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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:
key study
Study period:
08 Jan - 05 Mar 2009
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
All experiments for the test substance and for the positive controls were performed only in duplicates. All experiments for the solvent control were performed in triplicates, but no standard deviations were given. It should be noted that the study report is a translation from Japanese to English.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
Experiments for the test substance and for the positive controls were performed only in duplicates, for the solvent control in triplicates, but no standard deviations were given. Only 2-Aminoanthracene was used as positive control with S9-mix.
Principles of method if other than guideline:
This study was conducted in accordance with "Standards for Toxicity Investiagations" (Ministry of Labor, Notification No. 77, September 1, 1988 and Notification No. 67, June 2, 1997) "Procedures of Mutagenicity Test Using Microorganisms and Evaluation of Test Results" (Ministry of Labor, Offical Notification, February 8, 1999), "III Mutagenicity test" of "Reverse-Mutation Assay in Bacteria" prescribed in "Testing Methods Relating to the New Chemical Substances" (Notification No. 1121002 of the Pharmaceutical and Food Safety Bureau, MHLW, No.2 (2003.11.13) of the Manufacturing Industries Bureau, METI & No. 031121002 of the Environmental Health Department, MOE (November 21, 2003)).
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Target gene:
his operon for the S. typhimurium strains
trp operon for the E. coli strain
Species / strainopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital and 5,6-benzoflavon
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital and 5,6-benzoflavon
Test concentrations with justification for top dose:
Main test:
313, 625, 1250, 2500 and 5000 µg/plate with and without metabolic activation

Vehicle:
- Vehicle(s)/solvent(s) used: DMSO (Lot No. WF032, 100.0% in purity, spectrophotometric grade, DOJINDO Laboratories) was used.
- Justification for choice of solvent/vehicle:
The test substance is insoluble in distilled water (information provided by the sponsor), but soluble in DMSO (solubility ≥ 500 mg/mL).
Controlsopen allclose all
Negative controls:
no
Solvent controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene (0.5 µg/plate in water, TA98; 1 µg/plate in water, TA100: 2 µg/plate in water, TA1535, TA1537; 20 µg/plate in water, WP2uvrA)
Remarks:
with S9 mix
Negative controls:
yes
Solvent controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (0.01 µg/plate in DMSO, TA100, WP2uvrA; 0.1 µg/plate in DMSO, TA98); sodium azide (0.5 µg/plate in water,TA1535); 2-methoxy-6-chloro-9-[3-(2-chloroethyl)aminopropylamino]acridine*2HCl (0.5 µg/plate in DMSO,TA1537)
Remarks:
without S9 mix
Details on test system and conditions:
METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: All experiments with the negative control were performed in triplicates. All experiments with the positive controls and with the test substance were performed in duplicates.

DETERMINATION OF CYTOTOXICITY
- Method: Inspection of reduction in the number of revertant colonies
Evaluation criteria:
The test substance may be considered positive in this test system if the following criteria are met: The test substance is considered mutagenic, if two-fold (or more) increases in mean revertant numbers are observed at two consecutive dose levels or at the highest practicable dose level only. In addition, there must be evidence of a dose-response relationship showing increasing numbers of mutant colonies with increasing dose levels. All cultures must demonstrate the characteristic mean number of spontaneous revertants in the vehicle controls.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
other: TA 1535, TA 1537, TA 98, TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
for TA1537 with S9 mix at concentrations ≥ 2500 µg/plate
Vehicle controls valid:
yes
Negative controls valid:
not examined
Positive controls valid:
yes
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no, but tested up to limit concentrations
Vehicle controls valid:
yes
Negative controls valid:
not examined
Positive controls valid:
yes
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: The test substance is insoluble in distilled water (information provided by the sponsor), but soluble in DMSO (solubility ≥ 500 mg/mL).
- Precipitation: Precipitation of the test substance was observed at test concentrations ≥ 1250 µg/plate without metabolic activation for all tester strains (see Table 2).

RANGE-FINDING/SCREENING STUDIES: The test results of the dose finding study are shown in Table 1. No increases in the number of the revertant colonies of any tester strain were observed with or without metabolic activation. Cytotoxicity was detected by a reduction in the number of revertant colonies for the tester strain TA1537 at a test concentrations of 5000 µg/plate with S9 mix. Precipitation of the test substance was observed at a test concentrations ≥ 1250 µg/plate without metabolic activation for all tester strains.

COMPARISON WITH HISTORICAL CONTROL DATA: The values for the positive and the negative controls are in the range of the historical control data.

