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Genetic toxicity in vitro

Description of key information

An Ames test and a test on mammalian cell lines was conducted to determine the mutagenic potential of the test item. The test item showed no mutagenic potential in both tests.

The test item was assessed for its potential to induce structural chromosomal aberrations in human lymphocytes in vitro according to OECD guideline 473. The test item did not induce structural chromosomal aberrations.
The HPRT test was performed to investigate the potential of the test item to induce gene mutations at the HPRT locus in V79 cells. It can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells.

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Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015-01-14 to 2015-07-06
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
2014
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
Version / remarks:
1998
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test
Test material information:
Composition 1
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/β-naphthoflavone induced rat liver S9 was used as the metabolic activation system
Test concentrations with justification for top dose:
10.67, 18.67, 32.67, 57.17, 100.04, 175.07, 306.38, 536.16, 938.29, 1642.0 µg/mL (Exp I and IIA without S9 Mix)
100.04, 175.07, 201.11, 268.15, 306.38, 402.22, 536.29, 670.36 µg/mL (Exp. IIB without S9 Mix)
10.67, 18.67, 32.67, 57.17, 100.04, 175.07, 306.38, 536.16, 938.29, 1642.0 µg/mL (Exp I with S9 Mix)
100.04, 175.07, 306.38, 536.16, 938.29, 1642.0 µg/mL (Exp IIA with S9 Mix)
Vehicle:
DMSO
Negative controls:
no
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
Without metabolic activation
Negative controls:
no
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
With metabolic activation
Details on test system and conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: Without S9-Mix: 4 h, 22 h
With S9 Mix: 4 h
- Fixation time: The cultures were harvested by centrifugation 22 h after beginning of treatment

SPINDLE INHIBITOR: Colcemid (final concentration: 0.2 μg/mL)
STAIN: Giemsa

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: At least 150 metaphases per culture were evaluated

DETERMINATION OF CYTOTOXICITY
- Method: Percentages of mitotic suppression in comparison with the controls by counting 1000 cells per culture in duplicate

Evaluation criteria:
The chromosomal aberration assay will be considered acceptable if it meets the following criteria:
a) The rate of chromosomal aberrations in the solvent controls falls within the historical laboratory control data range.
b) The rate of chromosomal aberrations in the positive controls is statistically significant increased.

A test item can be classified as non-clastogenic if:
− the number of induced structural chromosomal aberrations in all evaluated dose groups is in the range of the historical laboratory control data and
− no statistically significant increase of the rate of structural chromosomal aberrations is observed in comparison to the respective solvent control.

A test item can be classified as clastogenic if:
− the number of induced structural chromosomal aberrations is not in the range of the historical laboratory control data and
− either a concentration-related or a statistically significant increase in the number of cells carrying structural chromosomal aberrations is observed.
Statistics:
The current historical data range together with the statistical significance, confirmed by the Fisher’s exact test (modified) (p < 0.05) using the validated R Script LM.Rnw, should be considered for classification of the test item.
Key result
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Vehicle controls valid:
yes
Negative controls valid:
not examined
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Additional information on results:
The test item, dissolved in DMSO, was assessed for its potential to induce chromosomal aberrations in human lymphocytes in vitro in the absence and presence of metabolic activation by S9 mix.
Three independent experiments were performed. In Experiment I the exposure period was 4 hours with and without S9 mix. In Experiment IIA the exposure period was 4 hours with S9 mix and 22 hours without S9 mix. In Experiment IIB (confirmatory experiment) the exposure period was 22 hours without S9 mix. The chromosomes were prepared 22 hours (in all experiments) after start of treatment with the test item.
In each experimental group two parallel cultures were analysed. At least 150 metaphases per culture were evaluated for structural chromosomal aberrations, except for the positive control in Experiment II, in the absence of S9 mix, where only 50 metaphases were evaluated due to strong clastogenic effects. 1000 cells were counted per culture for determination of the mitotic index.
The highest treatment concentration in this study, 1642.0 µg/mL (approx. 10 mM) was chosen with regard to the molecular weight of the test item and with respect to the OECD Guideline for in vitro mammalian cytogenetic tests.
In Experiment I and IIA, phase separation of the test item in the culture medium was observed at 536.16 µg/mL and above in the absence and presence of S9 mix at the end of treatment.
No relevant influence on osmolarity or pH was observed.
In Experiment I in the absence and presence of S9 mix, in Experiment IIB in the absence of S9 mix and in Experiment IIA in the presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration. In Experiment IIA in the absence of S9 mix no cytotoxicity was observed up to the highest evaluated concentration. Toxic effects, indicated by reduced mitotic indices were observed at higher concentrations.
In Experiments I and IIB, in the absence of S9 mix and Experiments I and IIA in the presence of S9 mix, no statistically significant or biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed. The aberration rates of the cells after treatment with the test item (0.3 – 3.0% aberrant cells, excluding gaps) exceeded the range of the solvent control values (1.0 – 2.3% aberrant cells, excluding gaps), but were within the range of the laboratory historical solvent control data. In Experiment IIA in the absence of S9 mix a minor but statistically significant increase was observed, following continuous treatment. Therefore a confirmatory experiment with closer concentrations was performed (reported as Exp. IIB). Since no increase was observed in this confirmatory experiment, the observed statistical significant values are judged as biological irrelevant.
Either EMS (660.0 or 770.0 µg/mL) or CPA (7.5 or 15.0 µg/mL) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations.

