Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

All required in vitro tests showed negative results for genetic toxicity.

The test substance is negative in Bacterial Reverse Mutation Assay for S. typhimurium TA 1535, TA 1537, TA 98 and TA 100, and E. coli WP2 uvr A with and without S9 metabolic activation.

3 -methylbutyl isovalerate as a Read-across substance did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes and therefore is considered to be non-mutagenic in this in vitro micronucleus test.

3 -methylbutyl isovalerate as a Read-across substance did not induce gene mutations at the HPRT locus in V79 cells and therefore is considered to be non-mutagenic in this HPRT assay.

Therefore, using a read across approach , the target substance is considered to be not mutagenic

According to Column 2 in Annex VIII (Section 8.4), an in vivo test is not necessary given the consistently negative results in multiple in vitro studies.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14.07.2015 - 15.10.2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. certificate)
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:
S9
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
3; 10; 33; 100; 333; 1000; 2500 and 5000 µg/plate (experiment 2); top dose chosen following the pre-experiment (experiment 1)
Vehicle:
- 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.
Negative controls:
yes
Remarks:
Medium
Solvent controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA with metabolic activation
Negative controls:
yes
Remarks:
Medium
Solvent controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
TA 1535; TA 100 without metabolic activation
Negative controls:
yes
Remarks:
Medium
Solvent controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylene-diamine
Remarks:
TA 1537, TA 98 without metabolic activation
Negative controls:
yes
Remarks:
Medium
Solvent controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
WP2 uvrA without metabolic activation
Details on test system and conditions:
METHOD OF APPLICATION: in DMSO

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

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: spontaneous reversion rates
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.
Key result
Species / strain:
other: TA 1535, TA 1537, TA 98 and TA 100 bacteria
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes

Experiment 1

Without metabolic activation

 

Dose/plate

TA 1535

TA 1537

TA 98

TA 100

WP2uvrA

DMSO

 

9±2

11±1

24±5

140±3

39±5

Untreated

 

11 ± 2

8 ± 1

27 ± 2

160 ± 10

44 ± 5

Test item

3 µg

8 ± 2

11 ± 2

24 ± 6

135 ± 5

40 ± 3

 

10 µg

11 ± 3

12 ± 2

24 ± 3

158 ± 11

39 ± 2

 

33 µg

10 ± 5

11 ± 3

19 ± 5

139 ± 13

43 ± 8

 

100 µg

8 ± 1

10 ± 2

18 ± 4

153 ± 9

38 ± 8

 

333 µg

9 ± 3

9 ± 3

21 ± 2

126 ± 5

42 ± 7

 

1000 µg

9 ± 1

11 ± 4

27 ± 6

127 ± 6

40 ± 3

 

2500 µg

7 ± 2

5 ± 2

20 ± 2

70 ± 4

38 ± 11

 

5000 µg

7 ± 1

5 ± 2

18 ± 4

46 ± 7*

28 ± 4

NaN3

10 µg

919 ± 66

 

 

2106 ± 122

 

4-NOPD

10 µg

 

 

413 ± 9

 

 

4-NOPD

50 µg

 

90 ±5

 

 

 

MMS

2.0 µL

 

 

 

 

783 ± 68

 

With metabolic activation

 

Dose/plate

TA 1535

TA 1537

TA 98

TA 100

WP2uvrA

DMSO

 

10 ± 2

11 ± 4

34 ± 8

117 ± 10

46 ± 8

Untreated

 

14 ± 4

9 ± 4

36 ± 2

144 ± 10

48 ± 10

Test item

3 µg

11 ± 3

12 ± 4

34 ± 5

115 ± 11

46 ± 7

 

10 µg

9 ± 1

12 ± 5

35 ± 3

107 ± 10

51 ± 7

 

33 µg

9 ± 1

12 ± 4

28 ± 6

110 ± 15

46 ± 6

 

100 µg

9 ± 1

13 ± 4

31 ± 4

116 ± 15

45 ± 4

 

333 µg

11 ± 2

12 ± 3

29 ± 3

101 ± 4

43 ± 2

 

1000 µg

8 ± 2

11 ± 2

34 ± 1

113 ± 6

43 ± 4

 

2500 µg

10 ± 3

9 ± 3

33 ± 2

86 ± 9

37 ± 7

 

5000 µg

9 ± 1

9 ± 2

26 ± 2

63 ± 11

44 ± 1

2-AA

2.5 µg

399 ± 8

225 ± 34

4168 ± 319

3201 ± 160

 

2-AA

10 µg

 

 

 

 

370 ± 37

 

