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Genetic toxicity in vitro

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Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1986
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine
Species / strain / cell type:
other: Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100.
Metabolic activation:
with and without
Metabolic activation system:
liver microsome fraction of Aroclor-induced rats for metabolic activation (S9-mix)
Test concentrations with justification for top dose:
0, 0.31, 0.93, 2.78, 8.33 and 25 µg/plate.
Details on test system and experimental conditions:
Salmonella typhimurium reverse mutation assay
Key result
Species / strain:
other: Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100.
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: >= 50.0 µg per plate
Conclusions:
Under the study conditions, the test substance was not considered to be mutagenic in the presence and absence of exogenous metabolic activation.
Executive summary:

A study was conducted to determine the in vitro genetic toxicity of the test substance according to OECD Guideline 471 (Ames test), in compliance with GLP. The substance was examined for mutagenic activity in the Ames test using the histidine-requiring Salmonella typhimurium mutant strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100, and a liver microsome fraction of Aroclor-induced rats for metabolic activation (S9-mix). The substance was tested at doses of 0, 0.31, 0.93, 2.78, 8.33 and 25 µg/plate. Based on preliminary test in TA 98, 25 µg/plate was chosen as the highest dose level. The test was carried out twice. Under the study conditions, the substance was considered to be non-mutagenic in the presence and absence of exogenous metabolic activation (Wilmer, 1986).

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From February 28, 2001 to March 26, 2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
- Physical state: Clear liquid
- Analytical purity: >93%
- Impurities (identity and concentrations): 0.8% Free Amine + Amine hydrochloride and ≤ 0.1% AAS
- Lot/batch No.: DEGE001033
- Expiration date of the lot/batch: January 2002
- Stability under test conditions: The test substance is hydrolytically and photolytically stable under the conditions of this study and has been shown to be stable in aqueous, alcohol and alcohol/aqueous solutions for extended periods, e.g. at least five years under standard laboratory conditions.
- Storage condition of test material: Room temperature in the dark
- pH (1% water): 6.5
Target gene:
Histidine
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbitone/β-Naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
Preliminary toxicity study: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate (With and without metabolic activation)
Main Experiment: Experiment 1 & 2: 0.15, 0.5, 1.5, 5, 15 and 50 µg/plate (With and without metabolic activation)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Sterile distilled water
Untreated negative controls:
yes
Remarks:
(concurrent untreated)
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
Without metabolic activation
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
Without metabolic activation
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
Without metabolic activation
Positive controls:
yes
Positive control substance:
other: Nitroquinoline-1-oxide (4NQO)
Remarks:
Without metabolic activation
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene (2AA)
Remarks:
With metabolic activation
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
With metabolic activation
Positive controls:
yes
Positive control substance:
other: 1,8-Dihydroxyanthraquinone (DANTHRON)
Remarks:
With metabolic activation
Details on test system and experimental conditions:
Method of application: In agar (direct plate incorporation)
Number of replications: Triplicates

Evaluation criteria:
The test substance was considered positive in the test system if the following criteria were met:
The test substance should have induced a reproducible, dose-related and statistically significant increase in the revertant count in at least one strain of bacteria.
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
- The test substance caused a visible reduction in the growth of the bacterial background lawn to all of the tester strains both with and without S9-mix beginning at 15µg/plate. The sensitivity of the bacterial tester strains to the toxicity of the test substance varied slightly between experiment number, strain type and exposures with or without S9-mix. The test substance was, therefore, tested up to the toxic limit. No test substance precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.
- No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test substance, either with or without metabolic activation.
- All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

Table 1. Cytotoxicity (Number of revertant colonies)

 

 

Test substance concentration (µg/plate)

With/ Without

S9-mix

Strain

0

0.15

0.5

1.5

5

15

50

150

500

1500

5000

Without

TA100

91

91

81

83

64

17S

0T

0T

0T

0T

0T

With

TA100

97

105

103

103

83

50S

0T

0T

0T

0T

0T

S=sparse bacterial background lawn

T= toxic, no bacterial lawn

Table 2. Genotoxicity (Mean number of revertant colonies)

Strain

TA100

TA1535

TA102

TA98

TA1537

Test substance concentration (mg/plate)

 

 

 

 

 

With S9

 

 

 

 

 

+ve control type (concentration (mg/plate))

2AA (1)

2AA (2)

DAN (10)

BP (5)

2AA (2)

Test number

1

2

1

2

1

2

1

2

1

2

+ve control

1772

2317

287

135

886

723

229

251

582

336

-ve control

143

137

17

17

349

308

36

25

12

22

0.15

129

137

13

13

357

315

26

24

13

18

0.5

134

11

10

14

346

313

33

25

15

14

1.5

129

127

15

14

373

307

32

26

16

14

5

142

143

10

13

369

341

32

23

17

16

15

132

0

12

2

363

151

37

11

17

6

50

28

0

10

0

276

0

22

0

10

0

Without S9

 

 

 

 

 

+ve control type (concentration (mg/plate))

ENNG (3)

ENNG (5)

MMC (0.5)

4NQO (0.2)

9AA (80)

Test number

1

2

1

2

1

2

1

2

1

2

+ve control

621

464

603

430

854

961

142

126

656

716

-ve control

153

134

12

18

316

336

38

22

16

17

0.15

150

123

13

15

340

308

29

18

18

19

0.5

132

114

11

21

331

339

30

18

17

18

1.5

154

111

19

16

339

232

25

14

15

11

5

145

111

9

10

326

325

28

19

12

18

15

84

0

11

0

320

0

23

0

0

0

50

0

0

0

0

0

0

0

0

0

0

Conclusions:
Under the study conditions, the test substance was found to be non-mutagenic with and without metabolic activation.

