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In vitro genotoxicity:

Several Fatty Acid Glycerides Category members were tested for their genotoxic potential in bacteria (Ames Test). In all testings Fatty Acid Glycerides were shown to be non-mutagenic in bacteria.

Salmonella typhimurium strains TA 97, TA 98 and TA100 were treated with the test material Glycerides, C12-18 (CAS No. 67701-26-2) diluted in EGDME using the Ames plate incorporation method according to OECD Guideline No. 471 (Kennelly, 1987). Test substance concentrations of 0, 1.6, 8, 40, 200, 1000 µg/plate were tested in triplicate, both with and without the addition of a rat liver homogenate metabolising system (S9). Limited solubility and precipitation of the test substance was observed at 1000 µg/plate, but at this dose the precipitated test agent did not interfere with the counting of colonies. Thus, 1000 µg/plate was chosen as the highest concentration fur mutation testing. The test material caused no cytotoxicity up to the highest, precipitating dose. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.

Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 were treated with the test material Glycerides, mixed decanoyl and octanoyl (CAS No. 73398-61-5) diluted in acetone according to EU Method B.13/14 (Schöberl, 1994). Test substance concentrations of 0, 8, 40, 200, 1000, 5000 µg/plate were tested in triplicate, both with and without the addition of a rat liver homogenate metabolising system (S9). Precipitation of the test substance was observed at 5000 µg/plate. The test material caused no cytotoxicity up to the highest, precipitating dose. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.

Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 were treated with the test material Castor oil, hydrogenated (CAS No. 8001-78-3) diluted in Tween 80/ bi-distilled water using the plate incorporation method according to OECD Guideline 471 (Banduhn, 1990). Test substance concentrations of 0, 8, 40, 200, 1000, 5000 µg/plate and 0, 6.25, 25, 100, 400, 1600 µg/plate (repetition test) were tested in triplicate, both with and without the addition of a rat liver homogenate metabolising system (S9). Precipitation of the test substance was observed, starting at a concentration of 200µg/plate. The test material caused no cytotoxicity up to the highest dose. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.

 

Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 were treated with the test material Glycerides, C14-18 and C16-22-unsatd. mono- and di- (CAS No. 91744-13-7) diluted in Tween 80/ bi-distilled water using the plate incorporation method according to OECD Guideline 471 (Banduhn, 1990). Test substance concentrations of 0, 8, 40, 200, 1000, 5000 µg/plate were tested in triplicate, both with and without the addition of a rat liver homogenate metabolising system (S9). The test material caused cytotoxicity in Salmonella strain TA 1535 at 5000 μg/plate. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.

 

An in vitro mammalian chromosome aberration test was performed with Castor oil (CAS No. 8001-79-4) in Chinese hamster Ovary (CHO) cells (Irwin, NTP report 1992). The occurrence of chromosome aberrations was investigated in the presence and absence of metabolic activation (rat liver S9-mix) with test substance concentrations of 0, 1600, 3000, 5000 µg/mL diluted with DMSO. Castor oil did not induce a significant increase in the number of phases with aberrations at any preparation time and dose level. No relevant cytotoxic effects were reported. Positive controls significantly increased the rate of chromosome aberrations indicating the sensitivity of the assay. In conclusion, Castor oil did not induce chromosome aberrations in Chinese hamster Ovary cells, neither in the presence nor in the absence of a metabolic activation system, under these experimental conditions.

An in vitro Sister Chromatid Exchange Assay was performed with Castor oil (CAS No. 8001-79-4) in Chinese hamster Ovary (CHO) cells (Irwin, NTP report 1992). The occurrence of sister chromatid exchanges was investigated in the presence and absence of metabolic activation (rat liver S9-mix) with test substance concentrations of 0, 160, 500, 1600 and 5000 µg/mL diluted with DMSO. Castor oil did not induce a significant increase in the number of sister chromatid exchanges at any dose level. No relevant cytotoxic effects were reported. Positive controls significantly increased the rate of sister chromatid exchanges indicating the sensitivity of the assay.

