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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:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
2012
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: well described GLP compliant study conducted to recognised international test guidelines. For read across justification see Section 13.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
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
Details on test material:
- Name of test material : CP12
- Physical state: Liquid
- Lot/batch No.: 0012
- Expiration date of the lot/batch: 2015-08-01
- Storage condition of test material: Room temperature

Method

Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
5000 , 2500, 1250, 625 ,313 and 156 µg/plate
Vehicle / solvent:
The substance was found to be sufficiently soluble in water for the highest concentration to be tested of 5000 μg/plate.
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
without S9, strains TA1535 and TA100
Untreated negative controls:
yes
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
without S9, strain TA1537
Untreated negative controls:
yes
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
without S9, strain TA98
Untreated negative controls:
yes
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without S9, strain WP2
Untreated negative controls:
yes
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoantracene
Remarks:
with S9, all strains
Details on test system and experimental conditions:
METHOD OF APPLICATION: In agar (plate incorporation) and preincubation

DURATION
- Preincubation period: 30minutes
- Exposure duration: 72 hours

SELECTION AGENT (mutation assays): Histidine requirement (S. thyphimurium) or tryptophan requirement (E. coli)

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: Reduction in number of spontaneous revertants, thinning of background lawn or microcolony formation
Evaluation criteria:
For a test to be considered valid, the mean of the vehicle control revertant colony numbers for each strain should lie within or close to the current historical control range of the laboratory unless otherwise justified by the Study Director. Also, the positive control compounds must induce an increase in mean revertant colony numbers of at least twice the concurrent vehicle controls.

For the tested substance to be considered mutagenic, two-fold (or more) increases in mean revertant numbers must be 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.
Statistics:
The mean number and standard deviation of revertant colonies were calculated for all groups. The “fold-increases” relative to the vehicle controls were calculated in order to compare the means for all treatment groups with those obtained for the vehicle control groups.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
not applicable
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
not applicable
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No
- Effects of osmolality: No
- Precipitation: No
- Other confounding effects: None

RANGE-FINDING/SCREENING STUDIES: Toxicity screening at concentration range of 50.0 - 5000 microgram/plate showed no toxicity

COMPARISON WITH HISTORICAL CONTROL DATA: Results show that mean plate counts for untreated and positive control plates fell within the test laboratory's acceptance criteria based on historical control data.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

ASSAY I

 

With (+) or

Dose

(µg/plate)

Number of revertants Mean (±SD)

without(-)

Base-pair substitution type

Frameshift type

S9 mix

TA100

TA1535

WP2 uvrA

TA98

TA1537

 

Untreated

132 (±11.1)

20 (±1.2)

30 (±2.8)

29 (±2.7)

18 (±1.7)

 

156

 

 

 

 

15 (±2.6)

S9 mix

313

168 (±4.9)

22 (±3.0)

30 (±1.5)

32 (±0.9)

20 (±1.5)

(-)

625

156 (±4.5)

24 (±3.5)

34 (±2.0)

30 (±1.2)

17 (±2.0)

 

1250

145 (±9.6)

27 (±1.0)

31 (±2.1)

31 (±2.9)

17 (±1.9)

 

2500

142 (±7.4)

24 (±2.1)

33 (±1.7)

32 (±2.2)

18 (±1.5)

 

5000

128 (±5.5)

17 (±1.0)

30 (±2.2)

26 (±0.6)

18 (±1.7)

 

Untreated

162 (±4.2)

19 (±0.3)

33 (±1.9)

45 (±2.3)

25 (±0.9)

 

313

176 (±8.1)

21 (±2.7)

34 (±0.7)

46 (±2.2)

22 (±1.5)

S9 mix

625

167 (±2.4)

23 (±2.3)

35 (±0.7)

41 (±5.2)

20 (±2.6)

(+)

1250

158 (±5.0)

21 (±1.2)

36 (±1.2)

47 (±4.1)

23 (±2.9)

 

2500

158 (±7.4)

21 (±0.9)

35 (±2.0)

42 (±2.8)

24 (±2.0)

 

5000

161 (±5.9)

21 (±3.2)

36 (±1.5)

50 (±2.3)

16 (±0.7)

