Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Genetic toxicity in vitro:

Ames test (OECD 471): negative in S. typhimurium TA 1535, TA 1537, TA 100 and TA 98, and E. coli WP2 uvr A , with and without metabolic activation

Chromosome aberration in mammalian cells (OECD 473): negative in Chinese hamster lung fibroblasts (CHL), without metabolic activation

Gene mutation in mammalian cells (OECD 476): negative in Chinese hamster lung fibroblasts, with and without metabolic activation

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21 Dec 2009 - 01 Feb 2010
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 Jul 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
The Department of Health of the Government of the United Kingdom, UK
Type of assay:
bacterial reverse mutation assay
Target gene:
his operon (S. typhimurium strains)
trp operon (E. coli strain)
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 80 mg/kg bw/day phenobarbitone and 100 mg/kg bw/day beta-naphthoflavone
Test concentrations with justification for top dose:
Experiment 1 and 2: 50, 150, 500, 1500 and 5000 μg/plate, with and without metabolic activation
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: the test substance was soluble in DMSO at 50 mg/mL
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
benzo(a)pyrene
other: 2-aminoanthracene
Remarks:
See 'Details on test system and conditions' for concentrations and strains
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation, experiment 1) and preincubation (experiment 2)

DURATION
- Preincubation period: 20 min (experiment 2)
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3 replication each in 2 independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: reduction in growth of bacterial background lawn

OTHER:
- positive control substances used: N-ethyl-N'-nitro-N-nitrosoguanidine (2 μg/plate in DMSO, -S9, WP 2urvA-; 3 μg/plate, -S9, TA 100; 5 μg/plate, -S9, TA 1535); 9-aminoacridine (80 μg/plate in DMSO, -S9, TA 1537); 4-nitroquinoline-1-oxide (0.2 μg/plate in DMSO, -S9, TA 98); 2-aminoanthracene (1 μg/plate in DMSO, +S9, TA 100; 2 μg/plate, +S9 TA 1535 and TA 1537; 10 μg/plate, +S9, WP 2urvA-); benzo-a-pyrene (5 μg/plate in DMSO, +S9, TA 98)
Evaluation criteria:
There are several criteria for determining a positive result, such as the dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolic activation. Biological relevance of the results will be considered first, statistical methods, as recommended by the UKEMS can also be used as an aid to evaluation, however, statistical significance will not be the only determining factor for a positive response.
A test material will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit a definitive judgement about the test material activity. Results of this type will be reported as equivocal.
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
precipitation was observed from 1500 μg/plate in experiment 1 and from 500 μg/plate in experiment 2, with and without metabolic activation
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
precipitation was observed from 1500 μg/plate in experiment 1 and from 500 μg/plate in experiment 2, with and without metabolic activation
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: precipitate was observed at concentrations at and above 1500 μg/plate in experiment 1 and concentrations at and above 500 μg/plate in experiment 2, with and without metabolic activation, respectively. This did not affect the scoring of revertant colonies.

RANGE-FINDING/SCREENING STUDIES: A toxicity screening study was performed to determine the toxicity of the test substance. 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate test substance was tested on TA 100 and E. coli WP2 uvrA using the plate incorporation method. Precipitation of the test material was observed at 5000 μg/plate. A range-finding study (plate incorporation method) was performed on all strains using dose levels of 50 - 5000 μg/plate. As the results for all the tested dose levels were valid, the results were used as part of the main study (experiment 1).

COMPARISON WITH HISTORICAL CONTROL DATA: yes, the results of the vehicle and positive controls fell within the range of the historical control data (see Table 3 under 'Any other information on results incl. tables')

ADDITIONAL INFORMATION ON CYTOTOXICITY: No cytotoxicity was observed at any dose level.

