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Apart from studies with the test substance geranyl acetate, studies using structurally similar compounds were taken into account to address the endpoint of genetic toxicity properly. Geranyl acetate and neryl acetate are trans/cis-isomers and therefore differ only in their relative orientation of functional groups within the molecule. Furthermore, relevant physicochemical parameters show comparability between geranyl acetate and neryl acetate. Relevant metabolites of geranyl acetate, i.e. hydrolysis product geraniol and the respective stereoisomer nerol are also suitable for read across. The hydrolysis and degradation of geranyl acetate into geraniol in compartments like plasma, liver and gastrointestinal tract was demonstrated in an in vitro study (for details see Chapter 7.1.1 Basic toxicokinetics, BASF SE 2013).

Mutagenicity in bacteria

In the chosen key study, a test performed by the National Toxicity Program of the US National Institutes of Health, bacteria strains S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 were treated with concentration of 0, 1, 3.3, 10, 33, 100, 333, 1000, 3333 µg (NTP, 1981) with and without metabolic activation by rat liver S9 mix and syrian hamster liver S9 mix. Incubation with geranyl acetate did not increase mutant frequencies. Therfore geranyl acaetate was found to be non-mutagenic under the chosen testing conditions.

In support, the stereoisomer neryl acetate has been tested in a study for mutagenicity in bacteria according to OECD 471 and GLP. Bacteria S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102 were treated with neryl acetate in DMSO at concentrations of 5 - 1500 ug/plate without metabolic activation and 5 – 5000 µg/plate with metabolic activation by an Aroclor 1254-induced rat liver S9 mix. Cytotoxicity was observed with or without metabolic activation. Neryl acetate did not induce any increase in the mutation frequency of the tester strains in the presence and absence of a metabolic activation system. In conclusion, these results indicate that neryl acetate under the experimental conditions described, was not mutagenic to Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and TA102 in the presence and absence of a metabolizing system.

 

Mutagenicity in mammalian cells in vitro

Conflicting data in literature are available to assess mutagenicity of geranyl acetate in mammalian cells in vitro. None of these studies have been conducted according to current OECD guidelines.

A mouse lymphoma assay with mouse lymphoma L5178Y cells was conducted according to the protocol of the US National Toxicity Program (NTP/Myhr, 1991). Cells were treated in three trials without metabolic activation and in 2 trials with metabolic activation by Aroclor 1254-induced male Fischer 344 rat liver S9 mix. No evident increase in mutant frequencies was observed after incubation with geranyl acetate without metabolic activation. In the presence of a metabolic system, geranyl acetate was evaluated as mutagenic. Two additional trials with the same test samples were performed 4 years later with different S9 mix cofactor concentrations. In these trials, no evident increase in mutant frequency was observed. Therefore the identified increase is mutant frequencies in the first 2 trials could not be reproduced. This increase also may have occurred only in the insoluble dose range, which was very difficult to determine because of the formation of minute droplets in the culture medium.

Further mouse lymphoma assays were published, showing increased mutant frequencies in geranyl acetate treated mouse lymphoma L5178Y cells in the presence of S9 mix, but not in the absence of S9 mix (Heck 1989, Caspary 1988, Tennant 1986). However, no increases in mutant frequencies were reported in geranyl acetate treated human lymphoblastoid (TK6) cells with or without metabolic activation.

In HPRT assays, reported in literature from short abstracts only, geranyl acetate was reported to be negative in the respective tests (Flowers 1988, Stankowski 1988).

In UDS tests in vitro reported in literature from short abstracts only, treatment of primary rat hepatocytes with geranyl acetate was reported to lead to no effects (Heck 1989, Mirsalis 1983).

Valid genotoxicity data from a study according OECD TG 476 and GLP is available for the relevant metabolite, i.e. hydrolysis product geraniol and the respective stereoisomer nerol. The reaction mass of geraniol and nerol (E- and Z-isomers) was tested in a HPRT test in CHO cells with and without metabolic activation (BASF SE, 2010). Cytotoxicity was found in the highest doses of 200 µg/ml for each condition. The test substance did not cause any relevant increase in the mutant frequencies in two independent experiments. Therefore, the reaction mass of geraniol and nerol is considered non-mutagenic under the chosen testing conditions.

 

Cytogenicity in mammalian cells in vitro.

Conflicting data in literature are available to assess cytogenicity of geranyl acetate in mammalian cells in vitro. None of these studies have been conducted according to current OECD guidelines.

A chromosomal aberration test with CHO cells was conducted according the protocol of the US National Toxicity Program (NTP/Galloway, 1987). No evident increase in chromosomal abberations was found after incubation with geranyl acetate (50-150 µg/ml in DMSO) in the presence or without metabolic activation. Geranyl acetate was found to be non-genotoxic under the chosen testing conditions.

