4-chlorobenzophenone

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames Assay:

Based on the data summarized and applying the weight of evidence approach, the test chemical did not induce gene mutation in the presence and absence of S9 metabolic activation system and hence it is not likely to be mutagenic in vitro.

In vitro chromosomal aberration study:

Based on the data summarized and applying the weight of evidence approach, the test chemical did not induce chromosomal aberrations in mammalian cells in the presence and absence of S9 metabolic activation system and hence it is not likely to be mutagenic in vitro.

In vitro mammalian cell gene mutation:

Based on the data summarized and applying the weight of evidence approach, the test chemical did not induce genotoxic effects in mammalian cells in the presence and absence of S9 metabolic activation system and hence it is not likely to be mutagenic in vitro.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

data is from peer reviewed journals

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD 471

Principles of method if other than guideline:

Ames Assay was performed to determine the mutagenic potential of the test chemical

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

Salmonella -Histidine
tryptophan - E.Coli

Species / strain / cell type:

S. typhimurium, other: S. typhimurium LT-2: TA1535, TA1537, TA100, and TA98

Species / strain / cell type:

E. coli WP2 uvr A

Cytokinesis block (if used):

no data available

Metabolic activation:

with and without

Metabolic activation system:

The test was also conducted with metabolic activation using a rat post-mitochondrial fraction (Aroclor 1254-induced S9) which was prepared by Lilly Research Laboratories or obtained from a commercial source (Molecular Toxicology, Annapolis, Maryland). The S9 was validated with appropriate positive controls prior to use

Test concentrations with justification for top dose:

100 to 1000 microgram/ml.

Vehicle / solvent:

solvent used name not mentioned

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

name not mentioned

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

2-acetylaminofluorene

9-aminoacridine

2-nitrofluorene

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate) : triplicates
- Number of independent experiments : single

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable):
- Test substance added in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk : agar medium

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 48 h
- Exposure duration/duration of treatment: 48h
- Harvest time after the end of treatment (sampling/recovery times): no data available


- OTHER: Five bacterial strains were used in both the Ames assay.They include four auxotrophs of S. typhimurium LT-2: TA1535, TA1537, TA100, and TA98 and a tryptophan auxotroph derived from E. coli: WP2uvrA . Salmonella typhimurium strains were obtained from Dr. Bruce N. Ames, and the E. coli strain was obtained from Dr. Brian A. Bridges of the MRC Cell Mutation Unit, Sussex, UK. The genotypes of these strains were confirmed using the procedure of Maron and Ames (1983). The overnight cultures were prepared from frozen stock (-150°C) by inoculating 0.2 ml of each tester strain in 20 ml of 2.5%
Oxoid Nutrient Broth No. 2 (Oxoid LTD). Minimal agar medium included 100 ml of 20% glucose; 80 ml of 10 x histidine-tryptophan-biotin(each his-tryp-bio, 0.5 mM); 40 ml of 25 × Vogel- Bonner salts (VB); and 280 ml Milli-Q water. The above mixture was warmed to 50°C and QS to 1 liter with melted 2.5% Difco Bacto agar. Final concentrations were 2% glucose, 1 × VB salts, and 0.04 mM of histryp-bio in 1.25% agar medium. A stock concentration of the test chemical was prepared at 100 mg/ml, and three 1:10 serial dilutions were performed. A concentration of 1000 microgram/ml of test compoundwas prepared by adding 10 ml of minimal agar to 0.1 ml of a 100 mg/ml solution of compound in the appropriate vehicle. The top agar was thoroughly mixed and poured onto the appropriate base layer plate. This resulted in a ten-fold concentration gradient across the plate ranging from 100 to 1000 microgram/ml.

Rationale for test conditions:

no data available

Evaluation criteria:

A test article was considered to have induced a positive response for bacterial mutation when a concentration-related increase in revertants was observed in which the number of revertants exceeded
the value of the vehicle control by at least two-fold (strains TA98, TA100, and WP2uvrA-) or a least three-fold (strains TA1535 and TA1537), for two successive concentrations of the test article.

Statistics:

no data available

Species / strain:

S. typhimurium, other: S. typhimurium LT-2: TA1535, TA1537, TA100, and TA98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not specified

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

no data available

Remarks on result:

other: not mutagenic

Conclusions:

The test chemical failed to induce genetic toxicity when tested on the Salmonella typhimurium LT-2: TA1535, TA1537, TA100, and TA98 and a tryptophan auxotroph derived from E. coli: WP2uvrA strains in the presence and absence of S9 metabolic activation system. Hence, the test chemical can be considered to be non-genotoxic in nature.

