[No public or meaningful name is available]

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test substance gave negative results in three in vitro genetic toxicology tests and is therefore considered to be non-mutagenic.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental phase: 23 August 2017 to 20 October 2017: Report issue: 13 February 2018.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine for Salmonella typhimuriun
Tryptophan for E.coli

Species / strain / cell type:

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

Remarks:

E. coli strain = WP2uvrA

Metabolic activation:

with and without

Metabolic activation system:

phenobarbitone / beta-naphthoflavone induced rat liver S9.

Test concentrations with justification for top dose:

Experiment one (plate incorporation method): 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate.

Experiment two (pre-incubation method):

1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate: WP2uvrA and TA98 with and without S9.
0.15, 0.5, 1.5, 5, 15, 50, 150 and 500 µg/plate: TA100 with and without S9 and TA1537 without S9 only.
0.5, 1.5, 5, 15, 50, 150, 500 and 1500 µg/plate; TA1537 with S9 only.
5, 15, 50, 100, 150, 250 and 500 µg/plate: TA1535 with S9.
0.15, 0.5, 1.5, 5, 15, 50 and 150 µg/plate: TA1535 without S9.

No correction for purity was made due to the UVCB nature of the test substance.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: dimethyl sulphoxide (DMSO)
- Justification for choice of solvent/vehicle: Choosen following solubility checks performed in-house.

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rate of TA98

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

Without S9

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rate of TA100

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Withuot S9

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rate of TA1535

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Without S9

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rate of TA1537

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Without S9

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rate of E. Coli WP2uvrA

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Without S9

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rate of TA98

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

With S9

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rate of TA100

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene

Remarks:

With S9

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rate of TA1535

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene

Remarks:

With S9

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rate of TA1537

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene

Remarks:

With S9

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rate of E. coli WP2uvrA

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene

Remarks:

With S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: iin agar with plate incorporation for experiment 1 and preincubation for experiment 2.

DURATION
- Preincubation period for bacterial strains: 10 hrs
- Exposure duration: 48 hrs

NUMBER OF REPLICATIONS: triplicate plating

DETERMINATION OF CYTOTOXICITY
- Method: plates were assessed for numbers of revertant colonies and examined for effects on the growth of the bacterial lawn.

Evaluation criteria:

Any one or all of the following criteria could be used to determine the overall result of the study:

1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
5. Fold increase greater than two times the concurrent solvent control for any tester strain.

A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met. Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgment about test item activity. Results of this type will be reported as equivocal.

Statistics:

Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control.

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Conclusions:

The test substance was considered to be non-mutagenic under the conditions of this test.

Executive summary:

Introduction

The mutagenicity of the test substance in bacteria was assessed using a test method was designed to be compatible with OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test".

Methods

Salmonella typhimuriums strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with solutions of the test substance using both the Ames plate incorporation and pre-incubation methods at up to eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors) over four separate experiments (two experiments with two further confirmatory experiments).

The dose range for Experiment 1 (plate incorporation) was 1.5 to 5000 mg/plate. The experiment was repeated on a separate day (pre-incubation method) using a dose range that ranged between 0.15 and 5000 µg/plate, depending on the strain type and presence or absence metabolic activation (S9). An expanded dose range was selected in Experiment 2 in order to achieve both four non-toxic dose levels and the toxic limit of the test item.

Due to differences in the results obtained in Experiments 1 and 2, a confirmatory test (Confirmatory Test 1) was dosed at 5 to 500 µg/plate using TA1535 (in the presence of S9-mix only) under pre-incubation conditions only. A second confirmatory experiment (Confirmatory Test 2) was dosed, again using TA1535, with and without S9-mix and using both plate incorporation and pre-incubation methods to confirm the difference in results obtained in Experiment 2 and Confirmatory Test 1. The dose range was the same as Confirmatory Test 1.

Results

The vehicle (dimethyl sulphoxide (DMSO)) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

Following analysis of the results of all the experiments, it was concluded that no biologically relevant increases in the frequency of revertant colonies were observed at any dose of the test item, either with or without metabolic activation (S9-mix).

No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix in any of the experiments detailed above.

