Dodecane-1,12-diol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation test in bacteria:

The test item was evaluated in an Ames Test on Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA, performed according to OECD TG 471. With the exception of S. typhimurium 98 no cytotoxocity was observed up to and including 5000 µg/plate. A single minor toxic effect, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in experiment I in strain TA 98 with S9 mix at 5000 µg/plate. The test item precipitated in the overlay agar in the test tubes from 100 to 5000 µg/plate in experiment I and from 1000 to 5000 µg/plate in experiment II. Precipitation of the test item in the overlay agar on the incubated agar plates was observed from 1000 to 5000 µg/plate in both experiments. The undissolved particles had no influence on the data recording.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

Therefore, the test item is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Chromosomal mutation test in mammalian cells:

The test item dissolved in DMSO, was assessed for its potential to induce micronuclei in human lymphocytes in vitro according to OECD TG 487. In the absence and presence of S9 mix, no cytotoxicity was observed up to the highest evaluated concentrations, which showed precipitation. In both independent experiments, neither a statistically significant nor a biologically relevant increase in the number of micronucleated cells was observed after treatment with the test item. Appropriate mutagens were used as positive controls. They induced statistically significant increases in cells with micronuclei. Therefore, the test item is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to the highest required concentration.

Gene mutation test in mammalian cells:

A study according to OECD TG 476 was conducted in order to investigate the potential of the test item to induce gene mutations at the HPRT locus in mammalian cells. For that purpose, V79 cells were exposed to the test item for 4 hours with and without metabolic activation. The highest applied concentration in the pre-test on toxicity (700 µg/mL) and in the main experiment (200 μg/mL) was chosen with regard to the OECD Guideline 476. No substantial and reproducible dose dependent increase of the mutation frequency was observed. 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. In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations in mammalian cells.

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:

April 2019

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:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

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

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Solubility and stability of the test substance in the solvent/vehicle: Stable in DMSO for 24 hours (Envigo Study Number XS21SQ, March 2019)


FORM AS APPLIED IN THE TEST (if different from that of starting material): On the day of the experiment, the test item was dissolved in DMSO.

Target gene:

mutant histidine gene

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:

Phenobarbital/β-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

plate incorporation assay (preexperiment/experiment I):
3, 10, 33, 100, 333, 1000, 2500, 5000 µg/plate with and without S9 mix
preincubation assay (experiment II):
33, 100, 333, 1000, 2500, 5000 µg/plate with and without S9 mix

The test item precipitated in the overlay agar in the test tubes from 100 to 5000 µg/plate in experiment I and from 1000 to 5000 µg/plate in experiment II. Precipitation of the test item in the overlay agar on the incubated agar plates was observed from 1000 to 5000 µg/plate in both experiments. The undissolved particles had no influence on the data recording.
The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used.
A single minor toxic effect, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in experiment I in strain TA 98 with S9 mix at 5000 µg/plate. No further toxic effects were observed in the remaining test groups.

Vehicle / solvent:

Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine (4-NOPD), 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 60 min
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: Each concentration, including controls, was tested in triplicate.

DETERMINATION OF TOXICITY
- Method: Toxicity of the test item can be evident as a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

with S9 mix at 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The test item precipitated in the overlay agar in the test tubes from 100 to 5000 µg/plate in experiment I and from 1000 to 5000 µg/plate in experiment II. Precipitation of the test item in the overlay agar on the incubated agar plates was observed from 1000 to 5000 µg/plate in both experiments. The undissolved particles had no influence on the data recording.

Conclusions:

negative

Executive summary:

The test item was evaluated in an Ames Test on Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA, performed according to OECD TG 471. With the exception of S. typhimurium 98 no cytotoxocity was observed up to and including 5000 µg/plate. A single minor toxic effect, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in experiment I in strain TA 98 with S9 mix at 5000 µg/plate. The test item precipitated in the overlay agar in the test tubes from 100 to 5000 µg/plate in experiment I and from 1000 to 5000 µg/plate in experiment II. Precipitation of the test item in the overlay agar on the incubated agar plates was observed from 1000 to 5000 µg/plate in both experiments. The undissolved particles had no influence on the data recording.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

Therefore, the test item is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

March 2019 - April 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

2016

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Solubility and stability of the test substance in the solvent/vehicle: Stable in DMSO at nominal concentrations of 0.03 mg/mL and 70 mg/mL during ambient temperature storage for up to 24 hours (Envigo CRS Limited, UK Study Number XS21SQ, 22nd of March 2019)


FORM AS APPLIED IN THE TEST (if different from that of starting material): Stock formulations of the test item and serial dilutions were made in DMSO. The final concentration of DMSO in the culture medium was 1.0 %.
All formulations were prepared freshly before treatment and used within two hours of preparation.