Executive summary:

Ames Assay was performed to determine the mutagenic potential of the test chemical.Five bacterial strains were used in the Ames assay. They include four auxotrophs of Salmonella typhimurium LT-2: TA1535, TA1537, TA100, and TA98 and a tryptophan auxotroph derived from E. coli: WP2uvrA.Salmonella typhimurium strains were obtained from Dr. Bruce N. Ames, and the E. coli strain was obtained from Dr. Brian A. Bridges of the MRC Cell Mutation Unit, Sussex, UK. The genotypes of these strains were confirmed using the procedure of Maron and Ames (1983). The overnight cultures were prepared from frozen stock (-150°C) by inoculating 0.2 ml of each tester strain in 20 ml of 2.5% Oxoid Nutrient Broth No. 2 (Oxoid LTD). Minimal agar medium included 100 ml of 20% glucose; 80 ml of 10 x histidine-tryptophan-biotin(each his-tryp-bio, 0.5 mM); 40 ml of 25 × Vogel- Bonner salts (VB); and 280 ml Milli-Q water. The above mixture was warmed to 50°C and QS to 1 liter with melted 2.5% Difco Bacto agar. Final concentrations were 2% glucose, 1 × VB salts, and 0.04 mM of histryp-bio in 1.25% agar medium. A stock concentration of the test chemical was prepared at 100 mg/ml, and three 1:10 serial dilutions were performed. A concentration of 1000 microgram/ml of test compound was prepared by adding 10 ml of minimal agar to 0.1 ml of a 100 mg/ml solution of compound in the appropriate vehicle. The top agar was thoroughly mixed and poured onto the appropriate base layer plate. This resulted in a ten-fold concentration gradient across the plate ranging from 100 to 1000 microgram/ml. The test was also conducted with metabolic activation using a rat post-mitochondrial fraction (Aroclor 1254-induced S9) which was prepared by Lilly Research Laboratories or obtained from a commercial source (Molecular Toxicology, Annapolis, Maryland). The S9 was validated with appropriate positive controls prior to use.In the Ames assay, 5 bacterial strains, 5 test concentrations, 1 solvent control, and 1 concentration per positive control were tested in triplicate, with and without metabolic activation (210 plates). Revertant colonies were counted using an Artek 880 Automated Colony Counter. A test article was considered to have induced a positive response for bacterial mutation when a concentration- related increase in revertants was observed in which the number of revertants exceeded the value of the vehicle control by at least two-fold (strains TA98, TA100, and WP2uvrA-) or a least three-fold (strains TA1535 and TA1537), for two successive concentrations of the test article. The test chemical failed to induce genetic toxicity when tested on the Salmonella typhimurium LT-2: TA1535, TA1537, TA100, and TA98 and a tryptophan auxotroph derived from E. coli: WP2uvrA strains in the presence and absence of S9 metabolic activation system. Hence, the test chemical can be considered to be non-genotoxic in nature.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

Data is from peer reviewed publication

Qualifier:

according to guideline

Guideline:

other: Similar to OECD 471

Principles of method if other than guideline:

Salmonella/Mammalian-Microsome Mutagenicity Assay was performed to determine the mutagenic nature of the test chemical

GLP compliance:

not specified

Type of assay:

bacterial gene mutation assay

Target gene:

Histidine

Species / strain / cell type:

S. typhimurium, other: TA98, TA100, TA1535, TA1537

Details on mammalian cell type (if applicable):

no data available

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Liver S9 homogenate was prepared from male Sprague-Dawley rats and Syrian golden hamsters that had been injected with Aroclor 1254 at 500 mg/kg body weight

Test concentrations with justification for top dose:

10-200 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: positive controls used details not mentioned

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: No data
- Exposure duration: 48 hrs
- Expression time (cells in growth medium): 48 hrs
- Selection time (if incubation with a selection agent): No data
- Fixation time (start of exposure up to fixation or harvest of cells): No data

SELECTION AGENT (mutation assays): No data
SPINDLE INHIBITOR (cytogenetic assays): No data
STAIN (for cytogenetic assays): No data

NUMBER OF REPLICATIONS: Five doses of test chemical were tested in triplicate on each tester strain without and with metabolic activation.

