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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
Study period:
Experimental starting date: 5th February 2014 Experimental completion date: 1st April 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

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
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
Qualifier:
according to
Guideline:
other: USA, EPA OCSPP harmonised guidelines
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
Identification: FAT 20341/A TE
Batch: BOP 02-12 (Navy PLK 241, BS)
Purity: 80.1% (w/w)
Physical State/Appearance: Black powder, solid at 20 °C
Expiry Date: 21 November 2017
Storage Conditions: Room temperature in the dark

Method

Target gene:
Histidine operon for Salmonella.
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Details on mammalian cell type (if applicable):
Not applicable.
Additional strain / cell type characteristics:
other: PLEASE REFER TO SECTION "ANY OTHER INFORMATION ON MATERIALS AND METHOD'
Metabolic activation:
with and without
Metabolic activation system:
phenobarbitone/beta­naphthoflavone induced rat liver, S9
Test concentrations with justification for top dose:
A pre-test for toxicity was conducted. The maximum concentration was 5000 µg/plate. The concentrations tested were 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate.

Test for Mutagenicity Range-finding Test (Experiemnt 1): 50, 150, 500, 1500 and 5000 µg/plate

Test for Mutagencity Main Test (Experiment 2): 500, 1000, 1500, 3000, 4000 and 5000 µg/plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Sterile distilled water.
- Justification for choice of solvent/vehicle: The test material was fully soluble in sterile distilled water at 50 mg/ml in solubility checks performed in-house
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rate
Negative solvent / vehicle controls:
yes
Remarks:
Sterile distilled water
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
Without S9 mix (0.5 µg/plate for TA102)
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rate
Negative solvent / vehicle controls:
yes
Remarks:
Sterile distilled water
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
Without S9 mix (3 µg/plate for TA 100, 5 µg/plate for TA 1535)
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rate
Negative solvent / vehicle controls:
yes
Remarks:
Sterile distilled water
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
Without S9 mix (80 µg/plate for TA 1537)
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rate
Negative solvent / vehicle controls:
yes
Remarks:
Sterile distilled water
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
Without S9 mix (0.2 µg/plate for TA 98)
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rate
Negative solvent / vehicle controls:
yes
Remarks:
Sterile distilled water
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
With S9 mix

Migrated to IUCLID6: Benzo(a)pyrene: 5 µg/plate
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rate
Negative solvent / vehicle controls:
yes
Remarks:
Sterile distilled water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene (2AA)
Remarks:
With S9 mix (1 µg/plate for TA 100, 2 µg/plate for TA 1535 and TA 1537) )
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rate
Negative solvent / vehicle controls:
yes
Remarks:
Sterile distilled water.
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 1,8-Dihydroxyanthraqiuinone (DAN)
Remarks:
With S9 mix (10 µg/plate for TA 102)
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rate
Negative solvent / vehicle controls:
yes
Remarks:
Sterile distilled water.
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
With S9 mix (5 µg/plate for TA 98)
Untreated negative controls:
yes
Remarks:
Spontaneous mutuation rates
Negative solvent / vehicle controls:
yes
Remarks:
Sterile distilled water
True negative controls:
no
Positive controls:
yes
Positive control substance:
congo red
Remarks:
Used in conjunction with the uninduced hamster S9 (50 µg/plate for TA 98 and TA 100)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

The Prival-Mitchell Modification to the Ames Test
The Prival-Mitchell modification to the standard Ames Test is necessary for the testing of azo dyes which can contain mutagenic aromatic amines which are not readily detected using the standard method (Prival and Mitchell (1982)). The modification differs in five key areas from the standard plate incorporation Ames Test:
• Uninduced hamster liver S9 rather than induced rat liver S9.
• 0.15 mL of S9 rather than the maximum of 0.05 mL of S9 in the standard Ames Test.
• The use of flavin mononucleotide (FMN), nicotinamide adenine dinucleotide (NADH), four times the standard amount of glucose-6-phosphate, and the inclusion of exogenous glucose 6 phosphate dehydrogenase in the co-factor mix.
• A 30 minute pre-incubation prior to the addition of the molten top agar.
• Vogel-Bonner plates containing 0.5% glucose instead of the standard 2% glucose.

Only the test item concentrations, vehicle and the positive control, Congo Red, were dosed using the Prival Mitchell modification

NUMBER OF REPLICATIONS: Three

All of the plates were incubated at 37 °C± 3 °C for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity). Manual counts were performed at and above 500 µg/plate because of test item induced colouration.

Evaluation criteria:
There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:

1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out of historical range response (Cariello and Piegorsch, 1996)).

A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.

Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgment about test item activity. Results of this type will be reported as equivocal.
Statistics:
Standard deviation

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Remarks:
Tested up to maximum recommended dose of 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Mutation Test
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid supplemented top agar and all of the S9-mixes used in both experiments were shown to be sterile. These data are not given in the report.

Results for the negative controls (spontaneous mutation rates) are presented in Table 1 and were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.

The test item caused no visible reduction in the growth of the bacterial background lawn at any dose level and was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. An intense test item coloration was noted at and above 500 µg/plate, this observation did not prevent the scoring of revertant colonies. No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

There were no toxicologically relevant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation in two separate experiments.

In Experiment 1 (range-finding test), there were small but statistically significant increases in revertant colony frequency in tester strains TA100 and TA98, in the absence of S9 only, at and above 1500 µg/plate. Although these increases were between 1.3 and 1.6 fold above the concurrent solvent controls, they were only at or below the maximum limit of the historical range (depending on the strain). There was also only moderate evidence of a dose response relationship. Therefore, the dose range used in Experiment 2 (main test) was amended in an attempt to replicate and magnify the response observed in Experiment 1. No increases in colony frequency, at any dose of the test item, either with or without metabolic activation were observed. Therefore, the response in the range-finding test was considered to have no biological or toxicological relevance because of the lack of reproducibility, the response was either below or at the maximum limit of the historical range and lacked a convincing dose-response relationship.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the induced rat liver and the uninduced hamster liver S9-mixes were validated.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1     Spontaneous Mutation Rates (Concurrent Negative Controls)

Range-finding Test (Experiment 1)

 

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

TA102

TA98

TA1537

91

 

13

 

328

 

18

 

19

 

102

(89)

13

(12)

299

(285)

13

(19)

8

(12)

75

 

9

 

227

 

25

 

8

 

 

Main Test (Experiment 2)

 

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

TA102

TA98

TA1537

99

 

9

 

279

 

23

 

9

 

82

(96)

13

(13)

311

(284)

19

(21)

7

(8)

108

 

18

 

261

 

21

 

9

 

 

Table2     Test Results: Range-finding Test (Experiment 1) – Without Metabolic Activation

Test Period

From: 28 February 2014

To: 03 March 2014

S9-Mix

(-)

Dose Level

Per Plate

Number of revertants (mean) +/- SD

Base-pair substitution strains

Frameshift strains

TA100

TA1535

TA102

TA98

TA1537

Solvent Control

(Water)

106

103

122

(110)

10.2#

22

27

15

(21)

6.0

323

295

252

(290)

35.8

18

25

16

(20)

4.7

13

10

11

(11)

1.5

50 µg

91

103

104

(99)

7.2

15

16

10

(14)

3.2

283

283

200

(255)

47.9

14

18

18

(17)

2.3

12

7

10

(10)

2.5

150 µg

81

93

99

(91)

9.2

9

16

12

(12)

3.5

227

224

205

(219)

11.9

19

27

26

(24)

4.4

12

10

10

(11)

1.2

500 µg

97 I

88 I

108 I

(98)

10.0

21 I

8 I

24 I

(18)

8.5

223 I

239 I

214 I

(225)

12.7

27 I

14 I

30 I

(24)

8.5

10 I

11 I

8 I

(10)

1.5

1500 µg

144 I

149 I

136 I

(143)

6.6

18 I

14 I

11 I

(14)

3.5

192 I

248 I

228 I

(223)

28.4

34 I

33 I

30 I

(32)

2.1

12 I

8 I

7 I

(9)

2.6

5000 µg

172 I

163 I

176 I

(170)

6.7

14 I

14 I

13 I

(14)

0.6

256 I

236 I

228 I

(240)

14.4

35 I

29 I

33 I

(32)

3.1

9 I

8 I

9 I

(9)

0.6

Positive controls

S9-Mix

(-)

Name

Dose Level

No. of Revertants

ENNG

ENNG

MMC

4NQO

9AA

3 µg

5 µg

0.5 µg

0.2 µg

80 µg

903

1074

833

(937)

124.0

891

967

1013

(957)

61.6

2077

1882

1695

(1885)

191.0

243

251

252

(249)

4.9

621

540

705

(622)

82.5

  Table 3     Test Results: Range-finding Test (Experiment 1) – With Metabolic Activation

Test Period

From: 28 February 2014

To: 03 March 2014

S9-Mix

(+)

Dose Level

Per Plate

Number of revertants (mean) +/- SD

Base-pair substitution strains

Frameshift strains

TA100

TA1535

TA102

TA98

TA1537

Solvent Control

(Water)

103

103

108

(105)

2.9#

17

13

18

(16)

2.6

321

304

327

(317)

11.9

36

32

27

(32)

