2-hydroxyethyl [(1R,2S,5R)-2-isopropyl-5-methyl-cyclohexyl] carbonate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial reverse mutation assay/Ames test: negative (equivalent or similar to OECD 471; GLP)

In vitro mammalian chromosome aberration test: negative (OECD 473; GLP)

In vitro mammalian cell gene mutation assay: negative (OECD 476; GLP)

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1992-08-26 to 1992-09-18

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1983-05-26

Deviations:

yes

Remarks:

toxic effects not appropriately considered in dose setting, occasionally leading to only 4 analysable concentrations.

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 1987-12-07

Type of assay:

bacterial reverse mutation assay

Target gene:

TA1537: his C 3076
TA98 and TA1538: his D 3052
TA1535 & TA100: his G 46

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

S. typhimurium TA 1538

Metabolic activation:

with and without

Metabolic activation system:

S9-mix (containing 10 % S9): distilled water (0.335 mL); phospahte buffer (0.2 M, pH 7.4; 0.5 mL); NADP (0.1 M; 0.04 mL); glucose-6-phosphate (1 M; 0.005 mL); MgCl2/KCl (0.4 M/1.65M; 0.02 mL; S9 fraction (0.1 mL)

Test concentrations with justification for top dose:

Experiment 1: 10, 33, 333, 1000 and 5000 µg/plate (with and without metabolic activation)
Experiment 2: 15, 50, 150, 500, and 1500 µg/plate (without metabolic activation)
Experiment 2: 15, 50, 150, 500, 1500, and 5000 (with metabolic activation)
The test concentrations were chosen according to an initial toxicity test performed with the test item.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

other: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)
- Cell titer (viable cells/mL):
Experiment 1 (with and without metabolic activation):
TA1535: 2.4 x10^9 (249, 235, 242)
TA1537: 2.1 x 10^9 (169, 204, 266)
TA1538: 2.4 x 10^9 (201,311,203)
TA98: 2.3 x 10^9 (214, 262, 223)
TA100: 1.2 x 10^9 (125, 105, 127)

Experiment 2 (with and without metabolic activation):
TA1535: 3.2 x10^9 (374, 361, 352)
TA1537: 1.0 x 10^9 (112, 91, 107)
TA1538: 0.7 x 10^9 (72, 67, 56)
TA98: 2.1 x 10^9 (229, 176, 224)
TA100: 1.4 x 10^9 (136, 137, 156)

MAIN EXPERIMENT (two independent experiments)
- Exposure duration: 48 hours at 37 °C in the dark
- Number of replications: 3 replicates for each experimental point
- the number of revertant colonies (his+ revertants) were counted and the plates were examined for the existence of a normal background lawn and/or precipitates as well as microscopically for microcolony growth.
- when there is any question about the nature of colonies scored as revertants and when positive mutagenic results are obtained, the genotype of revertant colonies are spot-checked by picking and streaking on histidine free plates.

DETERMINATION OF CYTOTOXICITY
An initial toxicity test was performed with the test item.

Rationale for test conditions:

The test concentrations were chosen according to an initial toxicity test performed with the test item.

Evaluation criteria:

not specified

Statistics:

χ²-test

Key result

Species / strain:

S. typhimurium, other: TA98, TA100, TA1535, TA1537, and TA1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

please refer to the field "Additional information on results" below.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

MAIN STUDY:
The number of spontanoeus revertants observed using each of the five strains was very close to those previously established in the laboratory and was within the range obtained by Ames et al. (1975)* as well as reported by De Serres and Shelby (1979)*.

The test compound increased slightly the spontaneous mutation frequency in some of the five tester strains and at some doses tested. However, the estimation of the statistical significane of the difference between the mean number of revertants ion the negative controls and the plates at each dosage level, using a χ²-test (Mohn and Ellenberger, 1977)*, revealed no significant difference at any one of the test points.

INFORMATION ON CYTOTOXICITY:
- with metabolic activation: bacteriotoxic effects were observed towards strains TA1535, TA 1537, and TA1538 at 1500µg/plate and towards TA98, and TA100 at 5000 µg/plate.
- without metabolica ctivation: a slight bacteriotoxic effect towards strains TA1537, TA1538, TA98, and TA100 was observed at 500 µg/plate and at 1500 µg the test compound was bacteriotoxic to alls strains.

