p-menth-1-en-8-yl acetate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Alpha-Terpinyl Acetate gave negative results in the Ames test with Salmonella typhimurium strains TA98, TA100, TA1535, TA1537, and E. coli strain WP2. Negative results were observed in the in vitro mouse lymphoma assay, both with and without metabolic activation with alpha-Terpineol. Negative results were also observed in the in vitro chromosome aberration assay both with and without metabolic activation with Terpineol multi. Based on these results alpha-Terpinyl Acetate is considered not to be genotoxic.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

between 31 August 2012 and 15 October 2012.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study is performed according to current guidelines and in compliance with GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Remarks:

TNO Triskelion, Utrechtsteweg 48 Zeist, The Netherlands

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver homogenate

Test concentrations with justification for top dose:

Test 1: 21, 62, 185, 556, 1667, 5000 µg/plate (TA 1535, TA 1537, TA 98, TA 100, E. Coli)
Test 2: 3.13, 6.25, 12.5, 25, 50, 100 µg/plate (TA 1535, TA 1537, TA 100)
Test 3: 7.8, 15.6, 31.3, 62.5, 125, 250 µg/plate (TA 100)

Vehicle / solvent:

dimethylsulphoxide (DMSO)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA 1535, TA 100 Without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

TA 1537 Without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

TA 98 Without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: N-ethyl-N-nitrosourea

Remarks:

WP 2 uvrA Without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene:

Remarks:

TA 1535, TA 98, TA 100 WP, 2 uvrA, With metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

TA 1537 With metabolic activation

Details on test system and experimental conditions:

- Characterization of the test system: The Salmonella typhimurium strains and the Escherichia coli WP2 uvrA strain are purchased from Trinova Biochem (Giessen, Germany) and are originally from Moltox Molecular Toxicology Incorporated, Boone, USA. Frozen stocks of each strain were checked for histidine or tryptophan requirement and for sensitivity to ampicillin, crystal violet and UV radiation. On the day of use, aliquots of S9 liver homogenate were thawed and mixed with a NADPH generating system. The final concentrations of the various ingredients in the S9-mix were: MgCl2 8 mM; KCl 33 mM; G-6-P 5 mM; NADP 4 mM; sodium phosphate 100 mM (pH 7.4), NaCl 46 mM, and S9 10 %. The S9-mix was kept on ice until use.
- Dose levels of the test substance: In the test, the plate-incorporation method with the histidine-requiring S. typhimurium mutants TA 1535, TA 1537, TA 98 and TA 100 and the tryptophan-requiring Escherichia coli mutant WP2 uvrA as indicator strains was applied. The assay has been described in detail by Ames et al. (1975) and by Maron and Ames (1983). A preliminary test to assess the toxicity of the test substance was not performed. Therefore the toxicity test was incorporated in the mutagenicity assay. Three bacterial reverse mutation tests were performed. DMSO was used as a diluent for the test substance. In the first test, just before use, a solution of 50 mg/mL was prepared. A clear, colourless solution was obtained. Six concentrations were tested, ranging from 21 to 5000 µg/plate with all strains. In the second and third test, just before use, a solution of 2.5 mg/mL was prepared. A clear, colourless solution was obtained. Six concentrations were tested, ranging from 3.13 to 100 µg/plate with strains TA 1535 and TA 1537 and ranging from 7.8 to 250 µg/plate with strain TA 100. Negative controls (DMSO) and positive controls were run simultaneously with the test substance. The actual concentrations of the test substance in the test solutions were not determined. Therefore, the concentrations quoted in this report are nominal concentrations.
- Mutation analysis: Fresh bacterial cultures were prepared by inoculation of nutrient broth with a thawed aliquot of the stock culture and subsequent incubation for 10-16 h at ca. 37°C while shaking. Briefly, the mutagenicity assay was carried out as follows. To 2 mL molten top agar (containing 0.6 % agar, 0.5 % NaCl and 0.05 mM L-histidine.HCl/0.05 mM biotin or 0.05 mM tryptophane for the S. typhimurium strains, and E. coli WP2 uvrA strain, respectively), maintained at ca. 46°C, were added subsequently: 0.1 mL of a fully grown culture of the appropriate strain, 0.1 mL of the test substance or of the negative control or of the positive control substance solution, and 0.5 mL S9-mix for the experiments with metabolic activation or 0.5 mL sodium phosphate 100 mM (pH 7.4) for the experiments without metabolic activation. The ingredients were thoroughly mixed and the mix was immediately poured onto minimal glucose agar plates (1.5 % agar in Vogel and Bonner medium E with 2 % glucose). All determinations were made in triplicate. The plates were incubated at ca. 37°C for approximately 48-72 hours. Subsequently, the his+ and trp+ revertants were counted. Toxicity was defined as a reduction (by at least 50%) in the number of revertant colonies and/or a clearing of the background lawn of bacterial growth as compared to the negative (vehicle) control and/or the occurrence of pinpoint colonies.