Table 1. Test results of experiment 1 (plate incorporation method)

With or without S9-mix

Test substance concentration

(μg/plate)

Mean number of revertant colonies per plate

(average of 3 plates ± Standard deviation)

Base-pair substitution type

Frameshift type

TA 100

TA1535

WP2 uvR

TA98

TA1537

DMSO

134 ± 5.7

27 ± 2.3

28 ± 0.6

26 ± 2.0

11 ± 5.0

50

136 ± 4.4

25 ± 0.0

26 ± 2.5

27 ± 0.6

14 ± 1.5

150

131 ± 6.4

29 ± 3.8

25 ± 6.7

24 ± 4.5

11 ± 3.8

500

115 ± 21.7

28 ± 1.5

27 ± 1.2

25 ± 1.0

17 ± 1.0

1500*

106 ± 18.0

25 ± 4.2

22 ± 4.7

22 ± 3.6

10 ± 2.6

5000*

116 ± 5.1

32 ± 2.1

16 ± 1.7

19 ± 1.2

10 ± 2.1

Positive controls,

-S9

Name

ENNG

ENNG

ENNG

4NQO

9AA

Concentrations

(μg/plate)

3

5

2

0.2

80

Mean No. of colonies/plate

(average of 3 ± SD)

429 ± 43.7

514 ± 43.1

726 ± 77.5

133 ± 11.0

884 ± 92.2

+

DMSO

102 ± 11.6

12 ± 2.5

25 ± 3.5

28 ± 2.3

12 ± 3.2

+

50

109 ± 10.4

12 ± 1.5

27 ± 0.6

28 ± 3.5

15 ± 1.0

+

150

106 ± 3.2

12 ± 1.0

29 ± 0.6

28 ± 1.2

11 ± 4.0

+

500

97 ± 1.5

14 ± 1.7

26 ± 5.0

25 ± 2.1

13 ± 2.6

+

1500*

101 ± 8.1

14 ± 1.2

24 ± 3.5

26 ± 2.6

11 ± 2.0

+

5000*

93 ± 3.0

13 ± 2.6

26 ± 2.5

27 ± 1.5

9 ± 2.6

Positive controls, +S9

Name

2AA

2AA

2AA

BaP

2AA

Concentrations

(μg/plate)

1

2

10

5

2

Mean No. of colonies/plate

(average of 3 ± SD)

1547 ± 34.6

223 ± 24.5

367 ± 41.1

218 ± 74.1

299 ± 29.5

ENNG: N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO: 4-nitroquinoline N-oxide

9AA:9-aminocridine

2AA: 2-amino-anthracene

BaP: benzo-a-pyrene

*: precipitation

 

Table 2. Test results of experiment 2 (plate incorporation method)

With or without S9-mix

Test substance concentration

(μg/plate)

Mean number of revertant colonies per plate

(average of 3 plates ± Standard deviation)

Base-pair substitution type

Frameshift type

TA 100

TA1535

WP2 uvR

TA98

TA1537

DMSO

102 ± 12.7

24 ± 2.5

22 ± 6.1

18 ± 4.4

16 ± 2.5

50

85 ± 12.4

24 ± 5.0

21 ± 2.6

15 ± 2.3

13 ± 1.2

150

94 ± 7.1

24 ± 2.9

18 ± 2.5

17 ± 5.1

13 ± 1.7

500*

90 ± 10.0

23 ± 4.0

21 ± 0.6

20 ± 2.1

15 ± 6.1

1500*

94 ± 5.1

26 ± 3.5

22 ± 1.5

20 ± 2.6

14 ± 1.5

5000*

96 ± 4.5

24 ± 2.3

23 ± 3.5

20 ± 2.0

14 ± 2.0

Positive controls,

-S9

Name

ENNG

ENNG

ENNG

4NQO

9AA

Concentrations

(μg/plate)

3

5

2

0.2

80

Mean No. of colonies/plate

(average of 3 ± SD)

