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Toxicological information

Genetic toxicity: in vitro

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Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD-GLP This study was selected as the key study because the information provided for the hazard endpoint is sufficient for the purpose of classification and labelling and/or risk assessment.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report Date:
2000

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
S. typhimurium tester strain TA 97a using ICR 191 as a positive control was used as a substitute for strain(s) TA 97 and/or TA 1537; E.coli WP uvrA (pKM 101) was the only E.coli tester used. (validity of the study not affected-as reported)
Qualifier:
according to
Guideline:
other: US EPA OPPTS Guidelines (Subpart H, 40CFR Part 799.9510, 1989)
Qualifier:
according to
Guideline:
other: Commission Directive 92/69/EEC, EEC Method B. 12
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Purity: 99.8%

Method

Species / strain
Species / strain / cell type:
other: S. typhimurium TA 97a, TA 98, TA 100 and TA 1535 and Escherichia coli strain WP2uvrA(pKM101)
Metabolic activation:
with and without
Metabolic activation system:
Induced rat liver S9
Test concentrations with justification for top dose:
First trial:
20%, 30%, 40%, 50%, 75%
[actual test substance conc: 16%, 22.5%, 31.4%, 35.4% , 40.1% in the absence of S9 and 18.9%, 25.7%, 32.6%, 40.2% , 60.7% in the presence of S9]
Second trial: 20%, 30%, 40%, 50%, 75% [actual test substance conc: 14.7%, 22.8%, 30.1%,, 42.9%, 61.3% in the absence of S9 and 15.5%, 22.9%, 32.8%, 48.4%, 68.3% in the presence of S9)
Controls
Untreated negative controls:
yes
Remarks:
filtered house-line air
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-nitrofluorene;N-Ethhyl-N-nitro-N-nitrosoguanidine;sodium azide; ICR 191 Acridine mutagen; 9,10-dimethyl-1,2-benzanthracene; 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: top agar plate incorporation method for gases (2 mL, 0.6% agar w/v, 0.6% NaCl w/v)

DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3

NUMBER OF CELLS EVALUATED: 10E8 (0.1 mL culture)

The study consisted of 2 independent trials with and without a metabolic activation system. A third trial, utilizing S. typhimurium TA98 with S9 was used to confirm the results. Three replicates were plated for each tester strain, test concentration and condition. Treatments with activation were conducted by adding 0.1 mL of positive control (positive control only), 0.5 mL of S9 mix and 0.1 mL (containing 10E8 bacteria) of an overnight culture to 2 mL of top agar supplemented with 0.05 mM L-histidine and 0.05 mM D-biotin for S. typhimurium or 0.05 mM L-tryptophan for the E. coli strain. These components were briefly mixed and poured onto a minimal glucose agar plate. Treatments in the absence of the metabolic activation system were identical to those in the presence of an exogenous metabolic activation system with the exception that 0.5 mL of sterile buffer was used as a replacement for the 0.5 mL volume of the exogenous metabolic activation system.

Plates were exposed to dilutions of the test gas after being placed on stainless steel racks specially designed to fit in 6-L glass chambers. Chambers were equipped with Teflon stopcocks and Viton o-ring gaskets. As gases, the test substance and filtered air flows were regulated using individual rotameters, and mixed prior to entry into chambers. A flow rate of approximately 6 L/minute for 5 minutes was used to create 5 volume changes to occur within the chambers to ensure homogeneous concentrations. Chambers were closed and 2-4 samplings of each chamber were taken and analyzed by GC to determine initial concentration of the test substance. Chambers were placed into an incubator at approximately 37°C for approximately 48 hours. Chambers were again sampled and analyzed to determine the ending test substance concentrations. The chambers were flushed with at least 5 chamber volumes of filtered air. The plates were then removed and refrigerated until evaluation.

DETERMINATION OF CYTOTOXICITY
Bacterial background lawns were evaluated for evidence of test substance toxicity and precipitation.

Evaluation criteria:
Any individual assay must have included a negative and positive control and at least three concentration levels per test substance for each tester strain and condition. A data point was excluded from analysis when acceptability criteria were not met. Such criteria are clearly defined as: tester strain integrity; tester strain titer; positive control values; revertant toxicity; rejection of plates, concentration levels, or assay.

Positive and negative classification are clearly stated in the classification criteria and are based on number of revertants in strains compared to the concurrent negative control. The test substance was classified as positive if the mean number of revertants in any strain (except TA1535) at any test substance concentration was at least two times greater than the mean number of revertants of the concurrent negative control, and there was a concentration-related increase in the mean number of revertants per plate in that same strain. For strain TA1535, there must have been a mean number of revertants that was at least three times greater than the mean of its concurrent negative control and a concentration-related increase in the mean number of revertants per plate. A test substance was classified as negative if all positive classification criteria for all strain were not met. Results not meeting criteria for either positive or negative classification were evaluated using scientific judgment and experience and may have been reported as equivocal.
Statistics:
Data for each tester strain were evaluated independently: the mean number of revertants and the standard deviation at each concentration in the presence of and absence of metabolic activation were calculated.

Results and discussion

Test results
Species / strain:
other: S. typhimurium TA 97a, TA 98, TA 100 and TA 1535 and Escherichia coli strain WP2uvrA(pKM101)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: no test substance related precipitate was observed at any concentration with any strain in any trial.