NUMBER OF CELLS EVALUATED: No data

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data

OTHER EXAMINATIONS:
- Determination of polyploidy: No data
- Determination of endoreplication: No data
- Other: No data

OTHER: No data

Rationale for test conditions:

no data available

Evaluation criteria:

For a test article to be considered positive, it had to induce at least a doubling (TA98, TA100, and TA1535) in the mean number of revertants per plate of at least one tester strain. This increase in the mean revertants per plate had to be accompanied by a dose response to increasing concentrations of the test chemical. If the study showed a dose response with a less than 3-fold increase on TA1537 or TA1538, the response had to be confirmed in a repeat experiment.

Statistics:

No data available

Species / strain:

S. typhimurium, other: TA98, TA100, TA1535, TA1537, and TA1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data
- Effects of osmolality: No data
- Evaporation from medium: No data
- Water solubility: No data
- Precipitation: No data
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES: The doses that were tested in the mutagenicity assay were
selected based on the levels of cytotoxicity observed in a preliminary dose range-finding study using strain TA100. Ten dose levels of the chemical, one plate per dose, were tested in both the presence and the absence of induced hamster S9. If no toxicity was observed, a total maximum dose of 10 mg of test chemical per plate was used.

COMPARISON WITH HISTORICAL CONTROL DATA: No data

ADDITIONAL INFORMATION ON CYTOTOXICITY: No data

Remarks on result:

other: not mutagenic

Results of the Salmonella Mutagenicity test:

Dose

TA98

TA100

TA102

TA1535

TA1537

TA1538

TA97

no S9

rat S9

Ham'r S9

no S9

rat S9

Ham'r S9

no S9

rat S9

Ham'r S9

no S9

rat S9

Ham'r S9

no S9

rat S9

Ham'r S9

no S9

rat S9

Ham'r S9

no S9

rat S9

Ham'r S9

Negative

Negative

Negative

Negative

Negative

Negative

Negative

Negative

Negative

Negative

Negative

Negative

DMSO

16 ± 6

23 ± 6

35 ± 7

84 ± 10

81 ± 8

87 ± 4

25 ± 5

8 ± 4

7 ± 1

5 ± 2

9 ± 6

8 ± 3

10ug

21 ± 2

27 ± 2

25 ± 6

82 ± 6

90 ± 13

86 ± 12

20 ± 4

15 ± 1

13 ± 6

5 ± 3

10 ± 2

9 ± 1

33ug

18 ± 7

29 ± 5

30 ± 4

84 ± 5

93 ± 12

79 ± 6

15 ± 3

10 ± 4

14 ± 3

8 ± 1

9 ± 3

6 ± 1

100ug

17 ± 5

25 ± 5

27 ± 4

88 ± 6

78 ± 6

69 ± 8

24 ± 6

8 ± 3

11 ± 3

4 ± 2

6 ± 2

6 ± 1

333ug

15 ± 6

21 ± 5

27 ± 4

77 ± 12

77 ± 3

76 ± 12

7 ± 3

7 ± 1

4 ± 2

3 ± 1

7 ± 4

0 ± 0

1000ug

5 ± 3

15 ± 4

15 ± 9

2 ± 1

33 ± 4

12 ± 5

3 ± 2

1 ± 1

0 ± 1

0 ± 0

1 ± 0

0 ± 0

Positive

845 ± 68

359 ± 77

2059 ± 66

1555 ± 55

600 ± 99

2706 ± 146

1269 ± 66

86 ± 8

205 ± 4

1372 ± 303

414 ± 30

291 ± 56

DMSO

17 ± 4

25 ± 5

28 ± 2

94 ± 5

91 ± 6

92 ± 13

7 ± 1

25 ± 5

8 ± 4

8 ± 3

5 ± 2

9 ± 6

33ug

20 ± 6

27 ± 8

28 ± 9

97 ± 12

88 ± 18

96 ± 8

13 ± 6

20 ± 4

15 ± 1

9 ± 1

5 ± 3

10 ± 2

100ug

20 ± 5

27 ± 2

33 ± 7

90 ± 13

90 ± 4

96 ± 8

14 ± 3

15 ± 3

10 ± 4

6 ± 1

8 ± 1

9 ± 3

333ug

16 ± 5

28 ± 6

30 ± 8

91 ± 9

80 ± 10

93 ± 7

11 ± 3

24 ± 6

8 ± 3

6 ± 1

4 ± 2

6 ± 2

1000ug

10 ± 2

18 ± 9

16 ± 2

4 ± 5

47 ± 10

26 ± 8

4 ± 2

7 ± 3

7 ± 1

0 ± 0

3 ± 1

7 ± 4

2000ug

2 ± 2

5 ± 3

3 ± 3

0 ± 0

0 ± 0

0 ± 0

0 ± 1

3 ± 2

1 ± 1

0 ± 0

0 ± 0

1 ± 0

Positive

794 ± 13

696 ± 80

1568 ± 474

1936 ± 22

769 ± 7

1286 ± 118

205 ± 4

1269 ± 66

322 ± 11

291 ± 56

715 ± 157

414 ± 30

Conclusions:

The test chemical failed to show any mutagenic activity in the presence or absence of activation by induced rat and hamster liver S9 preparations in Salmonella typhimurium strains TA 98,TA 100,TA1535,TA 1537.The test chemical was not considered to be mutagenic by AMES test.                                                           

Executive summary:

The test chemical was assessed for its possible mutagenic potential according to AMES test performed on Salmonella typhimurium strains TA 98,TA 100,TA1535,TA 1537and TA 1538 .

Salmonella typhimurium histidine auxotrophs TA98, TA100, TA1535, TA1537 were obtained from Dr. Bruce Ames, University of California (Berkeley, CA). Cultures were grown overnight in Oxoid nutrient broth no. 2 and were removed from incubation when they reached a density of (1-2)x10⁹ cells/mL. On the day of use, all tester strain cultures were checked for genetic integrity as recommended by Ames et al. Liver S9 homogenate was prepared from male Sprague-Dawley rats and Syrian golden hamsters that had been injected with Aroclor 1254 at 500 mg/kg body weight. The post-mitochondrial (microsomal) enzyme fractions were prepared as described by Ames et al. The components of the S9 mix were 8 mM MgCl2, 33 mM KCl, 5 mM glucose-6-phosphate, 4 mM NADP, 100 mM sodium phosphate (pH 7.4), and the appropriate S9 homogenate at a concentration of 0.1 mL/mL of mix. For each plate receiving microsomal enzymes, 0.5 mL of S9 mix was added. The doses that were tested in the mutagenicity assay were selected based on the levels of cytotoxicity observed in a preliminary dose range-finding study using strain TA100. Ten dose levels of the chemical, one plate per dose, were tested in both the presence and the absence of induced hamster S9. If no toxicity was observed, a total maximum dose of 10 mg of test chemical per plate was used. For testing in the absence of S9 mix, 100 microliters of the tester strain and 50 microliter of the solvent or test chemical were added to 2.5 mL of molten selective top agar at 45±2 °C. When S9 was used, 0.5 mL of S9 mix, 50 microliter of tester strain, and 50 microliter of solvent or test chemical were added to 2.0 mL of molten selective top agar at 45±2 °C. After it was vortexed, the mixture was overlaid onto the surface of 25 mL of minimal bottom agar. After the overlay had solidified, the plates were incubated for 48 h at 37±2°C. 0.3-100 µg/plate of the test chemical, together with the appropriate concurrent solvent and positive controls, were tested in triplicates on each tester strain without metabolic activation and also with activation by induced rat and hamster liver S9 preparations. The criteria used to evaluate a test were as follows: for a test article to be considered positive, it had to induce at least a doubling (TA98, TA100, and TA1535) in the mean number of revertants per plate of at least one tester strain. This increase in the mean revertants per plate had to be accompanied by a dose response to increasing concentrations of the test chemical. If the study showed a dose response with a less than 3-fold increase on TA1537 or TA1538, the response had to be confirmed in a repeat experiment. The test chemical failed to show any mutagenic activity in the presence or absence of activation by induced rat and hamster liver S9 preparations in Salmonella typhimurium strains TA 98,TA 100,TA1535,TA 1537.The test chemical was not considered to be mutagenic by AMES test.                                                           

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Ames Assay

Data available for the test chemicals were reviewed to determine the mutagenic nature of the test chemical. The studies are as mentioned below:

 

Study 1 : The test chemical was assessed for its possible mutagenic potential according to AMES test performed on Salmonella typhimurium strains TA 98,TA 100,TA1535,TA 1537and TA 1538 .

