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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
Mar to May 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guidelineopen allclose all
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
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid: viscous
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch No.of test material: P2D5000133
- Purity: 100 %
- Expiration date of the lot/batch: 2015-06-12

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Additional strain / cell type 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, 50, 160, 500, 1600, 5000 µg/plate (without and with S9 mix)

Vehicle / solvent:
Solvents used: demineralized water (test substance), deionized water (mitomycin C), DMSO (sodium azide, cumene hydroperoxide, 2-nitrofluorene, 4-nitro-1,2-phenylene diamine, 2-aminoanthracene)
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: sodium azide (TA 100, TA 1535), 2-nitrofluorene (TA 98), 4-nitro-1,2-phenylene diamine (TA 1537), mitomycin C (TA 102, plate incorp.), cumene hyd (TA 102, preincub. trials), 2-aminoanthracene (all strains)
Remarks:
The positive controls sodium azide, 2-nitrofluorene, 4-nitro-1,2-phenylene diamine, mitomycin C and cumene hydroperoxide were only used without S9 mix; the positive control 2-aminoanthracene was only used with S9 mix.
Details on test system and experimental conditions:
METHOD: Standard plate test and preincubation test
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 1537, TA 100 and TA 98 this increase should be about twice that of negative controls. 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

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium, other: TA 98, TA 100, TA 102, TA 1535, TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium, other: TA 98, TA 100, TA 102, TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
other: plate incorporation test

Any other information on results incl. tables

Table 1: Summary of results from the Salmonella mutagenicity assay (plate incorporation test) with KGD 1409 (mean values of revertants per plate)

Dose (µg per plate)

Without metabolic activation

 

 TA 1535

 TA 100

 TA 1537

 TA 98

 TA 102

Vehicle control (demin. water)

13

110

5

16

205

50

10

104

4

16

210

160

10

91

6

12

218

500

9

97

5

14

200

1600

10

92

6

15

201

5000

15

111

4

18

203

Positive control

660

1395

39

1167

782

Dose (µg per plate )

With metabolic activation (liver S9 mix)

TA 1535

TA 100

TA 1537

TA 98

TA 102

Vehicle control (demin. water)

12

154

9

28

296

50

11

139

9

29

329

160

11

164

10

26

321

500

13

134

8

31

301

1600

15

148

8

25

300

5000

20

144

8

26

272

Positive control

132

2980

279

2791

1158

Table 2: Summary of results from the Salmonella mutagenicity assay (independent preincubation test) with KGD 1409 (mean values of revertants per plate)

 

Dose (µg per plate)

Without metabolic activation

 

 TA 1535

 TA 100

 TA 1537

 TA 98

 TA 102

Vehicle control (demin. water)

10

114

6

16

240

50

10

114

7

21

245

160

8

111

7

20

261

500

10

105

6

13

253

1600

8

109

7

16

259

5000

20

118

8

17

265

Positive control

656

842

37

1101

550

Dose (µg per plate)

With metabolic activation (liver S9 mix)

TA 1535

TA 100

TA 1537

TA 98

TA 102

Vehicle control (demin. water)

10

156

10

27

334

50

10

150

8

28

343

160

11

162

9

27

341

500

9

164

9

28

339

1600

15

162

8

26

359

5000

25

178

8

28

337

Positive control

239

2204

382

2251

1013

 

Doses up to and including 5000 µg per plate did not cause any bacteriotoxic effects or precipitations.

Evidence of mutagenic activity of the test item was seen. On Salmonella typhimurium TA1535, a biologically relevant increase was found in the mutant count compared to the corresponding solvent control when using the preincubation method. Both with and without S9 mix, there was a positive response. The lowest reproducible effective dose was 5000 µg per plate for Salmonella typhimurium TA1535. The Salmonella/microsome test thus showed the test item to have a mutagenic effect.

In both experiments the positive controls sodium azide, 4-nitro-1,2-phenylene diamine, 2-nitrofluoren, mitomycin C, cumene hydroperoxide and 2-aminoanthracene increased mutant counts to well over those of the solvent controls, and thus demonstrated the system's sensitivity and the activity of the S9 mix.

Applicant's summary and conclusion

Conclusions:
positive
Executive summary:

The mutagenic potential of KGD 1409 was evaluated in a Salmonella/microsome test (plate incorporation test and preincubation methood) 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 5000 µg per plate did not cause any bacteriotoxic effects or precipitations.

Evidence of mutagenic activity of the test item was seen. On Salmonella typhimurium TA1535, a biologically relevant increase was found in the mutant count compared to the corresponding solvent control when using the preincubation method. Both with and without S9 mix, there was a positive response. The lowest reproducible effective dose was 5000 µg per plate for Salmonella typhimurium TA1535.

Therefore, the test item was considered to be mutagenic without and with S9 mix in the preincubation modification of the Salmonella/microsome test.