Please also refer to the field "Attached background material" below.

*References:
- Ames, B.N., J. McCann and E. Yamasaki (1975) Methods for detecting carcinogens and mutagens with the Salmonella/mammalian-microsome mutagenicity test, Mutation Res., 31: 347 - 364.
- De Serres, F.J., and M.D. Shelby (1979) Recommendations on data production and analysis using the Salmonella/microsome mutagenicity assay, Mutation Res., 64: 159 - 165.
- Mohn, G.R. and J. Ellenberger (1977) The use of E. coli K12/343/113 (lambda) as a multi-purpose indicator strain in various mutagenicity ttesting procedures, in: B.J. Kilbey (Ed.), Handbook of mutagenicity test procedures, Elsevier, Amsterdam, pp. 95-118.

Conclusions:

The substance tested non-mutagenic under the conditions of the study.
According to Regulation (EC) No 1272/2008 and subsequent adaptations, the substance should not be considered to have a mutagenic potential.

Executive summary:

A reliable bacterial reverse mutation assay equivalent or similar to OECD 471 (1983) was conducted. Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and TA1538 were treated with the substance using the plate incorporation method at five or six dose levels in triplicate with and without metabolic activation. Two experiments were conducted with the following concentrations:

Experiment 1: 10, 33, 333, 1000 and 5000 µg/plate (with and without metabolic activation)

Experiment 2: 15, 50, 150, 500, and 1500 µg/plate (without metabolic activation)

Experiment 2: 15, 50, 150, 500, 1500, and 5000 µg/plate (with metabolic activation)

In the concentration range investigated, the test item did not show mutagenic activity with and without a metabolic activation system. Precipitation of the test compound on the plates was not observed. In the presence of the metabolizing system bacteriotoxic effects were observed towards strains TA1535, TA1537, and TA1538 at 1500 µg/plate and towards TA98, and TA100 was observed at 500 µg/plate and at 1500 µg the test compound was bacteriotoxic to all strains.

Thus, the substance tested non-mutagenic under the conditions of this study.

According to Regulation (EC) No 1272/2008 and subsequent adaptations, the substance should not be considered to have a mutagenic potential.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1993-10-11 to 1994-01-26

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Deviations from the OECD 473 (2014): number of analysed cells was too low. Highest possible dose was not tested (without metabolic activation only). Short-term treatment without metabolic activation was missing. Tables not labelled appropriately. Historical control data is missing. Acceptability/evaluation criteria are missing.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

1983-05-26

Deviations:

yes

Remarks:

Highest possible dose was not tested (without metabolic activation only). Tables not labelled appropriately. Evaluation criteria missing.

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 1987-12-07

Type of assay:

other: in vitro mammalian chromosome aberration test

Target gene:

not applicable

Species / strain / cell type:

lymphocytes: human peripheral blood

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: healthy human donor
- Sex and number of blood donors: 1 male
- Whether whole blood or separated lymphocytes were used: whole blood (0.4 mL/culture tube)

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: each culture tube contained 5 mL of Ham's F10 complete medium (500 mL Ham's F10; 100 mL fetal calf serum (heat inactivated); 5 mL penicillin/streptomycin (10 000 IU/m1); 5 mL heparin (5000 IU/m1)), to which 0.1 mL of phytohaem-agglutinin was added. The tubes were sealed and incubated at 37 °C.

Metabolic activation:

with and without

Metabolic activation system:

S9-mix (containing 25 % S9): 4 mM NADP; 25 mM glucose-6-phosphate; 8 mM MgCl2; 33 mM KCl; 100 mM phosphate buffer pH 7.4

Test concentrations with justification for top dose:

Experiment 1/Experiment 2: 10, 30, and 100 µg/mL (without metabolic activation)
Experiment 1/Experiment 2: 30, 100, and 300 µg/mL (with metabolic activation)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

NOTE: two independent experiments were conducted separated by at least 14 days.

TOXICITY TEST:
- test was carried out with the same procedure as described below for the mutagenicity test.
- at least 2000 lymphocytes/ culture were examined
- mitotic index was calculated as the percentage of lymphocytes examined which were in mitosis.