Salmonella typhimurium histidine auxotrophs TA98, TA100, TA1535, TA1537 were obtained from Dr. Bruce Ames, University of California (Berkeley, CA). Cultures were grown overnight in Oxoid nutrient broth no. 2 and were removed from incubation when they reached a density of (1-2)x10⁹ cells/mL. On the day of use, all tester strain cultures were checked for genetic integrity as recommended by Ames et al. Liver S9 homogenate was prepared from male Sprague-Dawley rats and Syrian golden hamsters that had been injected with Aroclor 1254 at 500 mg/kg body weight. The post-mitochondrial (microsomal) enzyme fractions were prepared as described by Ames et al. The components of the S9 mix were 8 mM MgCl2, 33 mM KCl, 5 mM glucose-6-phosphate, 4 mM NADP, 100 mM sodium phosphate (pH 7.4), and the appropriate S9 homogenate at a concentration of 0.1 mL/mL of mix. For each plate receiving microsomal enzymes, 0.5 mL of S9 mix was added. The doses that were tested in the mutagenicity assay were selected based on the levels of cytotoxicity observed in a preliminary dose range-finding study using strain TA100. Ten dose levels of the chemical, one plate per dose, were tested in both the presence and the absence of induced hamster S9. If no toxicity was observed, a total maximum dose of 10 mg of test chemical per plate was used. For testing in the absence of S9 mix, 100 microliters of the tester strain and 50 microliter of the solvent or test chemical were added to 2.5 mL of molten selective top agar at 45±2 °C. When S9 was used, 0.5 mL of S9 mix, 50 microliter of tester strain, and 50 microliter of solvent or test chemical were added to 2.0 mL of molten selective top agar at 45±2 °C. After it was vortexed, the mixture was overlaid onto the surface of 25 mL of minimal bottom agar. After the overlay had solidified, the plates were incubated for 48 h at 37±2°C. 0.3-100 µg/plate of the test chemical, together with the appropriate concurrent solvent and positive controls, were tested in triplicates on each tester strain without metabolic activation and also with activation by induced rat and hamster liver S9 preparations. The criteria used to evaluate a test were as follows: for a test article to be considered positive, it had to induce at least a doubling (TA98, TA100, and TA1535) in the mean number of revertants per plate of at least one tester strain. This increase in the mean revertants per plate had to be accompanied by a dose response to increasing concentrations of the test chemical. If the study showed a dose response with a less than 3-fold increase on TA1537 or TA1538, the response had to be confirmed in a repeat experiment. The test chemical failed to show any mutagenic activity in the presence or absence of activation by induced rat and hamster liver S9 preparations in Salmonella typhimurium strains TA 98,TA 100,TA1535,TA 1537.The test chemical was not considered to be mutagenic by AMES test.                                    

                                                       

Study 2 : Ames Assay was performed to determine the mutagenic potential of the test chemical.Five bacterial strains were used in the Ames assay. They include four auxotrophs of Salmonella typhimurium LT-2: TA1535, TA1537, TA100, and TA98 and a tryptophan auxotroph derived from E. coli: WP2uvrA.Salmonella typhimurium strains were obtained from Dr. Bruce N. Ames, and the E. coli strain was obtained from Dr. Brian A. Bridges of the MRC Cell Mutation Unit, Sussex, UK. The genotypes of these strains were confirmed using the procedure of Maron and Ames (1983). The overnight cultures were prepared from frozen stock (-150°C) by inoculating 0.2 ml of each tester strain in 20 ml of 2.5% Oxoid Nutrient Broth No. 2 (Oxoid LTD). Minimal agar medium included 100 ml of 20% glucose; 80 ml of 10 x histidine-tryptophan-biotin(each his-tryp-bio, 0.5 mM); 40 ml of 25 × Vogel- Bonner salts (VB); and 280 ml Milli-Q water. The above mixture was warmed to 50°C and QS to 1 liter with melted 2.5% Difco Bacto agar. Final concentrations were 2% glucose, 1 × VB salts, and 0.04 mM of histryp-bio in 1.25% agar medium. A stock concentration of the test chemical was prepared at 100 mg/ml, and three 1:10 serial dilutions were performed. A concentration of 1000 microgram/ml of test compound was prepared by adding 10 ml of minimal agar to 0.1 ml of a 100 mg/ml solution of compound in the appropriate vehicle. The top agar was thoroughly mixed and poured onto the appropriate base layer plate. This resulted in a ten-fold concentration gradient across the plate ranging from 100 to 1000 microgram/ml. The test was also conducted with metabolic activation using a rat post-mitochondrial fraction (Aroclor 1254-induced S9) which was prepared by Lilly Research Laboratories or obtained from a commercial source (Molecular Toxicology, Annapolis, Maryland). The S9 was validated with appropriate positive controls prior to use.In the Ames assay, 5 bacterial strains, 5 test concentrations, 1 solvent control, and 1 concentration per positive control were tested in triplicate, with and without metabolic activation (210 plates). Revertant colonies were counted using an Artek 880 Automated Colony Counter. A test article was considered to have induced a positive response for bacterial mutation when a concentration- related increase in revertants was observed in which the number of revertants exceeded the value of the vehicle control by at least two-fold (strains TA98, TA100, and WP2uvrA-) or a least three-fold (strains TA1535 and TA1537), for two successive concentrations of the test article. The test chemical failed to induce genetic toxicity when tested on the Salmonella typhimurium LT-2: TA1535, TA1537, TA100, and TA98 and a tryptophan auxotroph derived from E. coli: WP2uvrA strains in the presence and absence of S9 metabolic activation system. Hence, the test chemical can be considered to be non-genotoxic in nature.

