Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames assay:

The test chemical did not induce mutation in the Salmonella typhimurium and E. coli strains both in the presence and absence of S9 metabolic activation system and hence is not likely to be mutagenic under the conditions of this study.

 

In vitro mammalian chromosome aberration study:

The test chemical did not induce chromosome aberrations in the mammalian cell line in the presence and absence of S9 metabolic activation system and hence it is not mutagenic in the chromosome aberration study performed.

 

In vitro gene mutation study in mammalian cells

Test chemical did not induce mutation in mammalian cell line in the presence and absence of metabolic activation and hence it is not likely to classify as a gene mutant in vitro.

Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Remarks:
Experimental data from various test chemicals
Justification for type of information:
Data for the target chemical is summarized based on the various test chemicals.
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
other: as mentioned below
Principles of method if other than guideline:
WoE for the target CAS is summarized based on data from various test chemicals.
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Target gene:
2. Histidine
3. Histidine for s. typhi and Tryptophan for E.coli
Species / strain / cell type:
S. typhimurium, other: TA98,TA 100,TA 1535 and TA 97
Remarks:
2
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Remarks:
3
Species / strain / cell type:
E. coli WP2 uvr A
Remarks:
3
Metabolic activation:
with and without
Metabolic activation system:
2. RLI = induced male Sprague Dawley rat liver S9; HLI = induced male Syrian hamster liver S9
3. Type and composition of metabolic activation system:
- source of S9
: Rat liver
- method of preparation of S9 mix :Not specified
- concentration or volume of S9 mix and S9 in the final culture medium
:Not specified
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability):Not specified

The activating mixture (S9) contained (v/v) water (70%), sodium phosphate buffer at pH 7.4 (10%), 0.2 M glucose 6-phosphate (2%), 0.1 M nicotinamide adenine dinucleotide phosphate (4%; NADP), 0.825 M potassium chloride (4%), 0.2 M magnesium chloride (4%), and 10% of rat liver homogenate (34 mg protein/ml) and was routinely checked for sterility.
Test concentrations with justification for top dose:
2. 0, 33, 100, 333, 1000, 3333, 10000 µg/plate
3. 0, 333, 667, 1000, 3330, 5000 μg/plate
Vehicle / solvent:
2. - Vehicle(s)/solvent(s) used: Distilled water
- Justification for choice of solvent/vehicle: The test substance is soluble in Distilled water
3. Not specified
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
Distilled water
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
other: 2-Aminoanthracene: 1.0 ug/Plate : TA 100, TA 97, TA 98 (+S9); 2.0 ug/Plate: TA 100, TA 97, TA 98, TA 1535 (+S9); 5.0 ug/Plate: TA 100, TA 97, TA 98, TA 1535 (+S9); 10.0 ug/Plate : TA 1535 (+S9). 4-Nitro-O-Phenylenediamine: TA 98 (-S9)
Remarks:
2
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
not specified
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
2-nitrofluorene
sodium azide
other: (+S9): 2-ammoanthracene (2.5 µg/plate for Salmonella, 25.0 µg/plate for E. coli) ; (-S9): ICR-191 (2.0 µg/plate for TA 1537)
Remarks:
3
Details on test system and experimental conditions:
2. NUMBER OF REPLICATIONS:
- Number of independent experiments: 3
3. NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): No data
- Number of independent experiments: triplicate
Other: Bacteria were prepared on master plates from frozen stocks, inoculated into flasks containing Vogel-Bonner salt solution, supplemented with 2.5% Oxoid nutrient broth No. 2, and harvested for use in late log phase. Strains were confirmed by routine testing for sensitivity to crystal violet (rfa wall mutation) or ampicillin resistance (pKMIOl plasmid). Experiments were conducted on two-layer plates. The lower layer was Vogel-Bonner minimal medium E plus 1.5% agar and 0.2% glucose, and the upper layer was 0.7% agar, 0.5% sodium chloride, supplemented with 10 ml of 0.5 mM histidine/ biotin, or 0.5 mM tryptophan, per 100 ml of agar when appropriate (Vogel and Bonner, 1956).
Evaluation criteria:
2. If the substance under test is mutagenic, the chemical-treated plates will have a much greater number of colonies than the negative control plates. A positive response is a reproducible, dose-related increase in mutant colonies in any single strain, with or without the addition of S9 metabolic enzymes. While there is no minimum percentage of increase required for a result to be considered positive, a twofold increase in mutant colonies in a treated plate is usually considered to be a positive (mutagenic) response. An equivocal response is any increase that is not reproducible, not dose-related, or not high enough in magnitude to be considered positive. A negative response occurs when no increases in mutant colonies are seen in the cultures treated with the test chemical, compared with the control.
3. Revertant colonies analyzed.
Statistics:
2. Mean or Mean ± Standard Error Mean.
3. Not specified
Species / strain:
S. typhimurium, other: TA 100, TA 98, TA 97 and TA 1535
Remarks:
2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
True negative controls validity:
not specified
Positive controls validity:
valid
Species / strain:
S. typhimurium, other: TA98, TA100, TA1535, TA1537
Remarks:
3
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
True negative controls validity:
not specified
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Remarks:
3
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
True negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
2. No data
3. Ames test:
- Signs of toxicity: Dose-ranging studies demonstrated no cytotoxic effects of test chemical up to 5 mg per plate using Salmonella strains TA100 and WP2uvrA.
- Individual plate counts: No data
- Mean number of revertant colonies per plate and standard deviation: No data
Information on bacterial strains: Salmonella typhimurium: Salmonella typhimurium Strains TA98, TA100, TA1535, and TA1537 have histidine synthesis mutations, the rfa wall mutation, and deletion of the uvrB gene (the latter two mutations enhance the sensitivity of these strains to detect mutagenic compounds). In addition, the TA98 and TA100 strains also have the plasmid pKM101, further increasing strain sensitivity. Histidine independence (the reverse mutation) is created by chemicals causing frame shifts in the TA98 and TA1537 strains, chemicals causing base deletions in the TA1535 strain, and by chemicals causing both frame shifts and base deletions in the TA 100 strain.
E. coli: A tryptophan-dependent E. coli strain WP2uvrA was used in the study. This strain has a uvrA DNA repair deficiency, increasing its sensitivity to detect mutagenic compounds.
Other:
-Aliquots of the culture were used to quantify the number of spontaneous revertants for both bacterial species.
-Background lawn was evaluated for evidence of cytotoxicity.
-Revertant colonies were counted manually, or using an automated colony counter.
Remarks on result:
other: No mutagenic potential observed

