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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
Study period:
2011-030-014 to 2011-05-26
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Followed guideline and GLP requirements. No analysis was conducted on formulations, but test item used within 4 hours of preparation and assumed stable.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
Test formulations not analyzed
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): SD10
- Physical state: clear, colorless liquid
- Analytical purity: 97.17%
- Purity test date: COA date 2010-11-04
- Lot/batch No.: 9147-192-3a
- Expiration date of the lot/batch: Reanalysis date 2012-11-04
- Storage condition of test material: Room temperature in the dark under nitrogen

Method

Target gene:
histidine or tryptopahn locus in genome of the 5 bacterial strains

Salmonella typhimurium strains Genotype Type of mutation indicated
TA1537 his C 3076; rfa-, uvrB- frame shift mutations
TA98 his D 3052; rfa-, uvrB-; R-factor frame shift mutations
TA 1535 his G 46; rfa-; uvrB- base pair mutations
TA100 his G 46; rfa-; uvrB-; R-factor base pair substitutions

Escherichia coli trp-; uvrA- base pair substitution
Species / strainopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell lines (if applicable):
- Type and identity of media: obtained on culture discs or frozen
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
-Source: University of California, Berkelyor Syngenta CTL, Alderley Ledge
Metabolic activation:
with and without
Metabolic activation system:
S9 microsomal fraction from rats induced with phenobarbitone/beta-naphthoflavone at 80/100 mg/kg/day orally for 3 days
Species / strain:
E. coli WP2 uvr A
Details on mammalian cell lines (if applicable):
- Type and identity of media: obtained on nutrient agar plate and stored frozen
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
- Source: British Industrial Biological Research Association
Metabolic activation:
with and without
Metabolic activation system:
S9 microsomal fraction from rats induced with phenobarbitone/beta-naphthoflavone at 80/100 mg/kg/day orally for 3 days
Test concentrations with justification for top dose:
Range-find test: five dose levels from 50 to 5000 ug/plate (50, 150, 500, 1500, 5000 ug/plate)
Main test: 5 dose levels from 50 to 5000 ug/plate (50, 150, 500, 1500, 5000 ug/plate)
Vehicle:
- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: immiscible in sterile distilled water and DMSO at 50 mg/ml but fully miscible in acetone at some concentration
Controlsopen allclose all
Negative controls:
no
Solvent controls:
yes
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
Migrated to IUCLID6: WP2uvrA, TA100, TA1535 without S-9
Negative controls:
no
Solvent controls:
yes
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
Migrated to IUCLID6: for TA1537 without S-9
Negative controls:
no
Solvent controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
Migrated to IUCLID6: for TA1537 without S-9
Negative controls:
no
Solvent controls:
yes
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene for TA100, TA1535, TA 1537 and WP2uvrA with S-9
Negative controls:
no
Solvent controls:
yes
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
Migrated to IUCLID6: For TA 98 with S-9
Details on test system and conditions:
METHOD OF APPLICATION: in agar (plate incorporation) for range finding test; preincubation for main test

DURATION
- Preincubation period: 20 minutes in preincubation main test
- Exposure duration: 48 hours incubation in both preincubation and plate test

SELECTION AGENT (mutation assays): histidine or tryptophan


NUMBER OF REPLICATIONS: procedures in triplicate for each bacterial strain and each test concentration, both with and without S-9. Two complete tests: plate incorporation and preincubation tests


DETERMINATION OF CYTOTOXICITY
- Method: preliminary toxicity test using ten concentrations of test article, and acetone control used to assess toxicity in TA100 and WP2uvrA both with and without S-9. Plates assessed for revertant colonies and growth on bacterial background lawn. The test article was non-toxic to strains of bacteria used.
In the range finding test, the test article caused no reduction in growth was noted in the growth of the bacterial background lawn, either with or without activation. In the main test, the test item induced toxicity as weakened bacterial background lawns in several tester strains were noted in presence and absence of S-9 at 5000 ug/plate. No test article precipitate was noted.