Target gene:

not applicable

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

- Type and identity of media: Dulbecco's Modified Eagle Medium/Ham's F12 (mixture 1:1) supplemented with 200 mM GlutaMAX TM, penicillin/streptomycin 100 U/mL/100 µg/mL, PHA 3 µg/mL, 10 % fetal bovine serum, 10 mM HEPES, and heparin 125 U.S.P.-U/mL.

Metabolic activation:

with and without

Metabolic activation system:

S9 mix from the liver of Phenobarbital/beta-naphthoflavone induced rats.

Test concentrations with justification for top dose:

Exp. I, (4 hrs): 4.5, 8.0,13.9, 24.4, 46.2, 74.6 (p), 131 (p), 229 (p), 400 (p) and 700 (p) µg/mL (without and with S9 mix*)
Exp. II, (20 hrs): 6.0, 10.4, 18.3, 32.0, 56.0, 98.0 (p), 171 (p) and 300 (p) µ/mL (without S9 mix)
Exp. II (4hrs): 6.0, 10.4, 18.3, 32.0, 56.0, 98.0 (p), 171 (p) and 300 (p) (with S9 mix)

The following concentrations were selected for reading:
Exp. I (without S9 mix): 24.4, 42.6 and 74.6 (p) µg/mL
Exp. II (without and with S9 mix): 32.0, 56.0 and 98.0 (p) µg/mL

*: was repeated due to technical error
(p): precipitation was observed at the end of treatment

Vehicle / solvent:

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

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: Demecolcin

Details on test system and experimental conditions:

Blood samples were drawn from healthy non-smoking donors not receiving medication. The lymphocytes of the respective donors have been shown to respond well to stimulation of proliferation with phytohemeagglutinine (PHA) and to positive control substances. All donors had a previously established low incidence of micronuclei in their peripheral blood lymphocytes. The lymphocytes were stimulated for proliferation by the addition of the mitogen PHA to the culture medium for a period of 48 hours.

Pulse exposure
About 48 hrs after seeding 2 blood cultures (10 mL each) were set up in parallel in 25 cm² cell culture flasks for each test item concentration. The culture medium was replaced with serum-free medium containing the test item. For the treatment with metabolic activation 50 μL S9 mix per mL culture medium was added. After 4 hrs the cells were gently centrifugated, separated from the supernatant, washed and resuspended in complete culture medium for a 16-hour recovery period. After this period Cytochalasin B (4 μg/mL) was added and the cells were cultured another approximately 20 hours until preparation.

Continuous exposure (without S9 mix)
About 48 hrs after seeding 2 blood cultures (10 mL each) were set up in parallel in 25 cm² cell culture flasks for each test item concentration. The culture medium was replaced with complete medium (with 10 % FBS) containing the test item. After 20 hours the cells were gently centrifugated, separated from the supernatant, washed and resuspended in complete culture medium. Cytochalasin B (4 μg/mL) was added and the cells were cultured another approximately 20 hours until preparation.

STAIN (for cytogenetic assays): Giemsa

NUMBER OF CELLS EVALUATED: At least 1000 binucleate cells per culture were scored for cytogenetic damage on coded slides.

DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxicity is characterized by the percentages of reduction in the CBPI (Cytokinesis-block proliferation index) in comparison with the controls (% cytostasis) by counting 500 cells per culture in duplicate.

DOSE SELECTION

Evaluation criteria:

Providing that all of the acceptability criteria are fulfilled, a test item is considered to be clearly negative if, in all of the experimental conditions examined:
− None of the test item concentrations exhibits a statistically significant increase compared with the concurrent solvent control
− There is no concentration-related increase
− The results in all evaluated test item concentrations should be within the range of the laboratory historical solvent control data
The test item is then considered unable to induce chromosome breaks and/or gain or loss in this test system.
Providing that all of the acceptability criteria are fulfilled, a test item is considered to be clearly positive if, in any of the experimental conditions examined:
− At least one of the test item concentrations exhibits a statistically significant increase compared with the concurrent solvent control
− The increase is concentration-related in at least one experimental condition
− The results are outside the range of the laboratory historical solvent control data
When all of the criteria are met, the test item is then considered able to induce chromosome breaks and/or gain or loss in this test system.
There is no requirement for verification of a clear positive or negative response.

