Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial Mutagenicity (Key, OECD 471), Ames: positive with and without metabolic activation

Mammalian Cytogenicity (Key, OECD 473), Chromosome Aberration: positive with and without metabolic activation

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:
18 Jan 2018 - 07 Feb 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted July 21, 1997
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
his operon for S. typhimurium strains
trp operon for E. coli strains

Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Arocolor 1254.
Test concentrations with justification for top dose:
Pre-experiment (dose-range finding test, Direct Plate Assay): 1.7, 5.4, 17, 52, 164, 512, 1600, 5000 µg/plate
Main-experiment (Pre-incubation Assay): 17, 52, 164, 512, 1600, 5000 µg/plate (Based on the results of the dose-range finding test)
Vehicle / solvent:
- Vehicle/solvent used: water (Milli-Q-water, Millipore Corp., Bedford, MA., USA).
- Justification for choice of solvent/vehicle: The test item formed a clear colorless solution in Milli-Q water.

- Vehicle/solvent used for positive control items: Saline = physiological saline (Eurovet Animal Health, Bladel, The Netherlands)
DMSO = dimethyl sulfoxide (Merck, Darmstadt, Germany)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: ICR-191, 2-aminoanthracene (2AA)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar, Direct Plate Assay, Pre-incubation assay

DURATION
- Preincubation period: 30 ± 2 min
- Exposure duration: at least 48 ± 4 h

NUMBER OF REPLICATIONS: 3 replications each in 2 independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: To determine the toxicity of the test item, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were observed.
Evaluation criteria:
ACCEPTABILITY CRITERIA
A Salmonella typhimurium reverse mutation assay and/or Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
a) The vehicle control and positive control plates from each tester strain (with or without S9-mix) must exhibit a characteristic number of revertant colonies when compared against relevant historical control data generated at Charles River Den Bosch.
b) The selected dose-range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.
c) No more than 5% of the plates are lost through contamination or some other unforeseen event. If the results are considered invalid due to contamination, the experiment will be repeated.
All results presented in the tables of the report are calculated using values as per the raw data rounding procedure and may not be exactly reproduced from the individual data presented.

EVALUATION CRITERIA
A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one follow up experiment. A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537 or TA98 is greater than three (3) times the concurrent control.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.

Statistics:
Mean values and standard deviations were calculated.
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Cytotoxicity was observed at 1600 μg/plate onwards without metabolic activation and at 5000 μg/plate with metabolic activation.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
without
Genotoxicity:
positive
Remarks:
2.3-fold dose-related increase in the number of revertant colonies in the absence of S9-mix.
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Cytotoxicity was observed at 1600 μg/plate onwards without metabolic activation and at 5000 μg/plate with metabolic activation.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with
Genotoxicity:
negative
Remarks:
1.9-fold dose-related increase in the number of revertant colonies in the presence of S9-mix. The increase observed was within the laboratory historical control data ranges and less than two-fold the concurrent solvent control
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Cytotoxicity was observed at 1600 μg/plate onwards without metabolic activation and at 5000 μg/plate with metabolic activation.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Cytotoxicity was observed at 1600 μg/plate onwards without metabolic activation and at 5000 μg/plate with metabolic activation.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Cytotoxicity was observed at 1600 μg/plate onwards without metabolic activation and at 5000 μg/plate with metabolic activation.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Direct Plate Assay
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation of the test substance on the plates was not observed at the start or at the end of the incubation period in any tester strain.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: Cytotoxicity, as evidenced by a decrease in the number of revertants and/or reduction of the bacterial background lawn, was observed in all tester strains. Except for tester strain WP2uvrA, where no toxicity was observed in the absence or presence of S9-mix.