Evaluation criteria:

- The mutagenicity study is considered valid if the mean colony counts of the vehicle control values of the strains are within acceptable ranges, if the results of the posi-tive controls meet the criteria for a positive response, if no more than 5 % of the plates are lost through contamination or other unforeseen events and if at least 3 doses are non toxic.
- A test substance is considered to be positive in the bacterial gene mutation test if the mean number of revertant colonies on the test plates increased in a concentration-related manner or if a two-fold or greater increase is observed compared to the negative control plates. A clear positive response does not need to be verified. Marginally or weakly positive results should be verified by additional testing.
- A test substance is considered to be negative in the bacterial gene mutation test if it produces neither a dose-related increase in the mean number of revertant colonies nor a reproducible positive response at any of the test concentrations.
- Positive results from the bacterial reverse mutation test indicate that a test substance induces point mutations by base pair substitutions or frameshifts in the genome of either Salmonella typhimurium and/or Escherichia coli. Negative results indicate that under the test conditions, the test substance is not mutagenic in the tested strains.
- Omission of a second test under these conditions is acceptable as a single test does not, or hardly ever results in false negative conclusions (TNO historical data in Appendix 5 and Kirkland and Dean, 1994).
- Both numerical significance and biological relevance are considered together in the evaluation.

Statistics:

No statistical analysis was performed.

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

GENOTOXICITY:
- In the first test, the mean numbers of his+ and trp+ revertant colonies of the negative controls in all strains used were within the acceptable range, except for the negative control for strain WP2 uvrA, in the presence of S9-mix. The number of revertants was just above the acceptable range; the possible occurrence of slight deviations from the acceptance criteria was described in the study plan and was considered to be acceptable and not to be of influence of the outcome of the study. In all strains the positive controls gave the expected increase in the mean numbers of revertant colonies.
- In the second test, the mean numbers of his+ revertant colonies of the negative controls in strains TA 1535 and TA 1537 were within the acceptable range. The negative control for strain TA 100 was outside the acceptable range, most likely due to a contamination. The second test with strain TA 100 was repeated in a third test.
- In the third test, the mean numbers of his+ revertant colonies of the negative control were within the acceptable range. In both tests, in all strains the positive controls gave the expected increase in the mean numbers of revertant colonies.
- In all tests, in all strains, in both the absence and presence of S9-mix, Terpinyl Acetate Alpha did not induce a more than 2-fold and/or dose related increase in the mean number of revertant colonies compared to the background spontaneous reversion rate observed with the negative control.
CYTOTOXICITY
- In the first test, the test substance was toxic to all strains, in both the absence and presence of S9-mix at various concentrations. Toxicity was evidenced by a decrease in the mean number of revertants and/or a (slightly) less dense background lawn of bacterial growth and pinpoint colonies. As a result, in strains TA 1535, TA 1537 and TA 100, in both the absence and presence of S9-mix, less than three non-toxic concentrations were tested which is considered to be the minimum for a valid test result. Therefore a second test with strains TA 1535, TA 1537 and TA 100 was performed.
- In the second and third test, the test substance was toxic to all strains tested, in both the absence and presence of S9-mix at various concentrations. Toxicity was evidenced by a decrease in the mean number of revertants and/or a (slightly) less dense background lawn of bacterial growth. For all strains tested, in both the absence and presence of S9-mix, three or more non-toxic concentrations could be evaluated.
TEST-SPECIFIC CONFOUNDING FACTORS
- In the first test, in all strains tested, a dose related precipitation of the test substance was observed in the final treatment mix, in both the absence and presence of S9-mix at and above 556 µg/plate.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative

It is concluded that the results obtained with the test substance in Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100, and in the Escherichia coli strain WP2 uvrA, in the absence and presence of the S9-mix, indicate that the test substance alpha-Terpinyl Acetate is not mutagenic under the conditions of this test.

Executive summary:

In a GLP compliant study, performed in accordance with OECD guideline 471, the test substance, alpha-Terpinyl Acetate, was examined for its possible mutagenic activity in the bacterial reverse mutation test using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and the Escherichia colistrain WP2uvrA, in the absence and presence of a liver fraction of Aroclor 1254-induced rats for metabolic activation (S9-mix). The test substance was diluted in dimethylsulphoxide (DMSO). Three bacterial reverse mutation tests were performed. In the first test, all strains were used, in the absence and presence of S9-mix, with six concentrations of the test substance, ranging from 21 to 5000 µg/plate. In the second test, in the absence and presence of S9-mix, strains TA 1535, TA 1537 and TA 100 were used. For each strain six concentrations of the test substance were used, ranging from 3.13 to 100 µg/plate for strains TA 1535 and TA 1537 and ranging from 7.8 to 250 µg/plate for strain TA 100. In the second test, the negative control for strain TA 100 was outside the acceptable range, and was there for regarded as invalid. A third test was performed with strain TA 100 identical to the second test. Negative controls (DMSO) and positive controls were run simultaneously with the test substance. In all tests, in all strains, in both the absence and presence of S9-mix, alpha-Terpinyl Acetate did not induce a more than 2-fold and/or dose related increase in the mean number of revertant colonies compared to the background spontaneous reversion rate observed with the negative control. It is concluded that the results obtained with the test substance in Salmonella typhimuriumstrains TA 1535, TA 1537, TA 98 and TA 100, and in the Escherichia colistrain WP2uvrA, in both the absence and presence of the S9-mix, indicate that the test substance alpha-Terpinyl Acetate is not mutagenic under the conditions employed in this study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

For alpha-Terpinyl Acetate a genotoxicity test in bacterial strains is available but no experimental information is available on cytogenicity or gene mutations in mammalian cells. Therefore, information on the genetic toxicity of alpha-Terpineol and Terpineol-multi will be used. This read-across information is described below.

Alpha Terpinyl Acetate and its genotoxic potential using read across information from alpha-Terpineol and Terpineol multi

Introduction and hypothesis for the analogue approach

For alpha-Terpinyl Acetate, a genotoxicity test in bacterial strains is available but no experimental information is available on cytogenicity or gene mutations in mammalian cells. This acetate is a tertiary-ester attached to an unsaturated cyclohexyl ring, with a methyl group attached to the unsaturated bond at the para-position (see section 1 on identity). It is liquid at room temperature with a melting point of < -20^oC and a molecular weight of 196 g/mol. The substance has a low volatility (3.5 Pa). In accordance with Article 13 of REACH, lacking information can be generated by means of applying alternative methods such as in vitro tests, QSARs, grouping and read-across. For assessing the genotoxicity of the alpha-Terpinyl Acetate the analogue approach is selected because for a closely related analogue alpha-Terpineol a gene-mutation study in human cells is available. Besides a large overlap in structure is alpha-Terpineol also the metabolite of alpha-Terpinyl Acetate. For covering cytogenicity the chromosomal aberration test with Terpineol multi is used, which mainly contains alpha-Terpineol.

Hypothesis: Alpha-Terpinyl Acetate has similar genotoxicity compared to alpha-Terpineol and Terpineol multi. The target and source substances are sufficiently similar in structure to be used for read across. In addition, systemically the target and the source substances are the same because during the first metabolic step the ester will be metabolised into the alcohol.

