 340, 1700 and 3400 mg/kg-bw (API 1989a).

Mutagenicity
Positive: Of 10 petroleum middle distillates, MD-7 LCO exhibited the highest mutagenicity index (14) in a modified Ames assay. The mutagenicity of the 10 distillates positively correlated with the percentage of three- to seven-ring hydrocarbons (PAHs) found in each distillate, and MD-7 LCO contained 8.7% PAHs (Nessel et al. 1998).

Mouse lymphoma assay
Positive: API 83-07 was tested at 5–80 nL/mL (without activation) and 2.5–30 nL/mL (with activation) in a forward mutation assay using the cell line L5178Y TK +/−. Cells were exposed for 4 h followed by a 2-day recovery period. API 83-07 was negative for mutagenicity without activation but positive with activation. When tested with activation, API 83-07 exhibited a positive response for mutant frequency (API 1985f). In another study, API 83-08 was positive both with and without activation (API 1985g).

Sister chromatid exchange assay
Equivocal: API 83-07 was tested at 5–80 µg/mL in Chinese hamster ovary cells with and without activation. In the absence of activation, API 83-07 produced a statistically significant increase in sister chromatid exchanges per cell at 10 and 20 µg/mL (the highest concentrations for which data were available), but no concentration–response was observed. In a repeat study, an increase was statistically significant only at 30 µg/mL. With activation, API 83-07 exhibited statistically significant increases in the frequency of sister chromatid exchanges at 10, 40 and 80 µg/mL, but a clear concentration–response was not observed (API 1988).

Mutagenicity
Positive: DGMK No. 8 was tested with S9 metabolic activation in Salmonella typhimurium TA98 using a modified Ames assay. A mutagenic index of 2.1 was observed, and the test substance contained 8% PAH content (Blackburn et al. 1984, 1986; DGMK 1991).

Positive: Test substance was positive when tested at concentrations of 0.26–42 mg/plate, with and without S9 metabolic activation, in S. typhimurium TA98 and TA100 (Conaway et al. 1984).