AVERAGE GENERATION TIME.
- method was carried out with the same procedure as described below for the mutagenicity test with the exception that 5-bromodeoxyuridine (BrdU, 30 µM) was added to the cultures, which were treated in the dark for 52 hours until the end of the incubation period.
- Staining technique: after the preparation of microscopic slides, sister chromatids were differentially stained using a modified fluorescence-plus-Giemsa technique (Perry and Wolff, 1974). Briefly, slides aged for 4 to 5 days were stained in 33258 Hoechst dissolved in NaCl/KCl solution, rinsed and mounted in M/15 Sörensen's phosphate buffer (pH 6.8) to irradiate them with a 254nm UV mineralogic lamp. After the exposure, the slides were incubated in 2xSSC solution at 59°C, rinsed in distilled water and stained with 6 % phosphate-buffered Giemsa solution and then mounted as usual.
- estimation of the average generation time: a replicative index based on 200 cells was calculated from the proportions of metaphase cells that completed one, two, or between one and two, or two and three cycles in BrdU. The average generation time (h) was calculated as the number of hours (52) in BrdU divided by the replicative index.
- Classification of metaphase cells:
M1. even, dark staining of both chromatids
M1+: 1 partial S phase in BrdU followed by 1 complete S phase gives regions with sister chromatid differentiation (SCD), and regions with both chromatids still dark
M2: 2 complete S phases in BrdU gives full SCD
M2+: 1 partial followed by 2 complete S phases gives regions with SCD and regions with even, light staining on both chromatids.

MUTAGENICITY TEST (Experiment 1 and Experiment 2):
- test item exposure without metabolic activation: cells were exposed to the test item concentrations for 24 hours.
- test item exposure with metabolic activation: cells were exposed to the test item concentrations and 0.5 mL of S9-mix for 3 hours (37 °C). After 3 hours the cells were centrifuged and washed to remove the test compound and S9-mix. After a further centrifugation the cell pellet was resuspended in complete medium and reincubated at 37°C to give a total of 24 hours incubation.

NUMBER OF REPLICATIONS: duplicate

CELL HARVESTING:
- 2 hours before the end of the incubation period, the cell division was arrested by the addition of 10 μg/ml Colcemid solution and incubated for further 2 hours.
- 24 hours after beginning of the treatment the cells were harvested by centrifugation.
- cells were resuspended in hypotonic potassium chloride solution and incubated at 37°C.
- then the cells were collected by centrifugation, resuspended, and fixed in 3:1, methanol : glacial acetic acid.
- cells were then washed at least twice in fresh fixative.

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
- cells were centrifuged and resuspended in fresh fixative.
- two or three drops of this suspension were transferred to a microscopic slide and left to air-dry.
- at least two slides/culture were prepared.
- slides were stained in Giemsa solution (10 % in 0.01 M phosphate buffer, pH 6.8) washed in buffer and distilled water and left to air-dry.
- permanent slides were made, after cleaning in xylene, using Eukitt mountant.

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE:
- slides were observed under a microscope with a 100x oil immersion objective lens at 1250 fold magnification.
- at least 100 well spread metaphases containing 46 chromosomes from each culture were analysed (total: at least 200 metaphases/test point).
- aberrations were classified using the nomenclature of Savage (1979)*.

*Reference:
- Savage, J. R. K. (1979) Annotation: Classification and relationships of induced chromosomal structural changes, J. Med. Genet., 13, 103- 122.

Rationale for test conditions:

According to the preliminary toxicity test the dose levels were chosen for the main study.

Evaluation criteria:

not specified

Statistics:

Statistical significance was determined according to the methods of Kastenbaum and Bowman (1970)*.
*Reference:
Kastenbaum, M. A., and K. O. Bowman (1970) Tables for determining the statistical significance of mutation frequencies, Mutation Res., 9, 527-549.

Key result

Species / strain:

lymphocytes: human peripheral blood

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

please refer to the field "Additional information on results" below

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

AVERAGE GENERATION TIME Of HUMAN PERIPHERAL BLOOD LYMPHOCYTES:
The average generation time was only slightly affected by the test substance both in the presence and absence of S9-mix. Thus a single sampling time at 1.5 times the normal cell-cyle lenth from the beginning of the treatment (24 hours) was used in all independent experiments.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: mitotic index
The highest test item concentration tested resulted in an average of 32 % cytotoxicity in the absence of S9-mix and 49 % in the presence of S9-mix.