Experiment 2

Without metabolic activation

 

Dose/plate

TA 1535

TA 1537

TA 98

TA 100

WP2uvrA

DMSO

 

11 ± 2

8 ± 3

22 ± 4

176 ± 13

34 ± 4

Untreated

 

11 ± 3

11 ± 2

32 ± 14

189 ± 23

40 ± 6

Test item

3 µg

12 ± 2

7 ± 1

23 ± 4

159 ± 12

35 ± 4

 

10 µg

10 ± 4

7 ± 1

22 ± 4

175 ± 23

36 ± 2

 

33 µg

10 ± 4

6 ± 1

26 ± 4

171 ± 20

33 ± 4

 

100 µg

10 ± 4

10 ± 1

24 ± 8

164 ± 6

34 ± 7

 

333 µg

9 ± 4

8 ± 1

22 ± 3

131 ± 14

37 ± 8

 

1000 µg

8 ± 2

7 ± 2

21 ± 5

91 ± 13

28 ± 4

 

2500 µg

6 ± 1*

7 ± 2

27 ± 1

72 ± 11

27 ± 1*

 

5000 µg

4 ± 2*

7 ± 2*

5 ± 2*

0 ± 0

22 ± 2*

NaN3

10 µg

1173 ± 24

 

 

1895 ± 196

 

4-NOPD

10 µg

 

 

428 ± 28

 

 

4-NOPD

50 µg

 

103 ± 19

 

 

 

MMS

2.0 µL

 

 

 

 

580 ± 90

 

With metabolic activation

 

Dose/plate

TA 1535

TA 1537

TA 98

TA 100

WP2uvrA

DMSO

 

9 ± 1

10 ± 3

28 ± 2

146 ± 14

43 ± 7

Untreated

 

12 ± 3

12 ± 2

40 ± 9

212 ± 8

49 ± 8

Test item

3 µg

9 ± 3

12 ± 2

31 ± 3

143 ± 6

44 ± 6

 

10 µg

10 ± 4

9 ± 5

33 ± 9

151 ± 40

47 ± 14

 

33 µg

13 ± 1

9 ± 4

33 ± 2

148 ± 23

50 ± 11

 

100 µg

9 ± 2

10 ± 4

29 ± 3

139 ± 8

52 ± 7

 

333 µg

10 ± 1

13 ± 4

33 ± 5

136 ± 14

50 ± 8

 

1000 µg

10 ± 4

8 ± 3

33 ± 4

109 ± 4

47 ± 12

 

2500 µg

13 ± 3

8 ± 3

24 ± 4

57 ± 7

32 ± 5

 

5000 µg

13 ± 1* 

7 ± 3*

26 ± 7

40 ± 4

21 ± 2* 

2-AA

2.5 µg

372 ± 72

174 ± 11

3958 ± 855

4259 ± 350

 

2-AA

10 µg

 

 

 

 

298 ± 8

 * Reduced background count

Conclusions:
The test substance is negative in Bacterial Reverse Mutation Assay for S. typhimurium TA 1535, TA 1537, TA 98 and TA 100, and E. coli WP2 uvr A with and without S9 metabolic activation, hence is classified as not mutagenic.
Executive summary:

This study was performed to investigate the potential of the test substance to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations in both experiments: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate. No precipitation of the test item occurred up to the highest investigated dose.

The plates incubated with the test item showed reduced background growth in all strains used. Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in all strains used except strains TA 1537 and WP2 uvrA. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test substance 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.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, the test substance is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
March - May 2016
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)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Qualifier:
according to
Guideline:
other: Japanese Guidelines: “Kanpoan No. 287 -- Environment Protection Agency“ “Eisei No. 127 -- Ministry of Health & Welfare“ “Heisei 09/10/31 Kikyoku No. 2 -- Ministry of International Trade & Industry“.
GLP compliance:
yes (incl. certificate)
Type of assay:
other: gene mutation assay in mammalian cells
Test material information:
Composition 1
Target gene:
HPRT
Species / strain:
other: Chinese hamster (V79)
Details on mammalian cell lines (if applicable):
CELLS USED
- Suitability of cells: Yes
- Cell cycle length, doubling time or proliferation index: doubling time 12 - 16 h in stock cultures


