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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:
from 6 October 2009 to 18 January 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: the study was performed according to internationally recognised guidelines and GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
mammalian liver post-mitochondrial fraction (S-9) prepared from male Sprague Dawley rats induced with Aroclor 1254
Test concentrations with justification for top dose:
- Range-Finder Experiment and Mutation Experiment 1: 1.6, 8, 40, 200, 1000, 5000 µg/plate
- Mutation Experiment 2: 156.25, 312.5, 625, 1250, 2500, 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: preliminary solubility data indicated that the test item was soluble in water for injection (purified water) at greater than 50 mg/mL.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
benzo(a)pyrene
mitomycin C
other: 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: direct plate incorporation; preincubation

DURATION
- Preincubation period: 1 hour (Experiment 2 only)
- Exposure duration: 3 days

NUMBER OF REPLICATES: triplicates (test item and positive control), quintuplicate (solvent control),

DETERMINATION OF CYTOTOXICITY
- Method: reduction in the number of revertants
Evaluation criteria:
For valid data, the test article was considered to be mutagenic if:
1. Dunnett's test gave a significant response (p ≤ 0.01) which was concentration related
2. the positive trend/effects described above were reproducible.
The test article was considered as positive in this assay if all of the above criteria were met.
The test article was considered as negative in this assay if none of the above criteria were met.
Results which only partially satisfied the above criteria were dealt with on a case-by-case basis. Biological relevance was taken into account.
Statistics:
Dunnett's test was used to compare the counts at each concentration with the control. The presence or otherwise of a concentration response was checked by non-statistical analysis, up to limiting levels (for example toxicity, precipitation or 5000 μg/plate).

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:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: the test item was completely soluble in the aqueous assay system at all concentrations treated, in each of the experiments performed.
- Effects of pH, effects of osmolality, evaporation from medium: no data

RANGE-FINDING/SCREENING STUDIES:
An initial toxicity Range-Finder Experiment was carried out in the absence and in the presence of S-9 in strain TA100 only, using test item concentrations at 1.6, 8, 40, 200, 1000 and 5000 μg/plate, plus negative (vehicle) and positive controls.
Following these treatments, evidence of toxicity (manifest as a reduction in the number of revertants) was observed in the presence of S-9 at 5000 μg/plate. Further evidence of probable toxicity was also apparent at this concentration in the absence of S-9.

COMPARISON WITH HISTORICAL CONTROL DATA:
Mean vehicle control counts fell within the normal historical ranges. The positive control chemicals all induced large increases in revertant numbers in the appropriate strains, which fell within the normal historical ranges.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Experiment 1: evidence of toxicity in the form of a slight thinning of background bacterial lawn or a reduction in revetant numbers was observed at 5000 μg/plate in strain TA1537 in the presence of S-9 and in strain TA102 in the absence and presence of S-9.
- Experiment 2: evidence of toxicity in the form of a slight thinning of the background bacterial lawn and/or a reduction of revertant numbers was observed in strains TA1537 and TA102 at 5000 μg/plate in the presence of S-9.

Any other information on results incl. tables

Table 1 : results of Experiment 1 without S-9

Compound

Concentration µg/plate

TA 98

TA 100

TA 1535

Revertant

Mean number/plate

Fold increase

Revertant

Mean number/plate

Fold increase

Revertant

Mean number/plate

Fold increase

water

-

25.4 ± 5.6

-

95.4 ± 2.9

-

20.6 ± 3.0

-

test item

1.6

22.3 ± 5.8

0.9

96.0 ± 1.7

1.0

24.7 ± 6.8

1.2

test item

8

30.7 ± 5.1

1.2

93.7 ± 1.2

1.0

26.7 ± 2.1

1.3

test item

40

21.0 ± 1.0

0.8

87.3 ± 6.0

0.9

21.0 ± 6.9

1.0

test item

200

23.7 ± 10.0

0.9

91.0 ± 19.5

1.0

20.3 ± 4.5

1.0

test item

1000

27.7 ± 1.2

1.1

104.0 ± 7.8

1.1

22.3 ± 4.7

1.1

test item

5000

30.0 ± 5.6

1.2

74.3 ± 16.1

0.8

19.0 ± 4.4

0.9

2-NF

5

1216.3 ± 61.5

47.9

-

-

-

-

NaN3

2

-

-

763.7 ± 69.9

8.0

27.3 ± 6.8

27.3

 

