(4S)-1-methyl-4-(6-methylhepta-1,5-dien-2-yl)cyclohexene

Endpoint summary

Additional information

Mutagenicity in bacteria

In the key study for genetic toxicity in vitro (mutagenicity) according to OECD TG 471 and GLP, beta-Bisabolene was investigated for its potential to induce gene mutations in 2 independent experiments according to the plate incorporation test protocol (Eurofins 2017; 175659). Several concentrations (0.00158 – 5 µL/plate) were tested in triplicates with and without metabolic activation using tester strains S. typhimurium TA 100, TA 1535, TA 98, TA 1537 and E.coli WP2 uvrA. No precipitation was observed and no bacteriotoxic effects were noted in any of the tester strains up to the highest concentration evaluated with metabolic activation. However, toxic effects were observed with beta-Bisabolene in strains TA 98, TA 100, and TA 1535 >=1.0 µL/plate (without metabolic activation) and in TA 1537 at concentrations >=0.1 µL/plate (without metabolic activation). No toxic effects were found in the second confirmatory experiment with or without metabolic activation. No biologically relevant increases in revertant colony numbers were observed following incubation with beta-Bisabolene at any concentration level with or without metabolic activation. Thus, beta-Bisabolene did not cause gene mutations by base pair changes or frameshifts in the genome and is considered non-mutagenic in this bacterial reverse mutation assay under the chosen testing conditions.

Several other AMES tests are available for the structurally related isomer alpha-Bisabolene:

In an AMES test acc. to OECD TG 471 and GLP, the test substance (i.e. a fermentation product containing 89.5% alpha-Bisabolene) was found to induce concentration-related increases of revertant colony numbers in a plate incorporation test in the tester strain E. coli WP2 uvrA (pKM101) at a concentration >= 2500 μg/plate with metabolic activation (Eurofins Munich; 2021; STUGC21AA0951-2).  Moreover, concentration-related increases were found in tester strain TA1537 at a concentration >= 2500 μg/plate, although these increases were found to be below the threshold value chosen (i.e. 3.0 fold increase). The authors concluded, that the fermentation product tested caused gene mutations in tester strain E. coli WP2 uvrA (pKM101) and that the test substance is considered to be mutagenic in this bacterial reverse mutation assay. The same batch of the test substance used was retested in a plate incorporation test (acc. to OECD TG 471) in the tester strain S. typhimurim TA1537 and E. coli WP2 uvrA up to 5000 μg/plate (BASF 2021; 40M0378/21M172). Revertant colony numbers were not increased with/without metabolic activation. Accordingly, alpha-Bisabolene with a purity of 98% did not increase the revertant colony numbers under comparable testing conditions (BASF 2021; 40M0379/21M174). A full GLP conform study acc. to OECD TG 471 in 5 tester strains with 98% alpha-Bisabolene is currently ongoing (BASF; 40M0379/21 M224). Preliminary results confirmed the absence of increases in revertant colonies in Salmonella strains TA 1535, TA100, TA1537, TA98 and E. coli WP2 uvrA up to 5000 μg/plate in the plate incorporation and preincubation test with and without metabolic activation. Since the increases were observed in a E. coli strain containing the plasmid pKM101, the relevance of this difference compared to the E. coli strain without the respective plasmid was investigated further. Accordingly, alpha-Bisabolene with a purity of 90% (BASF; 40M0378/21M172) and 98% (BASF; 40M0379/21M224) was tested in this strain in a plate incorporation test.  Both test substances did not increase the revertant colony numbers of E. coli WP2 uvrA (pKM101) with and without metabolic activation up to 5000 μg/plate.

Overall, the registered substance beta-Bisabolene was found to be non-mutagenic in the key study, i.e. a bacterial reverse mutation assay according to OECD TG 471 and GLP. Concerns related to increases in mutation frequencies by a fermentation product containing 89.5% of the structurally related isomer alpha-Bisabolene, were not confirmed by follow-up AMES tests with alpha-Bisabolene. The results of these studies are included in this endpoint summary as additional information. However, the studies were not included as full study entries into this dossier, since a valid key study with the registered substance beta-Bisabolene is available and presented to inform the endpoint genetic toxicity in vitro. 

 

Cytogenicity in mammals

In the key study for genetic toxicity in vivo (cytogenicity) according to OECD TG 474 and GLP, 5 male NMRI mice/dose were intraperitoneally treated with doses of 400, 1000, and 2000 mg/kg bw beta-Bisabolene (Eurofins 2017; 175660). Peripheral blood samples were collected 44 h and 68 h (negative control and high dose only) after a single application of the test item. The animals treated with doses of 400 and 1000 mg/kg bw showed no and slight signs of systemic toxicity. Animals of the highest dose showed clear toxic effects like reduction of spontaneous activity, prone position, constricted abdomen, ataxia, hunched posture, piloerection and half eyelid/eye closure.

For all dose groups, including positive and negative controls, 10000 polychromatic erythrocytes per animal were scored for incidence of micronucleated immature erythrocytes. The frequency of micronuclei in negative controls (44 h, 68 h) were within the negative historical control limits. Cyclophosphamide (40 mg/kg bw) as positive control induced a statistically significant increase in the micronucleus frequency, demonstrating the validity of the chosen testing conditions.

The mean MN frequency values for beta-Bisabolol treated dose groups were within the range of the concurrent negative control except for the value in the low dose group (400 mg/kg bw/d), which was slightly higher compared to the concurrent negative control (i.e. 0.29 % versus 0.21% in controls). However, all values of micronuclei frequencies were within the historical control limits of the negative control (0.14 - 0.31%). No statistically significant increases of cells with micronuclei were noted in any dose group in nonparametric (Mann-Whitney) or trend (Chi Square) tests. Thus, no biologically relevant increase of micronuclei was found after treatment with beta-Bisabolol in any of the dose groups evaluated.

In conclusion, it can be stated that during the study and under the experimental conditions reported, beta-Bisabolene did not induce structural and/or numerical chromosomal damage in the immature erythrocytes of the mouse. Therefore, beta-Bisabolene is considered to be non-mutagenic with respect to clastogenicity and/or aneugenicity in the Mammalian Erythrocyte Micronucleus Test.

 

Justification for classification or non-classification

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