 

A chromosome aberration study was performed with glycerides, C8 -18 and C18 -unsatd. mono- and di, acetates (CAS No. 91052 -13 -0) according to OECD guideline 473 with CHU/IU cells (Seki, 2010). The occurrence of chromosome aberrations was investigated in the presence and absence of metabolic activation (rat liver S9-mix) with test substance concentrations of 0.02, 0.39, 0.078 and 0.156 mg/mL for 6 hours with and without S9-mix and 0.078, 0.156, 0.313, 0.625, 1.25, 2.5 and 5.0 mg/mL for 24 and 48 hours without S9-mix diluted in acetone. The test substance did not induce a significant increase in the number of aberrations at any preparation time and dose level. No relevant cytotoxic effects were reported. The test substance precipitated at concentration ≥ 0.078 µg/mL. Positive controls significantly increased the rate of chromosome aberrations indicating the sensitivity of the assay. In conclusion, the test substance did not induce chromosome aberrations in CHU/IU cells, neither in the presence nor in the absence of a metabolic activation system, under these experimental conditions.

Structural analogue read-across from Glycerides, C8-18 and C18-unsatd. mono- and di-, acetates (CAS No. 91052-13-0) for mammalian toxicity was judged to be justified for the following reasons: This read across substance is also a glyceride, containing mainly C12-14 fatty acid and actetate moiety as well as glycerol. It’s an organic liquid with a pour point of -8 °C and a melting point of 357.85 °C and a vapour pressure < 0.01 Pa at room temperature. In contrast to the glycerides of the fatty acid glyceride category, it has a higher water solubility of 8.75 mg/L, which might influence its environmental distribution, but not the mammalian metabolism upon systemic uptake. Therefore it is expected to feed into the same mammalian physiological pathways as the members of the fatty acid glyceride category, like citric acid cycle, sugar synthesis and lipid synthesis. These processes are described in detail within the category justification.

 

An in vitro mammalian chromosome aberration test was performed with the SALATRIM (short- and long-chain acyl triglyceride molecules) family of triacylglycerols in Chinese hamster Ovary (CHO) cells (Hayes, 1994). The occurrence of chromosome aberrations was investigated in the presence and absence of metabolic activation (rat liver S9-mix) with test substance concentrations of 0, 250, 500 and 1000 µg/mL diluted with acetone. The high dose was limited by the low solubility of the fats in the assay medium. The test substance did not induce a significant increase in the number of phases with aberrations at any preparation time and dose level. No relevant cytotoxic effects were reported. Positive controls significantly increased the rate of chromosome aberrations indicating the sensitivity of the assay. In conclusion, the SALATRIM family of triacylglycerols did not induce chromosome aberrations in Chinese hamster Ovary cells, neither in the presence nor in the absence of a metabolic activation system, under these experimental conditions.

 

An in vitro mammalian cell gene mutation assay was performed with the SALATRIM (short- and long-chain acyl triglyceride molecules) family of triacylglycerols in Chinese hamster Ovary (CHO) cells (Hayes, 1994). Gene mutations in the HPRT locus were investigated in the presence and absence of metabolic activation (rat liver S9-mix) with test substance concentrations of 0, 31.25, 62.5, 125, 250, 500 and 1000 µg/mL diluted with acetone. The high dose was limited by the low solubility of the fats in the assay medium. The test substance did not induce a significant increase in the mutant frequency at any preparation time and dose level. No relevant cytotoxic effects were reported. Positive controls significantly increased mutant frequency indicating the sensitivity of the assay. In conclusion, the SALATRIM family of triacylglycerols did not induce the mutant frequency in Chinese hamster Ovary cells, neither in the presence nor in the absence of a metabolic activation system, under these experimental conditions.