Available experimental information

Alpha-Terpinyl Acetate (the target), alpha-Terpineol and Terpineol multi (sources) are negative in well conducted genotoxicity tests in bacterial strains (OECD TG 471).The source chemical alpha-Terpineol is negative in the mouse lymphoma assay (OECD TG 476). The source substance Terpineol multi, containing mainly alpha-Terpineol is negative in the in vitro cytogenicity assay (OECD TG 473). The genotoxicity tests from these source chemicals all receive reliability of 1.

Target chemical and source chemical(s)

Chemical structures of the target and source substances and their constituents are presented in the purity section below.

Purity / Impurities

The table with the constituents of the target and the source chemicals are presented in the toxico-kinetic section. Alpha-Terpinyl Acetate and its impurities will metabolise into alpha-Terpineol, which is the main constituent of Terpineol multi. Due to this similarity in structure and constituents of the target and the source the genotoxicity profile will be similar (see also the toxico-kinetic section for details on constituents). A more detailed discussion is presented below at analogue justification.

Analogue approach justification

According to Annex XI 1.5 read across can be used to replace testing when the similarity can be based on a common backbone and a common functional group and/or the likelihood of a similar metabolic pathway and similar metabolites. When using read across the result derived should be applicable for C&L and/or risk assessment and it should be presented with adequate and reliable documentation. The Terpinyl Acetates (all constituents) and the Terpineols (all constituents) have similar backbones and they have a similar metabolic pathway.

Structural similarities and differences: The target and the source chemicals (including their constituents) all belong to the terpinoids.

The backbone is the unsaturated cyclohexyl ring to which a tertiary-acetic ester is attached (target) or a tertiary alcohol (source). The target has an ester as the functional group while the source has an alcohol. The ester functionality and the alcohol both have a low order of reactivity, also because both do not have any adjacent groups (at the alpha or beta position) that may increase their (electrophilic) reactivity.Therefore this difference in functional groups between the target and source chemical is not expected to behave differently in relation to genotoxicity. In addition, the terpinyl-esters will be readily metabolised into their alcohols. The WHO (2000) when assessing these substances as flavouring additives, also considered the terpinoids, esters and their alcohols into one category. Also the constituents, the gamma and beta-versions of both the acetates and alcohols are structurally very similar with the same backbone and functional groups.

Toxico-kinetic:Terpinyl Acetates (all constituents) and Terpineols (all constituents) are expected to be readily absorbed via all routes based on information from WHO (2000), the human toxicological information and physico-chemical parameters (see also toxico-kinetic section).The Terpinyl Acetates (all constituents) will readily metabolise via oral, dermal and inhalation route into the respective alcohols, because carboxylesterases are abundantly available in all organs including skin and lungs. The metabolisation is presented in the toxico-kinetic section (IUCLID section 7.1).

Reactivity profile for genotoxicity:When running the OECD Toolbox (version 3.1.0.21) the Terpinyl Acetates and Terpineols, including their constituents, show a very low reactivity for DNA binding. There are two genotoxicity profilers which indicate slightly different results with some exceptions. For the Terpinyl Acetates (all constituents) the DNA profiling of OASIS detects three different mechanisms: Schiff base formers, SN1 and SN2 mechanisms. These mechanisms are considered for very specific acetates to which the Terpinyl Acetates do not belong (as presented in the details of this alert). Also there is an in vivo Micronucleus alert according to ISS, but it can be seen in the details of this alert that the applicability domain is very limitedly indicated. Therefore the overall genotoxicity profile of both the Terpinyl Acetates and the Terpineols are considered to be negative. This is also concluded by the WHO (2000).