 

Based on the data summarized and applying the weight of evidence approach, the test chemical did not induce gene mutation in the presence and absence of S9 metabolic activation system and hence it is not likely to be mutagenic in vitro.

 

In vitro chromosomal aberration study:

Data available for the test chemicals was reviewed to determine the mutagenic nature of the test chemical. The studies are as mentioned below:

 

Study 1: An in vitro mammalian chromosome aberration test was carried out on cells obtained from Chinese hamster Ovary. As some test chemicals may become potent clastogens when photosensitised, the clastogenic potential of the test article in this study was assessed by its effects on the chromosomes of CHO cells treated in the absence and presence of UV light. The study was performed according to EC Scientific Committee for Cosmetology Guideline CSC/803-5/90 (1990) Guidelines. A preliminary range-finding experiment, covering a broad range of doses, was performed in the presence of two doses of UV light to investigate the phototoxicity of the chemical and to determine the dose range to be used in the main study. The doses of UV used were 1500 and 750 mJ/cm2. There was no marked mitotic inhibition shown following irradiation at either of the two levels (1500 and 750 mJ/cm2), and only the higher UV dose was used in the main study. The highest concentration of the test material chosen for analysis was 3084 ug/ml (10mM) and a range of doses from this concentration were used in the absence and presence of UV light. Appropriate negative (solvent) and positive controls were included in the test system in the presence and absence of UV light. Treatment of cultures with chemical in the absence of UV light resulted in frequencies of cells with structural aberrations that were similar to and not significantly different from the frequency seen in concurrent, non irradiated, solvent control cultures. Aberrant cell frequency was within the historical negative control range. Treatment of cultures with test chemical in the presence of UV light resulted in frequencies of cells with structural aberrations that were not increased compared to the concurrent, irradiated, solvent control cultures. The test chemical tested negative for clastogenic effects in cultured CHO cells at doses up to 10 mM in the absence and presence of UV light.