Conclusion

The test substance was considered to be non-mutagenic under the conditions of this test.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental phase: 28 September 2017 to 06 February 2018. Report issue: 15 March 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

For each experiment, sufficient whole blood was drawn from the peripheral circulation of a non smoking volunteer (aged 18-35) who had been previously screened for suitability. The volunteer had not knowingly been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection. Based on over 20 years in house data for cell cycle times for lymphocytes using BrdU (bromodeoxyuridine) incorporation to assess the number of first, second and third division metaphase cells to calculate the average generation time (AGT) for human lymphocytes it is considered to be approximately 16 hours. Therefore using this average the in-house exposure time for the experiments for 1.5 x AGT is 24 hours.

The details of the donors used are:

Preliminary Toxicity Test: female, aged 26 years
Main Experiment (4(20)-hour without S9): male, aged 26 years
Main Experiment (4(20)-hour with S9): male, aged 32 years
Main Experiment (24-hour without S9): female, aged 20 years

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital / beta-naphthaflavone induced rat liver S9

Test concentrations with justification for top dose:

4(20)-hour without S9: 0, 39, 78, 117, 156, 208, 260, 312 µg/mL
4(20)-hour with S9 (2%): 0, 19.5, 39, 78, 117, 156, 234 µg/mL
24-hour without S9: 0, 10, 20, 40, 60, 80, 100, 120, 160 µg/mL

The top dose was selected based on toxicity observed in a preliminary toxicity study.

No adjustment for purity was made as the substance is a UVCB. Test concentrations therefore represent 100% of the substance.

There was no significant change in pH when the test item was dosed into media and the osmolality did not increase by more than 50 mOsm.

Vehicle / solvent:

Dimethyl Sulphoxide

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

METHOD OF APPLICATION: in culture medium

CELL CULTURE: Cells (whole blood cultures) were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented “in-house” with L-glutamine, penicillin/streptomycin, amphotericin B and 10 % foetal bovine serum (FBS), at approximately 37 ºC with 5 % CO2 in humidified air. The lymphocytes of fresh heparinised whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA).

DURATION
- Preincubation period: approximately 48 hours.
- Exposure duration:
4-hour exposure to test substance without S9 followed by a 20-hour treatment free incubation period
4-hour exposure to test substance with S9 followed by a 20-hour treatment free incubation period
20-hour exposure to test substance without S9.


- Fixation time (start of exposure up to fixation or harvest of cells):

SELECTION AGENT (mutation assays):

SPINDLE INHIBITOR (cytogenetic assays): Colcemid 0.1 µg/mL 2.5 hours before the required harvest time.

STAIN (for cytogenetic assays): 5% Giemsa for 5 minutes


METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: After incubation with demecolcine, the cells were centrifuged, the culture medium was drawn off and discarded, and the cells re-suspended in 0.075M hypotonic KCl. After approximately fourteen minutes (including centrifugation), most of the hypotonic solution was drawn off and discarded. The cells were re-suspended and then fixed by dropping the KCl cell suspension into fresh methanol/glacial acetic acid (3:1 v/v). The fixative was changed at least three times and the cells stored at approximately 4 ºC to ensure complete fixation prior to slide preparation.

NUMBER OF CELLS EVALUATED: A total of 2000 lymphocyte cell nuclei were counted and the number of cells in metaphase recorded and expressed as the mitotic index and as a percentage of the vehicle control value.

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): Where possible, 300 consecutive well-spread metaphases from each concentration were counted (150 per duplicate), where there were at least 15 cells with aberrations (excluding gaps), slide evaluation was terminated


DETERMINATION OF CYTOTOXICITY
- Method: other: Cytotoxicity was determined in a preliminary toxicity test by observation of heamolysis in cells and mitotic indices.
-

Evaluation criteria:

Criteria for determining the test result:

Providing that all of the acceptability criteria are fulfilled, a test item can be considered to be clearly negative if, in any of the experimental conditions examined:

1) The number of cells with structural aberrations in all evaluated dose groups are within the range of the laboratory historical control data.
2) No toxicologically or statistically significant increase of the number of cells with structural chromosome aberrations is observed following statistical analysis.
3) There is no concentration-related increase at any dose level

A test item can be classified as genotoxic if:

1) The number of cells with structural chromosome aberrations is outside the range of the laboratory historical control data.
2) At least one concentration exhibits a statistically significant increase in the number of cells with structural chromosome aberrations compared to the concurrent negative control.
3) The observed increase in the frequency of cells with structural aberrations is considered to be dose-related

When all of the above criteria are met, the test item can be considered able to induce chromosomal aberrations in human lymphocytes.