Executive summary:

A study was conducted to determine the in vitro genetic toxicity of the test substance according to OECD Guideline 471, EU Method B13/14 and US EPA OPPTS 850.5100 (Ames test), in compliance with GLP. The mutagenic potential was investigated in Salmonella typhimurium strains A1535, TA1537, TA102, TA98 and TA100 with and without metabolic activation. Six dose levels of the test substance for each bacterial strain were tested in triplicate with and without a metabolic activation system. The dose range was determined in a preliminary toxicity assay and was 0.15 to 50 µg/plate in the first experiment. The experiment was repeated on a separate day using the same dose range, fresh cultures of the bacterial strains and fresh test substance formulations. Additional dose levels were included in both experiments to allow for test substance-induced toxicity and to ensure there were a minimum of four non-toxic doses plated out. The vehicle (sterile distilled water) control plates produced counts of revertant colonies within the normal range. All the positive control chemicals used in the test produced marked increases in the frequency of revertant colonies, both with and without the S9 -mix. No test substance precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix. No significant increase in the frequency of revertant colonies was recorded for any of the bacterial strains with any dose of the test substance, either with or without metabolic activation. Under the study conditions, the test substance was found to be non-mutagenic with and without metabolic activation (Thompson, 2001).

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1985
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
5 strains were used, but not one of the strains having AT base pairs at the primary reversion site (such as TA 102 or E. coli WP2 strains).
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
- Batch no.: 15/83
- Storage: room temperature
- Appearance: pale yellow viscous liquid
- Content: 50% in water (technical quality)
- Specific gravity: 0.98
Target gene:
reverse mutation to histidine independency
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
S. typhimurium TA 1538
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver (S9-mix)
Test concentrations with justification for top dose:
RF test: 0, 5, 50, 500, 5000 µg/plate
main test 1: 1.5, 5, 15, 50, 150 µg/plate
main test 2: 0.5, 1.5, 5, 15, 50 µg/plate
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
other: N-ethyl-N'-nitro-N-nitroso-guanidine (TA1535 and TA100), 9-aminoacridine (TA1537), 2-nitrofluorene (TA1538 and TA98)
Remarks:
without S9-mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene (all strains)
Remarks:
with S9-mix
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:
cytotoxicity
Remarks:
50 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
not up to 150 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
No substantial increases in revertant colony numbers of any of the five tester strains were observed; the test substance was found to be non-mutagenic in the Ames test using strains TA1535, TA1537, TA1538, TA98 and TA100. Although these strains may not detect certain oxidising mutagens or cross-linking agents, it is not expected that the test substance will do so. Therefore, it is concluded that there is no evidence of mutagenic potential of the test substance in this bacterial test system.
Conclusions:
Under the study conditions, no significant increase in the numbers of revertant colonies of Salmonella typhimurium strains was recorded at any dose, either with or without metabolic activation. Therefore, the test substance was found to be non-mutagenic under the conditions of this test.
Executive summary:

A study was conducted to determine the in vitro genetic toxicity of the test substance according to OECD Guideline 471 (Ames test), in compliance with GLP. Salmonella typhimurium strains TA1535, TA1537, TA1538, TA98 and TA100 were treated with the test substance by the Ames plate incorporation method at five dose levels, in triplicate, with and without the addition of a rat liver homogenate metabolising system. The dose range was determined in a preliminary toxicity assay using 5 to 5,000 µg/plate. Doses in the first experiment were 1.5 -150 µg/plate. The experiment was repeated on a separate day. In this case, the dose range was 0.5 to 50 µg/plate. The solvent (DMSO) control plates gave counts of revertant colonies within the normal range. All positive control chemicals produced marked increases in the number of revertant colonies, both with and without the metabolising system. The test substance caused visible reduction in the growth of the bacterial lawn at 50 µg/plate (TA1535, TA100) and at 150 µg/plate (TA1535, TA98 and TA100). The test substance was, therefore, tested up to the maximally 150 µg/plate and 50 µg/plate in the first and second test, respectively. No significant increase in the numbers of revertant colonies was recorded for any of the bacterial strains with any dose, either with or without metabolic activation. Under the study conditions, the test substance was found to be non-mutagenic with and without metabolic activation (Jones, 1985).

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
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)
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Metabolic activation system:
S9 from rats treated with Aroclor 1254 (S-9 mix)
Test concentrations with justification for top dose:
Based on preliminary cytotoxicity test: 0, 0.36, 1.09, 3.27 and 9.80 µg/mL (without S9); 0, 6.13, 12.25, 24.5, 49.0 and 98.0 µg/mL (with metabolic activation of S9 from rats treated with Aroclor 1254).
Details on test system and experimental conditions:
Exposure times were 21h for the experiments without metabolic activation, and 3h for the experiments with metabolic activation, with harvesting times of 12 and 21h.
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: >= 15.68 µg/mL (- S9); >= 78.4 µg/mL (+S9)
Additional information on results:
- A preliminary toxicity test, showed that in the absence of S9-mix, the substance was toxic in concentrations of 15.68 µg/mL and above; in the presence of S9-mix, the substance was very toxic at 78.4 µg/mL and above at both harvesting times.
- The test substance did not induce a statistically significant increase in the number of cells with structural chromosome aberrations (breaks, exchanges and multiple aberrations) at any of the concentrations used, either in the absence or in the presence of the S-9 mix.
Conclusions:
Under the study conditions, the test substance was not clastogenic in Chinese hamster ovary cells with or without metabolic activation.
Executive summary:

A study was conducted to determine the in vitro genetic toxicity of the test substance according to OECD Guideline 473 (chromosome aberration test), in compliance with GLP. This experiment was realized in Chinese hamster ovary cells. Based on preliminary cytotoxicity test, 0, 0.36, 1.09, 3.27 and 9.80 µg/mL test substance (without S9) as well as 0, 6.13, 12.25, 24.5, 49.0 and 98.0 µg/mL (with metabolic activation of S9 from rats treated with Aroclor 1254) test substance were selected. The exposure times were 21h for the experiments without metabolic activation, and 3h for the experiments with metabolic activation, with harvesting times of 12 and 21h. In the preliminary toxicity test, in the absence of S9-mix, the substance was toxic at a concentration of 15.68 µg/mL and above; and in the presence of S9-mix, the substance was very toxic at 78.4 µg/mL and above at both harvesting times. The test substance did not induce a statistically significant increase in the number of cells with structural chromosome aberrations (breaks, exchanges and multiple aberrations) at any of the concentrations used, either in the absence or in the presence of the S-9 mix. Under the study conditions, the test substance was not clastogenic in Chinese hamster ovary cells with or without metabolic activation (Willmer, 1986).

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From March 22, 2001 to September 25, 2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
- Physical state: Extremely pale yellow, slightly viscous liquid with white precipitate
- Analytical purity: >93%
- Lot/batch No.: DEGE001033
- Expiration date of the lot/batch: January 2002
- Stability under test conditions: The test substance is hydrolytically and photolytically stable under the conditions of this study and has been shown to be stable in aqueous, alcohol and alcohol/aqueous solutions for extended periods, e.g. at least five years under standard laboratory conditions.
- Storage condition of test material: Room temperature in darkness

Species / strain / cell type:
lymphocytes: human
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbitone and ß-naphthoflavone - induced rat liver S9 fraction
Test concentrations with justification for top dose:
Preliminary toxicity test: 0, 19.5, 39, 78.1, 156.25, 312.5, 625, 1250, 2500 and 5000 µg/mL (with and without activation)
Chromosome aberration test:
Experiment 1: 0, 4, 8, 16, 20 µg/mL (with and without activation)
Experiment 2: 0, 4, 8, 12, 16, 24 µg/mL (with and without activation)
Vehicle / solvent:
Eagle’s minimal essential medium with HEPES buffer (MEM)
Untreated negative controls:
yes
Remarks:
Negative (media) control
Negative solvent / vehicle controls:
yes
Remarks:
Eagle’s minimal essential medium with HEPES buffer (MEM)
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
(Without S9, at 0.4 and 0.25 µg/mL in Experiment 1 and 2 respectively)
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
(With S9, at 12.5 and 10.0 µg/mL in experiment 1 and 2 respectively)
Details on test system and experimental conditions:
Method of application: In Eagle’s minimal essential medium with HEPES buffer (MEM)
Duration:
- Exposure duration:
Without metabolic activation: 4 and 24h
With metabolic activation: 4h
- Exposure procedure: The cultures were incubated at 37˚C for 4 or 24h (as appropriate) in the presence of the test substance at predetermined concentrations/vehicle/positive controls with or without the S9 reaction mixture.
- Expression time: Approximately 20h after initiation of treatment
- Fixation time: 4h
Spindle inhibitor: Demecolcine (colcemid, 0.1 μg/mL) was added approx. 2 h prior to harvest timeSpindle
Stain: When the slides were dry they were stained in 5% Gurrs Giemsa solution for 5 minutes, rinsed, dried and coverslipped using mounting medium.
Number of replications: At least 2 slides/ flask
Number of cell evaluated: 100 consecutive well-spread metaphase cells (if possible), from each culture were counted, and if the cell had 44-48 chromosomes, any gaps, breaks or rearrangements were noted.
Determination of the cytotoxicity: A total of 2000 lymphocyte cell nuclei were counted and the number of cells in metaphase recorded and expressed as the mitotic index and as a percentage of the vehicle control value.
Statistics:
The frequency of cells with aberrations (both including and excluding gaps) and the frequency of polyploid cells were compared, where necessary, with the concurrent vehicle control value using Fisher’s Exact test and chi-squared test.
Key result
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
slightly toxic at 20 µg/mL in experiment 1 (with S9 activation) and toxic at 16 µg/mL in experiment 1 and at 20 µg/mL in experiment 2 (without S9 activation))
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

Table 1. Results of chromosomal aberration in human lymphocytes (Experiment 1)

Treatment

(μg/mL)

S9

Activation

Treatment

Time

Mean

Mitotic

Index

Cells

Scored

Total Number of Aberrations
+Gaps - Gaps

Cells with Numerical

Aberrations

+ Gaps(%)

Cells with Structural Aberrations

- Gaps(%)

Vehicle

-

4

4.2

200

11    7

5.0

3.5

        Test substance

4

-

4

3.2

200

6   1

3.0

0.5

8

-

4

2.7

200

2   1

1.0

0.5

16

-

4

1.5

200

7   5

3.0

2.0

Positive control (MMC)

0.4

-

4

2.2

200

83   64

32.0

26.0**

 

Vehicle

+

4

3.9

200

5    1

2.5

0.5

      Test substance

8

+

4

2.3

200

4   1

2.0

0.5

16

+

4

2.1

200

7   4

2.5

1.0

20

+

4

1.6

200

7   3

3.5

1.5

Positive control (CP)

12.5

+

4

1.4

300

76   45

20.0

12.0**

Treatment: Cells from the 4-h treatment regimens were harvested 20h after the initiation of the treatments.

