Propanoic acid, 2-hydroxy-, (1R,2S,5R)-5-methyl-2-(1-methylethyl)cyclohexyl ester, (2S)-

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test: negative (±S9 mix) Mammalian gene mutation test: negative (±S9 mix) Chromosomal aberration test: negative (±S9 mix)

Link to relevant study records

Reference

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2008-02-14 to 2008-04-09

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was performed under GLP conditions and according to OECD guidelines

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

Not applicable

Species / strain / cell type:

lymphocytes: obtained from blood of healthy donors

Details on mammalian cell type (if applicable):

- Type and identity of media: blood cultures
- Properly maintained: yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Without S9 mix
Experiment I: 14.8, 25.9, 45.4, 79.4, 138.9, 243.1, 425.4, 744.5, 1302.9, 2280 µg/mL
Experiment II: 2.8, 4.8, 8.5, 14.8, 25.9, 45.4, 79.4, 138.9, 243.1, 425.4 µg/mL

With S9 mix
Experiment I: 14.8, 25.9, 45.4, 79.4, 138.9, 243.1, 425.4, 744.5, 1302.9, 2280 µg/mL
Experiment II: 138.9, 243.1, 425.4, 744.5, 1302.9, 2280 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: DMSO was chosen due to its solubility properties and its relative non-toxicity to the cell cultures

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: ethylmethane sulfonate (without S9 mix) and cyclophosphamide (with S9 mix)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration:
With S9 Mix: 4h / 22h for the Experiment I and 46h for the Experiment II
Without S9 Mix: 4h in Experiment I and II
- Fixation time (start of exposure up to fixation or harvest of cells): 22 and 46 hours after beginning of treatment

SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): Giemsa (MERCK, 64293 Darmstadt, Germany) or according to the Fluorescent plus Giemsa technique

NUMBER OF CELLS EVALUATED: 100 metaphase plates per culture, 1000 cells for determination of mitotic index

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: determination of polyploid cells in 250 metaphase cells
- Determination of endoreplication: not reported

Evaluation criteria:

A test item is classified as non-mutagenic if:
− the number of induced structural chromosome aberrations in all evaluated dose groups is in the range of our historical control data (0.0 - 4.0% aberrant cells, exclusive gaps).
− no significant increase of the number of structural chromosome aberrations is observed.

A test item is classified as mutagenic if:
− the number of induced structural chromosome aberrations is not in the range of our historical control data (0.0 - 4.0 % aberrant cells, exclusive gaps)
and
− either a concentration-related or a significant increase in number of structural chromosome aberrations is observed.

Although the inclusion of the structural chromosome aberrations is the purpose of this study, it is important to include the polyploids and endoreduplications. The following criteria is valid:
A test item can be classified as aneugenic if:
- the number of induced numerical aberrations is not in the range of our historical control data (0.0 – 0.8% polyploid cells).

Statistics:

Statistical significance was confirmed by means of the Fisher´s exact test (11) (p < 0.05). However, both biological and statistical significance should be considered together. If the above mentioned criteria for the test item are not clearly met, the classification with regard to the historical data and the biological relevance is discussed and/or a confirmatory experiment is performed.

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

cytotoxic in Experiment I, at highest evaluated concentration in the absence of S9 mix

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not reported
- Effects of osmolality: not reported
- Evaporation from medium: not reported
- Water solubility: 0.24g/L
- Precipitation: Precipitation was observed at the end of exposure to the test substance at dose levels above 425 µg/mL.

