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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information
Salmonella/microsome test (Ames test): positive with strains TA 98, TA 102 (+ S9 mix) and TA 100 (+/- S9 mix) and negative with strains TA 1535 and TA 1537 (+/- S9 mix)
Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
other information
Study period:
March 2009
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: guideline study with restriction (non-GLP)
Reference:
Composition 0
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:
no
Type of assay:
bacterial reverse mutation assay
Test material information:
Composition 1
Target gene:
Histidine gene locus
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Additional strain characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced male rat liver S9 mix
Test concentrations with justification for top dose:
0, 16, 50, 158, 500, 1581, 5000 µg/plate (first test, +/-S9 mix, all strains)
0, 500, 1000, 2000, 3000, 4000, 5000 and 6000 µg/plate (repeat test, +/-S9 mix, all strains)







Vehicle:
DMSO
Negative controls:
yes
Solvent controls:
no
Remarks:
No solvent control was used since sufficient evidence was available in the literature and from testing laboratory experience, indicating that the solvents used had no influence on the spontaneous mutant counts of the used strains.
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: sodium azide (only TA 1535), nitrofurantoin (only TA 100), 4-nitro-1,2-phenylene diamine (TA 1537 and TA 98), mitomycin C (only TA 102), 2-aminoanthracene (all strains).
Remarks:
The positive controls sodium azide, nitrofurantoin, 4-nitro-1,2-phenylene diamine and mitomycin C were only used without S9 mix; the positive control 2-aminoanthracene was only used with S9 mix.
Details on test system and conditions:
METHOD: each concentration including the controls was tested in triplicate.
Evaluation criteria:
A reproducible and dose-related increase in mutant counts of at least one strain is considered to be a positive result. For TA 1535, TA 100 and TA 98 this increase should be about twice that of negative controls, whereas for TA 1537 at least a threefold increase should be reached. For TA 102 an increase of about 100 mutants should be reached. Otherwise, the result is evaluated as negative. However, these criteria may be overruled by good scientific judgment. In case of questionable results, investigations should continue, possibly with modifications, until a final evaluation is possible.
Statistics:
not specified
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity:
no, but tested up to limit concentrations
Vehicle controls valid:
not examined
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: strain/cell type: TA 98, TA 102
Remarks:
Migrated from field 'Test system'.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity:
no, but tested up to limit concentrations
Vehicle controls valid:
not examined
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: strain/cell type: TA 98, TA 102
Remarks:
Migrated from field 'Test system'.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity:
no, but tested up to limit concentrations
Vehicle controls valid:
not examined
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: strain/cell type: TA 100
Remarks:
Migrated from field 'Test system'.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no, but tested up to limit concentrations
Vehicle controls valid:
not examined
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: strain/cell type: TA 1535, TA 1537
Remarks:
Migrated from field 'Test system'.

Table 1: Summary of results from the Salmonella mutagenicity assay (first test) with 4 -Hydrazo-6 -morph-pyrimidin (mean values of revertants per plate)

Dose (µg per plate)

Without metabolic activation

 

TA 1535

 TA 100

 TA 1537

 TA 98

 TA 102

0

9

110

9

24

245

16

5

98

6

28

234

50

9

107

6

25

228

158

8

116

5

25

235

500

7

114

6

29

186

1581

7

100

9

35

273

5000

 217

10

41 

318

 Positive control

1016

435

44

97

1397 

Dose ( µg per plate )

With metabolic activation (liver S9 mix)

 

TA 1535

 TA 100

 TA 1537

 TA 98

TA 102

0

7

191

10

38

350

16

9

177

7

31

309

50

6

148 

6

34

277

158

5

121

10

36

336

500

12

150

7

48

378

1581

11

194

13

56

403

5000

14 311 17 84 518

 Positive control

84

2472 

139 

2253 

1025 

 

Table 2: Summary of results from the Salmonella mutagenicity assay (repeat test with additional concentrations) with 4 -Hydrazo-6 -morph-pyrimidin (mean values of revertants per plate)

Dose (µg per plate)

With metabolic activation  (liver S9 mix)

 