COMPARISON WITH HISTORICAL CONTROL DATA: Historical control data are reported for tester strains used in the report study. Data reported are based on studies conducted in the period 1996-1998. All control solvents or diluents, metabolic activation based on Aroclor-induced rat liver S9 and all forms of study modification are included. The mean number of revertants observed in the negative control for each strain was within the prescribed acceptable historical control range.

Results:
In the first trial, there was an apparent leakage in one chamber at the high dose without S9. The other chamber concentrations decreased approximately 50% from the mean at 0-hr and 48-hr. Test substance-related toxicity, as evident by a concentration dependent reduction in mean revertant colonies per plate, was observed in all tester strains except S. typhimurium strains TA100 and TA1535 without S9. No evidence of mutagenicity was observed.

In a second trial, test substance related toxicity, as evidenced by a concentration-related reduction in the mean number of revertants per plate, was observed with all tester strains in the presence and absence of the metabolic activation system. The chamber concentrations were again decreased approximately 36% after 48 hours. Due to an equivocal response in S. typhimurium TA98 in the presence of S9 in trial 2, tester strain TA98 with a metabolic activation system was repeated. At one concentration level of strain TA98, a doubling of the mean revertant plate count was observed compared to the mean of the concurrent negative control. There was no concentration-related increase in the tester strain, and therefore the data was judged inconclusive.

A third trial was conducted with only TA98 with S9 at target concentrations of 45%, 55%, and 65%, again with a mean decrease in chamber concentration of 22% with no apparent chamber leakage. As this repeat of trial 2 was negative, again with evidence of toxicity, the conclusion that trial 2 also exhibited no evidence of mutagenicity was affirmed.

Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

 

Trial #1

(Without Activation)

 

                      (Histidine) + Revertants Per Plate

Strain TA97a

Strain TA98

Strain TA100

Strain TA1535

Strain E. coli pKM101

DME (target concentration)

 

 

 

 

 

0 %

137

18

123

12

187

20 %

122

16

127

16

136

30%

108

14

121

15

146

40%

88

10

101

13

68

50%

93

4

90

16

79

75%

71

8

108

14

98

 

 

 

 

 

 

ICR 191 (2 ug/plate)

2208

 

 

 

 

2NF(25 ug/plate)

 

1215

 

 

 

NAAZ(2 ug/plate)

 

 

658

588

 

ENNG(2 ug/plate)

 

 

 

 

1493

 

Trial #1

(With Activation)

 

                       (Histidine) + Revertants Per Plate

Strain TA97a

Strain TA98

Strain TA100

Strain TA1535

Strain E. coli pKM101

DME (target concentration)

 

 

 

 

 

0 %

172

20

167

18

167

20 %

178

18

152

17

179

30%

149

15

139

13

146

40%

173

13

138

15

98

50%

140

12

129

9

83

75%

63

4

115

9

19

 

 

 

 

 

 

DMBA(20 ug/plate)

1076

 

 

 

 

2AA(2 ug/plate)

 

1206

1839

287

1416

 

 

 

 

 

 

 

 

Trial #2

(Without Activation)

 

                          (Histidine) + Revertants Per Plate

Strain TA97a

Strain TA98

Strain TA100

Strain TA1535

Strain E. coli pKM101

DME (target concentration)

 

 

 

 

 

0 %

145

30

133

23

202

20 %

130

26

136

24

171

30%

140

19

133

22

164

40%

92

38

112

24

109

50%

33

31

100

26

33

75%

2

0

31

12

4

 

 

 

 

 

 

ICR 191(2 ug/plate)

2695

 

 

 

 

2NF(25 ug/plate)

 

1441

 

 

 

NAAZ(2 ug/plate)

 

 

1008

872

 

ENNG(2 ug/plate)

 

 

 

 

1714

 

Trial #2

(With Activation)

 

                        (Histidine) + Revertants Per Plate

Strain TA97a

Strain TA98

Strain TA100

Strain TA1535

Strain E. coli pKM101

DME (target concentration)

 

 

 

 

 

0 %

178

23

152

21

210

20 %

194

23

151

17

207

30%

148

25

144

33

159

40%

161

25

133

16

102

50%

32

48

110

37

26

75%

0

13

45

12

0

 

 

 

 

 

 

DMBA(20 ug/plate)

1503

 

 

 

 

2AA(2 ug/plate)

 

1192

1778

265

1798

 

 

 

 

 

 

 

 

Trial #3

(With Activation)

 

 

Strain TA98 

 

DME

 

 

0 %

25

 

45%

15

 

55%

8

 

65%

4

 

 

 

 

2AA

 

 

       25 ug/plate

1043

 

 

 

 

 

 

 

 

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

Under the conditions of the study, no evidence of mutagenic activity was detected in either trial with the substance. Based on the findings, the test substance was concluded to be negative for the induction of mutagenicity in the bacterial reverse mutation test in Salmonella typhimurium and Escherichia coli, with and without metabolic activation.

The study and the conclusions which are drawn from it fulfil the quality criteria (validity, reliability, repeatability).
Executive summary:

The test substance was evaluated in the bacterial reverse mutation test using Salmonella typhimurium strains TA97a, TA98, TA100, TA1535, and Escherichia coli strain WP2uvrA (pKM101) in the presence and absence of an exogenous metabolic activation system at target concentrations of 0, 20, 30, 40, 50, and 75%.

Under the conditions of the study, no evidence of mutagenic activity was detected in either trial with the substance. Based on the findings, the test substance was concluded to be negative for the induction of mutagenicity in the bacterial reverse mutation test in Salmonella typhimurium and Escherichia coli, with and without metabolic activation.