Benzenepropanoic acid, 3,5-bis(1,1-dimethylethyl)-4-hydroxy-, isotridecyl ester

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Key Information - In vitro: - Gene mutation (Bacterial reverse mutation assay / Ames test), Salmonella typhimurium TA 1535, TA 1537, TA 1538, TA 98 and TA 100: negative with and without metabolic activation [OECD TG 471, GLP; test item: structural analogue with EC 413-750-2] - Chromosome aberration, Chinese hamster Ovary (CHO): negative with and without metabolic activation [EU Method B.10 and OECD TG 473, GLP; test item: structural analogue with EC 413-750-2] - Gene mutation (mammalian cell gene mutation assay), mouse lymphoma L5178Y cells: negative with and without metabolic activation [OECD TG 476, GLP] Supporting information - In vivo: - Chromosome aberration, in vivo mammalian bone marrow cytogenetic test: negative [OECD TG 474, GLP; test item: structural analogue with EC 413-750-2]

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP - Guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

other: SOP 118 00 880, edition 4 valid from 27. Jan. 2014, “Mouse-Lymphoma-Test nach OECD 476"

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

Mutations at the thymidine kinase locus (Tk1) on chromosome 11 and/or structural chromosomal aberrations in mouse lymphoma L5178Y Tk+/- cells

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

Cleansed and for mycoplasma contamination screened stocks of cells were stored in liquid nitrogen in the cell bank of the laboratory to allow a continuous stock of cells, which guarantees similar parameters of the experiment and reproducible characteristics of the cells. Cleansed and for mycoplasma contamination screened cells were thawed 5 - 8 d prior treatment and cultivated in RPMI 1640 complete culture medium with 5 % HS in cell culture flasks at 37.0 ± 1.5 °C in a humidified atmosphere with 5.0 ± 0.5 % CO2. After thawing, the cells were again screened for mycoplasma contamination before they were used in the experiments.

Metabolic activation:

with and without

Metabolic activation system:

liver enzyme S9 fraction / “liver S9 mix from male rats, treated with Aroclor 1254”

Test concentrations with justification for top dose:

nominal concentrations in experiment I [mM]: 0.16*, 0.31*, 0.63, 1.25, 2.5, 5 and 10;
nominal concentrations in experiment II [mM]: 0.16*, 0.31*, 0.63*, 1.25, 2.5, 5 and 10;
* cultures were not continued since only 4 analysable concentrations are required by the guideline

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: DMSO is one of the organic vehicles compatible with this test system. A solubility test for the determination of a suitable solvent for the test item was performed in a non-GLP pre-test with RPMI 1640 and DMSO. The test item was soluble and stable in DMSO. Therefore, DMSO was used as solvent control in this study.

Untreated negative controls:

yes

Remarks:

solvent control (DMSO)

Negative solvent / vehicle controls:

yes

Remarks:

= negative control

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Remarks:

RPMI 1640 medium without supplements was used as solvent control for the positive control methyl methanesulphonate (MMS); 0.9% NaCl was used as solvent control for the positive control cyclophosphamide (CPA).

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 h (experiment I: +S9 and -S9, experiment II: +S9) and 24 h (experiment II: -S9)
- Expression time (cells in growth medium): 48 ± 4 h
- Selection time (if incubation with a selection agent): 7-10 days (pre-test), 7-15 days (main tests)

SELECTION AGENT (mutation assays): Selection Medium RPMI 1640 with TFT:
Horse Serum 15 %
Penicillin/Streptomycin (per mL: 10000 Units Pen/ 10 mg Strep) in H2O 1 %
Sodium pyruvate 2 %
Trifluorothymidine 5 µg/mL

NUMBER OF REPLICATIONS: two replicates per culture

NUMBER OF CELLS EVALUATED: 10^6

DETERMINATION OF CYTOTOXICITY
- Method: relative cloning efficiency (RCE, pre-test); relative total growth (RTG, main tests)

OTHER: Final conentrations of positive controls:
MMS: 19.5 µg/mL (Experiment I); due to the cytotoxic potential of MMS, a smaller concentration was included in experiment II because of the elongation of the exposure time. 15 µg/mL (Experiment II); as a back-up the concentrations 12.5 µg/mL and 19.5 µg/mL were also used in experiment II. However, since the lower concentration (12.5 µg/mL) did not induce a significant increase in mutant frequency, the next higher concentration was chosen for evaluation.
CPA: 4.5 µg/mL

Evaluation criteria:

A mutation assay is considered acceptable if it meets the following criteria (the current recommendations of the International Workshop on Genotoxicity Testing (IWGT)) (Moore et al., 2006, Moore et al., 2007):
- All plates, from either the cloning efficiency or the TFT resistance-testing portion of the experiment are analysable.
- The absolute cloning efficiency at the time of mutant selection (CE) of the solvent controls is 0.65 – 1.20.
- The total suspension growth of the solvent control calculated by the day 1 fold-increase in cell number multiplied by the day 2 fold-increase in cell number is 8 – 32. Following 24 h treatment the total suspension growth is 32 – 180.
- The mutant frequency of the solvent control is in the range of 50 – 170 per 106 cells or in the range of the historical data.
- The positive controls (MMS and CPA) should yield an absolute increase in total mutation frequency (MF) above spontaneous background MF (an induced MF [IMF]) of at least 300 per 106 cells or should be in the range of the historical data. At least 40 % of the IMF should be reflected in the small colony MF. Alternatively, the positive controls should increase the small colony MF of at least 150 per 106 small colonies in the concurrent vehicle control (a small colony IMF of 150 per 106).
- The upper limit of cytotoxicity observed in the positive control culture should be the same as for the experimental cultures (I.e. the relative total growth – RTG- should be greater than 10 % of the concurrent selective control group).
- The highest concentration of the test item should be 0.01 M or 5 mg/mL or 5 µL/mL, unless limited by toxicity or solubility of the test item. If toxicity occurs, the highest concentration should lower the relative total growth to approximately 10 to 20 % of survival. If precipitation is noted, the highest analysed concentration should be the lowest concentration where precipitation is observed by the naked eye.