2.

Table 1: TA100

Dose (ug/Plate)

Metabolic activation

Without S9

Without S9

With 10% Rat S9

With 30% Rat S9

With 10% Hamster S9

With 30% Hamster S9

Vehicle Control

124±7.1

124±3.2

133±9.7

144±6.9

119±7.7

130±9.6

33.0

120±5.8

123±7.7

 

 

 

 

100.0

118±11.1

133±7.7

131±3.5

135±5.8

124±7.4

129±2.6

333.0

106±4.3

127±9.3

124±5.0

134±3.8

123±8.4

139±2.1

1000.0

114±10.3

116±1.3

126±9.9

133±4.3

127±2.1

127±9.5

3333.0

95±4.0

110±5.9

132±8.4

140±10.2

118±5.5

135±4.2

10000.0

 

 

119±6.3

78±6.1

117±3.8

110±9.7

Positive control

951±25.6

974±25.5

593±13.8

629±50.7

641±19.1

1022±22.5

 

Table 2: TA1535

Dose (ug/Plate)

Metabolic activation

Without S9

Without S9

With 10% Rat S9

With 30% Rat S9

With 10% Hamster S9

With 30% Hamster S9

Vehicle Control

8±0.9

19±2.5

23±2.5

13±1.2

18±3.0

10±1.2

33.0

 

19±0.3

 

 

 

 

100.0

8±0.7

17±2.0

19±0.3

10±0.9

21±2.0

9±0.3

333.0

7±1.5

17±2.2

15±0.7

14±2.6

22±3.5

9±0.6

1000.0

7±0.0

16±1.7

23±4.1

12±2.3

17±4.2

6±0.7

3333.0

5±0.3

16±1.5

23±1.2

11±0.6

18±2.0

9±1.8

10000.0

Toxic

 

19±0.9

9±0.3

14±0.3

9±2.0

Positive control

980±23.7

1017±44.8

209±10.3

134±9.8

232±21.0

170±3.1

 

Table 3: TA97

Dose (ug/Plate)

Metabolic activation

Without S9

Without S9

With 10% Rat S9

With 30% Rat S9

With 10% Hamster S9

With 30% Hamster S9

Vehicle Control

144±9.4

156±3.9

181±8.1

173±11.9

185±4.4

170±4.8

33.0

 