Pre-Incubation Assay
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation of the test substance on the plates was not observed at the start or at the end of the incubation period in any tester strain.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: Cytotoxicity, as evidenced by a decrease in the number of revertants and/or reduction of the bacterial background lawn, was observed in all tester strains from 1600 μg/plate onwards without metabolic activation and at 5000 μg/plate with metabolic activation. Except for tester strain WP2uvrA in the presence of S9-mix, where no toxicity was observed

  Table 3: Dose-Range Finding Test (Direct-Plate assay)

 

EXPERIMENT 1 (Revertant colonies per plate ± SD)

 

S9-Mix

Without

 

 

Test item (µg/plate)

TA 100

 

WP2uvrA

 

Vehicle Control

 

113 ± 11

33 ± 11

Test Substance

1.7

121 ± 15

25 ± 5

5.4

123 ± 19

37 ± 2

17

115 ± 12

21 ± 1

52

134 ± 12

33 ± 6

164

141 ± 13

37 ± 6

512

166 ± 16

35 ± 0

1600

292 ± 22n 

41 ± 9

5000

0 ± 0aNP 

51 ± 4nNP

Positive Control

 

744 ± 65

1602 ± 77

 

S9-Mix

With

 

 

Test item (µg/plate)

TA 100

WP2uvrA

Vehicle Control

-

118 ± 6

43 ± 7

Test Substance

1.7

100 ± 8

50 ± 6

5.4

106 ± 13

40 ± 3

17

116 ± 13

35 ± 9

52

122 ± 6

38 ± 8

164

135 ± 18

43 ± 4

512

169 ± 21

58 ± 5

1600

310 ± 19

75 ± 4

5000

106 ± 941NP

52 ± 5nNP

Positive Control

 

2156 ± 227

558 ± 54

 

1=Revertants were only observed on a small part of the plate with normal background, no bacterial growth or background was observed on a large part of the plate

NP= No precipitate

a = Bacterial background lawn absent

n = Normal bacterial background lawn

s = Bacterial background lawn slightly reduced

 

Table 4: Direct-Plate assay

 

EXPERIMENT 1 (Revertant colonies per plate ± SD)

 

S9-Mix

Without

 

 

Test item (µg/plate)

TA 1535

TA 1537

TA 98

Vehicle Control

 

12 ± 2

3 ± 2

11 ± 1

Test Substance

17

12 ± 2

3 ± 0

17 ± 5

52

13 ± 5

4 ± 3

14 ± 3

164

14 ± 4

2 ± 1

11 ± 3

512

13 ± 2

2 ± 1

10 ± 6

1600

11 ± 3n

2 ± 1n

12 ± 6n

5000

0 ± 0aNP

2 ± 2eNP

0 ± 0aNP

Positive Control

 

1091 ± 65

834 ± 49

1141± 30

 

S9-Mix

With

 

 

Test item (µg/plate)

TA 1535

TA 1537

TA 98

Vehicle Control

-

11 ± 6

3 ± 4

16 ± 2

Test Substance

17

12 ± 5

4 ± 3

16 ± 3

52

10 ± 2

7 ± 4

22 ± 6

164

13 ± 5

4 ± 1

16 ± 4

512

17 ± 8

3 ± 2

17 ± 4

1600

18 ± 3

3 ± 2

19 ± 7

5000

5 ± 81NP

1 ± 21NP

3 ± 21NP

Positive Control

 

408 ± 32

374± 108

1298± 195

 

1=Revertants were only observed on a small part of the plate with normal background, no bacterial growth or background was observed on a large part of the plate

NP= No precipitate

a = Bacterial background lawn absent

n = Normal bacterial background lawn

s = Bacterial background lawn slightly reduced

 

Table 5: Plate-Incubation Test

 

EXPERIMENT 2 (Revertant colonies per plate ± SD)

 

S9-Mix

Without

 

 

Test item (µg/plate)

TA 1535

TA 1537

TA 98

TA 100

(fold)

WP2uvrA

(fold)

Vehicle Control

 

10 ± 2

3 ± 4

18 ± 3

106 ± 14

19 ± 5

Test Substance

17

13 ± 5

3 ± 1

17 ± 6

122 ± 15

28 ± 2

52

12 ± 2

3 ± 2

13 ± 5

130 ± 5

29 ± 15 (1.5)

164

19 ± 1

3 ± 2

14 ± 3

134 ± 16 (1.3)

35 ± 7 (1.8)

512

13± 3n

8 ± 2n

17 ± 3n

195 ± 5n (1.8)

44 ± 8n(2.3)

1600

0 ± 0a

0± 0a

0± 0a

0± 0a

1± 01n

5000

0 ± 0aNP

0 ± 0aNP

0 ± 0aNP

0 ± 0aNP

0 ± 0aNP

Positive Control

 