MEDIA USED
- Type and identity of media including CO2 concentration if applicable: MEM (minimal essential medium) containing Hank’s salts, neomycin (5 µg/mL), 10% FBS, and amphotericin B (1 %). During treatment no FBS was added to the medium. Cell culture incubation: humidified atmosphere with 1.5% CO2 at 37°C.
- Properly maintained: [yes]
- Checked for Mycoplasma contamination: [yes]
- Checked for karyotype stability: [yes)
- "Cleansed' against high spontaneous background: [yes]
Metabolic activation:
with and without
Metabolic activation system:
mammalian microsomal fraction S9 mix
Test concentrations with justification for top dose:
Doses applied in the gene mutation assay (concentrations marked with ¹ were chosen for the mutation rate analysis):

Experiment I (without S9, 4h exposure): 1.7, 3.4 ¹, 6.8 ¹, 13.5 ¹, 27.0 ¹, 54.0 ¹, 108.0 µg/mL

Experiment II (with S9, 4h exposure): 26.9 ¹, 53.8 ¹, 107.5 ¹, 215.0 ¹, 430.0* ¹, 860.0* µg/mL

* indicates Phase separation observed

The cultures at the lowest concentration without metabolic activation were not continued as a minimum of only four analysable concentrations is required by the guidelines. The cultures at the highest concentration without metabolic activation were not continued based on exceedingly severe cytotoxicity. The cultures at the highest concentration with metabolic activation were not continued to avoid analysis of too many insoluble concentrations.
Vehicle:
- 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.
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: 4h
- Expression time (cells in growth medium): 7d
- Selection time (if incubation with a selection agent): 8d


SELECTION AGENT (mutation assays): 6-TG (6-thioguanine)

NUMBER OF REPLICATIONS: 5 to determine mutation (culture flasks with medium containing 6-TG per concentration)

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Stained with 10% methylene blue in 0.01% KOH solution


DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
- Any supplementary information relevant to cytotoxicity: The experimental part of the main experiment without metabolic activation was prematurely terminated as exceedingly severe cytotoxicity occurred already at low concentrations. This experimental part was repeated as experiment IA in a lower concentration range. Experiment IA was again terminated due to severe cytotoxicity and repeated as experiment IB with even lower concentrations. The data of experiment IB are reported as first experiment without metabolic activation.
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:
Statistical Analysis: A linear regression analysis (least squares, calculated using a validated excel spreadsheet) 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.
experimental group p-value
experiment I, culture I without S9 mix, p-value: 0.492
experiment I, culture II without S9 mix, p-value: 0.025 S
experiment I, culture I with S9 mix, p-value: 0.453
experiment I, culture II with S9 mix, p-value: 0.855
S = significant trend
Key result
Species / strain:
other: Chinese hamster (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
without metabolic activation
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Additional information on results:
No relevant and reproducible increase in mutant colony numbers/10E6 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. A significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was solely detected in the second culture without metabolic activation. This trend however, was judged as irrelevant as all of the absolute values of the mutation frequency remained within the 95% confidence interval.
In the main experiment with and without S9 mix the range of the solvent controls was from 13.0 up to 18.1 mutants per 10E6 cells; the range of the groups treated with the test item was from 9.3 up to 26.9 mutants per 10E6 cells.
EMS (300 µg/mL) and DMBA (2.3 µg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies.
Conclusions:
The test item did not induce gene mutations at the HPRT locus in V79 cells and therefore is considered to be non-mutagenic in this HPRT assay.
Executive summary:

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 treatment period was 4 hours with and without metabolic activation. The maximum test item concentration of the pre-experiment (1723 µg/mL) was equal to a molar concentration of about 10 mM. The concentration range of the main experiment was limited by the cytotoxicity and the solubility of the test item.

The experimental part of the main experiment without metabolic activation was prematurely terminated as exceedingly severe cytotoxicity occurred already at low concentrations. This experimental part was repeated as experiment IA in a lower concentration range. Experiment IA was again terminated due to severe cytotoxicity and repeated as experiment IB with even lower concentrations. The data of experiment IB are reported as the first experiment without metabolic activation.

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 cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-03-02 to 2016-06-07
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell micronucleus test
Test material information:
Composition 1
Species / strain:
lymphocytes: human
Cytokinesis block (if used):
Cytochalasin B
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
3.65; 6.40; 11.2; 19.6; 34.3; 60.0; 105; 184; 322; 563 µg/L (experiment IA)
11.2, 19.6, 34.3, 60.0, 105, 184, 322, 563, 985, 1723 µg/L (experiment IB)
18.2, 27.3, 41.0, 61.5, 92.2, 138, 207, 311, 467 µg/L (experiment II); top dose chosen due to strong cytotoxic effects (low cell numbers) at higher concentrations
Vehicle:
- 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.
Solvent controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
mitomycin C
other: Demecolcin
Remarks:
Without metabolic activation
Solvent controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
With metabolic activation
Details on test system and conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 20 min
- Exposure duration: 4 hours (experiment I; with and without S9) ; 20 hours (experiment II; without S9)
- Selection time (if incubation with a selection agent): 20 hours Cytochalasin B exposure
- Fixation time (start of exposure up to fixation or harvest of cells): 40 hours after beginning of treatment

SELECTION AGENT (mutation assays): Cytochalasin B

SPINDLE INHIBITOR (cytogenetic assays): Giemsa

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: The slides were prepared by dropping the cell suspension in fresh fixative onto a clean microscope slide.