153±7.6

 

 

 

 

100.0

147±9.0

169±11.1

180±7.4

149±10.0

152±9.2

176±11.3

333.0

160±5.9

145±11.0

192±6.5

170±9.5

151±11.5

150±3.9

1000.0

172±3.0

169±2.3

180±8.9

172±10.5

182±8.2

165±2.0

3333.0

163±10.7

155±10.7

164±2.8

177±5.5

178±5.5

169±5.7

10000.0

Toxic

 

167±10.0

88±5.8

161±8.6

98±10.0

Positive control

491±14.6

499±6.6

462±20.5

544±17.2

590±16.6

605±4.9

 

Table 4: TA98

Dose (ug/Plate)

Metabolic activation

Without S9

Without S9

With 10% Rat S9

With 30% Rat S9

With 10% Hamster S9

With 30% Hamster S9

Vehicle Control

19±3.3

22±2.3

24±3.2

24±2.0

20±1.9

25±3.0

33.0

21±1.7

19±0.3

 

 

 

 

100.0

21±1.5

15±0.7

19±0.3

20±0.7

19±1.2

23±2.4

333.0

20±2.3

16±1.5

18±3.3

20±2.3

22±1.7

20±3.5

1000.0

23±2.6

21±4.2

26±2.4

23±1.3

22±1.0

21±1.5

3333.0

10±0.9

13±3.1

27±2.1

16±1.2

24±2.0

24±1.5

10000.0

 

 

21±1.7

9±0.9

20±2.7

10±1.5

Positive control

319±7.7

347±16.8

250±19.5

511±34.1

448±17.4

841±75.9

 

3.

 

Table: Control data for Ames Test Systems used to assess test chemical

 

Historical

Observed C-

Observed C+

P/O

Without S9

TA98

8-60

11±2 (n=6)

163±10 (n=6)

14.8

TA100

60-240

109±6 (n=3)

671±16 (n=3)

6.16

TA1535

4-45

11±1 (n=3)

483±19 (n=3)

43.9

TA1537

2-25

5.3±0.3 (n=3)

192±10 (n=3)

36.2

uvr

5-40

18±2 (n=3)

952±54 (n=3)

51.3

With S9

TA98

8-60

33±0 (n=3)

1087±31.8 (n=3)

32.9

TA100

60-240

138±4 (n=3)

1101±67.1 (n=3)

7.98

TA1535

4-45

14±1 (n=3)

123±3.5 (n=3)

8.79

TA1537

2-25

9.3±3 (n=3)

139±1.73 (n=3)

14.9

uvr

5-40

21±1 (n=3)

187±17.9 (n=3)

8.90

 

-Mean ± SEM revertants per plate are shown.

-Historical: range of values from most recent 25 negative control studies.

-P/O: ratio of observed mean.

-positive control/observed mean negative control ( a ratio > 3 is acceptable to validate the assay conditions)

-S9: rat microsomal 9000 g supernatant

-Activator: uvr, E. coli strain WP2urvA. Salmonella strains have the prefix TA.

-For positive (C+) and negative (C-) control test materials.

Conclusions:
The test chemical did not induce mutation in the Salmonella typhimurium and E.coli strains both in the presence and absence of S9 metabolic activation system and hence is not likely to be mutagenic under the conditions of this study.
Executive summary:

In different studies, the given test chemical has been investigated for the mutagenic nature. The studies are as mentioned below:

 

Genetic toxicity in vitro study was assessed for test chemical. For this purpose AMES test was performed. The test material was exposed to Salmonella typhimurium TA100, TA1535, TA98 and TA97 in the presence and absence of metabolic activation S9. The S9 mix were prepared from 10% and 30% induced male Sprague Dawley rat liver S9 and induced male Syrian hamster liver S9 repectively. The concentration of test material used in the presence and absence of metabolic activation were 0, 33, 100, 333, 1000, 3333, 10000 µg/plate. No mutagenic effects were observed in any strains, in the presence and absence of metabolic activation. Therefore, test chemical was considered to be not mutagenic in Salmonella typhimurium TA100, TA1535, TA98 and TA97 by AMES test. Hence, the substance cannot be classified as gene mutant in vitro.

