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Toxicological information

Genetic toxicity: in vitro

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Administrative data

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:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: pre GLP, no guideline

Data source

Reference
Reference Type:
publication
Title:
Salmonella mutagenicity test results for 250 chemicals
Author:
Haworth, S. et al
Year:
1983
Bibliographic source:
Environmental mutagenisis supplement, 1, 1983, 3-142

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
Bacterial reverse mutation assay (Ame's test) using strains TA1535 and TA100 and procedures comparable to OECD 471
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Purity 99,8%

Method

Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535
Species / strain / cell type:
S. typhimurium TA 100
Metabolic activation:
with and without
Test concentrations with justification for top dose:
0, 30, 300, 1000 and 3000 ug/plate Studies at Microbiological Associates (formerly EG&G Mason Research Institute)
0, 33.3, 100, 333.3, 1000, 3333 and 6666.6 (TA100 first experiment only) Microbial Genetics Department, SRI International
Vehicle / solvent:
H2O
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
other: 2-aminoanthracene
Remarks:
sodium Azide for -S9 and 2-aminoanthracene for +S9
Details on test system and experimental conditions:
These data were from studies carried out as part of the US EPA National Toxicology Program. Testing was also carried out in TA1537 and TA98, these strains gave consistently negative results with ethylene diamine in both laboratories and have not been included in the robust summary. The tests were carried out following a modification of the pre-incubation test of Yaahagi et. al. Liver S9 was prepared from male Sprague-Dawley rats (RLI) and Syrian Hamsters (HLI) that were induced with Aroclor 1254. Hamster liver was used as it was thought that some substances were detected using hamster liver but not rat liver. As rat liver S9 has become the accepted standard only the result using rat liver S9, are thought relevant.
To select the dose range for the mutagenicity assay, the test chemicals were checked for toxicity to TA100 up to a concentration of 10 mg/plate (10000ug/plate), both in the presence and absence of S9 mix. If toxicity was not apparent in the preliminary toxicity determination, the highest dose tested was 10 mg/plate; otherwise the upper limit of solubility was used. If toxicity was observed, the doses of test chemical were chosen so that the highest dose exhibited some degree of toxicity.
Positive Controls
The positive control chemicals were tested concurrently with each test chemical. 2-Aminoanthracene (2-AA) was tested on all strains in the presence of rat and hamster S-9. 4-Nitro-0-phenylenediamine (NOPD) was tested on TA98 without S-9. Also without S-9, sodium azide (SA) was tested on TAI00 and TA1535. and 9-aminoacridine (9-AAD) was tested on TA1537. The actual concentration for each positive control chemical used for each strain and activation condition was selected by the individual laboratory based on dose-response curves generated at the beginning of the testing program. The doses of the positive controls used by each laboratory are given in Table II.
Evaluation criteria:
Data Evaluation

Although procedures for the statistical analysis of Salmonella plate test data have been developed [Margolin et al, 1981], they were not incorporated into the initial data evaluations. The data were evaluated in an ad hoc manner by each testing laboratory and by NTP personnel. Prior to statistical analysis no formal rules were used; however, a positive response was indicated by a reproducible, dose-related increase, whether it be twofold over background or not. The matrix of test strains and activation systems used allowed the investigators to detect trends or patterns that might not be as evident if only one strain and activation system were examined. The data were subsequently evaluated using an analysis based on the models presented by Margolin et al [I981]; this analysis will be described elsewhere [Risko et al, manuscript in preparation]. As a result of these statistical analyses, 21 number of calls were changed from the original “negative" to “equivocal." The statistical analysis did not result in any “positive” or “equivocal" calls being called “negative”.
Statistics:
No statistical analysis was reported

Results and discussion

Any other information on results incl. tables

Dose

TA 100

TA 1535

 

N.A

Rat liver

Hamster Liver

N.A

Rat liver

Hamster Liver

0

122±3.3

130±13.3

98±2.4

19±1.0

19±6.7

15±0.9

30

126±5.8

135±6.7

133±24

19±3.8

9±0.9

11±3.5

100

117±8.4

116±9.3

109±8.4

22±1.5

10±4.7

13±0.0

300

111±4.8

142±5.6

119±7.5

23±2.0

18±2.4

17±1.0

1000

173±7.8

185±7.1

131±16.1

22±3.2

28±1.5

26±1.0

3000

158±1.5s

149±12.2s

185±11.8s

Toxic

29±4.9s

Toxic

Pos Control

1995±78

1256±5.8

3659±20.