1036 ± 33

138 ± 14

1146 ± 68

781 ± 11

152 ± 85

 

S9-Mix

With

 

 

Test item (µg/plate)

TA 1535

TA 1537

TA 98

TA 100

WP2uvrA

Vehicle Control

-

11 ± 2

5 ± 3

19 ± 6

102 ± 2

30 ± 7

Test Substance

17

13 ± 2

4 ± 2

15 ± 6

166 ± 18

36 ± 6

52

10 ± 5

7 ± 2

13 ± 2

180 ± 20

42 ± 6

164

14 ± 3

7 ± 2

19 ± 4

139 ± 7

39 ± 8

512

10 ± 0

5 ± 3n

13 ± 2

190 ± 26 (1.9)

42 ± 4 (1.4)

1600

14 ± 6

2 ± 1s

15 ± 3n

304 ± 22n(3.0)

56 ± 13 (1.9)

5000

0 ± 11nNP

0 ± 0aNP

0 ± 0aNP

0 ± 0aNP

57 ± 11nNP(1.9)

Positive Control

 

247 ± 22

230 ± 32

762 ± 14

1586 ± 89

587 ± 30

 

1=Revertants were only observed on a small part of the plate with normal background, no bacterial growth or background was observed on a large part of the plate

NP= No precipitate

a = Bacterial background lawn absent

n = Normal bacterial background lawn

s = Bacterial background lawn slightly reduced

 

Conclusions:
Under the conditions of the conducted test the substance induced significant dose-related increases in the number of revertant colonies in the tester strain TA100 and WP2uvrA both in the absence and presence of S9-mix. In the latter, only in the absence of S-9 mix in experiment 2, these increases exceeded the two-fold threshold (2.3-fold). Based on these results, the test substance is regarded to be mutagenic in bacteria.
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 April 1986 - 01 July 1986
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
1983
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
Not applicable
Species / strain / cell type:
lymphocytes: human
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Human peripheral blood was obtained by venepuncture from a healthy, non-smoking, male, human volunteer not currently taking any medication, and collected in heparinised vessels
- Sex, age and number of blood donors if applicable: One male volunteer, age not reported
- Whether whole blood or separated lymphocytes were used if applicable: whole blood

MEDIA USED
Complete culture medium: 418.0 mL RPMI 1640 medium with HEPES, sodium bicarbonate and L-glutamine; 75.0 mL fetal calf serum; 5.0 mL Heparin (100 i.u./mlL); and 2.0 mL Penicillin/streptomycin (5000 i.u./mL; 5000 ug/mL)
Treatment medium: 480.5 mL RPMI 1640 medium with HEPES, sodium bicarbonate and L-glutamine; 12.5 mL fetal calf serum; 5.0 mL Heparin (100 i.u./mlL); and 2.0 mL Penicillin/streptomycin (5000 i.u./mL; 5000 ug/mL)
Additional strain / cell type characteristics:
not applicable
Cytokinesis block (if used):
Cell division was arrested by the addition of the spindle poison, Colcemid (to a final concentration of 0.4 µg/mL)
Metabolic activation:
with and without
Metabolic activation system:
S9 liver microsomal fraction of Arcolor 1254 induced rats
Test concentrations with justification for top dose:
Preliminary experiment: 1.4, 7.2, 36, 180, 900 µg/mL, ± S9

Main experiment: 7.5, 15.0 and 30.0 ug/mL, -S9; 3.75, 7.5 and 15.0 µg/mL, +S9
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The compound was found to be soluble in dimethyl sulphoxide (DMSO) up to a maximum concentration of approximately 184 mg/mL.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
other:
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48 h
- Exposure duration: 24 h with and without metabolic activation during the first two hours of exposure
- Expression time (cells in growth medium): 24 h (including a 3 hour Colcemid treatment)

SPINDLE INHIBITOR (cytogenetic assays): Colcemid

STAIN (for cytogenetic assays): Giemsa stain

NUMBER OF REPLICATIONS: 2

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: After cells were fixed, the tubes were centrifuged, the supernatant removed and the cell pellet resuspended in a few drops of fresh fixative. Single drops of the cell suspension were
transferred to clean, moist, grease-free glass slides, and the slides were left to air-dry. Two or four slides (for the preliminary toxicity test or main cytogenetic test respectively) were made from each culture, stained for ten minutes in Giemsa stain (1 in 10 in Sorensen's buffer pH 6.8), washed in buffer and left to air-dry. Permanent mounts were made using DPX mountant after clearing in xylene.