613 ± 32.2

578 ± 58.0

861 ± 68.6

144 ± 9.5

354 ± 120.8

+

DMSO

99 ± 8.5

16 ± 2.1

28 ± 5.1

27 ± 6.7

14 ± 3.2

+

50

90 ± 4.2

15 ± 1.0

29 ± 1.5

25 ± 4.0

10 ± 1.5

+

150

94 ± 6.7

14 ± 1.5

25 ± 3.6

25 ± 8.2

14 ± 1.5

+

500*

98 ± 12.7

15 ± 1.2

24 ± 3.1

28 ± 1.7

14 ± 2.0

+

1500*

98 ± 2.1

16 ± 3.0

25 ± 3.6

26 ± 4.4

16 ± 2.5

+

5000*

97 ± 4.7

12 ± 1.5

26 ± 1.5

25 ± 2.3

12 ± 1.5

Positive controls, +S9

Name

2AA

2AA

2AA

BaP

2AA

Concentrations

(μg/plate)

1

2

10

5

2

Mean No. of colonies/plate

(average of 3 ± SD)

586 ± 96.2

235 ± 12.0

259 ± 36.7

387 ± 51.7

165 ± 8.4

ENNG: N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO: 4-nitroquinoline N-oxide

9AA:9-aminocridine

2AA: 2-amino-anthracene

BaP: benzo-a-pyrene

*: precipitation

Table 3. Historical controls

Strain

Control

without S9-mix

with S9-mix

Year

 

 

Mean ± SD

Mean ± SD

 

TA100

Solvent/untreated

94 ± 16.0

87 ± 14.8

2007

TA100

Positive

558 ± 313.5

1231 ± 651.1

2007

TA1535

Solvent/untreated

21 ± 5.2

13 ± 3.6

2007

TA1535

Positive

343 ± 864.2

231 ± 476.5

2007

WP2 uvR

Solvent/untreated

26 ± 5.8

28 ± 6.4

2007

WP2 uvR

Positive

652 ± 707.9

505 ± 535.2

2007

TA98

Solvent/untreated

21 ± 5.5

26 ± 6.0

2007

TA98

Positive

256 ± 289.7

308 ± 280.5

2007

TA1537

Solvent/untreated

11 ± 3.8

13 ± 4.1

2007

TA1537

Positive

1697 ± 1193.6

339 ± 254.4

2007

TA100

Solvent/untreated

105 ± 18.7

101 ± 18.5

2008

TA100

Positive

487 ± 184.7

1138 ± 517.2

2008

TA1535

Solvent/untreated

23 ± 5.3

15 ± 4.8

2008

TA1535

Positive

210 ± 107.7

272 ± 94.9

2008

WP2 uvR

Solvent/untreated

26 ± 5.9

29 ± 7.2

2008

WP2 uvR

Positive

548 ± 231.5

426 ± 273.9

2008

TA98

Solvent/untreated

19 ± 4.6

26 ± 5.3

2008

TA98

Positive

155 ± 70.8

247 ± 178.2

2008

TA1537

Solvent/untreated

13 ± 3.7

13 ± 3.8

2008

TA1537

Positive

930 ± 476.2

279 ± 101.7

2008

 
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24 May - 14 Sep 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
adopted Jul 2015
Deviations:
no
GLP compliance:
yes
Type of assay:
other: in vitro gene mutation study in mammalian cells
Target gene:
HPRT locus
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: JCRB Cell Bank, Japan
- Cell cycle length, doubling time or proliferation index: 13 h doubling time
- Modal number of chromosomes: 22

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: EMEM supplemented with 10% fetal calf serum, 5% CO2
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
Metabolic activation:
with and without
Metabolic activation system:
co-factor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital and 5,6-benzoflavone
Test concentrations with justification for top dose:
33.0, 65.0, 130 and 260 µg/mL (4 h), with and without metabolic activation. Precipitation of the test substance was observed in the culture medium at 260 µg/mL, which was selected as the highest dose level.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: the test substance was insoluble in water and acetone, and soluble in DMSO at concentrations up to 26 mg/mL.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-dimethylnitrosamine
ethylmethanesulphonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 h exposure with and without metabolic activation
- Expression time (cells in growth medium): 7-8 days
- Selection time (if incubation with a selection agent): 9 days