Study 2: The cytogenetic effect of the test chemical on cultured cells, chromosomal aberration test was performed using Chinese hamster cultured cells (CHL / IU). Chinese hamster lung-derived fibroblast cell line (CHL / IU) was used as the test cell line. Inactivated (56°C, 30 minutes) calf serum (Invitrogen) was added to Eagle-MEM liquid medium to a final concentration of 10 vol% and then used for the test. The prepared culture solution was stored in a cool dark place (4°C).The cells were cultured under conditions of 5% CO₂ and 37°C using a CO₂ incubator. The Kikkoman S9 mix within 6 months after production was used for the test. S9 in S9 mix was prepared from the liver of Sprague-Dawley male rats treated with phenobarbital and 5,6-benzoflavone as inducers.The cells were seeded on a 12-well multi-plate for cell culture, and after 3 days of culture, the test substance solution was treated. The test chemical is insoluble in water but readily soluble in acetone.To determine the treatment concentration of the test substance used in the chromosomal aberration test, the effect of the test substance on cell proliferation was investigated. The growth inhibitory effect of the test substance on CHL cells was determined by counting the number of viable cells in each group using a hemocytometer and the ratio of cell proliferation to the negative control group. As a result, the concentration of test chemical that showed about 50% growth inhibition was calculated from the value of 2 concentrations that sandwiched 50%. The cell viability at concentrations around this value was around 50%, but no difference was observed. On the other hand, growth inhibition exceeding 50% was not observed in any treatment concentration group (0.156 to 5.00 mg / ml) in the continuous treatment method for 24 hours and the short treatment method.From the results of the cell growth inhibition test, the high concentration group of the test substance used in the chromosomal aberration test was set to a high concentration of 5.00 mg / ml in the continuous treatment method and the short-time treatment method.The concentration of 1/ 4 was made low. In the 48 hour treatment, 4 concentrations were added, adding 0.625 mg / ml (high concentration 1/8), which showed the lowest cell viability.The doses used for the study were as follows: -S9 mix (24 h treatment): 0, 1250, 2500, 5000 μg/ml ; -S9 mix (48 h treatment): 0, 1250, 2500, 5000 μg/ml ; -S9 mix (6 h pulse treatment): 0, 1250, 2500, 5000 μg/ml ; +S9 mix (6 h pulse treatment): 0, 1250, 2500, 5000 μg/ml.In the short-time treatment method, after treatment for 6 hours in the absence (-S9 treatment) or presence (+ S9 treatment) of S9 mix, the culture medium was replaced with a fresh culture solution and culture was continued for 18 hours. In the case of the continuous treatment method, treatment was performed continuously for 24 hours.The cells were fixed with 10 vol% neutral buffered formalin solution (Wako Pure Chemical Industries, Ltd.), and stained with an aqueous solution of 0.1 w / v% crystal violet (Kanto Chemical) for 10 minutes. An appropriate amount of dye elution solution (30 vol% ethanol, 1 vol% acetic acid aqueous solution) is added, and left for about 5 minutes to elute the dye, and then at 580 nm using a spectrophotometer (type 105-50, Hitachi, Ltd.) Absorbance was measured. The ratio to the absorbance in the solvent control group, that is, the relative cell growth rate, was calculated for each dose group, and the 50% cell growth inhibitory concentration was calculated using the Probit method. As a result of continuous treatment of CHL cells with the test substance for 24 and 48 hours, the frequency of chromosomal structural abnormalities and ploidy cells in all treatment groups was less than 5%. In addition, chromosomal structural abnormalities and the frequency of ploidy cells were less than 5% in all treatment groups in the presence and absence of the short-time treatment S9 mix. Hence the test chemical can be considered to be non-mutagenic in nature.

 

Based on the data summarized and applying the weight of evidence approach, the test chemical did not induce chromosomal aberrations in mammalian cells in the presence and absence of S9 metabolic activation system and hence it is not likely to be mutagenic in vitro.

 

In vitro mammalian cell gene mutation:

 

Data available for the test chemicals was reviewed to determine the mutagenic nature of the test chemical. The studies are as mentioned below:

 

Study 1: L5178Y TK +/- mouse lymphoma assay was conducted to determine the mutagenic potential of the test chemical.L5178Y TK +/- mouse lymphoma cells were used for the study. The cells were grown in Fischer's medium for leukemic cells of mice supplemented with 10% horse serum, antibiotics (50 U penicillin/mi and 50 microgram streptomycin/ml), and 0.02% Pluronic F-68. All serum lots were pre-screened for their ability to support optimal growth. The cells were checked for the presence of mycoplasma by agar block isolation and Hoechst staining before and after cryopreservation.The toxicity of each chemical was first determined both with and without S9 prepared from Aroclor-1254-induced male Fischer 344 rats. Cells at a concentration of 6 × 105/ml were exposed for 4 h to a range of concentrations from 35 to 170 microgram/ml or the limit of solubility. The cells were then washed, re-suspended in growth medium, and incubated at 37 °C for 48 h. The rate of cell growth was determined for each of the treated cultures and compared to the rate of growth of the solvent controls. The doses of chemical selected for testing were within the range yielding approximately 0-90% cytotoxicity. For each assay there was a solvent control, a positive control for the test without metabolic activation and for the test with metabolic activation. The maximum solvent concentration was 1% for organic solvents and 10% for water. These levels had no effects on cell growth or spontaneous mutation frequency.The mutagenicity assay was performed according to the procedure described by Clive and Spector (1975). Cells in duplicate cultures were exposed to the test chemical, positive control, and solvent control for 4 h at 37°C; washed twice with growth medium; and maintained at 37 °C for 48 h in log phase growth to allow recovery and mutant expression. The cultures were adjusted to 0.3 × 106 cells/ml at 24-h intervals. They were then cloned in soft agar medium containing Fischer's medium, 20% horse serum, 2 mM sodium pyruvate, 0.02% Pluronic F-68 and 0.35% Noble agar. Resistance to trifluorothymidine (TFT) was determined by adding 3 microgram/ml TFT to one set of plates. The 100 × stock solution of TFT in saline was stored at -70°C and thawed immediately before use. Plates were incubated at 37°C in 5% CO2 in air for 12 days, and then counted with an automatic colony counter. Mutant frequencies were expressed as mutants per 104 surviving cells. In general, a response was considered positive if there was a dose-related increase in the mutant frequency above the spontaneous control frequency, with a 2-fold increase at more than 1 dose and relative total growth greater than 10%.The results in all the assays with test chemical were negative. Hence, the test chemical can be considered to be non-mutagenic to L5178Y TK +/- mouse lymphoma cells.