 

The above study is supported with another gene mutation study performed on Salmonella strains and E.Coli WP2uvrA to determine the mutagenic nature of test chemical. Bacterial strains used were, Salmonella typhimurium Strains TA98, TA100, TA1535, and TA1537 which have histidine synthesis mutations, the rfa wall mutation, and deletion of the uvrB gene, a tryptophan-dependent E. coli strain WP2uvrA was used in the study. This strain has an uvrA DNA repair deficiency, increasing its sensitivity to detect mutagenic compounds. The study was performed with and without S9 metabolic activation. The S9 mixture contained water, sodium phosphate buffer, glucose 6-phosphate, nicotinamide adenine dinucleotide phosphate (NADP), potassium chloride, magnesium chloride and 10% S9 from Rat liver. Positive control substances used in the experiment included 2-aminoanthracene, 2-nitrofluorene, sodium azide, ICR-191, and 4-nitroquinoline-N-oxide. The test concentrations used were 0, 333, 667, 1000, 3330, 5000 μg/plate. Bacteria were prepared on master plates from frozen stocks, inoculated into flasks containing Vogel-Bonner salt solution, supplemented with 2.5% Oxoid nutrient broth No. 2, and harvested for use in late log phase. Strains were confirmed by routine testing for sensitivity to crystal violet (rfa wall mutation) or ampicillin resistance (pKM101 plasmid). Experiments were conducted on two-layer plates. The lower layer was Vogel-Bonner minimal medium E plus 1.5% agar and 0.2% glucose, and the upper layer was 0.7% agar, 0.5% sodium chloride, supplemented with 10 ml of 0.5 mM histidine/ biotin, or 0.5 mM tryptophan, per 100 ml of agar when appropriate (Vogel and Bonner, 1956). All tests were conducted in triplicate. Aliquots of the culture were used to quantify the number of spontaneous revertants for both bacterial species. Background lawn was evaluated for evidence of cytotoxicity. Revertants colonies were counted manually, or using an automated colony counter. Dose-ranging studies demonstrated no cytotoxic effects of test chemical up to 5 mg per plate using Salmonella strains TA100 and WP2uvrA. Numbers of revertant colonies were present in the historical range with and without metabolic activation. Therefore, the test chemical can be considered as not mutagenic to Salmonella typhimurium Strains TA98, TA100, TA1535, and TA1537 and E. coli strain WP2uvrA.

 

Thus, based on the above summarized studies on test chemical, it can be concluded that the given test chemical did not induce mutation in the Salmonella typhimurium and E. coli strains both in the presence and absence of S9 metabolic activation system and hence is not likely to be mutagenic under the conditions of this study.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Gene mutation in vitro:

Ames assay:

In different studies, the given test chemical has been investigated for the mutagenic nature. The studies are as mentioned below:

 

Genetic toxicity in vitro study was assessed for test chemical. For this purpose AMES test was performed. The test material was exposed to Salmonella typhimurium TA100, TA1535, TA98 and TA97 in the presence and absence of metabolic activation S9. The S9 mix were prepared from 10% and 30% induced male Sprague Dawley rat liver S9 and induced male Syrian hamster liver S9 repectively. The concentration of test material used in the presence and absence of metabolic activation were 0, 33, 100, 333, 1000, 3333, 10000 µg/plate. No mutagenic effects were observed in any strains, in the presence and absence of metabolic activation. Therefore, test chemical was considered to be not mutagenic in Salmonella typhimurium TA100, TA1535, TA98 and TA97 by AMES test. Hence, the substance cannot be classified as gene mutant in vitro.

 