A sister chromatid exchange assay with CHO cells was conducted according the protocol of the US National Toxicity Program (NTP - Galloway, 1987). Slight increases in SCEs was seen in 2 trials without metabolic activation and in one trial with metabolic activation. A second trial with metabolic activation did not reproduces the findings from the first trial. According to the authors, the increase in SCEs without S9 was at cytotoxic levels that severely delayed the cell cycle. In the first trial with S9, cytotoxicity was mentioned but precipitation at the highest concentration tested resulted in reduced toxicity. However, a relationship between cytotoxicity and induced increases in SCEs cannot be ruled out.

In a publication summarizing genotoxicity data in a short table, geranyl acetate was reported to be negative in an AMES, UDS in primary F344 male rat hepatocytes, CA and SCE in CHO cells (Tennant 1986).

 

Cytogenicity in mammals in vivo

In a micronucleus test according to the protocol of the US National Toxicity Program, geranyl acetate was administered to B6C3F1 mice for 3 consecutive days i.p. at doses of 0, 450, 900, 1800 mg/kg bw/d in corn oil (NTP – Shelby 1993). No increase in the number of polychromatic erythrocytes containing micronuclei was observed up the highest dose tested. Therefore, geranyl acetate was found to show no cytogenic activity under the chosen testing conditions.

In support, valid genotoxicity data from a study according OECD TG 474 and GLP is available for the relevant metabolite, i.e. hydrolysis product geraniol and the respective stereoisomer nerol.

The reaction mass of geraniol and nerol (E- and Z-isomers) was also tested in a MNT in NMRI mice (BASF SE, 2010). For this purpose, the test substance, dissolved in DMSO and emulsified in corn oil, was administered once orally to male animals at dose levels of 375 mg/kg, 750 mg/kg and 1500 mg/kg body weight in a volume of 10 mL/kg body weight in each case. The animals were sacrificed and the bone marrow of the two femora was prepared 24 and 48 hours after administration in the highest dose group of 1500 mg/kg body weight and in the vehicle controls. In the test groups of 750 mg/kg and 375 mg/kg body weight and in the positive control groups, the 24-hour sacrifice interval was investigated only. After staining of the preparations, 2 000 polychromatic erythrocytes were evaluated per animal and investigated for micronuclei. The normocytes with and without micronuclei occurring per 2 000 polychromatic erythrocytes were also recorded. As vehicle control, male mice were administered merely the vehicle, DMSO/corn oil (ratio 2:3), by the same route and in the same volume as the animals of the dose groups, which gave frequencies of micronucleated polychromatic erythrocytes within the historical vehicle control data range. Both positive control substances, cyclophosphamide for clastogenicity and vincristine sulfate for spindle poison effects, led to the expected increase in the rate of polychromatic erythrocytes containing small or large micronuclei. No inhibition of erythropoiesis determined from the ratio of polychromatic to normochromatic erythrocytes was detected.

According to the results of the study, the single oral administration of the reaction mass of geraniol and nerol did not lead to any increase in the number of polychromatic erythrocytes containing either small or large micronuclei. The rate of micronuclei was within the range of the concurrent vehicle control in all dose groups and at all sacrifice intervals and within the range of the historical vehicle control data. Thus, under the experimental conditions of this study, the reaction mass of geraniol and nerol does not induce cytogenetic damage in bone marrow cells of NMRI mice in vivo.

In a chromosomal aberration test in B6C3F1 mice according to the protocol of the US National Toxicity Program, a single i.p. administration of geranyl acetate at doses ranging from 425 to 1700 mg/kg bw in corn oil was performed and bone marrow has been assessed (NTP 1987). An increase in percent cells with chromosomal abberations has been observed at the highest dose tested. No further data on toxicity is available. The test substance has been retested under same treatment conditions but a dose range of 250 -1500 mg/kg bw. The authors stated, that the chosen testing conditions were according to a non-standard protocol. An increase in cells with chromosomal aberrations was not reproduced in these tests.

In a sister chromatid exchange (SCE) assay in mice according to the protocol of the US National Toxicity Program, a single i.p. administration of geranyl acetate at doses ranging from 425 to 1700 mg/kg bw in corn oil was performed and bone marrow has been assessed (NTP 1987). No evident increase in SCEs has been observed up to the highest dose tested. Therefore geranyl acetate did not show any cytogenic activity under the chosen testing conditions.

In a comet assay in mice, reported from literature (results from summary table), geranyl acetate was found to be negative (Sasaki, 2000).

Overall in a weight of evidence, the majority of test data incl. studies with higher validity demonstrated, that geranyl acetate is not genotoxic.


Endpoint Conclusion:

Justification for classification or non-classification

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