METHOD OF APPLICATION: in agar (plate incorporation) and preincubation;
DURATION
- Preincubation period: 20 minutes, preincubation test
- Exposure duration: 48 hours incubation both tests

SELECTION AGENT (mutation assays): histidine or tryptophan

NUMBER OF REPLICATIONS: Each concentration tested in triplicate, with and without activation, and each positive or vehicle control. Two independent tests: plate incorporation and pre-incubation on separate days.


DETERMINATION OF CYTOTOXICITY
- Method: relative total growth (bacterial background lawn)


OTHER:

S-9 produced from Sprague Dawley male rats, 6 to 8 weeks of age, weighing approximately 250 grams. Induction was with oral phenobarbitone/beta-naphthoflavone given orally for 3 days at a dose of 80/100 mg/kg/day.
Evaluation criteria:
Acceptance criteria for valid test: all strains meet required characteristics, all tester strains exhibit characteristic number of spontaneous revertants per plate in the vehicle and untreated controls; all tester strain cultures should be in the range of 0.9 to 9 x 10^9 bacteria per ml; positive control chemicals must be included to demonstrate sensitivity of test strainsto mutagen exposure and the integrity of the S-9 mix. All positive controls should produce marked increases in frequency of revertant colonies, both with and without metabolic activation. There should be no evidence of excessive contamination, and there should be a minimum of 4 non-toxic dose levels.

Evaluation criteria: Any one or all of the following can be used to determine overall result: dose-related increase in mutant frequency over dose range tested; reproducible increase at one or more concentrations; biological relevance against in house historical control ranges; statistical analysis of data as determined by UKEMS; fold increase greater than two times the concurrent solvent control for any tester strain particularly if outside historical range.

Results and discussion

Test resultsopen allclose all
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
other: Weakened bacterial background lawns in preincubation test in presence and absence of S-9 at high dose.
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
other: Weakened bacterial background lawns in preincubation test in presence and absence of S-9 at high dose.
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Ames Test Results: Range Finding (Plate Incorporation) and Main Test (Preincubation)

Range Finding Test, Without Metabolic Activation

Test article ug/plate

TA100

TA1535

WP2uvrA

TA 98

TA 1537

0

112 (20.0)

21 (1.7)

28 (8.7)

24 (2.1)

12 (2.3)

50

102 (8.6)

21 (2.0)

24 (3.0)

22 (1.0)

10 (1.7)

150

110 (8.4)

22 (2.5)

27 (2.0)

17 (3.1)

12 (1.0)

500

99 (6.6)

22 (0.0)

25 (4.0)

21 (1.0)

9 (1.0)

1500

102 (6.7)

23 (2.3)

24 (7.0)

22 (0.6)

10 (3.8)

5000

99 (16.6)

22 (0.0)

23 (1.5)

23 (2.9)

12 (1.2)

Positve control, concentration, colonies per plate (SD)

ENNG

3 ug/plate

573 (65.4)

ENNG

5 ug/plate

383 (72.7)

ENNG

2 ug/plate

943 (107.5

4NQO

0.2 ug/plate

177 (21.2)

9AA

80 ug/plate

757 (122.2)

 (Mean Number of Revertant Colonies per Plate and Standard Deviation)

Range Finding Test, with Metabolic Activation

 (Mean Number of Revertant Colonies per Plate and Standard Deviation)

Test article ug/plate

TA100

TA1535

WP2uvrA

TA 98

TA 1537

0

97 (2/1)

14 (1.7)

31 (7.8)

25 (2.6)

12 (3.5)

50

92 (2.6)

14 (1.0)

31 (1.5)

21 (2.5)

11 (1.0)

150

82 (5.5)

14 (1.5)

30 (3.5)

23 (1.5)

12 (4.0)

500

88 (2.1)

13 (0.6)

27 (4.0)

23 (3.6)

10 (0.6)

1500

90 (4.6)

13 (0.6)

28 (2.9)

26 (4.6)

9 (1.0)

5000

91 (5.6)

14 (1.2)

28 (3.1)

21 (1.5)

13 (3.1)

Positive control, concentration, colonies per plate (SD)

