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Mutagenicity in bacteria

Mutagenicity of citronellol in bacteria was analyzed in a study performed under GLP according to OECD guideline 471 and EU method B.13/14 (BASF, 1991). In this study bacteria strains S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 were treated with citronellol at concentrations of 8 - 500 µg/plate with and without metabolic activation by S9 fraction from Aroclor induced Sprague-Dawley rats. The strains S. typhimurium TA 100 and TA 98 were also exposed to 5000 µg/plate citronellol. Although bacteriotoxic effects at doses >= 500 µg/plate were noted, no increase in revertant colonies was observed. Therefore citronellol was not mutagenic in bacteria under the chosen testing conditions.

In a study in literature, performed by the National Toxicology Program (NTP) of the US National Institute of Health according to internal guidelines, bacteria strains S. typhimurium TA 1535, TA 97, TA 98 and TA 100 were treated with concentrations of 0, 1, 3, 10, 33, 100, 333 mg/plate citronellol in DMSO with and without metabolic activation by induced male Sprague Dawley rat liver S9 mix and induced male Syrian hamster liver S9 mix (NTP, 2002). Although bacteriotoxic effects were seen in the highest dose experiments, no increase in revertant colonies was observed. Therefore citronellol was not mutagenic in bacteria under the chosen testing conditions.

A variety of studies in literature are available, but were not taken into account for assessment due to methodological deficits:

Citronellol at concentrations of 8-16 ppm was not mutagenic in Salmonella typhimurium strains TA98 or TA100 (Kono, 1995). Thereby, strain TA98 was tested with and without S9 while TA100 was only tested without activation. Another Ames test was conducted where Salmonella typhimurium strains TA98 or TA100 were treated with urine fractions of two Sprague-Dawley rats gavaged with 0.5 ml citronellol (Rockwell, 1979). The tests were reported to be negative. A Japanese study reported a Bacillus subtilis recombination assay with Bacillus subtilis strains H17 and M45 which were treated with 17 µg/disc citronellol (Oda, 1978). As a result no genetic toxicity was found.

In order to support the data available for citronellal, data from an AMES test performed with the structurally comparable compound nerol (cis-3,7-dimethyl-2,6-octadien-1-ol) are included into the weight of evidence. Inclusion of nerol into this assessment is justified by the structural similarity between nerol and citronellol. Both structures are identical except for an additional double bond found in nerol. The structure of nerol is considered to represent a worst case compared to citronellol (3,7-dimethyloct-6-en-1-ol) based on this second double bond as possible additional reactive feature. A putative steric hindrance to the action of cytochromes such as those present in the S9 mix due to the additional double bond is not to be expected (see Chapter “Toxicokinetics, metabolism and distribution”). Furthermore, relevant physicochemical parameters show comparability between nerol and citronellol (molecular weight of 154.2 and 156.3 ; log Pow at 2.6 and 3.41; vapour pressure of 1 and 8.6 Pa; water solubility of 769 and 307 mg/l respectively).

Genetic toxicity of nerol was analyzed in a bacterial reverse mutation assay performed under GLP according to OECD guideline 471 (Symrise, 2000). Bacteria strains S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102 were treated with nerol at concentrations of 5, 15, 50, 150, 500, 1500 and 5000 µg/plate with and without metabolic activation by Aroclor-induced rat liver S9 mix. As a result, cytotoxicity was observed at 1500 ug/plate in TA100 and TA 102, and in TA98, TA1535 and TA1537 at a concentration of 5000 µg/plate. However, nerol was found to be not mutagenic under conditions of the study

Overall in a weight of evidence and on the basis of the data available, citronellol is considered to be not mutagenic in bacteria.

Mutagenicity in mammalian cells

No valid key study is available for the assessment of the mutagenicity of citronellol in mammalian cells in vitro.

However, data from a key study performed with a reaction mass consisting of the structurally comparable compounds nerol (cis-3,7-dimethyl-2,6-octadien-1-ol) and the respective stereoisomer geraniol (trans-3,7-dimethyl-2,6-octadien-1-ol) were used to address this endpoint via read across. Inclusion of nerol/geraniol into this assessment is justified by the structural similarity with citronellol (3,7-dimethyloct-6-en-1-ol). These structures are identical except for an additional double bond found in nerol/geraniol. The structure of nerol/geraniol is considered to represent a worst case when compared to citronellol based on this second double bond as possible additional reactive feature. A putative steric hindrance to the action of cytochromes such as those present in the S9 mix due to the additional double bond is not to be expected (see Chapter “Toxicokinetics, metabolism and distribution”). Furthermore, relevant physicochemical parameters show comparability between nerol/geraniol and citronellol (molecular weight of 154.2 and 156.3 ; log Pow at 2.6 and 3.41;  vapour pressure of 1 and 8.6 Pa; water solubility of 769 and 307 mg/l respectively).

In the chosen key study according to OECD TG 476 and GLP, the reaction mass of geraniol and nerol 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 testing 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 was found to be not mutagenic under the chosen testing conditions.

Based on these data, citronellol is considered to be non-mutagenic in mammalian cells.

 

Cytogenicity in mammalian cells/mammals

No valid key study is available for the assessment of cytogenicity of citronellol in vitro or in vivo.

However, data from tests performed with the structurally comparable compounds nerol (cis-3,7-dimethyl-2,6-octadien-1-ol) or the respective stereoisomer geraniol (trans-3,7-dimethyl-2,6-octadien-1-ol) were used to address this endpoint via read across. Inclusion of nerol/geraniol into this assessment is justified by the structural similarity with citronellol (3,7-dimethyloct-6-en-1-ol). These structures are identical except for an additional double bond found in nerol/geraniol. The structure of nerol/geraniol is considered to represent a worst case when compared to citronellol based on this second double bond as possible additional reactive feature. A putative steric hindrance to the action of cytochromes such as those present in the S9 mix due to the additional double bond is not to be expected (see Chapter “Toxicokinetics, metabolism and distribution”). Furthermore, relevant physicochemical parameters show comparability between nerol/geraniol and citronellol (molecular weight of 154.2 and 156.3 ; log Pow at 2.6 and 3.41;  vapour pressure of 1 and 8.6 Pa; water solubility of 769 and 307 mg/l respectively).

In a chromosomal aberration test, concentrations of 0.0313, 0.0625, 0.125 mg/ml geraniol were tested in mammalian CHL cells 1-164 cells for clastogenic effects (Ishidate, 1984). After 48 h, 8% of the cells were polyploid (slightly, but significant elevated compared to control), and 4% of the cells showed chromosomal aberrations (not elevated compared to control). Thus, the test results were regarded as ambiguous.

In the other study, mammalian cells concentrations of 0, 33.3, 100, 333 and 1000 µM were tested in CHO cells during a sister chromatid exchange assay (Sasaki, 1989). Although cytotoxicity was noted at a concentration of 1000 µM, no substance induced chromatid exchange was detected.

 

The ambiguous results found in vivo could not be confirmed in vivo.

The reaction mass of geraniol and nerol was 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 1 500 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 1 500 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.

Overall in a weight of evidence, the data of the structural analogue provide evidence for an absence of any cytogenic activity of citronellol.


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.