MUTAGENICITY TEST (Experiment 1 and Experiment 2):
The test item did not induce an increase in the number of chromosome aberrations in cultured human blood lymphocytes in the absence and presence of a metabolizing system as compared to the controls.

CONTROLS:
- spontaneous value of aberrations observed were within the range previously established in the laboratory.

Please also refer for further information on results to the field "Attached background material" below.

Conclusions:

The substance was non-clastogenic under the conditions of the study.
According to Regulation (EC) No 1272/2008 and subsequent adaptations, the substance should not be considered to have a mutagenic potential.

Executive summary:

An in vitro mammalian chromosomal aberration test was performed with the test item dissolved in DMSO using human peripheral blood lymphocytes in the absence and presence of metabolic activation. The test procedure was equivalent or similar to the method described in the OECD guideline 473 (1983). Two independent experiments investigating induction of chromosomal aberration were performed.

Cells were treated in the absence of metabolic activation at test item concentrations of 10, 30, and 100 µg/mL for 24 hours (experiment 1 and experiment 2). With metabolic activation, cells were treated with the test item at concentrations of 30, 100, and 300 µg/mL for three hours (experiment 1 and experiment 2). After 3 hours. the test item and S9 -mix were removed and the cells were resuspended in complete medium and reincubated to give a total of 24 hours incubation. Each treatment was established in duplicate. Negative and positive controls were also run concurrently. The effect of the test item on the cell cycle time of human peripheral blood lymphocytes both in the presence and absence of metabolic activation was investigated.

The highest concentration tested resulted in an average of 32 % cytotoxicity in the absence of S9-mix and 49 % in the presence of S9-mix. Furthermore, the test compound affected only slightly the cell-cycle time of human peripheral blood lymphocytes both in the presence and absence of S9-mix. Therefore, a sampling time of 1.5 times the normal cell-cycle length from the beginning of the treatment (24 hours) was used in all independent experiments. The test item did not induce an increase in the number of chromosome aberrations in cultured human blood lymphocytes in the absence and presence of a metabolizing system.

Thus, the substance was non-clastogenic under the conditions of this study.

According to Regulation (EC) No 1272/2008 and subsequentadaptations, the substance should not be considered to have a mutagenic potential.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2016-06-30 to 2016-11-02

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2016-07-29

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2015-09-14

Type of assay:

other: in vitro mammalian cell gene mutation assay

Specific details on test material used for the study:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: on the day of the experiment (immediately before treatment), the test material was heated to a maximum of 60 °C in a water bath until completely molten. Afterwards, warm DMSO (vehicle) was added to dissolve the test item.

Target gene:

HPRT

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Laboratory for Mutagenicity Testing; Technical University, 64287 Darmstadt, Germany
- Suitability of cells: the high proliferation rate and a good cloning efficiency of untreated cells (as a rule more than 50 %) both necessary for appropriate performance of the study, recommended the use of this cell line.
- Doubling time: 12 - 16 hours in stock cultures
- Methods for maintenance in cell culture: thawed stock cultures were propagated at 37 °C in plastic flasks. About 2-3×10^6 cells were seeded into each flask with MEM (minimal essential medium) containing Hank’s salts supplemented with 10% foetal bovine serum (FBS), neomycin and amphotericin B. The cells were sub-cultured once or twice weekly. All incubations were done at 37 °C with 1.5 % carbon dioxide (CO2) in humidified air.
- Modal number of chromosomes: 22

MEDIA USED
- Type and identity of media: for seeding of the cell cultures the complete culture medium was MEM (minimal essential medium) containing Hank’s salts, neomycin, 10% FBS, and amphotericin B (1 %). During treatment no FBS was added to the medium. For the selection of mutant cells the complete medium was supplemented with 11 μg/mL 6-thioguanine. All cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % CO2 (98.5 % air).

- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically 'cleansed' against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

S9-mix: S9 supernatant, MgCl2 (8 mM), KCl (33 mM), glucose-6-phosphate (5 mM), and NADP (4 mM) in sodium-ortho-phosphate buffer (100 mM, pH 7.4)

Test concentrations with justification for top dose:

Pre-experiment: 16.0, 32.0, 64.1, 128.1, 256.3, 512.5, 1025.0, and 2050.0 µg/mL (4 hour treatment; with and without metabolic activation)
Experiment 1: 4.0, 8.0, 16.0, 32.0, 64.0, 96.0, and 128.0 µg/mL (4 hour treatment; without metabolic activation)
Experiment 1: 8.0, 16.0, 32.0, 64.0, 128.0, 192.0, and 256.0 µg/mL (4 hour treatment; with metabolic activation)
Experiment 2: 32.0, 48.0, 64.0, 72.3, 80.6, and 88.0 µg/mL (4 hour treatment; without metabolic activation)
Experiment 2: 32.0, 64.0, 80.6, 96.0, 112.0, and 128.0 µg/mL (4 hour treatment; with metabolic activation)
Please also refer for information on the justification for the top dose to the field "Rationale for test conditions" below.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO (final concentration in the culture medium: 0.5 % (v/v))
- Justification for choice of solvent/vehicle: the solvent was chosen based on solubility properties and compatibility with the cell cultures.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

PRE-TEST ON TOXICITY
- a pre-test was performed in order to determine the concentration range for the mutagenicity experiment.
- general culture conditions and experimental conditions were the same as described for the mutagenicity experiment below.
- pre-experiment was performed in the presence and absence (4 h treatment) of metabolic activation.
- test medium was checked for precipitation or phase separation at the beginning and at the end of treatment (4 hours) prior to removal to the test item.
- colony forming ability of approx. 500 single cells (duplicate cultures/concentration level) after treatment with the test item was observed and compared to the controls.
- toxicity of the test item is indicated by a reduction of the cloning efficiency (CE).

- osmolarity and the pH-value were determined in culture medium of the solvent control and of the maximum concentration in the pre-experiment without metabolic activation.

MAIN EXPERIMENT - Experiment 1 and 2
Seeding:
- two to four days after sub-cultivation stock cultures were trypsinized at 37 °C.
- then, complete culture medium with 10% FBS was added and a single cell suspension was prepared.
- trypsin concentration for all sub-culturing steps was 0.2% in saline.
- prior to the trypsin treatment the cells were rinsed with PBS.
- approx. 0.7 to 1.2×10^7 were seeded in flasks.
- cells were grown for 24 hours prior to treatment.

Treatment:
- after 24 hours the medium was replaced with serum-free medium containing the test item, solvent, or positive controls, either without S9 mix or with S9 mix.
- 4 hours after treatment, the medium was replaced with complete medium following washing steps with "saline G".
- after the end of treatment the cells were trypsinised as described above and sub-cultivated.
- at least 2.0×10^6 cells/experimental point (concentration series plus controls) were subcultured in flasks containing medium.
- two flasks were seeded per experimental point with approx. 500 cells each to determine the relative survival (cloning efficiency I) as measure of test item induced cytotoxicity. The cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % CO2.
- colonies used to determine the cloning efficiency I were fixed and stained 6 to 8 days after treatment as described below.
- three or four days after first sub-cultivation approx. 2.0×10^6 cells/ experimental point were sub-cultivated in flasks containing medium.
- following the expression time of 7 days five flasks were seeded with about 4 to 5×10^5 cells each in medium containing 6-TG.
- two flasks were seeded with approx. 500 cells each in non-selective medium to determine the viability (cloning efficiency II).
- cultures were incubated at 37 °C in a humidified atmosphere with 1.5% CO2 for about 8 days.
- colonies were stained with 10% methylene blue in 0.01% KOH solution.
- stained colonies with more than 50 cells were counted. In doubt the colony size was checked with a preparation microscope.

NUMBER OF REPLICATIONS: duplicates

Rationale for test conditions:

In the pre-experiment a relevant cytotoxic effect was observed at 128.1 μg/mL and above in the absence of metabolic activation and at 256.3 μg/mL and above in the presence of metabolic activation. Furthermore, precipitation occurred at 512.5 μg/mL and above after 4 hours treatment with and without metabolic activation.
The dose range of the first main experiment was set according to data generated in the pre-experiment. Based on the steep gradient of toxicity, the recommended cytotoxic range of approx. 10 - 20 % relative adjusted cloning efficiency I was not covered. Therefore, the dose range of the second main experiment was fine-tuned to cover the toxic range with very closely spaced concentrations.