1305±40.0

82±5.5

143±18

 

 

 

 

 

 

 

0

115±5.6

117±11.6

109±4.9

19±1.0

9±1.2

13±1.0

30

118±8.4

103±1.8

112±10.7

19±3.8

15±2.6

8±1.0

100

126±12.5

88±8.4

111±6.1

22±1.

15±5.1

12±0.3

300

130±4.5

133±2.4

114±12.6

23±2.0

13±1.7

10±0.3

1000

156±5.5

153±2.0

146±7.1

22±3.2

28±1.9

19±3.5

3000

155±19.7s

164±4.9s

160±4.2s

Toxic

29±4.8s

20±2s

Pos Control

1864±48.3

1264±86.8

2739±115

1305±40

81±3.3

123±8.8

Studies conducted at Microbiological Associates, Bethesda Md, formerly EG&G Mason Research Institute.

Dose

TA 100

TA 1535

 

N.A

Rat liver

Hamster Liver

N.A

Rat liver

Hamster Liver

0

112±10.7

107±4.6

112±5.0

11±2.3

9±2.1

7±1.5

33.3

125±2.1

 

145±3.4

18±1.8

 

8±0.7

100

110±2.7

127±12.5

151±9.8

17±1.2

12±1.3

5±1.5

333.3

127±9.2

142±4.6

149±2.8

18±3.7

16±0.9

9±2.0

1000

102±4.5

148±4.2

178±8.3

22±4.7

23±2.4

16±1.2

3333

159±10.0

159±5.8

148±7.4s

47±4.5

43±4.7

15±3s

6666.6

 

  219±6.7

 

 

 

 

6666.7

 

 

 

 

      78±3.5

 

Pos Control

281±4.9

633±31.8

1464±84.3

170±3.9

246±19

333±16

 

 

 

 

 

 

 

0

98±10.1

86±3.8

134±4.0

18±0.6

8±1.9

8±1.9

33.3

 

123±7.6

 

 

  6±1.2

 

100

136±8.5

131±15.3

135±7.2

 9±1.8

5±1.7

11±2.8

333.3

138±6.2

166±4.1

151±4.6

12±2.2

  9±1.8

9±1.8

1000

122±9.9

168±10.3

145±14.0

18±1.8

19±2.5

21±1.5

3333.3

168±10.3

166±2.1s

174±17.4

38±1.7

29±5.1s

47±2.5

6666.6

206±3.8

 

221±9.0

34±8.4

 

82±2p

 

 

 

 

 

 

 

Pos Control

217±3.5

571±39.1

1741±54.0

126±5.6

266±8.7

400±25

 

                                                                                                                                                                          

Studies conducted at Microbial Genetics Department, SRI International, Menlo Park, CA                                         

                                                                                                                                                                            

s = Slight clearing of background lawn growth                                                                                                        

p = precipitate                

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
positive weak response mainly in TA1535

Slightly positive results were obtained in an Ames Salmonella Reverse Mutation Assay for Salmonella strains TA 100 and TA1535
Executive summary:

These data were from studies carried out as part of the US EPA National Toxicology Program. Testing was also carried out in TA1537 and TA98, these strains gave consistently negative results with ethylene diamine in both laboratories and have not been included in the robust summary. The tests were carried out following a modification of the pre-incubation test of Yaahagi et. al. Liver S9 was prepared from male Sprague-Dawley rats (RLI) and Syrian Hamsters (HLI) that were induced with Aroclor 1254. Hamster liver was used as it was thought that some substances were detected using hamster liver but not rat liver. As rat liver S9 has become the accepted standard only the result using rat liver S9, are considered to be relevant. 

In general in the studies the negative and positive control culture produced the expected results, supporting the validity of the studies. Ethylene diamine was considered to have produced weak positive responses in T100 and TA1535 by the criteria of dose related increases. When tested at Microbiological Associates TA 100 showed a small dose related increase in revertants both with and without S9 (RLI) however for TA1535 the dose related increase over the solvent control was only seen in the presence of S9. In either case none of the increases approached the two fold increase criteria originally suggested for a positive result in the Ames test. In the Microbial Genetics Department, SRI International for TA100 in the absence of S9, there were increases particularly at the top dose but no clear dose responses. For TA100 in the presence of rat liver S9 there were reproducible dose related increases in revertants, which just attained a twofold increase over the solvent controls. . For TA1535 there were dose related increases in revertants which exceeded a twofold increase both in the presence and absence of rat S9.

Slightly positive results were obtained in an Ames Salmonella Reverse Mutation Assay for Salmonella strains TA 100 and TA1535