SELECTION AGENT (mutation assays): 6-thioguanine

NUMBER OF REPLICATIONS: four dishes in two independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
- Any supplementary information relevant to cytotoxicity: for the cytotoxicity evaluation, cells were fixed with formaldehyde and stained with crystal violet, after which the cells were washed and relative cell survival measured.
Evaluation criteria:
When the mutation frequency (MF) in each test article treatment group satisfied all criteria described below, the results were judged to be positive. When any criterion was not satisfied, the results were judged to be negative. When different results between cultures (dish 1 and dish 2) were obtained, the results were comprehensively evaluated by considering their differences.
The result was judged to be positive:
1) When the average MFs in the test article treatment groups were three times or more than that of the concurrent negative (solvent) control group. When the MFs in the concurrent negative (solvent) control are zero, MFs in the test article treatment groups were three times or more than the mean MFs of the historical data of the negative control group.
2) When the average MFs in treatment groups were over ranges of the historical data of the negative control group.
Key result
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 examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: In the main study, precipitation was observed at 260 µg/mLwith and without metabolic activation at the beginning and at the end of the treatment period, and at 130 µg/mL with metabolic activation at the end of the treatment period. This was not considered to affect the results.

RANGE-FINDING/SCREENING STUDIES:
A preliminary cytotoxicity test was performed to determine the concentration range to be used in the main study. Precipitation of the test substance was observed in the culture medium at 260 µg/mL, which was selected as the highest dose level. The cytotoxicity test was performed using 6 concentrations in the range 8.1 - 260 µg/mL. No cytotoxicity was observed at any concentration level and the highest concentration was therefore selected to be the highest concentration in the main study.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
The results of the positive controls fell within the range of the historical control data with and without metabolic activation, and the results of the negative control fell within the range of the historical control data without metabolic activation (see table 4 under "Any other information on results incl. tables"). The mutation frequency in the negative control group with metabolic activation was higher than the historical control data range and the experiment was therefore repeated. In the 2nd experiment, the mutation frequency in the negative control group with metabolic activation fell within the range of the historical control data.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: A preliminary cytotoxicity test was perfomed at concentrations of 8.1, 16.0, 33.0, 65.0, 130 and 260 µg/mL. The relative cell survival was shown to be comparable between the test substance and the solvent control.

Table 1: test results for 4-h treatment without metabolic activation

Group

Concentration (µg/mL)

Cytotoxicity

Mutation

 

 

 

Average in %

6-TG resistant colonies/dish

(mean)

Mutation frequency (10-6)

DMSO

1 vol%

100.0

1.3

15.2

Test substance

33.0

114.0

0.8

7.8

Test substance

65.0

105.0

0.9

9.8

Test substance

130

97.7

0.9

10.7

Test substance

260*

103.6

1.2

12.9

EMS

1000

77.8

91.4

1413

EMS: ethylmethanesulphonate

*precipitation observed

Table 2: test results for 4-h treatment with metabolic activation (1st experiment)

Group

Concentration (µg/mL)

Cytotoxicity

Mutation

 

 

 

Average in %

6-TG resistant colonies/dish

(mean)

Mutation frequency (10-6)

DMSO

1 vol%

100.0

1.3

14.8**

Test substance

33.0

98.5

0.6

7.1

Test substance

65.0

101.0

0.4

4.6

Test substance

130*

106.7

1.3

14.2**

Test substance

260*

97.6

1.4

16.8**

NDMA

1000

68.4

37.8

642.2

NDMA: N-dimethylnitrosamine

*precipitation observed

**The mutation frequency was higher than the upper limit of the historical control data range

 

Table 3: test results for 4-h treatment with metabolic activation (2nd experiment)

Group

Concentration (µg/mL)

Cytotoxicity

Mutation

 

 

 

Average in %

6-TG resistant colonies/dish

(mean)

Mutation frequency (10-6)

DMSO

1 vol%

100.0

0.7

6.6

Test substance

33.0

95.8

0.3

2.6

Test substance

65.0

98.2

0.3

3.1

Test substance

130*

99.6

0.4

3.6

Test substance

260*

97.4

0.6

5.7

NDMA

1000

73.5

39.0

532.0

NDMA: N-dimethylnitrosamine

*precipitation observed

 