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

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: The criteria for the evaluation of micronuclei are described in the publication of Countryman and Heddle (1976).

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

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 was confirmed by the Chi square test (a < 0.05), using a validated test script of “R”, a language and environment for statistical computing and graphics. Within this test script a statistical analysis was conducted for those values that indicated an increase in the number of cells with micronuclei compared to the concurrent solvent control.
Key result
Species / strain:
lymphocytes: human
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity:
no
Remarks:
no Cytotoxicity observed in experiment I up to phase separation
Vehicle controls valid:
yes
Remarks:
DMSO
Positive controls valid:
yes
Key result
Species / strain:
lymphocytes: human
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
at highest concentration moderate cytotoxicity was observed
Vehicle controls valid:
yes
Remarks:
DMSO
Positive controls valid:
yes

Summary of results

Experiment

Preparation interval

Test item concentration in µg/mL

Proliferation index CBPI

Cytostasis in %*

Micronucleated cells in %**

 

 

Exposure period 4 hrs without S9 mix

IB

40 hrs

Solvent control1

2.01

 

0.15

 

 

Positive control2

1.81

19.9

10.70S

 

 60.0

 2.01

 0.6

 0.10

 

 105

 1.86

 14.9

 0.25

 

 184PS

 1.86

 15.3

 0.20

 

 

Exposure period 20 hrs without S9 mix

II

40 hrs

Solvent control1

1.95

 

0.75

 

 

Positive control3

1.54

42.5

3.90S

 

 

92.2

1.92

3.0

0.75

 

 

138#

1.72

23.5

1.45S

 

 

207

1.51

46.0

0.90

 

 

Exposure period 4 hrs with S9 mix

IA

40 hrs

Solvent control1

1.88

 

0.20

 

 

Positive control4

1.49

44.3

5.85S

 

 

105

2.02

n.c.

0.20

 

 

184

2.00

n.c.

0.35

 

 

322PS

1.91

n.c.

0.35

 

* For the positive control groups and the test item treatment groups the values are related to the solvent controls

** The number of micronucleated cells was determined in a sample of 2000 binucleated cells

#The number of micronucleated cells was determined in a sample of 4000 binucleated cells

PS Phase separation occurred at the end of treatment

S The number of micronucleated cells is statistically significantly higher than corresponding control values

n.c. Not calculated as the CBPI is equal or higher than the solvent control value

1 DMSO 1.0 % (v/v)

2 MMC 1.0 µg/mL

3 Demecolcin 125.0 µg/mL

4 CPA 15.0 µg/mL

Toxicity – Experiment IA

Concentration (µg/mL)

Exposure time

Preparation interval

CBPI per 500 cells*

Cytostasis (%)

 

With S9 mix

Solvent control

4 hrs

40 hrs

1.88

 

3.65

4 hrs

40 hrs

n.d.

n.d.

6.40

4 hrs

40 hrs

n.d.

n.d.

11.2

4 hrs

40 hrs

n.d.

n.d.

19.6

4 hrs

40 hrs

n.d.

n.d.

34.3

4 hrs

40 hrs

1.91

n.c.

60.0

4 hrs

40 hrs

1.98

n.c.

105

4 hrs

40 hrs

2.02

n.c.

184

4 hrs

40 hrs

2.00

n.c.

322PS

4 hrs

40 hrs

1.91

n.c.

563PS

4 hrs

40 hrs

1.94

n.c.

985PS

4 hrs

40 hrs

1.90

n.c.

1723PS

4 hrs

40 hrs

1.88

n.c

 

Experimental groups evaluated for cytogenetic damage are shown in bold characters

* Mean value of two cultures in %

PS Phase separation occurred at the end of treatment

n.d. Not determined

n.c. Not calculated as the CBPI was equal or higher than solvent control value

Toxicity – Experiment IB

 

Concentration (µg/mL)

Exposure time

Preparation interval

CBPI per 500 cells*

Cytostasis (%)

 

Without S9 mix

Solvent control

4 hrs

40 hrs

2.01

 

11.2

4 hrs

40 hrs

2.05

n.c.