 

Study 2: An in vitro mammalian cell gene mutation study was designed and conducted to determine the genotoxicity profile of the test chemical when administered to Chinese Hamster Ovary (CHO) cells.The study was performed as per OECD 476 Guidelines. A preliminary dose-finding study was conducted prior to the main study. A range of different concentrations of the test chemical were tested in 96-well plates and analyzed by two commonly used assays, i.e. the colorimetric assay of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)and the bicinchoninic acid(BCA) assay to assess cell viability and protein concentration, respectively.From the basis of the results from the MTT and BCA assays, test concentrations of the test chemical was chosen to be included in the gene toxicity test.Cells (0.5 x 10⁵per well) were seeded in fresh medium (2 ml per well) in 6 well-plates and were incubated overnight at 37°C, 5% CO₂. On the day of exposure, the test chemical was added to each applicable well to give a final concentrations of 0, 1.0, 2.5, 5.0 or 10.0 mM and in the absence or 4% (80ml) presence of S9-induced metabolic activation. Negative controls (cell medium), solvent/vehicle controls (PBS) and positive control substances were also included in each experiment.N-ethyl-N-nitrosourea (ENU) (2.5 mM) was used as positive control substance in experiments without metabolic activation, while 7,12-dimethylbenz(a) anthracene (8mM) was used in experiments with metabolic activation. pH and osmolality was not determined in the preliminary dose-finding/toxicity test. During the exposure period, the treated cultures were incubated at 37°C, 5% CO₂.After the 3 hour exposure period, cells were washed several times with sterile PBS to remove the test chemical. The cultures were then incubated for 7 days at 37°C, 5% CO₂. During the expression period, the cell medium was exchanged every second day. However, if the cell population increased so that cells covered more than 90% of the well surface, cells were sub-cultured.At the end of the expression period, cells were trypsinized and the number cells per ml and well was calculated manually using a heamocytometer. Cells were transferred to new 6 well plates with or without TG added as a selection/restrictive agent. Four 6-well plates were prepared per culture, i.e. duplicate plates for viable count evaluation and duplicate plates for TG-treatment. The plates were incubated for 14 days at 37°C in a humidified atmosphere enriched with 5% CO₂. At the end of the 14 day incubation period, the cells were fixed with 100% methanol for 10 min. and then stained with a 0.5% Crystal Violet solution. The plates were scored for total number of colonies by manual counting. As a result, the mutation frequency could be calculated (see equation below).A minimum of 2 replicates per dose concentration, including negative and positive control, were performed for each experiment. Positive controls, such as N-ethyl-N-nitrosourea (ENU) experiments without metabolic activation and 7,12-dimethylbenz(a) anthracene in experiments with metabolic activation, were also included in each test. Treatment with N-ethyl-N-nitrosourea produced a significant increase in the number of revertant colonies whereas 7,12-dimethylbenz(a) anthracene did not. As such, the performance of the metabolic activation system could not be ascertained in the experiment. Without metabolic activation, the test chemical failed to produce a significant increase in the number of revertant colonies when compared to control data. No conclusion could be drawn regarding the mutagenicity of the test chemical in CHO cells in the presence of metabolic activation because of invalid positive control data. No cytotoxic effects were observed when CHO cells were exposed to the test chemical for 3 hrs.

 

Based on the data summarized and applying the weight of evidence approach, the test chemical did not induce genotoxic effects in mammalian cells in the presence and absence of S9 metabolic activation system and hence it is not likely to be mutagenic in vitro.

Justification for classification or non-classification

Based on the data summarized and applying the weight of evidence approach, the test chemical is not likely to be mutagenic in vitro. Comparing the above annotations with the criteria of CLP Regulations, the test chemical can be classified under the category "Not Classified".