Table 4: Historical control data of gene mutation test (4-hour treatment) using V79 cells

S9-mix

Control

Mutation frequency (10-6)

 

 

No. of data

Mean ± SD

Minimum

Maximum

-

Negative control

38

3.9 ± 4.9

0

21.0

-

EMS (1 mg/mL)

27

1002 ± 249

588

1530

+

Negative control

44

3.6 ± 2.7

0

10.2

+

NDMA (2 mg/mL)

26

601 ± 359

229

1453

SD: standard deviation

NDMA: N-dimethylnitrosamine

EMS: ethylmethanesulphonate

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Remarks:
Guideline study with acceptable restrictions. The sampling time for 6 h treatment is not equivalent to 1.5 normal cell cycle length, no concurrent measures of cytotoxicity for all cultures in the main experiment.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
adopted Sep 2014
Deviations:
yes
Remarks:
The sampling time for 6 h treatment is not equivalent to 1.5 normal cell cycle length, no concurrent measures of cytotoxicity for all cultures in the main experiment.
Qualifier:
according to
Guideline:
other: The Guidelines for the Testing of Chemicals (State Environmental Protection Administration of China) 2004.5
Deviations:
no
GLP compliance:
not specified
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
Not applicable
Species / strain / cell type:
other: Chinese hamster lung fibroblasts (CHL)
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI 1640 medium, supplemented with 10% fetal bovine serum, 1000 U/mL penicillin, 100 µg/mL streptomycin, 2 mM/L L-glutamine
- Properly maintained: yes
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
6 h treatment with and without metabolic activation: 156, 312 and 625 µg/mL
24 h treatment, without metabolic activation: 156, 312 and 625 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Remarks:
10 µg/mL mitomycin C in physiological saline, -S9; 1 mg/mL cyclophosphamide in physiological saline, +S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 6 and 24 h
- Fixation time (start of exposure up to fixation or harvest of cells): 6 h treatment: 6 h; 24 h treatment: 24 h

SPINDLE INHIBITOR (cytogenetic assays): 0.2 µg/mL colchicine, treatment 4 h prior to harvesting
STAIN (for cytogenetic assays): 10% Giemsa

NUMBER OF REPLICATIONS: 3

NUMBER OF CELLS EVALUATED: 200 per culture

DETERMINATION OF CYTOTOXICITY
- Method: MTT cytotoxicity assay

OTHER EXAMINATIONS:
- Determination of polyploidy: no
- Determination of endoreplication: no
Evaluation criteria:
The tested concentrations were considered positive when statistically significant increases in total alterations frequency exceeded the historical control mean.
Statistics:
The data was analysed using the chi-square test with the software SPSS 11.5 for Windows.
Key result
Species / strain:
other: Chinese hamster lung fibroblasts (CHL)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: the IC50 was calculated to be 0.640 mg/mL in the preliminary study
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES: An MTT cytotoxicity assay was performed to select suitable dose levels for the main study. The concentrations 0.078, 0.156, 0.312, 0.625, 1.25, 2.50 and 5.0 mg/mL were used (no metabolic activation reported). Four replications per dose level were used. The IC50 was calculated using the SPSS16.0 software, resulting in an IC50 of 0.640 mg/mL (See table 2 under ' Any other information on results incl. tables').

COMPARISON WITH HISTORICAL CONTROL DATA: No

Table 1: Chromosome aberrations, summarised data

 

Metabolic activation

Test substance concentration (μg/mL)

Total number of metaphases analysed

Chromosome aberration cell count

Chromosome aberration type*

6 h treatment, 6h harvesting time

-

Vehicle control

(DMSO)

200

4

b/p

-

156

200

3

b

-

312

200

3

b

-

625

200

3

b

-

mitomycin C

200

45

b/p

24 h treatment, 24h harvesting time

-

Vehicle control

(DMSO)

200

4

b/p

-

156

200

4

b/p

-

312

200

5

b/p

-

625

200

6

b/p

-

mitomycin C

200

54

b/p/l

6 h treatment, 6h harvesting time

+

Vehicle control

(DMSO)

200

3

b/p

+

156

200

2

b

+

312

200

4

b

+

625

200

4

b

+

cyclophosphamide

200

51

b/p/l

* b: break, d: deletion, t: translocation, r: ring, f: fragment, other: dicentric, triradial, quadriradial etc.