Statistics:

A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. With the assessment of this regression, it can be evaluated whether mutations increase with increasing dose of the test item. A p-value of 0.05 or lower (significance level 95%) is considered as critical. p-values from 0.208 - 0.580 were obtained.
The statistical significance values of the positive controls were <0.001. The chi-square test was used. Statistically significant increase in mutants is considered as given if p is below 0.01.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

other: negative control was vehicle control

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH and osmolality: None of the tested solvent controls, positive controls or test item concentrations showed a critical value of the osmolality and the pH value. A negative influence of these parameters on the assay can be excluded therefore.
- Precipitation: As the test item is an oily substance, precipitation was visible at the concentrations 10 mM and 5 mM. However, the substance could be easily removed from the cells during the washing step.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

The study was performed according to the OECD Guideline 476 without deviations and according to the principles of the good laboratory practice and therefore considered to be of the highest quality (reliability Klimisch 1). The validity criteria of the test system were fulfilled. No substantial and reproducible dose dependent increase in mutant colony numbers was observed in both experiments. No relevant shift of the ratio of small versus large colonies was observed up to the maximal concentration of the test item. Hence, it can be stated that under the experimental conditions, the test item did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic activation.

Executive summary:

The OECD 476 study (GLP, 2016) was performed to investigate the potential of the test item to induce mutations at the thymidine kinase locus (Tk1) on chromosome 11 and/or structural chromosomal aberrations in mouse lymphoma L5178Y Tk+/- cells. The assay was performed in two independent experiments, using two parallel cultures each (replicates). In the experiments, 7 concentrations of the test item were used and tested with and without metabolic activation. The exposure times were 4 h (experiment I: +S9 and -S9, experiment II: +S9) and 24 h (experiment II: -S9). The following real concentrations of the test item were investigated in experiments I and II: 10 mM, 5 mM, 2.5 mM, 1.25 mM, 0.63 mM, 0.31 mM, and 0.16 mM. Precipitation of the test item was visible at the concentrations 10 mM and 5 mM in both approaches and experiments. However, the substance could be easily removed from the cells during the washing step.

MMS (19.5 µg/mL in experiment I and 15.0 µg/mL in experiment II) and CPA (4.5 µg/mL) were used as positive controls.

No significant reduction of growth was observed with and without S9 after 4 and 24 h treatment with test item (all concentrations) in all experimental parts. Therefore, all tested concentrations could be evaluated of mutagenicity.

The mutant frequencies for the solvent control DMSO in both experiments (+ S9 and -S9) were in the range of 50 - 170 colonies per 10⁶ cells (experiment I: +S9: 83 and -S9: 81; experiment II: +S9: 51 and -S9: 66). The mutant frequencies of the solvent control NaCl 0.9 % were also in the normal range between 50 - 170 colonies per 10⁶ cells (experiment I: 77, experiment II: 59). The mutant frequencies of the solvent control RPMI 1640 were also in the normal range between 50 - 170 colonies per 10⁶ cells (experiment I: 80, experiment II: 79).

The positive control CPA showed in both experiments a distinct increase in induced total mutant frequency and exceeded the number of mutant colonies of the corresponding solvent control of more than 300.

Also the positive control MMS showed a distinct increase in induced total mutant colonies, but did not exceed the threshold of more than 300 colonies in comparison to the solvent control in experiment I. However, the mutation frequency of the small colonies was increased over 150 in comparison to the solvent control. In experiment II the mutant frequency was increased to more than 300 mutant colonies in comparison to the solvent control. As all further acceptability criteria of the assay were also met, the study was valid.

In all tested concentrations of the test item, no substantial and reproducible dose dependent increase in mutant colony numbers was observed in both main experiments. No relevant shift of the ratio of small versus large colonies was observed up to the maximal concentration of the test item. The mutation frequency did not reach or exceed the threshold of 126 above the corresponding solvent control.

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the L5178Y Tk+/- cell line in the absence and the presence of metabolic activation; under the conditions of the test, the test item is declared as a non-mutagen.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

Since EC 700-397-7 is a near analogue to the test substance (EC 413-750-2), the experimental data (Ames test, Chromosome abberation assay and in vivo data) from this substance were used in a read-across approach in addition to the MLA on the test substance itself: The structural analogue was negative in an Ames test (bacterial reverse mutation assay according to OECD Guideline 471, GLP) and in a chromosomal aberrations assay (Chinese hamster Ovary cells according to EU Method B10 and OECD Guideline No. 473, GLP) with and without metabolic activation. Further, it was also negative in an in vivo mammalian bone marrow cytogenetic test (OECD Guideleine No. 474, GLP). The substance with EC 700 -397 -7 itself has been tested in a mammalian cell gene mutation assay (MLA, OECD Guideline No. 476, GLP) and did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic activation. No substantial and reproducible dose dependent increase in mutant colony numbers was observed. No relevant shift of the ratio of small versus large colonies was observed up to the maximal concentration of the test item.

Justification for selection of genetic toxicity endpoint
GLP and guideline study

Justification for classification or non-classification

The test material does not meet the criteria for classification and will not require labelling as a mutagen in accordance with European Regulation (EC) No. 1272/2008.