Evaluation criteria:

A test item is classified as positive if it induces a concentration-related increase of the mutant frequency exceeding the historical solvent control range.
A test item producing no concentration-related increase of the mutant frequency above the historical solvent control range is considered to be non-mutagenic in this system.
A mutagenic response is described as follows:
The test item is classified as mutagenic if it induces with at least one of the concentrations in both parallel cultures a mutation frequency that exceeds the historical negative and solvent control data range (95% confidence interval limits).
The increase should be significant and dose dependent as indicated by statistical analysis (linear regression, least squares).

Statistics:

A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. The numbers of mutant colonies generated with the test item were compared to the solvent control groups (significance if p-value < 0.05). Both, biological and statistical significance was considered together.
A t-test was performed to evaluate a possible significant increase of the mutation frequency at test points exceeding the 95% confidence interval (significance if p-value < 0.05).

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Please refer to the field "Additional information on results" below.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

NOTE: the main experiments were evaluated at the following concentrations:
Experiment 1: 4.0, 8.0, 16.0, 32.0, and 64.0 µg/mL (4 hour treatment; without metabolic activation)
Experiment 1: 8.0, 16.0, 32.0, and 64.0 µg/mL (4 hour treatment; with metabolic activation)
Experiment 2: 48.0, 64.0, 72.3, 80.6, and 88.0 µg/mL (4 hour treatment; without metabolic activation)
Experiment 2: 64.0, 80.6, 96.0, 112.0 and 128.0 µg/mL (4 hour treatment; with metabolic activation)

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH (pre-experiment): no relevant shift of pH of the medium even at the maximum concentration of the test item.
- Effects of osmolarity (pre-experiment): no relevant shift of osmolarity of the medium even at the maximum concentration of the test item.

- Precipitation:
Pre-experiment: precipitation occurred at 512.5 μg/mL and above after 4 hours treatment with and without metabolic activation.
Experiment 2: precipitation was noted at 128 µg/mL with metabolic activation.

RANGE-FINDING/SCREENING STUDIES:
In the pre-experiment a relevant cytotoxic effect (relative cloning efficiency of 50% or below) was observed at 128.1 μg/mL and above in the absence of metabolic activation and at 256.3 μg/mL and above in the presence of metabolic activation.

INFORMATION ON CYTOTOXICITY (MAIN EXPERIMENT):
- Experiment 1: relevant cytotoxic effects indicated by an adjusted cloning efficiency I below 50% occurred at 64.0 μg/mL without metabolic activation. Based on a rather steep gradient of cytotoxicity the recommended cytotoxic range of approximately 10-20% relative adjusted cloning efficiency I was not covered in the first experiment. Therefore, a second experiment was performed using very closely spaced concentrations.
- Experiment 2: the recommended cytotoxic range of approximately 10-20% relative adjusted cloning efficiency I was not covered, but the test was analysable showing very high toxicity at 88.0 μg/mL without metabolic activation with one of the parallel cultures. In the presence of metabolic activation precipitation occurred at the maximum concentration of 128 μg/mL.

GENOTOXICTY (MAIN EXPERIMENT):
No relevant and reproducible increase in mutant colony numbers/10^6 cells was observed in the main experiments up to the maximum concentration with and without metabolic activation.

The 95% confidence interval was exceeded at several experimental points. The t-test run at every one of the experimental points exceeding the 95% confidence interval was not-significant at all occasions with a single exception. A significant t-test was noted at 112.0 μg/mL with metabolic activation. This result was judged as biologically irrelevant as it was not reproduced at any other, even higher concentration. Furthermore, the 95% confidence interval was solely exceeded in one of both parallel cultures at this concentration.

A significant dose dependent trend of the mutation frequency indicated by p <0.05 was determined in the second culture of experiment 1 and 2 with metabolic activation. These trends were judged as irrelevant as they were not reproduced in the parallel cultures.

In the main experiment with and without S9 mix the range of the solvent controls was from 9.6 up to 31.5 mutants/10^6 cells; the range of the groups treated with the test item was from 4.0 up to 44.0 mutants/10^6 cells. The highest solvent control value of 31.5 mutants/10^6 cells exceeded the 95% confidence interval but the mutation frequency of the mean value of both parallel cultures (31.5 and 16.3 equal to a mean of 23.9) was fully acceptable.

Ethylmethane sulfonate (300 μg/mL) and 7,12-dimethylbenz(a)anthracene (2.3 μg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies.