 

 

Table 2: Cytotoxicity

Dose mg/mL

Number of wells

Optical Density

DMSO

4

1.013 ± 0.0387

0.078

4

0.978 ± 0.0318

0.156

4

0.908 v 0.0331

0.312

4

0.683 ± 0.0849

0.625

4

0.486 ± 0.0319

1.25

4

0.283 ± 0.0427

2.50

4

0.161 ± 0.0134

5.0

4

0.091 ± 0.0142

 

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

Genetic toxicity in vivo

Description of key information

Micronucleus test (OECD 474): negative in mouse bone marrow cells

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
No historical negative or positive control data were reported, dose volume was 20 mL/kg bw instead of max. 10 mL/kg bw, 2000 immature erythrocytes/animal were scored instead of 4000.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
adopted Sep 2014
Deviations:
yes
Remarks:
No historical negative or positive control data were reported, dose volume 20 mL/kg bw, 2000 immature erythrocytes/ animal were scored
Qualifier:
according to
Guideline:
other: The Guidelines for the Testing of Chemicals (State Environmental Protection Administration of China) 2004.5
Deviations:
not specified
Qualifier:
according to
Guideline:
other: The Guidelines for the Hazard Evaluation of New Chemical Substances (HJ/T 154-2004)
Deviations:
no
GLP compliance:
not specified
Type of assay:
micronucleus assay
Species:
mouse
Strain:
other: Kunming (SPF grade)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Vital River Laboratory Animal Technology Co. Ltd., Beijing, China
- Weight at study initiation: 25 - 28 g (females, main study), 26 - 28 g (males, main study); 18.1 - 20.4 (females, preliminary study), 18.7 - 20.1 (males, preliminary study)
- Fasting period before study: from 12 h before dosing until 3 h after (preliminary study only)
- Housing: the animals were housed in clear plastic cages with stainless steel tops
- Diet: feed (SCXK (JIN) 2012-0001, Tianjin Huarong Laboratory Animal Science and Technology Co., Ltd., Tianijin, China), ad libitum
- Water: tap water, ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 25
- Humidity (%): 50 ± 20
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: vegetable oil
- Justification for choice of solvent/vehicle: the test substance formed a dosable and apparently miscible suspension in vegetable oil
- Concentration of test material in vehicle: approximately 100 mg/mL; administered as 2 mL/100 g bw
Duration of treatment / exposure:
2 days
Frequency of treatment:
daily (2 doses in total)
Post exposure period:
24 h
Dose / conc.:
2 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Route of administration: intraperitoneal injection
- Doses / concentrations: 40 mg/kg bw
Tissues and cell types examined:
Tissue: bone marrow
Cell type: bone marrow cells
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: No mortality or signs of toxicity were noted in mice administered a single dose of 5000 mg/kg bw in the range-finding study. The recommended limit dose according the OECD guideline 474 is 2000 mg/kg bw for administration periods of less than 14 days. Therefore the limit dose of 2000 mg/kg bw/day was selected.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): the bone marrow cells were harvested 24 h after the last test substance administration.

DETAILS OF SLIDE PREPARATION: Slides were fixed with methanol and stained with Giemsa solution.