Compound

Concentration µg/plate

TA 1537

TA 102

Revertant

Mean number/plate

Fold increase

Revertant

Mean number/plate

Fold increase

water

-

10.4 ± 0.9

-

321.6 ± 31.1

-

test item

1.6

11.3 ± 8.7

1.1

301.3 ± 7.0

0.9

test item

8

8.7 ± 1.5

0.8

314.7 ± 20.8

1.0

test item

40

10.0 ± 4.6

1.0

327.0 ± 28.9

1.0

test item

200

8.7 ± 2.5

0.8

316.7 ± 8.5

1.0

test item

1000

11.3 ± 4.6

1.1

280.7 ± 8.0

0.9

test item

5000

9.0 ± 2.0

0.9

192.3 ± 9.3

0.6

AAC

50

108.7 ± 32.0

10.4

-

-

MMC

0.2

-

-

883.7 ± 68.5

2.7

   

Table 2 : results of Experiment 1 with S-9

Compound

Concentration µg/plate

TA 98

TA 100

TA 1535

Revertant

Mean number/plate

Fold increase

Revertant

Mean number/plate

Fold increase

Revertant

Mean number/plate

Fold increase

water

-

32.0 ± 4.2

-

99.8 ± 13.5

-

20.2 ± 6.7

-

test item

1.6

26.0 ± 4.6

0.8

95.0 ± 2.6

1.0

26.3 ± 4.0

1.3

test item

8

35.7 ± 8.5

1.1

100.7 ± 10.2

1.0

20.0 ± 2.6

1.0

test item

40

37.7 ± 2.3

1.2

82.0 ± 10.6

0.8

23.0 ± 6.6

1.1

test item

200

31.3 ± 2.3

1.0

102.3 ± 7.1

1.0

23.7 ± 5.5

1.2

test item

1000

27.3 ± 4.2

0.9

82.0 ± 15.7

0.8

19.0 ± 3.5

0.9

test item

5000

29.0 ± 2.0

0.9

40.7 ± 3.1

0.4

18.7 ± 2.9

0.9

B[a]P

10

285.3 ± 16.8

8.9

-

-

-

-

AAN

5

-

-

1682.0 ± 109.8

16.9

263.3 ± 19.1

13.0

 

Compound

Concentration µg/plate

TA 1537

TA 102

Revertant

Mean number/plate

Fold increase

Revertant

Mean number/plate

Fold increase

water

-

18.6 ± 2.1

-

244.6 ± 18.2

-

test item

1.6

16.7 ± 5.8

0.9

247.7 ± 24.0

1.0

test item

8

19.0 ± 3.6

1.0

257.0 ± 5.6

1.1

test item

40

17.0 ± 3.0

0.9

265.3 ± 10.4

1.1

test item

200

16.3 ± 1.2

0.9

248.3 ± 2.3

1.0

test item

1000

13.0 ± 5.3

0.7

214.0 ± 10.8

0.9

test item

5000

10.3 ± 1.5

0.6

143.0 ± 7.2

0.6

AAN

5-20

69.0 ± 7.2

3.7

2169.3 ± 285.8

8.9

 

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

The test item did not induce mutation in five histidine-requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium when tested under the conditions of this test.
Executive summary:

In an in-vitro bacterial reverse gene mutation assay according to OECD guideline 471 and GLP, scored as validity 1 according to Klimisch criteria, the test item was assayed for mutation in five histidine-requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium, both in the absence and in the presence of metabolic activation by an Aroclor 1254-induced rat liver post-mitochondrial fraction (S-9), in two separate experiments. All test item treatments were performed using formulations prepared in purified water at concentration up to 5000 µg/plate.

 

The test item was completely soluble in the aqueous assay system at all concentrations treated, in each of the experiments performed.

Evidence of toxicity was observed at 5000 µg/plate in several strains: TA100 in the presence of S-9 and possible in the absence of S-9, TA1537 in the presence of S-9 and in strain TA102 in the absence and presence of S-9.

The mean numbers of revertant colonies on negative control plates all fell within acceptable ranges, and were significantly elevated by positive control treatments.

Following treatments with test item of all the test strains in the absence and presence of S-9, no increases in revertant numbers were observed that were statistically significant when the data were analysed at the 1% level using Dunnett’s test. This study was considered therefore to have provided no evidence of any test item mutagenic activity in this assay system.

 

It was concluded that the test item did not induce mutation in five histidine-requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium when tested under the conditions of this study. These conditions included treatments at concentrations up to 5000µg/plate,in the absence and in the presence of a rat liver metabolic activation system (S-9).