HISTORICAL CONTROL DATA
Please refer to the field "Any other information on results incl. tables" below

Historical Data 

2012 – 2015
Number of mutant colonies per 10^6cells
without metabolic activation (4 hours treatment time)
	Positive control ethylmethane sulfonate 150 and 225 µg/mL	Solvent control (medium, acetone, water, DMSO, ethanol, THF)
Range:	53.9 - 889.0	1.6 - 42.8
Mean value:	153.0	15.0
Standard deviation:	88.5	7.4
95% confidence interval	--	0.2 - 29.7
Number of studies:	147	147
with metabolic activation (4 hours treatment)
	Positive control 7,12-dimethylbenz(a)anthracene 1.1 and 2.2 µg/mL	Solvent control (medium, acetone, water, DMSO, ethanol, THF)
Range:	59.6 - 2042.6	2.4 - 44.2
Mean value:	424.6	14.6
Standard deviation:	291.4	7.0
95% confidence interval	--	0.6 - 28.7
Number of studies:	142	142
without metabolic activation (24 hours treatment)
	Positive control ethylmethane sulfonate 150 and 225 µg/mL	Solvent control (medium, acetone, water, DMSO, ethanol, THF)
Range:	108.5 – 786.1	2.4 – 41.8
Mean value:	341.6	14.2
Standard deviation:	125.5	6.8
95% confidence interval	--	0.6 – 27.8
Number of studies:	129	129

The 95% confidence interval is derived from the mean value plus/minus 2 times the standard deviation.

Conclusions:

The substance tested non-mutagenic under the conditions of the study.
According to Regulation (EC) No 1272/2008 and subsequent adaptations, the substance should not be considered to have a mutagenic potential.

Executive summary:

An in vitro mammalian cell gene mutation assay was performed with the test item dissolved in DMSO using Chinese hamster lung fibroblasts (V79). The test procedure was according to the method described in OECD guideline 476 (2016). The assay was performed in two independent experiments, using two parallel cultures/concentration. The first main experiment was performed with and without metabolic activation and a treatment period of 4 hours (test item concentrations: 4.0, 8.0, 16.0, 32.0, 64.0, 96.0, and 128.0 µg/mL (without S9) or 8.0, 16.0, 32.0, 64.0, 128.0, 192.0 and 256.0 µg/mL (with S9)). The second experiment was also performed with a treatment period of 4 hours in the presence and absence of metabolic activation (test item concentrations: 32.0, 48.0, 64.0, 72.3, 80.6, and 88.0 µg/mL (without S9) or 32.0, 64.0, 80.6, 96.0, 112.0 and 128.0 µg/mL (with S9)). Solvent control and positive controls were also run concurrently.

Relevant cytotoxic effects occurred in the first experiment at 64.0 μg/mL without metabolic activation. Based on a rather steep gradient of cytotoxicity the recommended cytotoxic range of approximately 10 - 20 % relative adjusted cloning efficiency I was not covered in the first experiment. Therefore, the second experiment was performed using very closely spaced concentrations. Even in the second experiment this range was not covered but the test was analysable showing very high toxicity at 88.0 μg/mL without metabolic activation with one of the parallel cultures. In the presence of metabolic activation precipitation occurred at the maximum concentration of 128 μg/mL.

No relevant and reproducible increase in mutant colony numbers/10^6 cells was observed in the main experiments up to the maximum concentration with and without metabolic activation.

The substance tested non-mutagenic under the conditions of the study.

According to Regulation (EC) No 1272/2008 and subsequent adaptations, the substance should not be considered to have a mutagenic potential.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Genetic toxicity in vitro

The substance was not observed to be mutagenic in bacterial reverse mutation assay (equivalent or similar to OECD 471), in vitro mammalian chromosomal aberration test (equivalent or similar to OECD 473), and in vitro mammalian cell gene mutation assay (OECD 476).

Justification for classification or non-classification

Genetic toxicity in vitro

The substance should not be considered to have a mutagenic potential based on a bacterial reverse mutation assay (OECD 471), an in vitro mammalian chromosomal aberration test (OECD 473), and an in vitro mammalian cell gene mutation assay (OECD 476). The substance does not require classification according to Regulation (EC) No 1272/2008 and subsequent adaptations.