Experiment

Preparation interval

Test item concentration in µg/mL

Mitototic indices in % of control

incl. Gaps

aberrant cells in % excl. Gaps

with exchanges

Exposure period 4 h without S9-Mix

I

22 h

solvent control

100.0

2.7

2.3

0.0

Positive control

56.2

8.7

8.3

1.7

306.38

98.2

2.7

2.7

0.0

536.16

81.2

1.3

1.0

0.0

938.29

118.5

3.0

2.3

0.3

1642.00

71.0

1.7

1.7

0.0

Exposure period 22 h without S9 Mix

IIA

22 h

Solvent control

100.0

1.7

1.0

0.0

Positive control

33.4

51.9

51.9

9.0

175.07

111.8

1.3

1.3

0.0

306.38

114.5

3.2

3.0

0.2

536.16

96.3

3.5

3.5

0.0

Exposure period 22 h without S9 Mix

IIB

22 h

Solvent control

100.0

1.3

1.3

0.0

Positive control

53.7

20.3

20.0

5.7

402.22

81.1

1.0

0.7

0.0

536.29

86.3

3.0

3.0

0.3

670.36

67.7

2.0

1.7

0.0

Exposure period 4 h with S9 Mix

I

22 h

Solvent control

100.0

1.0

1.0

0.0

Positive control

41.6

11.0

10.7

1.7

306.38

89.0

0.3

0.3

0.0

536.16

97.6

2.7

2.3

0.0

938.29

114.8

2.3

2.3

0.0

1642.00

117.7

2.0

1.7

0.3

IIA

22 h

Solvent control

100.0

2.3

2.3

0.0

Positive control

85.2

13.0

13.0

0.7

175.07

80.8

2.0

1.3

0.3

306.38

87.9

2.7

1.7

0.0

536.16

86.3

3.0

2.7

0.0

Conclusions:
Interpretation of results (migrated information):
negative

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce structural chromosomal aberrations in human lymphocytes in vitro.
Therefore, the test item is considered to be non-clastogenic in this chromosome aberration test, when tested up to insoluble concentrations (phase separation) and in Experiment IIA up to the highest evaluable concentration.
Executive summary:

The test item, dissolved in DMSO, was assessed for its potential to induce structural chromosomal aberrations in human lymphocytes in vitro in three independent experiments. 150 metaphases were evaluated for structural chromosomal aberrations, except for the positive control in Experiment II, in the absence of S9 mix, where only 50 metaphases were evaluated.

The highest applied concentration in this study (1642.0 µg/mL of the test item, approx. 10 mM) was chosen with regard to the molecular weight of the test item and with respect to the current OECD Guideline 473.

