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Key value for chemical safety assessment

Additional information

Genotoxicity data for retinyl acetate and structurally and metabolically related substances, i.e. retinol, and retinyl palmitate are used to support the conclusions on genotoxicity. 

In the GLP key study for bacterial mutagenicity similar to OECD TG 471 , no mutagenic effects were noted in Salmonella typhimurium strains (TA 1535, TA 1537, TA 1538, TA 98, TA 100) in the presence or absence of metabolic activation for retinyl acetate up to 750 µg/plate (Chételat 1982 B-45410).

Several non-GLP screening studies for bacterial mutagenicity in line with OECD TG 471 were performed with samples from the technical synthesis process of vitamin A, namely “vitamin A Rohöl”, “vitamin A Endöl” or “vitamin A raffiniertes Öl” (BASF 2006 Rohöl-Endöl; BASF 40M0532/064088; BASF 40M0532/064126). Retinyl acetate is considered to be the main component in these samples, however, no detailed information of its composition is available. No increase in the number of revertants was observed with tester strains TA 1535, TA 1537, TA 98 and E. coli WP2 uvrA, whereas slight mutagenic effects  were observed in the tester strain TA 100. Impurities as cause for these findings cannot be ruled out.

 

Further supportive mutagenicity studies in bacteria with retinol and retinyl plamitate confirmed the absence of a mutagenic potential of retinol and respective esters.

In the GLP studies for bacterial mutagenicity similar to OECD TG 471 , no mutagenic effects were noted in Salmonella typhimurium strains (TA 1535, TA 1537, TA 1538, TA 98, TA 100) in the presence or absence of metabolic activation for retinyl palmitate (Chételat 1982 B-45409).

In the study for bacterial mutagenicity on retinol (47% in Polysorbate 20) according to OECD TG 471 and GLP, no mutagenic effects were noted in Salmonella typhimurium strains (TA 1535, TA 1537, TA 98, TA 100) and the E. coli strain (WP2 uvrA) in the presence or absence of metabolic activation (BASF 2000, 40M0097/994030).

 

In a HGPRT gene mutation assay, retinol was found to be non mutagenic with and without metabolic activation with S9 mix prepared from liver homogenate of Aroclor 1254-pretreated rats (Budroe 1988).

 

In a collaborative study with 10 participating laboratories to evaluate a test protocol of the in vitro micronucleus (MN) test with V79 cells, retinyl acetate has been tested in 3 laboratories without metabolic system (von der Hude 2000). An increase of the frequency of micronuclei has been observed in 1/3 laboratories only at the highest concentration tested (31.6 μg/ml), resulting also in remarkable precipitation, a decrease in proliferation index and an increase in the mitotic index. No information on test substance specification, i.e. impurities is given. 

 

In a chromosomal abberation test and SCE test in vitro using human fibroblasts (HE2144) without metabolic activation, retinyl acetate was found positive at the highest concentration (65.6 µg/ml) (Sasaki 1980). Furthermore, a chromosomal aberration test and SCE test without  metabolic activation using a fetal Syrian hamster (female) pulmonary epithelial cell line (M3E3/C3) showed a dose dependent increase in the presence of retinol (Mohr 1991). For both studies, no data on cytotoxicity or substance specification are available to confirm the validity of the chosen experimental conditions.

In contrast, a chromosomal abberation test using Chinese hamster fibroblasts without metabolic activation found retiny acetate to be negative (Ishidate 1978).

Furthermore, retinol was found negative in a chromosomal abberration test in vitro using primary human lymphocytes without metabolic activation (Cohen 1970) and both, a chromosomal abberation test and sister chromatid exchange test using Chinese hamster V79 cells showed no genotoxic potenial of retinol in the presence and absence of a metabolic system (Qin 1985).

 

Retinol and retinyl acetate were negative in a sister chromatid exchange test in vitro in Chinese hamster V79 cells without metabolic activation (Sirianni 1981).

An unscheduled DNA synthesis test in primary rat hepatocytes using retinol and retinyl acetate was negative up to cytotoxic concentrations in the absence of a metabolic system (Budroe 1987, Althaus 1982). 

 

In in vivo erythrocyte micronucleus tests in mice according to OECD Guideline 474, no statistically significant or biologically relevant differences in the frequency of erythrocytes containing micronuclei were found between the vehicle control and the 2 dose groups with “vitamin A Rohöl” or “vitamin A raffiniertes Öl” (1000, 2000 mg/kg bw; BASF 26M0378/064145; BASF 26M0532/064147). The test substances represent samples from the technical synthesis process of vitamin A and retinyl acetate is considered to be the main component. However, no detailed information of its composition is available. In conclusion, no chromosome-damaging (clastogenic) effect nor an impairment of chromosome distribution in the course of mitosis (aneugenic activity) in bone marrow cells in vivo was observed for these test samples, being indicative for an absence of clastogenicity/ aneugenicity of retinol and the respective ester.

A micronucleus test in ddY mice, scoring MNPCEs and total erythrocytes from bone marrow, is available in literature (Hayashi 1988). A single (37.5, 75, 150, 300 mg/kg bw) injection or 4 daily intraperitoneal injections of retinyl palmitate (300 mg/kg bw/d) did not increase the frequency of MNPCEs in any dose group, providing further evidence for the absence of a clastogenic potential of retinyl palmitate in vivo.

 

Taken all relevant studies with retinyl acetate or structural and metabolic analoges (i.e. retinol, retinyl palmitate,) retinyl acetate is not considered to be genotoxic in a weight of evidence.

Justification for classification or non-classification

The present data on genetic toxicity do not fulfill the criteria laid down in 67/548/EEC and 1272/2008/EEC and therefore, a non-classification is warranted.