Uncertainty of the prediction:The genotoxicity profile has a high certainty because of close similarity in chemical structure, toxico-kinetic behaviour and the similar metabolic pathway between the Terpinyl Acetates and Terpineols, including their constituents. At the site of contact the ester functionality could, theoretically be somewhat more reactive compared to the alcohol. Due to the absence of activating adjacent groups in the near vicinity of the ester-bond this will have a minimal effect on the genotoxic profile. The absence of genotoxicity is further supported by the negative results in the genotoxicity information in bacterial strains, which has been conducted for both the Terpineol and Terpinyl Acetates. Therefore also the cytogenicity data and the genotoxicity data in mammalian cells of the Terpineol can be used for the Terpinyl Acetates, which is also concluded by the WHO (2000).

Data matrix

The relevant information on physico-chemical properties and toxicological characteristics are presented in the data matrix below.

Conclusions per endpoint for C&L, PBT/vPvB and dose descriptor

When using read across the result derived should be applicable for C&L and/or risk assessment and be presented with adequate and reliable documentation.

For alpha-Terpinyl Acetate a well conducted genotoxicity test in bacterial strains test is available which is clearly negative (OECD TG 471). For the source substance alpha-Terpineol a well conducted negative gene mutation in mammalian cells is available (OECD TG 476). For the source substance Terpineol multi (containing mainly alpha-Terpineol) a well conducted mammalian in vitro cytogenicity assay is available (OECD TG 476) showing absence of cytogenicity. Therefore also alpha-Terpinyl Acetate is considered to be negative for these endpoints, because the ester-bond is only slightly more reactive at the site of contact and systemically only the alcohol will exists.

Final conclusion on hazard, C&L, DNEL and risk characterisation

Alpha-Terpinyl Acetate is negative in the Ames test and Terpineol (including its constituents) is negative for genotoxicity. Therefore, alpha-Terpinyl Acetate (including its constituents) will be negative for this endpoint. This means that alpha-Terpinyl Acetate does not need to be classified for genotoxicity according to EU Directive 67/548 (DSD) and EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008. In view of the absence of genotoxicity a risk characterization for this endpoint is not needed.

References

WHO, 2000, Evaluation of certain food additives, Technical Report Series 891, page 53/54,http://whqlibdoc.who.int/trs/WHO_TRS_891.pdf.

Data matrix

Common names	Alpha-Terpinyl Acetate (Target)	Alpha-Terpineol (Source)	Terpineol multi (Source)
Chemical structures*
CAS	80-26-2	98-55-5	8000-41-7
Physico-chemical data
Molecular weight	196	154	154
Physical state	Liquid	Liquid	Liquid
Melting point °C	< -20	< -20	< -36
Vapour pressure Pa (measured)	3.5	6.5	300^
Water solubility mg/l (measured)	23	2870	2540
Log Kow (measured)	4.4	2.6	2.6
Human health endpoints
Genotoxicity -Ames test	Negative (OECD TG 471)	Negative (OECD TG 471)	Negative (OECD TG 471)
In vitro cytogenicity	Read across	No data	Negative (OECD TG 473)
Genotoxicity in vitro MLA	Read across	Negative (OECD TG 476)

 *(see attached document at the end of the section: IUCLID, Genetic toxicity, 7.6:Endpoint summary; ^This 300 Pa may be due to volatile impurities because also the model of MPBPVP of EpiSuite is predicting a value of 2.62, which is in line with the experimental value of alpha-Terpineol.

alpha-Terpinyl Acetate: Ames test

In a GLP compliant study, performed in accordance with OECD guideline 471, the test substance, alpha-Terpinyl Acetate, was examined for its possible mutagenic activity in the bacterial reverse mutation test using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and the Escherichia coli strain WP2 uvrA, in the absence and presence of a liver fraction of Aroclor 1254-induced rats for metabolic activation (S9-mix) (TNO Triskelion 2012). The test substance was diluted in dimethylsulphoxide (DMSO). Three bacterial reverse mutation tests were performed. In the first test, all strains were used, in the absence and presence of S9-mix, with six concentrations of the test substance, ranging from 21 to 5000 µg/plate. In the second test, in the absence and presence of S9-mix, strains TA 1535, TA 1537 and TA 100 were used. For each strain six concentrations of the test substance were used, ranging from 3.13 to 100 µg/plate for strains TA 1535 and TA 1537 and ranging from 7.8 to 250 µg/plate for strain TA 100. In the second test, the negative control for strain TA 100 was outside the acceptable range, and was there for regarded as invalid. A third test was performed with strain TA 100 identical to the second test. Negative controls (DMSO) and positive controls were run simultaneously with the test substance. In all tests, in all strains, in both the absence and presence of S9-mix, alpha-Terpinyl Acetate did not induce a more than 2-fold and/or dose related increase in the mean number of revertant colonies compared to the background spontaneous reversion rate observed with the negative control.It is concluded that the results obtained with the test substance in Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100, and in the Escherichia coli strain WP2uvrA, in both the absence and presence of the S9-mix, indicate that the test substance is not mutagenic under the conditions employed in this study.