Dose selection of the cytogenetic experiment was performed considering the toxicity data and the solubility of the test item in accordance with the OECD Guideline 473. In Experiment I in the absence and presence of S9 mix, in Experiment IIB in the absence of S9 mix and in Experiment IIA in the presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration. In Experiment IIA in the absence of S9 mix no cytotoxicity was observed up to the highest evaluated concentration. Toxic effects, indicated by reduced mitotic indices were observed at higher concentrations.

In Experiments I and IIB, neither a statistically significant nor a biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item. Only in Experiment IIA a minor but statistically significant increase was observed in the absence of S9 mix following continuous treatment, therefore a confirmatory experiment with closer concentrations was performed (reported as Exp. IIB).

No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures. Appropriate mutagens were used as positive controls. They induced statistically significant increases in cells with structural chromosome aberrations. In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce structural chromosomal aberrations in human lymphocytes in vitro.

Therefore, is considered to be non-clastogenic in this chromosome aberration test, when tested up to insoluble concentrations (phase separation) and in Experiment IIA up to the highest evaluable concentration.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015-11-10 to 2016-01-26
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
2015
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Version / remarks:
1998
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay
Test material information:
Composition 1
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/β-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
25.6, 51.3, 102.5, 205.0, 410.0, 820.0 µg/mL
Vehicle:
DMSO
Negative controls:
no
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
Without metabolic activation
Negative controls:
no
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
With metabolic activation
Details on test system and conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 hours
- Expression time: Approx. 7 days
- Fixation time: Approx. 8 days

SELECTION AGENT: 11 μg/mL 6-thioguanine.

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

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).
However, in a case by case evaluation this decision depends on the level of the corresponding solvent control data. If there is by chance a low spontaneous mutation rate within the laboratory´s historical control data range, a concentration-related increase of the mutations within this range has to be discussed. The variability of the mutation rates of solvent controls was also taken into consideration.
Statistics:
A linear regression 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. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological and statistical significance was considered together.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
not examined
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Additional information on results:
No relevant cytotoxic effect indicated by an adjusted relative cloning efficiency below 50% in both cultures occurred up to the maximum concentration with and without metabolic activation.
No relevant and reproducible increase in mutant colony numbers/106 cells was observed in the main experiment up to the maximum concentration. The 95% confidence interval was not exceeded.
A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. No significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in any of the experimental groups.
In the main experiment with and without S9 mix the range of the solvent controls was from 7.4 up to 24.7 mutants per 106 cells; the range of the groups treated with the test item was from 6.9 up to 25.9 mutants per 106 cells.
EMS (150 μg/mL) and DMBA (2.2 μg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies.

Conc. µg/mL

PS

S9 mix

Relative cloning
efficiency I %

Relative cell
density %

Relative adjusted
cloning efficiency I %

mutant colonies
/ 10 6

95 % confidence interval

relative cloning efficiency I %

relative cell density %

relative adjusted cloning efficiency %

mutant colonies
/ 10 6

95 % confidence interval

Column

1

2

3

4

5

6

7

8

9

10

11

12

13

Exp I/4 h
treatment

culture I

culture II

Solvent control with DMSO

-

100.0

100.0

100.0

20.9

0.0-29.7

100.0

100.0

100.0

24.7

0.0-29.7

Positive control (EMS)

150.0

-

101.5

68.9

69.9

176.1

0.0-29.7

105.9

79.6

84.3

185.3

0.0-29.7

Test item

25.6

-

101.6

77.2

78.5

11.8

0.0-29.7

103.5

95.0

98.3

12.9

0.0-29.7

Test item

51.3

-

100.2

105.4

105.6

13.1

0.0-29.7

99.8

98.9

98.7

25.9

0.0-29.7

Test item

102.5

-

93.1

96.5

89.8

22.4

0.0-29.7

101.7

97.7

99.4

20.9

0.0-29.7

Test item

205.0

-

102.7

103.8

106.5

13.2

0.0-29.7

114.4

84.8

97.0

18.0

0.0-29.7

Test item

410.0

PS

-

86.9

56.2

48.8

15.9

0.0-29.7

80.6

96.3

77.6

9.6

0.0-29.7

Test item

820.0

PS

-

culture was not continued #

culture was not continued #

Solvent control with DMSO

+

100.0

100.0

100.0

7.4

0.0-27.7

100.0

100.0

100.0

13.7

0.0-27.7

Positive control (DMBA)