2AA

1 ug/plate

1421 (218.7)

2AA

2 ug/plate

260 (23.6)

2AA

10 ug/plate

268 (31.5)

BP

5 ug/plate

284 (31.5)

2AA

2 ug/plate

231 (17.7)

Main Test, Without Activation

Test article ug/plate

TA100

TA1535

WP2uvrA

TA 98

TA 1537

0

116 (24.1)

23 (5.6)

37 (5.5)

17 (4.6)

9 (0.6)

50

95 (1.2)

21 (3.6)

38 (5.8)

15 (2.3)

4 (2.1)

150

110 (12.3)

23 (7.4)

33 (2.3)

14 (3.5)

6 (3.0)

500

112 (4.0)

21 (7.6)

33 (5.9)

13 (0.0)

7 (0.6)

1500

106 (106)

27 (6.4)

33 (0.6)

22 (3.0)

7 (1.5)

5000

122 (13.3)

23 (5.0)

25 (5.3)

8 (3.2)

4 (1.7)

Positive control, concentration, colonies per plate (SD)

ENNG

3 ug/plate

614 (60.1)

ENNG

5 ug/plate

258 (6.0)

ENNG

2 ug/plate

909 (80.5)

4NQO

0.2 ug/plate

134 (16.4)

9AA

80 ug/plate

836 (10.7)

 (Mean Number of Revertant Colonies per Plate and Standard Deviation)

Main Test, With Activation

Test article ug/plate

TA100

TA1535

WP2uvrA

TA 98

TA 1537

0

108 (1.7)

18 (3.6)

35 (34)

24 (4.4)

8 (1.2)

50

114 (14.6)

17 (3.2)

27 (1.5)

22 (4.4)

9 (0.6)

150

97 (17.9)

13 (0.6)

28 (2.0)

17 (3.5)

7 (2.9)

500

101 (2.1)

15 (4.9)

22 (4.4)

21 (1.0)

8 (3.2)

1500

93 (1.5)

13 (4.0)

31 (2.1)

19 (1.0)

8 (3.6)

5000

94 (4.4)

12 (4.0)

22 (2.1)

18 (7.8)

6 (0.0)

Positive control, concentration, colonies per plate (SD)

2AA

1 ug/plate

1249 (106.2)

2AA

2 ug/plate

440 (44.8)

2AA

10 ug/plate

345 (24.4)

BP

5 ug/plate

510 (7.5)

2AA

2 ug/plate

194 (38.9)

 (Mean Number of Revertant Colonies per Plate and Standard Deviation)

Applicant's summary and conclusion

Conclusions:
The test article was considered non-mutagenic under the conditions of the test.
Executive summary:

Test article was tested in a reverse mutation assay (Ames test) using Salmonella typhimurium and Escherichia coli following OECD Guideline 471 and complying with GLP requirements. Salmonella typhimurium (strains TA 1535, 1537, TA 98 and Ta 100) and Escherichia coli (WP2uvrA) were treated with the test article using both the Ames plate incorporation and pre-incubation methods at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard cofactors). Dose range for the range finder test based on a preliminary toxicity test was 50 to 5000 ug/plate. The experiment was repeated on a separate day (pre-incubation method) using the same dose range as the range-finding test, fresh cultures of bacteria and fresh test article fomulations. The vehicle (acetone) gave counts of revertant colonies within normal range. All positive control chemicals induced marked increases in the frequency of revertant colonies, both with and without metabolic activation. Thus validity was assured. The test item caused no significant reduction in growth of the bacterial background lawn in a toxicity test so it was tested at maximum recommended dose level of 5000 ug/plate.

In the range finding test, no visible reduction of background lawn was seen at any dose. In the main test, weakened bacterial background lawn was seen in several tester strains with and without activation at the high dose. These results were not sufficiently severe to prevent the test item being tested up to the maximum dose of 5000 ug/plate. No precipitate of test article was seen in any plates.

No significant increase in frequency of revertant colonies was recorded for any of the strains with any dose of the test article, with or without metabolic activation. The test article, SD10, was considered non-mutagenic under the conditions of the test.