Although the inclusion of the structural chromosome aberrations is the purpose of this study, it is important to include numerical aberrations in the form of polyploidy and endoreduplicated cells.

Statistics:

The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test.

A toxicologically significant response is recorded when the p value calculated from the statistical analysis of the frequency of cells with aberrations excluding gaps is less than 0.05 when compared to its concurrent control and there is a dose-related increase in the frequency of cells with aberrations which is reproducible. Incidences where marked statistically significant increases are observed only with gap-type aberrations will be assessed on a case by case basis.

Key result

Species / strain:

lymphocytes: derived from the blood of human volunteers.

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxicity was observed in a preliminary toxicity test and doses for the main test were selected based on the results of this test.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

All the positive control chemicals induced a demonstrable positive response (p≤0.01) and confirmed the validity and sensitivity of the assay and the integrity of the S9-mix.

The frequency of cells with chromosome aberrations (excluding gaps) in the vehicle control cultures were within the current historical control data range fro the laboratory.

The test item did not induce any statistically significant increases in the frequency of cells with aberrations either in the absence or presence of metabolic activation.

Conclusions:

The test substance was considered to be non-clastogenic to human lymphocytes in vitro.

Executive summary:

Introduction

The study was conducted to assess the potential of the test substance to cause chromosomal damage. It was designed to meet the requirements of the following guideline: OECD Guidelines for Testing of Chemicals N. 493 “ In Vitro Mammalian Chromosome Aberration Test2 adopted 29 July 2016.

Method

Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for chromosome aberrations at up to four dose levels, together with vehicle and positive controls. 

In this study, three exposure conditions were investigated; 4 hours exposure in the presence of an induced rat liver homogenate metabolising system (S9), at a 2% final concentration with cell harvest after a 20-hour expression period, 4 hours exposure in the absence of metabolic activation (S9) with a 20-hour expression period and a 24-hour exposure in the absence of metabolic activation.

The dose levels used in the Main Experiment were selected using data from the Preliminary Toxicity Test where the results indicated that the maximum concentration should be limited by toxicity. The dose levels selected for the Main Experiment were as follows:

Exposure Group	Concentration of Test Substance (µg/mL)
4(20)-hour without S9	0, 39, 78, 117, 156, 208, 260, 312
4(20)-hour with S9 (2%)	0, 19.5, 39, 78, 117, 156, 234
24 -hour without S9	0, 10, 20, 40, 60, 80, 100, 120, 160

Results

The test substance did not induce any statistically significant increases in the frequency of cells with aberrations, using a dose range that included a dose level that induced 55±5% mitotic inhibition or greater. The test item demonstrated marked toxicity in all three exposure groups.

Vehicle and positive controls all responded as expected demonstrating that the assay was responding as expected. 

Conclusion

The test substance was considered to be non-clastogenic to human lymphocytes in vitro.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental phase: 28 September 2017 to 06 February 2018. Report issue: 05 March 2018.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Target gene:

Thymidine kinase Locus (TK+/-)

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: The L5178Y TK+/- 3.7.2c mouse lymphoma cell line was obtained from Dr. J. Cole of the MRC Cell Mutation Unit in Brighton, UK. The cells were originally obtained from Dr. D. Clive of Burroughs Wellcome (USA) in October 1978 and were frozen in liquid nitrogen at that time.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Stock cultures were propagated in plastic flasks in RPMI 1640 complete culture medium. The cells were subcultured two times prior to treatment. The cell cultures were incubated at 37 +/- 1.5°C in a humidified atmosphere with 4.5 % carbon dioxide and 95.5 % ambient air.

-Selective Media: RPMI 1640 (complete culture medium) with the addition of 5 µg/mL of trifluorothymidine (TFT).

- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Yes
- Periodically checked for karyotype stability: Yes
- Periodically 'cleansed' against high spontaneous background: Yes

Metabolic activation:

with and without

Test concentrations with justification for top dose:

Main Test Doses Applied
4-hours without S9: 0, 0.7, 1.3, 2.5, 5.0, 10, 20, 40, 60 µg/mL
4-hours with S9: 0, 1.3, 2.5, 5, 10, 20, 40, 60, 80, 100 µg/mL
24-hour without S9: 0, 0.9, 1.9, 3.8, 7.5, 15, 30, 40, 50 µg/mL

The doses for the main test were based on the results of a preliminary range-finding toxicity test.

No adjustment for purity was made as the test substance is a UVCB. Test concentrations therefore represent 100% of test substance.

Both, pH value and osmolarity were determined at the maximal concentration of the test substance and in the solvent control without metabolic activation. For the dose selection the solubility of the test substance and changes of the pH-value and the osmolarity due to the test substance were taken into consideration.

All formulations were prepared freshly before treatment and used within two hours of preparation. The formulation was assumed to be stable for this period.

Vehicle / solvent:

Dimethyl Sulphoxide

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl Sulphoxide

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium (suspension growth)

Evaluation criteria:

The following were the test criteria for concluding if a mutagenic response has occurred:

A relevant increase of the mutation frequency should be dose-dependent.

A mutagenic response is considered to be reproducible if it occurs in both parallel cultures.

A test item is considered equivocal in this assay if the threshold is reproducibly exceeded but the increase of the mutation frequency is not dose dependent. However, in the evaluation of the test results the historical variability of the mutation rates in the solvent controls of this study are taken into consideration.

Results of test groups are generally rejected if the relative total growth is less than 10 % of the vehicle control.

Whenever a test item is considered mutagenic according to the above mentioned criteria, the ratio of small versus large colonies is used to differentiate point mutations from clastogenic effects. If the increase of the mutation frequency is accompanied by a reproducible and dose dependent shift in the ratio of small versus large colonies clastogenic effects are indicated.

A test item is classified as non-mutagenic if the induced mutation frequency does not reproducibly exceed a threshold of 126 colonies per 106 cells above the corresponding solvent control.

A test item not meeting the conditions for a classification as mutagenic or non-mutagenic will be considered equivocal in this assay.

Statistics:

A linear regression will be performed using a validated test script of "R", a language and environment for statistical computing and graphics (p < 0.05), to assess a possible dose dependent increase of mutant frequencies. The number of mutant colonies obtained for the groups treated with the test item will be compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological relevance and statistical significance are considered together.

Key result

Species / strain:

mouse lymphoma L5178Y cells

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

Conclusions:

Under the conditions of the assay the test substance did not induce gene mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y. Therefore, the test substance is considered to be non-mutagenic in this mouse lymphoma thymidine kinase locus assay using the cell line L5178Y.

Executive summary:

Introduction

The study was performed to investigate the potential of the test substance to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y. The method was designed to meet the requirements of the following guidelines:  

•       OECD Guidelines for the Testing of Chemicals, adopted 28 July 2015, Guideline No. 490 "In Vitro Mammalian Cell Gene Mutation Tests using the Thymidine Kinase Gene“.

•       Commission Regulation (EC) No. 440/2008 B.17: ”Mutagenicity – In vitro Mammalian Cell Gene Mutation Test“, dated May 30, 2008.

Method

The assay was performed in two independent experiments, using two parallel cultures each. The first experiment was performed with and without liver microsomal activation and a treatment period of 4 hours. The second experiment was solely performed in the absence of metabolic activation with a treatment period of 24 hours.

The maximum concentration in the pre-experiment (5000 µg/mL, UVCB substance) was chosen with respect to the OECD guideline 490. The concentration range of the main experiments was limited by cytotoxicity of the test item.

Results

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.

Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system.

Conclusion

Under the conditions of the assay the test substance did not induce gene mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y. Therefore, the test substance is considered to be non-mutagenic in this mouse lymphoma thymidine kinase locus assay using the cell line L5178Y.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)