METHOD OF ANALYSIS: 2000 polychromatic erythrocytes (PCE) were scored per animal to determine the frequency of micronucleated polychromatic erythrocytes (MNPCE). The ratio of polychromatic to monochromatic erythrocytes (PCE/NCE) was determined.
Statistics:
The statistical significance between groups was analysed using ANOVA. All results were expressed as mean ± standard deviation; values were considered significant at p < 0.05.
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 5000 mg/kg bw
- Clinical signs of toxicity in test animals: none
- Rationale for exposure: the range-finding study was performed to establish the highest dose level at which no or low systemic toxicity was observed.
- Other: 5 mice/sex were administered 5000 mg/kg bw by gavage as a single dose. There was no mortality during the 14-day observation period. No adverse clinical signs were observed and the body weight gain was within the expected range for this species and strain. The histopathological examination did not show any abnormal results.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): the induction of micronuclei was not significantly increased in the treatment group, compared with the control group (see Table 1 - 3 under 'Any other information on results incl. tables').
- Ratio of PCE/NCE (for Micronucleus assay): For females, the ratio of PCE/NCE was 1.06, 1.06 and 1.05 for the vehicle, treatment and positive control group, respectively. For males, the ratio of PCE/NCE was 1.05, 1.03 and 1.02 for the vehicle, treatment and positive control group, respectively.
- Appropriateness of dose levels and route: the route and dose level was suitable under the conditions of the study.
- Statistical evaluation: the difference between the vehicle control and test substance group was not significant.

OTHER:
No effects on body weight were observed in any of the control or treatment groups.

Table 1: Results of the in vivo micronucleus assay in male animals

 

 

 

Total micronuclei

per 2000

PCEs at

sampling time

Exp group

Number

of animals/sex

Dose [mg/kg bw]

24 h

Vehicle control

(vegetable oil)

5

0

7

Test substance

 

5

2000

11

Positive control

(cyclophosphamide)

5

40

192*

*statistically significant (p<0.05);

  Table 2: Results of the in vivo micronucleus assay in female animals.

 

 

 

Total micronuclei

per 2000

PCEs at

sampling time

Exp group

Number

of animals/sex

Dose [mg/kg bw]

24 h

Vehicle control

(vegetable oil)

5

0

10

Test substance

 

5

2000

8

Positive control

(cyclophosphamide)

5

40

205*

*statistically significant (p<0.05);

 

Table 3: Results of the in vivo micronucleus assay

Treatment group

Dose

[mg/kg bw]

Sampling time

[h]

Mean frequency of PCE with MN

 

PCE/NCE ratio

Females

Vehicle control (vegetable oil)

0

24

0.01

1.06 ± 0.04

Test substance

2000

24

0.01

1.06 ± 0.04

Positive control (cyclophosphamide)

40

24

2.05

1.05 ± 0.02

Males

Vehicle control (vegetable oil)

0

24

0.07

1.05 ± 0.03

Test substance

2000

24

0.08

1.03 ± 0.03

Positive control (cyclophosphamide)

40

24

1.92

1.02 ± 0.04

 

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

Additional information

Genetic toxicity in vitro

An in vitro bacterial reverse mutation assay (Ames test) was performed with Reaction mass of 1,1'-(isopropylidene)bis[3,5-dibromo-4-(2,3-dibromo-2-methylpropoxy)benzene] and 1,3-dibromo-2-(2,3-dibromo-2-methylpropoxy)-5-{2-[3,5-dibromo-4-(2,3,3-tribromo-2-methylpropoxy)phenyl]propan-2-yl}benzene (Sanders, 2010). The study was performed according to OECD guideline 471 and under GLP conditions using S. typhimurium strains TA 1535, TA 1537, TA 98 and TA 100, and E. coli WP2 uvR at concentrations up to 5000 µg/plate. Experiment 1 was carried out as a plate incorporation assay, while experiment 2 was performed as a pre-incubation assay. No cytotoxicity was observed. Precipitation was noted from 500 µg/plate in experiment 1 and from 1500 µg/plate in experiment 2, respectively, with and without metabolic activation. This did not affect the scoring of revertant colonies. The positive and vehicle (DMSO) controls were valid. The test substance did not induce reversions in any of the S. typhimurium or E. coli strains with or without metabolic activation.