RANGE-FINDING/SCREENING STUDIES: A preliminary cytotoxicity test was performed to determine the concentrations to be used in the mutagenicity assay. Cytotoxicity is characterized by the percentages of mitotic suppression in comparison to the controls by counting 1000 cells per culture in duplicate. The experimental conditions in this pre-test phase were identical to those described for the mutagenicity assay.
The pre-test phase was performed with 10 concentrations of the test item and a solvent and positive control. All cell cultures were set up in duplicate. Exposure times were 4 hrs (with and without S9 mix) and 22 hrs (without S9 mix). The preparation interval was 22 hrs after start of the exposure. Additional solvent control cultures (with and without S9 mix) were used in the presence of BrdU (5-bromodeoxyuridine; 6 µg/mL) to reassure the replication time of the cultured lymphocytes.
Using reduced mitotic indices as an indicator for toxicity in Experiment I, clear toxic effects were observed after 4 hrs treatment with 425.4 µg/mL and above in the absence of S9 mix. In addition, 22 hrs treatment with 243.1 µg/mL and above in the absence of S9 mix induced strong toxic effects. In the presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration. Considering the toxicity data of the pre-test, 425.4 µg/mL (without S9 mix) and 2280.0 µg/mL (with S9 mix) were chosen as top concentrations in the second experiment

COMPARISON WITH HISTORICAL CONTROL DATA: The incidences of chromosome aberrations in the positive control and negative control were both within the range of the test laboratory's historical data, indicating that this study is valid.

ADDITIONAL INFORMATION ON CYTOTOXICITY: Clear cytotoxicity was observed in Experiment I, after continuous treatment at the highest evaluated concentration of 243.1 µg/mL (44.1 % of control). In the presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration. In experiment I, after pulse treatment in the absence of S9 mix, and in Experiment II, concentrations showing clear cytotoxic effects were not scorable for cytogenetic damage

Remarks on result:

other: strain/cell type: human lymphocytes

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative

The test item did not induce structural chromosomal aberrations as determined by the chromosome aberration test in human lymphocytes in vitro.

Executive summary:

This in vitro assay with human lymphocytes was performed to assess the potential of the test item Menthyl lactate to induce structural chromosomal aberrations in the absence and presence of an exogenous metabolic activation system (liver S9 mix from phenobarbital/ß-naphthoflavone treated male rats).The study was performed under GLP and followed the method described in OECD TG 473.

In each experimental group two parallel cultures were analysed. Per culture 100 metaphases were evaluated for structural chromosomal aberrations. The highest applied concentration in this study (2280 µg/mL of the test item) was chosen with regard to the molecular weight and the purityof the test item and with respect to the current OECD Guideline 473. Dose selection of the cytogenetic experiments was performed considering the toxicity data and test item precipitation.

In Experiment I, after continuous treatment in the absence of S9 mix, clear cytotoxicity was observed at the highest evaluated concentration. In the presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration. In Experiment I, after pulse treatment, in the absence of S9 mix and in Experiment II, concentrations showing clear cytotoxic effects were not scorable for cytogenetic damage.

In both independent experiments, neither a statistically significant nor a biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item. No relevant increase in the frequencies of polyploid metaphases was found after treatment with the test item as compared to the frequencies of the controls. Appropriate mutagens were used as positive controls. They induced statistically significant increases (p < 0.05) in cells with structural chromosome aberrations.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

Three genetic toxicity in vitro assays were conducted with the substance covering different modes of action of genetic toxicity. The substance did not produce significant genetic toxicity in a reliable bacterial reverse mutation assay with S. typhimurium and E. coli, in a in vitro micronucleus assay with human lymphocytes and in an in vitro Gene Mutation Assay in mouse lymphoma L5178Y cells. It is therefore concluded that the substance is not genotoxic. Further testing on the genetic toxicity of Menthyl lactate is not required.

Justification for selection of genetic toxicity endpoint
GLP and guideline study performed in mammalian cells to detect the activity of clastogenic and aneugenic chemicals.

Justification for classification or non-classification

Based on the above stated assessments on genotoxicity the test substance Menthyl lactate is not considered to be genotoxic and does not need to be classified according to Council Directive 2001/59/EC (28th ATP of Directive 67/548/EEC) and according to CLP (Regulation (EC) No 1272/2008 of the European Parliament and of the Council) as implementation of UN-GHS in the EU.