The above study is supported with another gene mutation study performed on Salmonella strains and E.Coli WP2uvrA to determine the mutagenic nature of test chemical. Bacterial strains used were, Salmonella typhimurium Strains TA98, TA100, TA1535, and TA1537 which have histidine synthesis mutations, the rfa wall mutation, and deletion of the uvrB gene, a tryptophan-dependent E. coli strain WP2uvrA was used in the study. This strain has an uvrA DNA repair deficiency, increasing its sensitivity to detect mutagenic compounds. The study was performed with and without S9 metabolic activation. The S9 mixture contained water, sodium phosphate buffer, glucose 6-phosphate, nicotinamide adenine dinucleotide phosphate (NADP), potassium chloride, magnesium chloride and 10% S9 from Rat liver. Positive control substances used in the experiment included 2-aminoanthracene, 2-nitrofluorene, sodium azide, ICR-191, and 4-nitroquinoline-N-oxide. The test concentrations used were 0, 333, 667, 1000, 3330, 5000 μg/plate. Bacteria were prepared on master plates from frozen stocks, inoculated into flasks containing Vogel-Bonner salt solution, supplemented with 2.5% Oxoid nutrient broth No. 2, and harvested for use in late log phase. Strains were confirmed by routine testing for sensitivity to crystal violet (rfa wall mutation) or ampicillin resistance (pKM101 plasmid). Experiments were conducted on two-layer plates. The lower layer was Vogel-Bonner minimal medium E plus 1.5% agar and 0.2% glucose, and the upper layer was 0.7% agar, 0.5% sodium chloride, supplemented with 10 ml of 0.5 mM histidine/ biotin, or 0.5 mM tryptophan, per 100 ml of agar when appropriate (Vogel and Bonner, 1956). All tests were conducted in triplicate. Aliquots of the culture were used to quantify the number of spontaneous revertants for both bacterial species. Background lawn was evaluated for evidence of cytotoxicity. Revertants colonies were counted manually, or using an automated colony counter. Dose-ranging studies demonstrated no cytotoxic effects of test chemical up to 5 mg per plate using Salmonella strains TA100 and WP2uvrA. Numbers of revertant colonies were present in the historical range with and without metabolic activation. Therefore, the test chemical can be considered as not mutagenic to Salmonella typhimurium Strains TA98, TA100, TA1535, and TA1537 and E. coli strain WP2uvrA.

 

Thus, based on the above summarized studies on test chemical, it can be concluded that the given test chemical did not induce mutation in the Salmonella typhimurium and E. coli strains both in the presence and absence of S9 metabolic activation system and hence is not likely to be mutagenic under the conditions of this study.

 

In vitro mammalian chromosome aberration study:

In different studies, the given test chemical has been investigated for the chromosomal aberration in mammalian cell lines. The studies are as mentioned below:

 

In-vitro mammalian chromosome aberration test was performed to evaluate the mutagenic nature of the test chemical. Cloned Chinese hamster ovary cells (CHO-W-B1) were cultured in Mc-Coy’s 5a medium with 10% fetal calf serum, L-glutamine, and antibiotics. Test was carried out with and without an in vitro metabolic activation system (S9 mix). The S9 mix consisted of 15µl/ml liver homogenate (from male Sprague-Dawley rats, induced with Aroclor 1254), 2.4 mg/ml NADP, and 4.5 mg/ml isocitric acid in serum-free medium. In without metabolic activation, the test chemical was left in culture until colcemid addition, whereas with activation the test chemical was added along with S9 mix for only 2 hr at the beginning of the test period. The doses used for the study were Without S9: 455-506 µg/ml and With S9: 348-443 µg/ml. The test chemical did not induce chromosome aberrations in the Chinese hamster ovary cell line in the presence and absence of S9 metabolic activation system and hence it is not mutagenic in the chromosome aberration study performed.

 

The above study is supported with another In-vitro mammalian chromosome aberration test performed to evaluate the mutagenic nature of the test chemical using Chinese hamster ovary cells (CHO-W-B1) with and without an in vitro metabolic activation system. Cells were harvested after 19.5 to 26 hours incubation with the test material. The test material precipitated from the culture medium at concentration higher than 653 µl/ml. Slides were stained with Giemsa a coded. One hundred cells were scored from each concentration group having sufficient metaphases. Positive control and control solvent were used. The doses used for the study were without S9: 453-653 µl/ml; with S9: 503-653 µl/ml. The test chemical did not induce chromosome aberrations in the Chinese hamster ovary cell line in the presence and absence of S9 metabolic activation system and hence it is not mutagenic in the chromosome aberration study performed.

 

Thus, based on the above summarized studies on test chemical, it can be concluded the test chemical did not induce chromosome aberrations in the mammalian cell line in the presence and absence of S9 metabolic activation system and hence it is not mutagenic in the chromosome aberration study performed.

