Linalyl acetate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation in bacteria:
- S. typhimurium TA98, TA100, TA1535, TA1537, with and without metabolic activation (Ames test, OECD 471, GLP): negative (BASF 40M0244/914163)

Gene mutation in mammalian cells:
L5178Y cells, with and without metabolic activation (Mouse lymphoma assay): negative (Lorillard 15880-0-4-311R; Read-Across to CAS No. 78-70-6)

Cytogenicity in mammalian cells:
- Peripheral human lymphocytes with and without metabolic activation (in vitro chromosomal aberration assay, OECD 473, GLP): negative (DSM 289968)

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Cytogenicity in vivo:
- CD-1 mouse, up to 1500 mg/kg (micronucleus test OECD 474, GLP), : negative (DSM 328826; Read-Across to CAS No. 78-70-6)

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Valid experimental data were available to assess the genetic toxicity of linalyl acetate in vitro. Additionally, there are studies available for genotoxicity that were conducted with the structurally relevant metabolite linalool (CAS 78-70-6).

 

In-vitro:

Gene mutation in bacteria:

In the key study, linalyl acetate (97.3 % purity) was not mutagenic in an Ames test according to OECD TG 471 and GLP with and without metabolic activation when tested up to 5000μg/plate in Salmonella typhimurium strains TA 98, TA100, TA1535 and TA1537 (BASF 40M0244/914163).

In literature, a non-GLP Ames test was reported, using the S. typhimurium strains TA 98, TA100 and E. coli WP2 uvr A and linalyl acetate (98% purity) up to 9000 µg per plate (Di Sotto 2008). No increase in revertants was observed with and without metabolic activation in the Salmonella strains, but a weak and concentration dependent increase was reported (up to 2.8 fold vs control) for E. coli. However, doubling of the spontaneous mutation rate compared to control was observed at high test substance concentrations (> 5000 µg/plate or >10 mM) only, exceeding maximum test concentrations according to current standard protocols.

Three additional AMES tests using the Salmonella strains TA1535, TA1537, TA1538, TA98 and TA100 were reported as short study summaries in literature (Wahler 2016). In these studies, Linalyl acetate did not induce significant number of revertant colonies with any of the five strains either in the presence or absence of S-9 mix. It was concluded that no evidence of mutagenic potential of Linalyl acetate was obtained in the tests at the concentrations used.

 

Gene mutation in mammalian cells:

In the key study similar to OECD 476 and according to GLP, linalool was tested for induction of forward mutations at the thymidine kinase (TK) locus in the presence and absence of rat liver S9 metabolic activation using the mouse lymphoma L5178Y cell line (Lorillard 15880-0-4-311R). No reproducible increase in mutant frequencies were observed with and without metabolic activation up to cytotoxic concentrations. Therefore, linalool was evaluated as negative in the L5178Y mouse lymphoma forward mutation assay under the test conditions chosen.

In a scarcly reported unschedueld DNA sythesis assay from secondary source, linaly acetate was tested negative results using primary rat hepatocytes without metabolic activation (Heck 1989).

 

Cytogenicity in mammalian cells:

In the key study according to OECD 473 and GLP, linalyl acetate (>96% purity) was tested for its potential to induce chromosome aberrations in cultured peripheral human lymphocytes (DSM 289968). Linalyl acetate (33 - 180 µg/ml in DMSO) was applied to human primary lymphocytes. Both in the absence and presence of a metabolic system (Aroclor-1254 induced rat liver S9-mix), linalyl acetate did not induce a biologically relevant increase in the number of cells with chromosome aberrations up to cytotoxic concentrations. It is concluded that linalyl acetate is not clastogenic in human lymphocytes under the experimental conditions chosen.

 

In literature, a non-GLP and non-guideline micronucleus test in human lymphocytes was reported (Di Sotto 2011). Linalyl acetate (98% purity) was applied (0.5 - 300 µg/ml in DMSO) for 24 hours (without S9) and cytotoxicity (decrease of the nuclear division index) was observed at 300 µg/ml only. A significant and concentration dependent increase of micronuclei (MN) frequency was observed (10-100 µg/ml). Frequencies of nucleoplasmic bridges (NPBs) or nuclear buds (NBUDs) were not affected. The authors concluded that linalyl acetate seems to be responsible for genotoxic damages in lymphocytes under the chosen experimental conditions, albeit a confirmation of this effect in other biological systems and in in vivo studies is required. Further, the authors speculate, that an increase in MN frequency but not of NPBs could be indicative of an aneugenic but no clastogenic effect.

 

Certain limitations question the MN test published. No detailed information on the preliminary toxicity study is provided by the authors to justify the test concentrations used, the study lacked experiments with metabolic activation and MN analysis was not done using DNA specific staining. Furthermore, the strength of effects observed (Linalyl acetate was more potent than the positive contro ethyl methanesulfonate) and a potential misinterpretation of stained non-DNA material questions the results published. As outlined above, the putative cytogenic effects have not been confirmed in human lymphocytes in the chosen key study acc. to OECD TG and GLP at a comparable test concentration range. Therefore the published in vitro MNT has been disregarded.

 

The positive in vitro results published for linalyl acetate have little implication for in vivo genotoxicity. The authors of the in vitro MNT acknowledge, that linalyl acetate is converted in vivo into linalool and acetate and consider the implication of the positive in vitro results for the in vivo genotoxicity as limited (Di Sotto 2016).

 

 

In vivo:

No reliable data for genetic toxicity in-vivo are available for linalyl acetate. In a Drosophila wing spot test, published in literature, Linalyl acetate has been fed to larvae for approximately 48 hours at concentrations of 2.5, 5.0, 7.5 and 10.0 µl/ml (Mademtzoglou 2013). Wings of the respective adult flies were scored for the presence of mosaic spots. As reflected in the frequency of small and total spots, linalyl acetate induced weak effects after chronic treatment at all concentrations tested. The induced increase of spot frequencies was not concentration-dependent. Due to the weak increase compared to the concurrent control, which was independent from the concentration used and the fact, that the study did not follow any regulatorily accepted standard protocol to assess genotoxicity in vivo, the study has been disregarded.

There is one study which was conducted with the structurally relevant metabolite linalool (linalool and acetic acid are the resulting hydrolyzation products of linalyl acetate). The genetic toxicity study in-vivo for linalool was included into this assessment by read-across.

 

In the key study according to OECD 474 and GLP, linalool was tested in the micronucleus test in CD1 mice (DSM 328826). Four groups each comprising 5 males and 5 females, received a single oral intubation. Two groups were dosed with 1500 mg/kg body weight, one group was dosed with 1000 mg/kg body weight and one group was dosed with 500 mg/kg body weight. All animals of the 1500 and 1000 mg/kg dose group and one male/two females of the 500 mg/kg dose group showed lethargy and ataxia. No decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the vehicle controls was observed in the linalool treated groups, reflecting a lack of toxic effects on erythropoiesis. No increase in the frequency of micronucleated polychromatic erythrocytes was observed in the polychromatic erythrocytes of the bone marrow of all linalool treated animals. Positive and negative controls produced appropriate responses. It is concluded that linalool is not mutagenic in the micronucleus test under the chosen test conditions.

 

Taken together, linalyl acetate is non-genotoxic in mammalian cells and bacteria in vitro and in vivo.

Justification for classification or non-classification

The present data on genetic toxicity do not fulfill the criteria laid down in 67/549/EEC and 1272/2008/EC and therefore a non-classification is warranted.