The potential of Reaction mass of 1,1'-(isopropylidene)bis[3,5-dibromo-4-(2,3-dibromo-2-methylpropoxy)benzene] and 1,3-dibromo-2-(2,3-dibromo-2-methylpropoxy)-5-{2-[3,5-dibromo-4-(2,3,3-tribromo-2-methylpropoxy)phenyl]propan-2-yl}benzene to induce chromosomal aberrations was tested in an assay with Chinese hamster lung fibroblasts (CHL), performed according to OECD 473 (Zhang, 2014). CHL cells were exposed to test substance concentrations of 156, 312 and 625 µg/mL. The upper concentration was determined in a range-finding study, in which an IC50 value of 0.640 was calculated. A short-term treatment (6 hours) was performed with a 6-hour harvest time, with and without metabolic activation. In addition, a 24-hour treatment with 24-hour harvest time without metabolic activation was performed. There were no increases in the number of chromosome aberrations at any dose level. The positive controls induced a significant increase in the number of chromosomal aberrations. Therefore the test substance is not considered to cause cytogenetic effects under the conditions of this test. As the results of the test are only valid for the 24-hour exposure duration without metabolic activation, the study cannot be used for classification purposes.

An in vitro gene mutation test was performed with Reaction mass of 1,1'-(isopropylidene)bis[3,5-dibromo-4-(2,3-dibromo-2-methylpropoxy)benzene] and 1,3-dibromo-2-(2,3-dibromo-2-methylpropoxy)-5-{2-[3,5-dibromo-4-(2,3,3-tribromo-2-methylpropoxy)phenyl]propan-2-yl}benzene in Chinese lung fibroblasts (V79) (Yamakage, 2016). The study was conducted according to OECD guideline 476 and GLP. The cells were treated with 33.0, 65.0, 130 and 260 µg/mL for 4 h, with and without metabolic activation. Precipitation of the test substance was observed from the start of the treatment at 260 µg/mL with and without metabolic activation, and at 130 µg/mL with metabolic activation at the end of the treatment. The precipitation was not considered to affect the results. No cytotoxicity was observed at any concentration. The results of the positive controls with and without metabolic activation, and the results of the negative control without metabolic activation fell within the range of the historical control data. The mutation frequency in the negative control group with metabolic activation was higher than the historical control data range and the experiment was therefore repeated. In the 2nd experiment, the mutation frequency in the negative control group with metabolic activation fell within the range of the historical control data. The test substance did not cause an increase in mutation frequency with and without metabolic activation.

 

Genetic toxicity in vivo

The potential for in vivo genetic toxicity of Reaction mass of 1,1'-(isopropylidene)bis[3,5-dibromo-4-(2,3-dibromo-2-methylpropoxy)benzene] and 1,3-dibromo-2-(2,3-dibromo-2-methylpropoxy)-5-{2-[3,5-dibromo-4-(2,3,3-tribromo-2-methylpropoxy)phenyl]propan-2-yl}benzene was assessed in a micronucleus test performed according to the Guidelines for the Testing of Chemicals (State Environmental Protection Administration of China) 2004.5 and similar to OECD guideline 474 (Li, 2015). 5 Kunming mice/sex/dose were administered 2000 mg/kg bw/day test substance in vegetable oil by gavage for 2 days. The treatment and vehicle animals were sacrificed 24 hours after dosing. Bone marrow cells from the femur were extracted, and slides were prepared and stained with a Giemsa solution. No effects on body weight were observed in any of the control or treatment groups. The vehicle control group and positive control group were shown to be valid. For the treatment group, no increase in the frequency of micronucleated polychromatic erythrocytes was observed in the isolated polychromatic erythrocytes and no decrease in the ratio of polychromatic to normochromatic erythrocytes was noted, compared with the vehicle control group.

 

Overall conclusion for genetic toxicity

Based on the available data, Reaction mass of 1,1'-(isopropylidene)bis[3,5-dibromo-4-(2,3-dibromo-2-methylpropoxy)benzene] and 1,3-dibromo-2-(2,3-dibromo-2-methylpropoxy)-5-{2-[3,5-dibromo-4-(2,3,3-tribromo-2-methylpropoxy)phenyl]propan-2-yl}benzene is considered to be not mutagenic in vitro, and not clastogenic in vivo.

Justification for classification or non-classification

Based on the available data, the results on genetic toxicity for the test substance does not meet the classification criteria according to Regulation (EC) 1272/2008 and are therefore conclusive but not sufficient for classification.