 

In vitro gene mutation study in mammalian cells;

In different studies, the given test chemical has been investigated for the mutagenic nature. The studies are as mentioned below:

In vitro mammalian cell gene mutation assay was performed to determine the mutagenic nature of the test chemical. The study was performed using L5178Y TK+/- 3.7.C mouse lymphoma cells in the presence and absence of S9 metabolic activation system at test concentrations of 0.21, 0.26, 0.33, 0.41, 0.51, and 0.64 uL/mL for the first lab with and without S9, and 0.05, 0.1, 0.2, 0.4, and 0.5 uL/mL for without S9 and 0.0078 to 0.5 uL/mL for with S9 for the second lab. The high dose of chemical was determined by solubility and toxicity. L5178Y mouse lymphoma cells were maintained at 37 degree C as suspension cultures in supplemented Fischer's medium; normal cycling time was approximately 10 hours. To reduce the number of spontaneously occurring cells resistant to trifluorothymidine (TFT), subcultures were exposed once to medium containing thymidine, hypoxanthine, methotrexate, and glycine for 1 day; to medium containing thymidine, hypoxanthine, and glycine for 1 day; and to normal medium for 3 to 5 days. For cloning, the horse serum content was increased and Noble agar was added. All treatment levels were replicated, including concurrent positive and solvent (dimethylsulfoxide) controls. Ethyl methanesulfonate (without S9) and methylcholanthrene (with S9) were used as positive controls. Treated cultures contained 6 x 10E6 cells in 10 mL medium. Incubation with test chemical continued for 4 hours, at which time the medium plus chemical was removed, and cells were resuspended in fresh medium and incubated for an additional 2 days to express the mutant phenotype. Cell density was monitored so that log phase growth was maintained. After the 48 hour expression period, cells were plated in medium and soft agar supplemented with TFT for selection of TFT-resistant cells, and cells were plated in nonselective medium and soft agar to determine cloning efficiency. Plates were incubated at 37 degree C in 5% carbon dioxide for 10 to 12 days. All data were evaluated statistically for both trend and peak responses. Both responses had to be significant (P<=0.05) for test chemical to be considered capable of inducing TFT resistance. A single significant response led to a questionable conclusion, and the absence of both a trend and a peak response resulted in a negative call. Hence, test chemical gave a negative response at one laboratory, with and without S9 and an equivocal (weakly positive) response at a second laboratory in the presence of induced S9. At the second laboratory, the response was negative without S9. Appropriate responses were obtained with the positive controls indicating the test was sensitive and valid.

 

The above study is supported with another In vitro mammalian cell gene mutation assay performed to determine the mutagenic nature of the test chemical. The study was performed using L5178Y TK+/- 3.7.C mouse lymphoma cells in the presence and absence of S9 metabolic activation system. The mouse lymphoma cells were grown in Fischer’s medium for leukemic cells of mice supplemented with 10% horse serum and 0.02% pluronic F-68. The test chemical was dissolved in DMSO and the cells at a concentration of 1.2 ×107/mL were exposed for 4 h to a range of concentrations from 0.5-30 µg/ml. Concurrent solvent and positive control chemicals were also included in the study. The cells were washed twice with growth medium, and maintained at 37 ± 1 °C for 48 h in log-phase growth to allow recovery and mutant expression. Cells in the cultures were adjusted to 3 X 105/mL at 24 h intervals. They were then cloned 1 X 106 cells/plate for mutant selection and 200 cells/plate for viable count determinations) in soft agar medium containing Fischer’s medium, 20% horse serum, 2 mM sodium pyruvate, 0.02% pluronic F-68, and 0.23% granulated agar. Resistance to trifluorothymidine (TFT) was determined by adding TFT (final concentration, 3 g/mL) to the cloning medium for mutant selection. Plates were incubated at 37 ± 1 °C in 5% CO2 in air for 10-12 days and then counted with an Artek automated colony counter. Only colonies larger than approx. 0.2 mm in diameter were counted. Mutant frequencies were expressed as mutants per 106 surviving cells. Although there are several different methods for evaluating mouse lymphoma data, results from this study were interpreted using a doubling of the mutant frequency over the concurrent solvent-treated control value as an indication of a positive effect, together with evidence of a dose-related increase. Doubling of the mutant frequency was previously reported as representing a positive effect. Only doses yielding total growth values of 10% were used in the analysis of induced mutant frequency. Doses yielding less than 10% total growth were used in determining dose response. The test chemical was considered to have no mutagenic inducing potency on the mammalian cell line in presence and absence of S9 metabolic activation system.

 

Thus, based on the above summarized studies on test chemical, it can be concluded that the given test chemical did not induce mutation in mammalian cell line in the presence and absence of metabolic activation and hence it is not likely to classify as a gene mutant in vitro.

 

Justification for classification or non-classification

Based on the available data, the given test chemical does not exhibit gene mutation by Ames assay, In vitro mammalian chromosome aberration study and In vitro gene mutation study in mammalian cells. Hence, it is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.