+ve control

Chemical

SA

SA

MMS

2NF

9-AA

S9 mix(-)

Doseµg/plate

1.0

1.0

500

2.0

50

 

Colonies/plate

617 (±21.9)

494 (±10.1)

173 (±7.9)

138 (±4.1)

215 (±12.5)

+ve control

Chemical

2-AA

2-AA

2-AA

2-AA

2-AA

S9 mix(+)

Doseµg/plate

1.0

1.0

10

1.0

1.0

 

Colonies/plate

1234 (±42.8)

107 (±5.2)

264 (±17.3)

535 (±36.4)

118(±4.1)

 

SA = sodium azide; MMS = methylmethanesulphonate; 2NF = 2-nitrofluorene; 9-AA = 9-aminoacridine; 2-AA = 2-aminoanthracene

 

ASSAY II

 

With (+) or

Dose

(µg/plate)

Number of revertants Mean (±SD)

without(-)

Base-pair substitution type

Frameshift type

S9 mix

TA100

TA1535

WP2 uvrA

TA98

TA1537

 

Untreated

145 (±2.9)

19 (±1.0)

29(±1.2)

30 (±0.9)

21 (±0.9)

 

156

140 (±9.8)

19 (±1.7)

27 (±1.5)

26 (±2.6)

19 (±1.7)

S9 mix

313

127 (±3.6)

16 (±1.8)

34 (±2.3)

26 (±1.5)

18 (±1.5)

(-)

625

123 (±7.1)

16 (±2.1)

27 (±2.7)

30 (±2.9)

16 (±0.9)

 

1250

103 (±9.3) *

17 (±1.5)

34 (±2.4)

30 (±0.9)

12 (±1.8) *

 

2500

107 (±4.3) *

25 (±1.9)

28 (±2.0)

33 (±2.0)

6 (±22.3 *

 

5000

86 (±3.8) *

17 (±1.5)

30 (±0.3)

30 (±3.5)

6 (±1.8) *

 

Untreated

130 (±2.3)

17 (±2.0)

34 (±1.2)

43 (±1.9)

24 (±1.2)

 

156

118 (±2.0)

18 (±0.9)

36 (±1.7)

45 (±2.0)

26 (±2.8)

 

313

128 (±5.5)

23 (±1.5)

36 (±1.9)

50 (±2.8)

25 (±0.6)

S9 mix

625

126 (±8.2)

19 (±2.1)

37 (±0.6)

45 (±2.6)

26 (±1.5)

(+)

1250

101 (±3.4) *

18 (±1.5)

35 (±2.1)

38 (±2.3)

23 (±2.3)

 

2500

100 (±6.0) *

18 (±1.7)

29 (±2.8)

38 (±0.9)

21 (±1.9)

 

5000

106 (±5.2) *

21 (±0.7)

34 (±0.7)

42 (±1.8)

14 (±0.7) *

+ve control

Chemical

SA

SA

MMS

2NF

9-AA

S9 mix(-)

Doseµg/plate

1.0

1.0

500

2.0

50

 

Colonies/plate

662 (±32.1)

492 (±41.1)

197 (±12.2)

147 (±4.2)

167 (±53.8)

+ve control

Chemical

2-AA

2-AA

2-AA

2-AA

2-AA

S9 mix(+)

Doseµg/plate

2.0

1.0

20

2.0

1.0

 

Colonies/plate

912 (±44.6)

107 (±5.3)

240 (±11.9)

636 (±39.7)

112 (±10.1)

 

SA = sodium azide; MMS = methylmethanesulphonate; 2NF = 2-nitrofluorene; 9-AA = 9-aminoacridine; 2-AA = 2-aminoanthracene; * = thinning of background lawn

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

It is concluded that the substance showed no evidence of mutagenic activity in this bacterial system under the test conditions employed.
Executive summary:

Gene mutation has been investigated in bacteria using strains of Salmonella typhimurium and Escherichia coli, in accordance with OECD/EU test methods. Five tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA were used and experiments were performed both in the absence and presence of metabolic activation, using liver S9 fraction from rats pre-treated with phenobarbital and 5,6-benzoflavone. The tested substance did not induce reverse mutation in the tester strains, neither in the absence nor presence of S9 metabolism.