2.2

+

66.8

171.4

114.5

151.3

0.0-27.7

79.4

104.8

83.2

140.4

0.0-27.7

Test item

25.6

+

104.5

137.4

culture was not continued ##

103.5

108.1

culture was not continued ##

Test item

51.3

+

102.1

104.1

106.3

23.6

0.0-27.7

104.9

109.3

114.7

7.5

0.0-27.7

Test item

102.5

+

96.4

124.6

120.1

11.7

0.0-27.7

107.3

105.0

112.6

6.6

0.0-27.7

Test item

205.0

+

103.6

145.5

150.8

24.5

0.0-27.7

98.2

101.2

99.4

14.5

0.0-27.7

Test item

410.0

PS

+

99.4

102.1

101.5

16.8

0.0-27.7

96.3

103.3

99.5

6.9

0.0-27.7

Test item

820.0

PS

+

98.8

122.6

121.2

21.6

0.0-27.7

103.3

96.9

100.1

11.6

0.0-27.7

PS= phase separation visible at the end of treatment

# = culture was not continued due to exceedingly severe cytotoxic effects

## = culture was not continued since a minimum of only four analysable concentrations is required

Conclusions:
Interpretation of results: negative

It can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, the test item is considered to be non-mutagenic in this HPRT assay.
Executive summary:

The study was performed to investigate the potential of test item to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster. The treatment period was 4 hours with and without metabolic activation. The maximum test item concentration of the pre-experiment (1642 μg/mL) was equal to a molar concentration of about 10 mM. The concentration range of the main experiment was limited by phase separation of the test item and cytotoxicity. No substantial and reproducible dose dependent increase of the mutation frequency was observed in the main experiment. 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 at the HPRT locus in V79 cells. Therefore, the test item is considered to be non-mutagenic in this HPRT assay.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Reference:
Composition 0
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Test material information:
Composition 1
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9 activation system
Species / strain:
other: TA92
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9 activation system
Species / strain:
other: TA94
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9 activation system
Species / strain:
other: TA2637
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9 activation system
Test concentrations with justification for top dose:
Tested at 6 doses with a maximum of 5 mg/plate
Vehicle:
DMSO
Negative controls:
no
Solvent controls:
yes
Remarks:
This conclusion can be drawn because of the validity of the Ames test.
True negative controls:
no
Positive controls:
yes
Remarks:
This conclusion can be drawn because of the validity of the Ames test.
Details on test system and conditions:
METHOD OF APPLICATION: According to Ames, McCann and Yamasaki (1975)

DURATION
- Preincubation period: Cells cultured overnight were pre-incubated with both the test sample and the S-9 mix for 20 min at 37 °C
- Exposure duration: 2 days

Evaluation criteria:
The result was considered positive if the number of colonies found was twice the number in the control.
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
not determined
Vehicle controls valid:
yes
Negative controls valid:
not specified
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Key result
Species / strain:
other: TA92
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
not determined
Vehicle controls valid:
yes
Negative controls valid:
not specified
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Key result
Species / strain:
other: TA94
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
not determined
Vehicle controls valid:
yes
Negative controls valid:
not specified
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Key result
Species / strain:
other: TA2637
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
not determined
Vehicle controls valid:
yes
Negative controls valid:
not specified
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Additional information on results:
Up to 5 mg per plate no effects were seen.
Conclusions:
Interpretation of results
negative

In a mutagenesis study with the Salmonella strains TA92, TA1535, TA100, TA1537, TA94, TA98 and for some samples TA2637 with and without metabolic activation no mutagenic effects were observed.
Executive summary:

The test item was tested at 6 different doses by using Salmonella strains TA92, TA1535, TA100, TA1537, TA94, TA98 and for some samples TA2637 with and without S-9 liver microsome fraction. Each sample was tested at 6 doses; only maximum dose reported. The result was considered positive if the number of colonies found was twice the number in the control (exposed to the appropriate solvent or untreated). As vehicle DMSO was used. At 5 mg per plate no effects were observed.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
abstract
Remarks:
Only one strain tested, only short abstract available
Reference:
Composition 0
Principles of method if other than guideline:
Suspensions of bacterial cells are exposed to the test substance in the presence and in the absence of an exogenous metabolic activation system. Revertant colonies are counted and compared to the number of spontaneous revertant colonies.
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Test material information:
Composition 1
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
not specified
Vehicle:
DMSO
Details on test system and conditions:
METHOD OF APPLICATION: in agar

Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
not specified
Genotoxicity:
negative
Cytotoxicity:
not specified
Vehicle controls valid:
not specified
Negative controls valid:
not specified
Positive controls valid:
not specified
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Additional information on results:
No effects at 0.2-1.6 mg/plate.
Conclusions:
Interpretation of results (migrated information):
negative

The genotoxic potential of the test item was determined in a reverse mutation test in bacteria. No mutagenic potential was found.
Executive summary:

A mutagenic test was conducted with E.coli WP2 uvrA on 18 rec assay positive agents. DMSO was used as vehicle. No effects were observed at a concentration of 0.2 to 1.6 mg/plate.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

A weight of evidence approach was performed to cover the endpoint in vitro gene mutation in bacteria.

Ames Test

Weight of evidence

Ames study of Ishidate, 1984 (Food Chem Toxicol 22 pg 623-636).

The test item was tested at 6 different doses by using Salmonella strains TA92, TA1535, TA100, TA1537, TA94, TA98 and for some samples TA2637 with and without S-9 liver microsome fraction. Each sample was tested at 6 doses; only maximum dose reported. The result was considered positive if the number of colonies found was twice the number in the control (exposed to the appropriate solvent or untreated). The test substance was tested in a purity of 99.9%. DMSO was used as vehicle at 5 mg per plate: No effects were observed

(Anti)Mutagenic acitivity test of Yoo, 1986 (J Osaka City Med Cent 34 (3-4) pg 1986).

A mutagenic test was conducted with E.coli WP2 uvrA on 18 rec assay positive agents. DMSO was used as vehicle. No effects were observed at concentrations from 0.2 to 1.6 mg/plate.

Conclusion: The mutagenic potential of the test item in bacteria was tested on 6 Salmonella strains and in E.coli WP2 uvrA. No mutagenic potential was found in these bacterias. In conclusion it can be stated the test substance is not mutagenic in bacteria.

Chromosome aberration test

The test item, dissolved in DMSO, was assessed for its potential to induce structural chromosomal aberrations in human lymphocytes in vitro in three independent experiments. In Experiment IIA in the absence of S9 mix no cytotoxicity was observed up to the highest evaluated concentration. Toxic effects, indicated by reduced mitotic indices were observed at higher concentrations. No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures. In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce structural chromosomal aberrations in human lymphocytes in vitro. Therefore, it is considered to be non-clastogenic in this chromosome aberration test, when tested up to insoluble concentrations (phase separation) and in Experiment IIA up to the highest evaluable concentration.

HPRT test

The study was performed to investigate the potential of the test item to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster. The concentration range of the main experiment was limited by phase separation of the test item and cytotoxicity. No substantial and reproducible dose dependent increase of the mutation frequency was observed in the main experiment. In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, the test item is considered to be non-mutagenic in this HPRT assay.

Conclusion: 4 genetic toxicity studies were performed to determine the genetic toxicity potential of the test item. No genetic toxicity was observed in all studies.


Justification for selection of genetic toxicity endpoint
Evaluation of genetic toxicity was done based on the GLP and guideline in vitro studies (Ames, CA and HPRT)

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. As a result the substance is not considered to be classified for genetic toxicity under Regulation (EC) No 1272/2008, as amended for the tenth time in Regulation (EU) No 2017/776.