                         Frequency of Aberrant Cells: **, p ≤ 0.001

Table 2. Results of chromosomal aberration in human lymphocytes (Experiment 2)

Treatment

(μg/mL)

S9

Activation

Treatment

Time

Mean

Mitotic

Index

Cells

Scored

Total Number of Aberrations
+Gaps - Gaps

Cells with Numerical

Aberrations

+ Gaps(%)

Cells with Structural Aberrations

- Gaps(%)

Vehicle

-

4

7.0

200

4    1

2.0

0.5

        Test substance

4

-

24

4.9

200

6   4

2.0

1.0

8

-

24

2.7

200

6   6

2.5

2.5

12

-

24

2.6

200

12   2

6.0

1.0

Positive control (MMC)

0.2

-

24

2.3

200

115   86

37.0

30.0**

 

Vehicle

+

4

5.9

200

8    4

2.5

0.5

       Test substance

8

+

4

5.2

200

7   3

3.5

1.5

16

+

4

3.3

200

0   0

0

0

24

+

4

3.4

200

1   1

0.5

0.5

Positive control (CP)

12.5

+

4

1.4

200

108   79

33.5

27.0**

Treatment: Cells from both the 4-h and 24 h treatment regimens were harvested 20 h after the initiation of the treatments.

Frequency of Aberrant Cells: **, p ≤ 0.001

Conclusions:
Under the study conditions, the test substance was found to be non-clastogenic to human lymphocytes with and without metabolic activation.
Executive summary:

A study was conducted to determine the in vitro genetic toxicity of the test substance according to OECD Guideline 473 and EU Method B.10 (chromosome aberration test), in compliance with GLP. This experiment was performed in human lymphocyte cells. Duplicate cell cultures of human lymphocytes, treated with the test substance, were evaluated for chromosome aberrations at three dose levels, together with vehicle and positive controls (mitomycin-C (without S9) and cyclophosphamide (with S9)). Four treatment conditions were used for the study. Experiment 1 and 4 h exposure with and without metabolic activation was followed by a 20 h expression period. In Experiment 2, the 4 h exposure with metabolic activation was repeated while in the absence of metabolic activation the exposure time was increased to 24 h. The doses studied were 0, 4, 8, 16, 20 µg/mL (with and without activation) in Experiment 1 and 0, 4, 8, 12, 16, 24 µg/mL (with and without activation) in Experiment 2. The test substance was considered negative for chromosomal aberrations in human lymphocytes in vitro under the S9 metabolic activation and non-activation conditions of the assay. There was no indication of chromosomal ploidy changes in cultures exposed to the test substance in either the presence or absence of S9 mix. Mutant frequencies of all cultures treated with the test substance were within the acceptable range for background mutant frequencies. Under the test conditions, the test substance was found to be non-clastogenic to human lymphocytes with and without metabolic activation (Durward, 2001).

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1986
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Target gene:
HGPRT locus
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
Based on a preliminary cytotoxicity test:
0, 5, 10, 12.5, 15, 17.5 and 20 nL/mL (without metabolic activation) and
0, 15, 30, 45, 60, 75 and 90 nL/mL (with metabolic activation)
Positive controls:
yes
Positive control substance:
N-dimethylnitrosamine
ethylmethanesulphonate
Details on test system and experimental conditions:
- The point mutation test was carried out in duplicate both in the presence and the absence of a metabolic activation system.
- Exposure duration of 5h.
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: > 17.5 nL/mL (- S9), >= 70 nL/mL (+ S9)
Additional information on results:
- Concentration-dependent increases in the number of mutants at the HGPRT locus were not observed either with or without metabolic activation. Exposure to the highest concentrations tested resulted in significant (initial) toxicity for the CHO cells; this indicates that the test was carried out with appropriate concentrations of the test substance. Exposure to either EMS or DMN gave the expected increases in the mutation frequency. 
The toxicity of the test substance for CHO cells was clearly reduced in the presence of metabolic activation.
Conclusions:
Under the study conditions, the test substance did not show any mutagenic activity at the HGPRT locus in CHO cells with or without metabolic activation.

Executive summary:

A study was conducted to determine the in vitro genetic toxicity of the test substance according to OECD Guideline 476 (cell gene mutation test), in compliance with GLP. This study was performed to evaluate the potential to induce mutations at the HGPRT locus in Chinese hamster ovary (CHO) cells. Based on a preliminary toxicity test, 0, 5, 10, 12.5, 15, 17.5 and 20 nL/mL (without metabolic activation) and 0, 15, 30, 45, 60, 75 and 90 nL/mL (with metabolic activation) were selected for the experiment. The entire experiment was repeated to confirm the results of the first experiment. Five h exposure was used both with and without S9-mix. The test substance did not induce dose-related increase in the mutant frequency at the HGPRT locus at any dose level, either with or without metabolic activation. Exposure to the highest concentrations tested resulted in significant (initial) toxicity for the CHO cells; this indicates that the test was carried out with appropriate concentrations of the test substance. Exposure to positive control substances gave the expected increases in the mutation frequency. The toxicity of the test substance for CHO cells was clearly reduced in the presence of metabolic activation. Under the study conditions, test substance did not show any mutagenic activity at the HGPRT locus in CHO cells with and without metabolic activation (Davis, 1986).