NUMBER OF CELLS EVALUATED: For the preliminary experiment, approximately 1000 lymphocytes per culture were examined using a light microscope, and the mitotic index was calculated as the percentage. For the main experiment, approximately 1000 cells were scored and the mitotic index calculated.

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): 100 metaphases (with 46 centromeres)

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes
- Methods: Scoring followed the recommendations of the Ad Hoc committee of the Environmental Mutagen Society and the Institute for Medical Research, 1972
Evaluation criteria:
According to the OECD guidelines, the biological relevance of the results will be the criterion for the interpretation of the results, a statistical evaluation of the results is not regarded as necessary. However, for the interpretation of the data both, biological and if evaluated, statistical significance should be considered together. A test item is considered to be negative if there is no biologically relevant increase in the percentage of aberrant cells above concurrent control levels, at any dose group.
Statistics:
The Fisher Exact Probability test is a useful nonparametric technique for analysing data when comparing two small independent samples. It is used when the scores for the samples all fall into one or other of two mutually exclusive classes. The test determines whether the two groups differ in the proportions with which they fall into the two classifications.

Data from replicate cultures with or without S-9 mix were compared. If significant differences were found, each treatment was then compared with the respective negative control - with or without activation. Where no significant differences were found, data from activated and non-activated cultures were pooled for subsequent analysis.
Key result
Species / strain:
lymphocytes: human
Metabolic activation:
without
Genotoxicity:
positive
Remarks:
15 and 30 µg/mL
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
lymphocytes: human
Metabolic activation:
with
Genotoxicity:
positive
Remarks:
7.5 and 15 µg/mL
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
26% reduction at 15 µg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES: The preliminary test showed marked depression of cell division at an test item concentration of 36.0 µg/mL (65% reduction of mitotic index over control values without S-9 mix, 93% reduction with S-9 mix).

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: In the absence of S-9 mix mean mitotic indices were 3.3, 2.7 and 2.4 for treatment levels of 7.5, 15.0 and 30.0 µg/mL respectively, compared with the solvent control value of 2.4 showing no reduction in mitotic activity at any level tested. In the presence of S-9 mix mean mitotic indices were 7.6, 7.3 and 6.1 for treatment levels of 3.75, 7.5 and 15 µg/mL, showing an approximately 26% reduction in mitotic activity at the highest test item concentration tested compared to the control value of 8.2.

Table 1: Main cytogenetic test – number and types of chromosomal aberrations

 

Group

Concentration (µg/mL)

S-9 Mix

Numbers of cells scored

Mean mytotic index

Cells with aberrations

Cells with aberrations

 

 

 

 

 

Total

Range %

Mean %*

Total

Range %

Mean %*

DMSO

/

-

300

2.4

9

1-5

3.0

3

0-2

1.0

 

 

+

300

8.2

6

1-4

2.0

0

0-0

0.0

Test substance

3.75

-

/

/

/

/

/

/

/

/

 

+

300

7.6

14

3-7

4.7

4

0-2

1.3

 

7.5

-

300

3.3

14

3-6

4.7

5

1-2

1.7

 

+

300

7.3

14

2-7

4.7

6

1-3

2.0

 

15.0

-

212

2.7

31

14-15.4

14.6

17

7-10

8.0

 

+

300

6.1

23

5-10

7.7

9

2-4

3.0

 

30.0

-

60

2.4

9

0-26.1

15.0

9

0-26.1

15.0

 

+

/

/

/

/

/

/

/

/

Cyclophosphamide

6

-

300

5.3

4

0-4

1.3

0

0-0

0.0

 

 

+

300

2.9

188

56-68

62.7

149

43-58

49.7

Chlorambucil

2.5

-

300

2.9

123

32-48

41.0

80

23-33

26.7

 

 

+

/

/

/

/

/

/

/

/

*Mean% = (Number aberrant metaphases/ Total cells scored) x 100

Conclusions:
Under the conditions of the conducted test, the test substance induced damage to chromosomal structure in peripheral human lymphocytes. In the main experiment statistically significant increase in % aberrant cells was observed at test substance concentration of 15 and 30 µg/mL without metabolic activation and at 7.5 and 15 µg/mL with metabolic activation (cytotoxicity detected at 15 µg/mL). Therefore, the test substance is regarded to be clastogenic.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

A testing proposal for an in vivo somatic cell study according to OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test) is provided.