In vivo genotoxicity:

An in vivo Mammalian Erythrocyte Micronucleus Test was performed with the SALATRIM (short- and long-chain acyl triglyceride molecules) family of triacylglycerols in Crl:CD BR VAF/Plus rats according to OECD Guideline 474 (Hayes, 1994). Groups of 20 male and 20 female animals were exposed to either of two SALATRIM fats or corn oil at 10% (w/w) of the diet (equivalent to 7000 mg/kg bw/day) for at least 13 weeks. Erythrocytes were scored to determine the frequency of micro-nucleated erythrocytes. No signs of systemic toxicity in any of the treated animals were observed. No increases in the frequency of micronuclei in polychromatic erythrocytes of the femoral bone marrow of Crl:CD BR VAF/Plus rats exposed to 7000 mg/kg bw SALATRIM occurred. Because these data were collected from a 13-week sub-chronic toxicity study, a positive control for micronuclei formation was not included.

An in vivo Mammalian Erythrocyte Micronucleus Test was performed with Castor oil (CAS No. 8001-79-4) in B6C3F1 mice similar to OECD Guideline 474 (Irwin, NTP report 1992). 10 animals per group were treated with test substance concentrations of 0, 0.62, 1.25, 2.50, 5.00, 10.0 % (w/w) in the diet by oral feeding for 13 weeks (approx. 0, 917, 2022, 3800, 7823, 15017 mg/kg bw/day). Blood smears were prepared from peripheral blood samples obtained by cardiac puncture of dosed and control animals at the termination of the 13 week study. At least 2000 PCE and 10000 NCE from each animal were scored to determine the frequency of micro-nucleated erythrocytes. No signs of systemic toxicity in any of the treated animals were observed. No increases in the frequency of micronuclei in Peripheral Blood Erythrocytes of B6C3F1 mice exposed to Castor Oil in doses up to approx. 15000 mg/kg bw fed for 13 weeks occurred, whereas the positive control substance (0.2 % urethane) significantly increased the number of normochromatic and polychromatic erythrocytes with micronuclei in three control animals.

An in vivo Mammalian Erythrocyte Micronucleus Test was performed with Glycerides, C16-18 and C18-hydroxy mono- and di- (CAS No. 91845-19-1) in CFW 1 mice according to OECD Guideline 474 (Wallat, 1985). 7 animals received a single dose of the test substance diluted in peanut oil at a concentrations of 10000 mg/kg bw by oral gavage. 24 hours after application the femoral bone marrow was taken from both femurs. 1000 erythrocytes per animal were scored to determine the frequency of micro-nucleated erythrocytes. No signs of systemic toxicity in any of the treated animals were observed. No increases in the frequency of micronuclei in polychromatic erythrocytes of the femoral bone marrow of CFW 1 mice exposed to 10000 mg/kg bw Glycerides, C16-18 and C18-hydroxy mono- and di- occurred, whereas the positive control substance (10 mg cyclophosphamide/kg bw) significantly increased the number of polychromatic erythrocytes with micronuclei.

Considering the clearly negative results of all available in vitro and in vivo genotoxicity testings of Fatty Acid Glycerides and the proven physiological function of triglycerides, it can be assumed that all members of this category are not genotoxic, neither in vitro nor in vivo.

 


Short description of key information:
All available in vitro and in vivo genotoxicity studies were found negative, indicating that Fatty Acid Glycerides have no genotoxic potential.
Available in vitro genotoxicity studies on Fatty Acid Glycerides (CAS No.):
- Ames Test: 67701-26-2, 8001-78-3, 91744-13-7, 73398-61-5 and medium and long-chain triglycerides
- Chromosome Aberration: 8001-79-4, 91052-13-0 and medium and long-chain triglycerides
- Mammalian gene mutation test in vitro (HPRT): medium and long-chain triglycerides
- Sister Chromatide Exchange: 8001-79-4
Available in vivo genotoxicity studies on Fatty Acid Glycerides (CAS No.):
- Micronucleus assay: 8001-79-4, 91845-19-1 and medium and long-chain triglycerides

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

According to DSD (67/548/EEC) or CLP (1272/2008/EC) classification criteria for genetic toxicity, no classification is required.