alpha-Terpineol: MLA

In a mammalian cell gene mutation assay conducted similarly to OECD guideline 476, mouse lymphoma L5178Y cells cultured in vitro were exposed to alpha-Terpineol at concentrations between 0.14 µg/mL and 0.65 µg/mL in the presence and absence of metabolic activation with liver S9 prepared from Aroclor 1254-induced male Sprague-Dawley rats (Seifried 2006). Alpha-Terpineol was tested for cytotoxic concentration up to an upper limit of 10000 µg/plate. In both non-activated and S9-activated conditions, response was negative at a dose 0.14-0.65 µg/mL The positive controls ethylmethylsulfonate (without metabolic activation) and 3-methylcholanthrene (with metabolic activation) induced the appropriate response.

Terpineol multi: Chromosome aberration

In an in vitro chromosome aberration test performed according to OECD guideline 473 and in compliance with GLP, human primary lymphocyte cultures were exposed to Terpineol multi in DMSO at concentration range of 5.598-1543 μg/mL, for 3 + 17 h (treatment + recovery) with metabolic activation (2% S-9 fraction of Aroclor 1254-induced male Sprague-Dawley rats), and for 3 + 17 h or 20 + 0 h (treatment + recovery) without metabolic activation for a preliminary cytotoxicity test (Lloyd 2010). In the main test, two experiments were performed at concentrations up to 600 µg/mL without S-9 and up to 800 µg/mL with S-9 and the following concentrations were selected for analysis: Experiment 1: Without S-9 (treatment: 3 h): 0, 350, 425 and 450 μg/mL; with S-9 (treatment: 3 h): 0, 300, 550 and 625 μg/mL. Experiment 2: Without S-9 (treatment: 20 h): 0, 75, 200 and 225 μg/mL; with S-9 (treatment: 3 h): 0, 400, 550, 625 and 650 μg/mL. Proportion of cells with structural aberrations in negative control cultures fell within historical vehicle control ranges. Positive controls (4-nitroquinoline-N-oxide at 2.5 and 5 µg/mL without S-9 and cyclophosphamide at 10, 20 and 30 µg/mL with S-9) induced the appropriate response. Treatment of cells with Terpineol multi in the presence or absence of S-9 in both experiments resulted in frequencies of cells with structural or numerical aberrations that were generally similar to those observed in concurrent vehicle controls for all concentrations analysed. Numbers of aberrant cells (excluding gaps) in treated cultures fell within the normal range with the exception of one culture at the highest concentration analysed with S-9 in experiment 1 (625.0 µg/mL). However, the aberration frequency (excluding gaps) in the replicate culture at 625.0 µg/mL in experiment 1 and in all other cultures analysed in experiments 1 and 2 fell within the normal range. Under the test conditions, Terpineol multi is not considered as clastogenic in human lymphocytes.

Justification for selection of genetic toxicity endpoint
The information from the three standard in vitro genotoxicity tests is available using the substance as such or from its analogues. This information is sufficiently adequate to cover this endpoint.

Justification for classification or non-classification

Based on the negative results of the Ames test with alpha-Terpinyl Acetate, the negative result of the mouse lymphoma assay with alpha-Terpineol and the negative result of the in vitro chromosome aberration assay with Terpineol multi, alpha-Terpinyl Acetate does not need to be classified for genotoxicity according to EU Directive 67/548 and EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.