Statistics:

Statistical significance will be confirmed by using the Chi-squared test (α < 0.05) using the validated R Script CHI2.Rnw for those values that indicate an increase in the number of cells with micronuclei compared to the concurrent solvent control. Other statistical methods may be used if appropriate.

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Two independent experiments were performed. In Experiment I, the exposure period was 4 hours with and without S9 mix. In Experiment II, the exposure period was 20 hours without S9 mix. The cells were prepared 40 hours after start of treatment with the test item.
In each experimental group two parallel cultures were analysed. 1000 binucleate cells per culture were scored for cytogenetic damage on coded slides. To determine a cytotoxic effect the CBPI was determined in 500 cells per culture and cytotoxicity is described as % cytostasis.

In Experiment I, precipitation of the test item in the culture medium was observed at 74.6 µg/mL and above in the absence of S9 mix at the end of treatment. In addition, precipitation occurred in Experiment II in the absence and presence of S9 mix at 98.0 µg/mL and above at the end of treatment.

No relevant influence on osmolarity or pH was observed. The osmolarity is generally high compared to the physiological level of approximately 300 mOsm. This effect however, is based on a final concentration of 1% DMSO in medium. As the osmolarity is measured by freezing point reduction, 1% of DMSO has a substantial impact on the determination of osmolarity.
In the absence and presence of S9 mix, no cytotoxicity was observed up to the highest evaluated concentrations, which showed precipitation.

Conclusions:

negative

Executive summary:

The test item dissolved in DMSO, was assessed for its potential to induce micronuclei in human lymphocytes in vitro according to OECD TG 487. In the absence and presence of S9 mix, no cytotoxicity was observed up to the highest evaluated concentrations, which showed precipitation. In both independent experiments, neither a statistically significant nor a biologically relevant increase in the number of micronucleated cells was observed after treatment with the test item. Appropriate mutagens were used as positive controls. They induced statistically significant increases in cells with micronuclei. Therefore, the test item is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to the highest required concentration.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

28 March 2019 - 17 April 2019

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)

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Solubility and stability of the test substance in the solvent/vehicle: Stable in DMSO at nominal concentrations of 0.03 mg/mL and 70 mg/mL during ambient temperature storage for up to 24 hours (Envigo CRS Limited, UK Study Number XS21SQ, 22nd of March 2019)


FORM AS APPLIED IN THE TEST (if different from that of starting material): On the day of the experiment (immediately before treatment), the test item was dissolved in DMSO. The final concentration of DMSO in the culture medium was 1% (v/v). All formulations were prepared freshly before treatment and used within two hours of preparation.

Target gene:

HPRT locus

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: MEM containing Hank's salts supplemented with 10 % FBS (except during 4 hour treatment), neomycin (5 µg/mL) and amphotericin B (1 %); for the selection of mutant cells the complete medium was supplemented with 11 µg/mL 6-thioguanine.
- 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 from the liver of phenobarbital/beta-naphthoflavone induced rats.

Test concentrations with justification for top dose:

Pre-experiment (4 hours exposure): without and with S9-mix: 5.5, 10.9, 21.9, 43.8, 87.5 (p), 175.0 (p), 350.0 (p) and 700.0 (p) mg/mL

Main- experiment (4 hours exposure): without and with S9-mix: 3.2, 6.3, 12.5, 25.0, 50.0, 100.0 (p) and 200.0 (p) µg/mL

The following concentrations were selected for reading:
Exp. I with and wihout S9-mix: 6.3, 12.5, 25.0, 50.0, 100.0 (p) ug/mL

(p) = precipitation visible at the end of treatment

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen based on its compatibility with the test item, its solubility properties and its relative non-toxicity to the cell cultures.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

A pre-test was performed in order to determine the toxicity of the test item. In addition the pH-value and the osmolarity were measured. The general culturing and experimental conditions in this pre-test were the same as described below for the mutagenicity experiment.
In this pre-test approximately 1.5 million cells were seeded in 25 cm² flasks 24 hours prior to treatment. After approximately 24 hours the test item was added and the treatment proceeded for 4 hours (duplicate cultures per concentration level). Immediately after treatment the test item was removed by rinsing with PBS. Subsequently, the cells were trypsinized and suspended in complete culture medium. After an appropriate dilution the cell density was determined with a cell counter. Toxicity of the test item is evident as a reduction of the cell density compared to a corresponding solvent control. A cell density of approximately 1.5 million cells in 25 cm² flasks is about the same as approximately 10 million cells seeded in 175 cm² bottles 24 hours prior to treatment with the main experiment.
The pre-experiment was performed in the presence and absence (4 h treatment) of metabolic activation. Test item concentrations between 5.5 µg/mL and 700 µg/mL were used. The highest concentration of the pre-experiment was based on the solubility properties of the test item.