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From May 11, 1987 to September 28, 1988
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
equivalent or similar to
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Deviations:
no
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Specific details on test material used for the study:
- Name of test material (as cited in study report): Alkyldimethylbenzylammonium chloride (ADBAC)
- Physical state: Slightly viscous, clear yellow liquid
- Analytical purity: 81.09% active substance in aqueous/ethanol solution.
- Impurities (identity and concentrations): 0.2% Free amine and 0.1% amine hydrochloride
- Lot/batch No.: 7293K
- Stability under test conditions: The test substance is hydrolytically and photolytically stable under the conditions of this study and has been shown to be stable in aqueous, alcohol and alcohol/aqueous solutions for extended periods, e.g. at least five years under standard laboratory conditions.

Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
Cell Type: CHO-K1-BH4
Metabolic activation:
with and without
Metabolic activation system:
Aroclor-induced rat liver S9 fraction
Test concentrations with justification for top dose:
Preliminary cytotoxicity assay: 1, 2, 5, 10, 20, 50, 100, 200, 500 and 5000 µg/mL (With and without metabolic activation)

Mutation assay (Without S-9)
Trial I: 1, 5, 10, 13, 16, 20, 25, 35, 50, and 65 µg/mL
Trial II: 1, 5, 10, 12, 14, 16, 18, 20, and 24 µg/mL

Mutation assay (With S-9)
Trial I: 1, 5, 10, 20, 30, 40, 50, 65, 85 and 100 µg/mL
Trial II: 10, 20, 22, 24, 26, 28, 30, 40 and 50 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Sterile deionized water
Untreated negative controls:
yes
Remarks:
(Negative (media) controls)
Negative solvent / vehicle controls:
yes
Remarks:
(10% water in culture medium)
Positive controls:
yes
Positive control substance:
other: 5-Bromo-2’-deoxyuridine (BrdU)
Remarks:
(without metabolic activation)
Positive controls:
yes
Positive control substance:
other: 3-Methycholanthrene (MCA)
Remarks:
(with metabolic activation)
Details on test system and experimental conditions:
Method of applicfation: Single monolayer culture of CHO cells (in medium).

Duration:
- Exposure duration: 4h (with and without metabolic activation)
- Exposure procedure: The cells were quantitatively seeded at 200 cells/dish (cytotoxicity assay) and at about 3 X 10 (6) cells/T-75 (75 cm2) tissue culture flask on the day before dosing. After18h, cell cultures were exposed to test or control substances for 4h.
- Expression time: Approximately 7d after initiation of treatment
- Fixation time: 7d after treatment initiation.

Stain: Colonies were fixed in alcohol, stained with Giemsa solution and counted by eye, excluding those with approximately 50 cells or less.

Number of cells evaluated: 200 cells/dish

Determination of cytotoxicity
- Method: Following parameters were calculated in the study:
1) Relative survival to treatment: The average number of colonies in three dishes (seeded at 200 cells each) was determined.
Relative survival (%) = (Average number of colonies per treated culture/average number of colonies per vehicle control dish) X 100%
2) Relative population growth: This parameter showed the cumulative growth of the treated cell population, relative to the vehicle control growth, over the entire expression period and prior to mutant selection.
Relative population growth (%) = (Treated culture population increase over the expression period/vehicle control population increase over the expression period) X 100%
3) Absolute cloning efficiency: The ability of the cells to form colonies at the time of mutant selection is measured by the absolute cloning efficiency (CE).
Absolute CE (%) = [Average number of viable colonies per dish/200] x 100%
4) Mutant frequency: The mutant frequency is calculated as the ratio of colonies found in thioguanine-selection medium to the total number of cells seeded, adjusted by the absolute CE.
Mutant Frequency = Total mutant clones/[number of dishes X 2x10(5) X absolute CE]
Evaluation criteria:
Evaluation of a positive response:
To evaluate the test substance as a mutagen following criterion should be met:
- The mutant frequency must meet or exceed 15 x 10E-6 in order to compensate for random fluctuations in the 0 to 10E-6 background mutant frequencies that are typical for this assay.
- A dose related or toxicity related increase in mutant frequency for at least 3 doses should be observed.
- If an increase in mutant frequency is observed for a single dose near the highest testable toxicity, as defined previously and the number of mutant colonies is more than twice the value needed to indicate a significant response, the test substance generally will be considered mutagenic.

Evaluation of a Negative Response:
A test substance is evaluated as nonmutagenic in a single assay only if the minimum increase in mutant frequency is not observed for a range of applied concentrations that extends to concentrations causing about 10 to 15% survival or extends to a concentration at least 75% of that causing excessive toxicity.
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
completely toxic at 20 µg/mL and higher concentration (without activation) and at 40 µg/mL and higher concentration (with activation)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
- Mutant frequencies of all cultures treated with the test material were within the acceptable range for background mutant frequencies (0 to 15E-06 without S9 mix and 0 to 13.5E-06 with S9 mix).
- Mutation Frequency/10(6) cells for trial I and II (without S9 mix) were as follows:
Trial I:
Control: 0, 7.2
Treated: 0 – 4.0
Positive Control (BrdU): 133.3
Trial II:
Control: 8.9, 2.9
Treated: 0.8 – 14.6 (all within historical control range)
Positive Control (BrdU): 114.0
- Mutation Frequency/10(6) cells for trial I and II (with S9 mix) were as follows:
Trial I:
Control: 6.9, 0
Treated: 0.8 – 6.6
Positive Control (3-MCA): 235.3
Trial II:
Control: 1.3, 2.9
Treated: 0.8 – 6.5 (all within historical control range)
Positive Control (3-MCA): 131.7
Conclusions:
Under the study conditions, the test substance was not found to induce any forward mutations at the HGPRT locus in CHO cells with and without metabolic activation.
Executive summary:

A study was conducted to determine the in vitro genetic toxicity of the test substance according to a method similar to US EPA OPPTS 870.5300, in compliance with GLP. The study was performed on the HGPRT locus in Chinese hamster ovary (CHO) cells at test substance concentrations ranging from 0 to100 µg/mL. Preliminary cytotoxicity test showed the test substance to be slightly more toxic without S9 metabolic activation than with activation. The test substance was completely toxic at 20 µg/mL and higher without activation and completely toxic at 40 µg/mL and higher with activation. Dose levels selected for the first trial of the mutation assays covered nontoxic and highly toxic doses. Two independent non-activation and S9 metabolic activation assays were performed. Mutant frequencies of all cultures treated with the test substance were within the acceptable range for background mutant frequencies (0 to 13.5 x 10-6 with S9 mix and 0 to 15 x 10-6 without S9 mix). Under the study conditions, the test substance was not found to induce any forward mutations at the HGPRT locus in CHO cells with and without metabolic activation (Young, 1989).

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

Genetic toxicity in vivo

Description of key information

The test substance was not mutagenic in bacterial reverse mutation assays or in the HPRT locus in Chinese hamster ovary cells, either in the presence or absence of metabolic activation. The number of chromosomal aberrations was not increased by the test substance in the Chinese hamster ovary cells or human lymphocytes. Further, no increase in the frequency of micronucleated polychromatic erythrocytes was observed in peripheral blood samples from male and female mice treated orally with 400 mg of test substance / kg bw. Based on the overall weight of evidence, the test substance is non-genotoxic.

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
Study period:
1985
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: EEC Directive 79/831, Annex V, Method No. 431
Deviations:
no
GLP compliance:
not specified
Type of assay:
micronucleus assay
Specific details on test material used for the study:
- Analytical purity: 80.2% w/w s. in ethanol solution
- Impurities (identity and concentrations): 0.2% free amine and 0.1% amine hydrochloride
- Lot/batch No.: L- 5383
- pH (1% water): 6.3
Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals and environmental conditions:
Test animals
- Source: Breeders were purchased from G1. Bomholtgard Ltd., but the mice used were born in the Scantox Laboratories
- Age at study initiation: 6-7 weeks
- Weight at study initiation: 25-30g
- Assigned to test groups randomly: Yes, in groups of 5
- Fasting period before study: No
- Housing: 5 animals/cage, males and females separately in type III Macrolone cages, bedding used was special softwood sawdust "Spanvall Special White " from Spanvall Ltd., DK-4535 Vallekilde
- Diet: Complete rodent diet "Altromin 1314" from Chr. Petersen Ltd., DK-4100 Ringsted, ad libitum
- Water: Drinking water adjusted to pH 2.5 with hydrochloric acid, ad libitum
- Acclimation period:

Environmental conditions
- Temperature: 21 ±2°C
- Humidity: 55 ± 15%
- Air changes: 10/h
- Photoperiod: 12h dark /12h light

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: Distilled water
- Concentration of test material in vehicle: 400 mg/10 mL distilled water for the dose level of 400 mg/kg bw.
- Amount of vehicle: The vehicle was administered orally at a volume of 10 mL/kg bw.
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Dilution of the test substance in distilled water

Frequency of treatment:
Once
Post exposure period:
24, 48 and 72 h


Dose / conc.:
400 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide (positive control):
- Route of administration: Oral
- Doses / concentrations: 30 mg/10 mL equivalent to 30 mg/kg bw


Tissues and cell types examined:
Bone marrow erythrocytes




Details of tissue and slide preparation:
Dose selection:
Preliminary investigations:
- A few mice were treated orally with various concentrations of the test substance diluted with distilled water. Thereby the maximum tolerated dose was estimated at 400 mg/kg bw. At this dosage bone marrow smears showed a reduced number of polychromatic erythrocytes (PCE) as compared with normochromatic erythrocytes (NCE).

Details of slide preparations: Immediately after sacrifice, femurs of a mouse were dissected free of muscle, and by a 1 mL syringe with needle the bone marrow was flushed out into 5 mL of fetal calf serum. After thorough shaking, the mixture was centrifuged for 10 min. at about 1000 rpm. Thereafter, smears were made after removal of the supernatant. The specimens were fixed in methanol and stained with May-Grunwald/Giemsa.

Method of analysis: Prior to microscopic assessment, all slides were furnished with code numbers, so that the counting was blind.
The following counts were made:
Number of normochromatic erythrocytes (NCE) per 1000 erythrocytes
Number of polychromatic erythrocytes (PCE) per 1000 erythrocytes
Number of micronuclei (MN) in 1000 normochromatic erythrocytes
Number of micronuclei (MN) in 1000 polychromatic erythrocytes.
Evaluation criteria:
Increase in the frequency of micronucleated polychromatic erythrocytes in treated animals as compared to controls


Statistics:
The statistical difference was analysed by one-way ANOVA. In the case of PCE (%) the test was performed on the values observed, and for the MN (per thousand) the test was done on computed rank values transformed to normal scores according to Blom's method (Blom, 1958).

Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
one mortality in the 72h test group on Day 3 after treatment
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Results of the dose-range finding study
- Dose range: Up to 400 mg/kg bw
- Clinical signs of toxicity in test animals: Maximum tolerated dose was estimated at 400 mg/kg bw. At a dose exceeding 400 mg/kg bw, the mortality was too high.
- Evidence of cytotoxicity in tissue analyzed: At 400 mg/kg bw, bone marrow smears showed a reduced number of polychromatic erythrocytes (PCE) as compared with normochromatic erythrocytes (NCE).
Results of the main test
- Induction of micronuclei: No significant difference as compared to controls.
- Appropriateness of dose levels and route: Yes
- Statistical evaluation: Yes

Conclusions:
Under the test conditions, the test substance did not induce an increase in the frequency of micronucleated polychromatic erythrocytes in peripheral blood samples from both male and female mice.
Executive summary:

A study was conducted to determine the in vivo toxicity of the test substance according to OECD Guideline 474, in compliance with GLP. This study was performed to evaluate the chromosome-damaging effect of the test substance in mice. The experimental animals were 50 NMRI mice, divided into 5 groups. Of the 5 groups, three were test groups, one negative control group and one positive control group. The test groups were treated with 400 mg test substance/kg bw, the negative control group with distilled water and the positive control group with 30 mg cyclophosphamide/kg bw. The mice were killed 24, 48 and 72 h, respectively after treatment. From bone marrow smears micronucleus counts were made per 1000 polychromatic erythrocytes. Under the test conditions, the test substance did not induce an increase in the frequency of micronucleated polychromatic erythrocytes in peripheral blood samples from both male and female mice (Kallesen, 1985).

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

Additional information

In vitro

Study 1. A study was conducted to determine the in vitro genetic toxicity of the test substance according to OECD Guideline 471 (Ames test), in compliance with GLP. The substance was examined for mutagenic activity in the Ames test using the histidine-requiring Salmonella typhimurium mutant strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100, and a liver microsome fraction of Aroclor-induced rats for metabolic activation (S9-mix). The substance was tested at doses of 0, 0.31, 0.93, 2.78, 8.33 and 25 µg/plate. Based on preliminary test in TA 98, 25 µg/plate was chosen as the highest dose level. The test was carried out twice. Under the study conditions, the substance was considered to be non-mutagenic in the presence and absence of exogenous metabolic activation (Wilmer, 1986).

Study 2. A study was conducted to determine the in vitro genetic toxicity of the test substance according to OECD Guideline 471, EU Method B13/14 and US EPA OPPTS 850.5100 (Ames test), in compliance with GLP. The mutagenic potential was investigated in Salmonella typhimurium strains A1535, TA1537, TA102, TA98 and TA100 with and without metabolic activation. Six dose levels of the test substance for each bacterial strain were tested in triplicate with and without a metabolic activation system. The dose range was determined in a preliminary toxicity assay and was 0.15 to 50 µg/plate in the first experiment. The experiment was repeated on a separate day using the same dose range, fresh cultures of the bacterial strains and fresh test substance formulations. Additional dose levels were included in both experiments to allow for test substance-induced toxicity and to ensure there were a minimum of four non-toxic doses plated out. The vehicle (sterile distilled water) control plates produced counts of revertant colonies within the normal range. All the positive control chemicals used in the test produced marked increases in the frequency of revertant colonies, both with and without the S9 -mix. No test substance precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix. No significant increase in the frequency of revertant colonies was recorded for any of the bacterial strains with any dose of the test substance, either with or without metabolic activation. Under the study conditions, the test substance was found to be non-mutagenic with and without metabolic activation (Thompson, 2001).

Study 3. A study was conducted to determine the in vitro genetic toxicity of the test substance according to OECD Guideline 471 (Ames test), in compliance with GLP. Salmonella typhimurium strains TA1535, TA1537, TA1538, TA98 and TA100 were treated with the test substance by the Ames plate incorporation method at five dose levels, in triplicate, with and without the addition of a rat liver homogenate metabolising system. The dose range was determined in a preliminary toxicity assay using 5 to 5,000 µg/plate. Doses in the first experiment were 1.5 -150 µg/plate. The experiment was repeated on a separate day. In this case, the dose range was 0.5 to 50 µg/plate. The solvent (DMSO) control plates gave counts of revertant colonies within the normal range. All positive control chemicals produced marked increases in the number of revertant colonies, both with and without the metabolising system. The test substance caused visible reduction in the growth of the bacterial lawn at 50 µg/plate (TA1535, TA100) and at 150 µg/plate (TA1535, TA98 and TA100). The test substance was, therefore, tested up to the maximally 150 µg/plate and 50 µg/plate in the first and second test, respectively. No significant increase in the numbers of revertant colonies was recorded for any of the bacterial strains with any dose, either with or without metabolic activation. Under the study conditions, the test substance was found to be non-mutagenic with and without metabolic activation (Jones, 1985).