19.6

4 hrs

40 hrs

2.03

n.c.

34.3

4 hrs

40 hrs

2.08

n.c.

60.0

4 hrs

40 hrs

2.01

0.6

105

4 hrs

40 hrs

1.86

14.9

184PS

4 hrs

40 hrs

1.86

15.3

322PS

4 hrs

40 hrs

1.61

39.6

563PS

4 hrs

40 hrs

1.86

15.5

985PS

4 hrs

40 hrs

1.65

36.1

1723PS

4 hrs

40 hrs

n.e.

n.e.

 

Experimental groups evaluated for cytogenetic damage are shown in bold characters

* Mean value of two cultures in %

PS Phase separation occurred at the end of treatment

n.c. Not calculated as the CBPI was equal or higher than solvent control value

n.e. Not evaluable due to strong cytotoxic effects

 

Toxicity – Experiment II

 

Concentration (µg/mL)

Exposure time

Preparation interval

CBPI per 500 cells*

Cytostasis (%)

 

Without S9 mix

Solvent control

20 hrs

40 hrs

1.95

 

18.2

20 hrs

40 hrs

n.d.

n.d.

27.3

20 hrs

40 hrs

n.d.

n.d.

41.0

20 hrs

40 hrs

n.d.

n.d.

61.5

20 hrs

40 hrs

1.96

n.c.

92.2

20 hrs

40 hrs

1.92

3.0

138

20 hrs

40 hrs

1.72

23.5

207

20 hrs

40 hrs

1.51

46.0

311

20 hrs

40 hrs

1.52

45.0

467PS

20 hrs

40 hrs

n.e.

n.e.

 

Experimental groups evaluated for cytogenetic damage are shown in bold characters

* Mean value of two cultures in %

PS Phase separation occurred at the end of treatment

n.d. Not determined

n.c. Not calculated as the CBPI was equal or higher than solvent control value

n.e. Not evaluable due to strong cytotoxic effects

 

Conclusions:
The test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes and therefore is considered to be non-mutagenic in this in vitro micronucleus test.
Executive summary:

The test item, dissolved in DMSO, was assessed for its potential to induce micronuclei in human lymphocytes in vitro in three independent experiments. In each experimental group two parallel cultures were analyzed. Per culture at least 1000 binucleated cells were evaluated for cytogenetic damage. The highest applied concentration in this study (1723 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 487. Dose selection of the cytogenetic experiment was performed considering the toxicity data and the occurrence of test item phase separation in accordance with OECD Guideline 487.

In the experiments IA and IB in the absence and presence of S9 mix, no cytotoxicity was observed up to the highest evaluated concentration, which showed phase separation. In Experiment II in the absence of S9 mix after continuous treatment moderate cytotoxicity of 46.0 % cytostasis was observed at the highest evaluable concentration. In the experiments IA and IB in the absence and presence of S9 mix, no relevant increase in the number of micronucleated cells was observed after treatment with the test item. In Experiment II in the absence of S9 mix after continuous treatment one statistically significant increase (1.45 %) in micronucleate cells was observed after treatment with 138 µg/mL. The value exceeded the 95 % control limit of the historical control data range (0.05 – 1.05% micronucleate cells). This can be declared as biologically irrelevant because the increase is not dose-related. Appropriate mutagens were used as positive controls. They induced statistically significant increases in cells with micronuclei.

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes. Therefore, the test item is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to phase separating or the highest evaluable concentration.

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

Genetic toxicity in vivo

Link to relevant study records
Reference
Endpoint:
genetic toxicity in vivo, other
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Information are available from reliable studies for all the required in vitro endpoints. The results of all studies were in agreement.

Where there was more than one reliable study for an endpoint the most recent studies were selected as key studies.

In vitro key and supporting studies indicate that the test substance is negative in the gene mutation (Bacterial reverse mutation assay / Ames test) test and it is predicted from a read a cross substance 3 -methylbutyl isovalerate in the key mammalian cell gene mutation test/ HPRT, with and without metabolic activation.

One key in vitro mammalian micronucleus study for a read a cross substance 3 -methylbutyl isovalerate as well as one supporting for the test item are available for in vitro mammalian chromosome aberration and, respectively, to determine the genetic toxicity of the substance. The results show that the substance does not induce chromosome aberration or micronuclei in mammalian cells.

Justification for classification or non-classification

Based on the available data, the substance does not require classification for mutagenicity according to Regulation (EC) No 1272/2008.