Culture Medium:
For seeding and treatment of the cell cultures the complete culture medium was MEM (minimal essential medium) containing Hank’s salts, 10% FBS (except during 4 hour treatment), neomycin (5 μg/mL) and amphotericin B (1%). 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.
Seeding:
Two to four days after sub-cultivation stock cultures were trypsinized at 37 °C for approximately 5 to 10 minutes. Then the enzymatic digestion was stopped by adding complete culture medium with 10% FBS and a single cell suspension was prepared. The trypsin concentration for all sub-culturing steps was 0.2% in saline. Prior to the trypsin treatment the cells were rinsed with PBS. Approximately 0.7 to 1.2 ×10+E7 were seeded in plastic flasks. The 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, either without S9 mix or with 50 µl/mL S9 mix. Concurrent solvent and positive controls were treated in parallel. 4 hours after treatment, this medium was replaced with complete medium following two washing steps with PBS.
Immediately after the end of treatment the cells were trypsinised as described above and sub-cultivated. At least 2.0 x 106 cells per experimental point (concentration series plus controls) were subcultured in 175 cm² flasks containing 30 mL medium.
Two additional 25 cm² flasks were seeded per experimental point with approx. 500 cells each to determine the relative survival (RS) as measure of test item induced cytotoxicity. The cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % CO2.
The 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 approximately 2.0 x 106 cells per experimental point were sub-cultivated in 175 cm² flasks containing 30 mL medium.
Following the expression time of 7 days five 75 cm² cell culture flasks were seeded with about 4 to 5 x 10+E5 cells each in medium containing 6-TG. Two additional 25 cm² flasks were seeded with approx. 500 cells each in non-selective medium to determine the viability (cloning efficiency II).
The cultures were incubated at 37 °C in a humidified atmosphere with 1.5% CO2 for about 8 days. The colonies were stained with 10% methylene blue in 0.01% KOH solution.
The stained colonies with more than 50 cells were counted. In doubt the colony size was checked with a preparation microscope.

Evaluation criteria:

A test item is classified as clearly mutagenic if, in any of the experimental conditions examined, all of the following criteria are met:
a) at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b) the increase is dose-related when evaluated with an appropriate trend test,
c) any of the results are outside the distribution of the historical negative control data (e.g. Poisson-based 95% control limits).

A test item is classified as clearly non-mutagenic if, in all experimental conditions examined, all of the following criteria are met:
a) none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b) there is no concentration-related increase when evaluated with an appropriate trend test,
c) all results are inside the distribution of the historical negative control data (e.g. Poisson-based 95% control limits).

There is no requirement for verification of a clearly positive or negative response. In case the response is neither clearly negative nor clearly positive as described above or in order to assist in establishing the biological relevance of a result, the data should be evaluated by expert judgement and/or further investigations.
In rare cases, even after further investigations, the data set will preclude making a conclusion of positive or negative results, and therefore the test chemical response will be concluded to be equivocal.

Statistics:

A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. The number of mutant colonies obtained for the groups treated with the test item were 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 and statistical significance were considered together.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Pre-experiment:
No relevant cytotoxic effects indicated by a cloning efficiency I or relative cell density below 50% occurred up to the maximum concentration (the highest concentration was based on the solubility properties of the test item) with and without metabolic activation.
The 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. Precipitation occurred at 87.5 µg/mL and above after 4 hours treatment with and without metabolic activation.
There was no relevant shift of pH and osmolarity of the medium even at the maximum concentration of the test item.

Conclusions:

negative

Executive summary:

A study according to OECD TG 476 was conducted in order to investigate the potential of the test item to induce gene mutations at the HPRT locus in mammalian cells. For that purpose, V79 cells were exposed to the test item for 4 hours with and without metabolic activation. The highest applied concentration in the pre-test on toxicity (700 µg/mL) and in the main experiment (200 μg/mL) was chosen with regard to the OECD Guideline 476. No substantial and reproducible dose dependent increase of the mutation frequency was observed. 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. In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations in mammalian cells.

 

 

 

 

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the available studies no classification according to EU Regulation 1272/2008 is required for genotoxicity.