TA 1535

 TA 100

 TA 1537

 TA 98

 TA 102

0

10

146

9

38

333

500

6

148

10

40

340

1000

8

133

9

43

334

2000

7

166

12

59

327

3000

11

170

8

52

354

4000

16

210

13

56

457

5000

14 

 227

17

49 

525

 6000

 18

 291

 20

 68

 534

 Positive control

71

2337

122

2305

1296 

Dose ( µg per plate )

Without metabolic activation

 

TA 1535

 TA 100

 TA 1537

 TA 98

TA 102

0

8

119

7

28

244

500

9

127

7

39

220

1000

10

123 

6

24

226

2000

6

170

7

28

248

3000

9

157

7

38

191

4000

8

181

5

39

260

5000

 7

208

9

44

285

6000

1150

245

8

46

284

 Positive control

84

366 

58 

89

1128

 

There was no indication of a bacteriotoxic effect of 4-Hydrazo- 6-morph-pyrimidin at doses of up to and including 6000 µg per plate. The total bacteria counts consistently produced results comparable to the negative controls, or differed only insignificantly. No inhibition of growth was noted as well.

Three of the five strains concerned revealed a relevant increase in mutant counts. Strains TA 100, TA 98 and TA 102 were affected. The findings for Salmonella typhimurium TA 100, TA 98 and TA 102 were confirmed by further repeat tests. The lowest dose at which this finding was observed was 4000 µg per plate for Salmonella typhimurium TA 102 and 6000 µg per plate for Salmonella typhimurium TA l 00 and T A 98. Positive findings for TA 98 and TA l02 were obtained only with S9 mix. Positive findings for TA l00 were obtained with and without S9 mix, the effects being comparable.

The positive controls sodium azide, nitrofurantoin, 4-nitro-1,2-phenylene diamine, mitomycin C and 2-aminoanthracene had a marked mutagenic effect, as was seen by a biologically relevant increase in mutant colonies compared to the corresponding negative controls.

Executive summary:

The mutagenic potential of the test material was evaluated in a Salmonella/microsome test with the S. typhimurium strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 in the presence and absence of S9 mix according to OECD TG 471. Doses up to and including 6000 µg per plate did not cause any bacteriotoxic effects. Total bacteria counts remained unchanged and no inhibition of growth was observed. Evidence of mutagenic activity of the test material was seen. On Salmonella typhimurium TA 102 and TA 98, a biologically relevant increase was found in the mutant count compared to the corresponding negative control. Positive response was found only with S9 mix. For TA 100 there was a positive response without and with S9 mix, and the effect was comparable. The lowest effective dose was 4000 µg per plate for Salmonella typhimurium TA 102 and 6000 µg per plate for TA 100 and TA 98. The Salmonella/microsome test thus showed the test material to have a mutagenic effect.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Additional information

Additional information from genetic toxicity in vitro:

The mutagenic potential of the test material was evaluated in a Salmonella/microsome test with the S. typhimurium strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 in the presence and absence of S9 mix according to OECD TG 471. Doses up to and including 6000 µg per plate did not cause any bacteriotoxic effects. Total bacteria counts remained unchanged and no inhibition of growth was observed. Evidence of mutagenic activity of the test material was seen. On Salmonella typhimurium TA 102 and TA 98, a biologically relevant increase was found in the mutant count compared to the corresponding negative control. Positive response was found only with S9 mix. For TA 100 there was a positive response without and with S9 mix, and the effect was comparable. The lowest effective dose was 4000 µg per plate for Salmonella typhimurium TA 102 and 6000 µg per plate for TA 100 and TA 98. The Salmonella/microsome test thus showed the test material to have a mutagenic effect.


Justification for selection of genetic toxicity endpoint
The second study is restricted to three Salmonella typhimurium L T2 mutants (strains TA 98; TA 100 and TA 102) which showed a positive response in a previous study (= rel 2, Ames, Herbold 2011)

Justification for classification or non-classification

Based on the available information (restricted to in vitro studies detecting gene mutations in bacteria) a classification according to Directive 67/548/EEC or Regulation (EC) No. 1272/2008 (CLP) is not required although the test result is positive.