ADDITIONAL INFORMATION ON CYTOTOXICITY: Cytotoxicity was detected by a reduction in the number of revertant colonies for the tester strain TA1537 at test concentrations ≥ 2500 µg/plate with S9 mix (see Table 2).

Any other information on results incl. tables

Table 1. Test results of the dose finding study (preincubation method).

Test substance concentration

(μg/plate)

Mean number of revertant colonies per plate

(average of 2 or 3 plates*)

Base-pair substitution type

Frameshift type

TA 100

TA1535

WP2uvrA

TA98

TA1537

– S9 -mix

0

97

13

29

22

15

– S9 -mix

4.88

116

7

35

21

13

– S9 -mix

19.5

100

9

29

17

14

– S9 -mix

78.1

99

11

26

21

19

– S9 -mix

313

117

13

26

21

17

– S9 -mix

1250

101 P

8 P

32 P

20 P

8 P

– S9 -mix

5000

105 P

6 P

38 P

22 P

7 P

Positive controls, – S9 -mix

Name

AF-2

NaN3

AF-2

AF-2

ICR-191

Concentrations

(μg/plate)

0.01

0.5

0.01

0.1

0.5

Mean No. of colonies/plate

(average of 2 plates)

603

468

311

517

1580

+ S9 -mix

0

112

13

33

39

30

+ S9 -mix

4.88

102

11

33

23

18

+ S9 -mix

19.5

100

10

39

25

21

+ S9 -mix

78.1

104

8

33

28

20

+ S9 -mix

313

127

12

30

31

25

+ S9 -mix

1250

118

13

43

29

11

+ S9 -mix

5000

104

6

34

31

7

Positive controls, + S9 -mix

Name

2AA

2AA

2AA

2AA

2AA

Concentrations

(μg/plate)

1

2

10

0.5

2

Mean No. of colonies/plate

(average of 2 plates)

962

246

634

278

234

 * All experiments of the solvent control were performed in triplicates. All experiments for the positive controls and for the test substance were performed in duplicates.

AF-2 = 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide

NaN3= Sodium azide

ICR-191 = 2-Methoxy-6-chloro-9-[3-(2-chloroethyl)aminopropylamino]acridine*2HCl

2AA = 2-Aminoanthracene

P = Precipitate

 

Table 2. Test results of the main study (preincubation method).

Test substance concentration

(μg/plate)

Mean number of revertant colonies per plate

(average of 2 or 3 plates*)

Base-pair substitution type

Frameshift type

TA 100

TA1535

WP2uvrA

TA98

TA1537

– S9 -mix

0

103

10

28

25

10

– S9 -mix

313

117

7

26

21

9

– S9 -mix

625

108

9

31

23

5

– S9 -mix

1250

104 P

8 P

32 P

26 P

6 P

– S9 -mix

2500

110 P

12 P

32 P

15 P

6 P

– S9 -mix

5000

108 P

8 P

26 P

24 P

6 P

Positive controls, –S9 -mix

Name

AF-2

NaN3

AF-2

AF-2

ICR-191

Concentrations

(μg/plate)

0.01

0.5

0.01

0.1

0.5

Mean No. of colonies/plate

(average of 2 plates)

674

344

377

516

1441

+ S9 -mix

0

114

7

33

32

24

+ S9 -mix

313

105

7

30

28

16

+ S9 -mix

625

140

7

29

22

11

+ S9 -mix

1250

113

8

28

30

14

+ S9 -mix

2500

112

6

30

37

7

+ S9 -mix

5000

117

11

35

31

7

Positive controls, +S9 -mix

Name

2AA

2AA

2AA

2AA

2AA

Concentrations

(μg/plate)

1

2

10

0.5

2

Mean No. of colonies/plate

(average of 2 plates)

1039

202

657

371

284

 * All experiments with the solvent control were performed in triplicates. All experiments with the positive controls and with the test substance were performed in duplicates.

AF-2 = 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide

NaN3= Sodium azide

ICR-191 = 2-Methoxy-6-chloro-9-[3-(2-chloroethyl)aminopropylamino]acridine*2HCl

2AA = 2-Aminoanthracene

P = Precipitate

The test result of the main test are shown in Table 2. No increases in the number of the revertant colonies of any test strain were observed with or without metabolic activation. Cytotoxicity was detected by a reduction in the number of revertant colonies for the tester strain TA1537 at a test concentrations 2500 µg/plate with S9 mix. Precipitation of the test substance was observed at test concentrations ≥ 1250 µg/plate without metabolic activation for all tester strains.

Applicant's summary and conclusion

Conclusions:
Under the conditions of the Ames test the test substance was not mutagenic in any of the five strains (TA 1535, TA 1537, TA 98, TA 100 and WP2 uvr A) tested with and without metabolic activation.