Endpoint conclusion
Endpoint conclusion:
no study available (further information necessary)

Additional information

Genetic toxicity (mutagenicity) in bacteria in vitro

A bacterial gene mutation assay (Ames test) was performed according to OECD TG 471 under GLP conditions (Groot, 2018). The strains Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 and Escherichia coli WP2 uvr A were tested according to the direct plate and the pre-incubation method in the absence and presence of a metabolic activation system (Aroclor 1254-induced rat liver S9-mix). A dose-range finding experiment (direct plate assay) at concentrations from 1.7 to 5000 µg/plate were performed. The main experiments (direct plate assay, pre-incubation assay) were conducted at concentrations from 17 to 5000 µg/plate (vehicle: water), including positive controls. The test substance induced significant dose-related increases in the number of revertant colonies in the tester strain TA 100 both in the absence and presence of S9-mix. The increases were observed in both experiments. In tester strain WP2 uvrA, dose-related increases in the number of revertant colonies both in the absence and presence of S9-mix were observed. However, these increases remained mostly below the two-fold threshold. Only in the absence of S-9 mix in experiment 2, the test item induced an up to 2.3-fold dose-related increase in the number of revertant colonies in tester strain WP2uvrA. However, the increase was more than two-fold the concurrent solvent control. Cytotoxicity, evident by a decrease in the number of revertants and/or reduction of the bacterial background lawn, was observed in all tester strains from 1600 µg/plate onwards without metabolic activation and at 5000 µg/plate with metabolic activation, except for tester strain WP2uvrA, where no toxicity was observed. The included positive and negative controls showed the expected results. Hence, the test substance was mutagenic in the TA 100 strain tested with and without metabolic activation and in WP2uvrA without metabolic activation.

 

Genetic toxicity (cytogenicity) in mammalian cells in vitro

An in vitro mammalian chromosome aberration test was conducted with the test substance in accordance with OECD TG 473 (Bootman, 1986). The induction of structural chromosome aberrations was evaluated in vitro in lymphocytes of freshly heparinised human whole blood cultures, with and without a metabolic activation system (S9 -mix from rats treated with Arocolor 1254). Test substance concentrations of 1.4 - 900 µg/mL (preliminary experiment), 7.5 - 30 µg/mL (main experiment: without metabolic activation) and 3.75 -15 µg/mL (main experiment: with metabolic activation) were applied for 24 hours. Cell division was arrested by the addition of colcemid, three hours before the cells were harvested. In the preliminary study marked depression of cell division was observed at concentrations of 36 µg/mL (65% reduction of mitotic index over control values without S-9 mix, 93% reduction with S-9 mix). In the main experiment statistically significant increase in % aberrant cells was observed at test substance concentration of 15 and 30 µg/mL without metbaolic activation and at 7.5 and 15 µg/mL with metabolic activation. In the absence of S-9 mix no reduction in mitotic activity were observed. In the presence of S-9 mix mean mitotic indices were 7.6, 7.3 and 6.1 for treatment levels of 3.75, 7.5 and 15 µg/mL, showing an approximately 26% reduction in mitotic activity at the highest test substance concentration tested compared to the control value of 8.2. The negative as well as the positive controls showed the expected results. Therefore, under the conditions of the study, the test substance showed clastogenic activity in this chromosomal aberration test with and without metabolic activation performed in peripheral human lymphocytes in vitro.

 

Conclusion

Taken together, the available in vitro data on genetic toxicity and mutagenicity indicate a mutagenic and clastogenic potential of the test substance.

Justification for classification or non-classification

Data on genetic toxicity in vivo are missing. Therefore the available data on genotoxicity and mutagenicity do not meet criteria for classification according to Regulation (EC) No. 1272/2008 (CLP).