4.5

23

16

20

(20)

3.5

50 µg

96

95

96

(96)

0.6

13

8

11

(11)

2.5

309

324

301

(311)

11.7

29

31

34

(31)

2.5

21

15

15

(17)

3.5

150 µg

104

97

110

(104)

6.5

12

11

13

(12)

1.0

319

229

313

(287)

50.3

33

30

23

(29)

5.1

16

16

19

(17)

1.7

500 µg

95 I

111 I

114 I

(107)

10.2

13 I

20 I

15 I

(16)

3.6

371 I

353 I

253 I

(326)

63.6

25 I

26 I

26 I

(26)

0.6

16 I

16 I

14 I

(15)

1.2

1500 µg

111 I

100 I

102 I

(104)

5.9

18 I

12 I

12 I

(14)

3.5

274 I

360 I

284 I

(306)

47.0

27 I

19 I

33 I

(26)

7.0

9 I

13 I

12 I

(11)

2.1

5000 µg

116 I

101 I

119 I

(112)

9.6

16 I

17 I

20 I

(18)

2.1

264 I

228 I

288 I

(260)

30.2

26 I

21 I

28 I

(25)

3.6

12 I

9 I

13 I

(11)

2.1

Positive controls

Rat S9-Mix

(+)

Name

2AA

2AA

DAN

BP

2AA

Dose Level

1 µg

2 µg

10 µg

5 µg

2 µg

No. of Revertants

522

528

521

(524)

3.8

257

266

245

(256)

10.5

1455

1302

1388

(1382)

76.7

108

120

136

(121)

14.0

294

267

200

(254)

48.4

Positive controls

Hamster S9-Mix

(+)

Name

CR

CR

Dose Level

50 µg

50 µg

No. of Revertants

231

587

665

(494)

231.4

146

209

234

(196)

45.3

 

 

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

The test material was considered to be non-mutagenic under the conditions of this test.
Executive summary:

Introduction

The test method was designed to be compatible with the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF, the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Regulation (EC) number 440/2008 of 30 May 2008 and the USA, EPA OCSPP harmonized guideline - Bacterial Reverse Mutation Test.

 

 

Methods

This study was designed to be compatible with the procedures indicated by the following internationally accepted guidelines and recommendations:

 

·     OECD Guidelines for Testing of Chemicals No. 471 (1997) "Bacterial Reverse Mutation Test".

·     Method B13/14 of Commission Regulation (EC) number 440/2008 of 30 May 2008.

·     USA, EPA OCSPP harmonized guidelines.

·     Japanese Ministry of Economy, Trade and Industry, Japanese Ministry of Health, Labour and Welfare and Japanese Ministry of Agriculture, Forestry and Fisheries. 

….

Salmonella typhimuriumstrains TA1535, TA1537, TA102, TA98 and TA100 were treated with the test item using the Ames plate pre-incubation method (Prival and Mitchell modification) at up to six dose levels, in triplicate, both with and without the addition of a hamster liver homogenate metabolizing system (30% liver S9 in modified co‑factors). The dose range for the range-finding test (Experiment 1) was determined in a Preliminary Toxicity Test and was 50 to 5000 µg/plate. The experiment was repeated on a separate day using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was based on the results of the range-finding test and was 500, 1000, 1500, 3000, 4000 and 5000 µg/plate.

 

 

Results…….

The vehicle (sterile distilled water) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the induced rat liver S9-mix and the uninduced hamster liver S9-mix were validated.

 

The test item caused no visible reduction in the growth of the bacterial background lawn at any dose level and was, therefore, tested up to the maximum recommended dose level of 5000 mg/plate. An intense test item coloration was noted at and above 500 mg/plate, this observation did not prevent the scoring of revertant colonies. No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

 

There were no toxicologically relevant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation in two separate experiments. 

 

In Experiment 1 (range-finding test), there were small but statistically significant increases in revertant colony frequency in tester strains TA100 and TA98, in the absence of S9 only, at and above 1500 µg/plate. Although these increases were between 1.3 and 1.6 fold above the concurrent solvent controls, they were only at or below the maximum limit of the historical range (depending on the strain). There was also only moderate evidence of a dose response relationship. Therefore, the dose range used in Experiment 2 (main test) was amended in an attempt to replicate and magnify the response observed in Experiment 1. No increases in colony frequency, at any dose of the test item, either with or without metabolic activation were observed. Therefore, the response in the range-finding test was considered to have no biological or toxicological relevance because of the lack of reproducibility, the response was either below or at the maximum limit of the historical range and lacked a convincing dose-response relationship. 

 

 

Conclusion

FAT 20341/A TEwas considered to be non-mutagenic under the conditions of this test.