Study 4. A study was conducted to determine the in vitro genetic toxicity of the test substance according to OECD Guideline 473 (chromosome aberration test), in compliance with GLP. This experiment was realized in Chinese hamster ovary cells. Based on preliminary cytotoxicity test, 0, 0.36, 1.09, 3.27 and 9.80 µg/mL test substance (without S9) as well as 0, 6.13, 12.25, 24.5, 49.0 and 98.0 µg/mL (with metabolic activation of S9 from rats treated with Aroclor 1254) test substance were selected. The exposure times were 21h for the experiments without metabolic activation, and 3h for the experiments with metabolic activation, with harvesting times of 12 and 21h. In the preliminary toxicity test, in the absence of S9-mix, the substance was toxic at a concentration of 15.68 µg/mL and above; and in the presence of S9-mix, the substance was very toxic at 78.4 µg/mL and above at both harvesting times. The test substance did not induce a statistically significant increase in the number of cells with structural chromosome aberrations (breaks, exchanges and multiple aberrations) at any of the concentrations used, either in the absence or in the presence of the S-9 mix. Under the study conditions, the test substance was not clastogenic in Chinese hamster ovary cells with or without metabolic activation (Willmer, 1986).

Study 5. A study was conducted to determine the in vitro genetic toxicity of the test substance according to OECD Guideline 473 and EU Method B.10 (chromosome aberration test), in compliance with GLP. This experiment was performed in human lymphocyte cells. Duplicate cell cultures of human lymphocytes, treated with the test substance, were evaluated for chromosome aberrations at three dose levels, together with vehicle and positive controls (mitomycin-C (without S9) and cyclophosphamide (with S9)). Four treatment conditions were used for the study. Experiment 1 and 4 h exposure with and without metabolic activation was followed by a 20 h expression period. In Experiment 2, the 4 h exposure with metabolic activation was repeated while in the absence of metabolic activation the exposure time was increased to 24 h. The doses studied were 0, 4, 8, 16, 20 µg/mL (with and without activation) in Experiment 1 and 0, 4, 8, 12, 16, 24 µg/mL (with and without activation) in Experiment 2. The test substance was considered negative for chromosomal aberrations in human lymphocytes in vitro under the S9 metabolic activation and non-activation conditions of the assay. There was no indication of chromosomal ploidy changes in cultures exposed to the test substance in either the presence or absence of S9 mix. Mutant frequencies of all cultures treated with the test substance were within the acceptable range for background mutant frequencies. Under the test conditions, the test substance was found to be non-clastogenic to human lymphocytes with and without metabolic activation (Durward, 2001).

Study 6. A study was conducted to determine the in vitro genetic toxicity of the test substance according to OECD Guideline 476 (cell gene mutation test), in compliance with GLP. This study was performed to evaluate the potential to induce mutations at the HGPRT locus in Chinese hamster ovary (CHO) cells. Based on a preliminary toxicity test, 0, 5, 10, 12.5, 15, 17.5 and 20 nL/mL (without metabolic activation) and 0, 15, 30, 45, 60, 75 and 90 nL/mL (with metabolic activation) were selected for the experiment. The entire experiment was repeated to confirm the results of the first experiment. Five h exposure was used both with and without S9-mix. The test substance did not induce dose-related increase in the mutant frequency at the HGPRT locus at any dose level, either with or without metabolic activation. Exposure to the highest concentrations tested resulted in significant (initial) toxicity for the CHO cells; this indicates that the test was carried out with appropriate concentrations of the test substance. Exposure to positive control substances gave the expected increases in the mutation frequency. The toxicity of the test substance for CHO cells was clearly reduced in the presence of metabolic activation. Under the study conditions, test substance did not show any mutagenic activity at the HGPRT locus in CHO cells with and without metabolic activation (Davis, 1986).

Study 7. A study was conducted to determine the in vitro genetic toxicity of the test substance according to a method similar to US EPA OPPTS 870.5300, in compliance with GLP. The study was performed on the HGPRT locus in Chinese hamster ovary (CHO) cells at test substance concentrations ranging from 0 to100 µg/mL. Preliminary cytotoxicity test showed the test substance to be slightly more toxic without S9 metabolic activation than with activation. The test substance was completely toxic at 20 µg/mL and higher without activation and completely toxic at 40 µg/mL and higher with activation. Dose levels selected for the first trial of the mutation assays covered nontoxic and highly toxic doses. Two independent non-activation and S9 metabolic activation assays were performed. Mutant frequencies of all cultures treated with the test substance were within the acceptable range for background mutant frequencies (0 to 13.5 x 10-6 with S9 mix and 0 to 15 x 10-6 without S9 mix). Under the study conditions, the test substance was not found to induce any forward mutations at the HGPRT locus in CHO cells with and without metabolic activation (Young, 1989).

In vivo

Study 1. A study was conducted to determine the in vivo toxicity of the test substance according to OECD Guideline 474, in compliance with GLP. This study was performed to evaluate the chromosome-damaging effect of the test substance in mice. The experimental animals were 50 NMRI mice, divided into 5 groups. Of the 5 groups, three were test groups, one negative control group and one positive control group. The test groups were treated with 400 mg test substance/kg bw, the negative control group with distilled water and the positive control group with 30 mg cyclophosphamide/kg bw. The mice were killed 24, 48 and 72 h, respectively after treatment. From bone marrow smears micronucleus counts were made per 1000 polychromatic erythrocytes. Under the test conditions, the test substance did not induce an increase in the frequency of micronucleated polychromatic erythrocytes in peripheral blood samples from both male and female mice (Kallesen, 1985).

Justification for classification or non-classification

Based on the available data, no classification is required for genotoxicity according to CLP (EC 1272/2008) criteria.