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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:
10 Aug 2012 - 07 Sep 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
(adopted 21 Jul 1997)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
(adopted 30 May 2008)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Bayerisches Landesamt fuer Gesundheit und Lebensmittelsicherheit, Erlangen, Germany
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid

Method

Target gene:
his operon
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:
Co-factor supplemented liver S9 homogenate from phenobarbital and ß-naphthoflavone induced male Wistar rats
Test concentrations with justification for top dose:
- pre-test (toxicity): 3.16, 10.0, 31.6, 100, 316, 1000, 2500, and 5000 µg/plate
- main test (mutagenicity): 31.6, 100, 316, 1000, 2500, and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-nitro-o-phenylene-diamine; 2-aminoanthracene
Remarks:
+S9: 2-aminoanthracene 2.5 µg/plate (TA 98, TA 100, TA 1535, TA 1537) 10 µg/plate (TA 102); -S9: 4-nitro-o-phenylene-diamine 10 µg/plate (TA 98) 40 µg/plate (TA 1537), sodium azide 10 µg/plate (TA 100, TA 1535), methylmethanesulfonate 1 µL/plate (TA 102)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 60 min
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3 plates each in 2 independent tests

DETERMINATION OF CYTOTOXICITY
- Method: evaluation of background lawn, reduction in the number of revertants down to a mutation factor ≤0.5 in relation to the solvent control
Evaluation criteria:
The Mutation Factor is calculated by dividing the mean value of the revertant counts through the mean values of the solvent control.
A test item is considered as mutagenic if a clear and dose-related increase in the number of revertants occurs and/or a biologically relevant positive response for at least one of the dose groups occurs in at least one tester strain with or without metabolic activation.
A biologically relevant increase is described as follows:
- if in tester strains TA 98, TA 100, TA 102 the number of reversions is at least twice as high
- if in tester strains TA 1535, TA 1537 the number of reversions is at least three times higher
than the reversion rate of the solvent control.
According to the OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.
A test item producing neither a dose related increase nor a reproducible biologically relevant response at any of the dose groups is considered to be non-mutagenic in this system.

Results and discussion

Test results
Key result
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
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: no
No further information given in the study report.

RANGE-FINDING/SCREENING STUDIES:
The test item was not toxic to the tester strains TA 98 and TA 100 up to limit concentrations.

COMPARISON WITH HISTORICAL CONTROL DATA:
The results of the positive and solvent controls are within the range of the historical control data.

Any other information on results incl. tables

Tab.1: Experiment 1 (plate incorporation) without metabolic activation - Number of revertants per plate (mean of 3 plates ± standard deviation)

Conc. [µg/plate]

TA 98

Mutation Factor

TA 100

Mutation Factor

TA 1535

Mutation Factor

TA 1537

Mutation Factor

TA 102

Mutation Factor

Solvent control

22±5.5

1.0

98±11.9

1.0

6±0.6

1.0

8±2.0

1.0

211±22.9

1.0

31.6

15±2.5

0.7

88±7.2

0.9

9±2.3

1.4

8±1.7

1.0

189±7.4

0.9

100

17±5.0

0.8

92±9.5

0.9

5±2.6

0.8

8±2.6

1.0

184±16.0

0.9

316

20±3.8

0.9

112±16.5

1.1

7±0.6

1.1

9±3.8

1.1

186±20.0

0.9

1000

23±5.5

1.1

95±15.2

1.0

4±1.2

0.7

8±1.7

1.0

176±6.7

0.8

2500

16±2.5

0.7

88±7.8

0.9

5±0.6

0.7

7±1.0

0.9

187±20.3

0.9

5000

20±3.6

0.9

87±15.1

0.9

6±4.0

1.0

5±1.2

0.7

190±8.7

0.9

Positive control

251±17.6

11.6

642±137.8

6.5

996±137.1

157.2

78±10.7

9.8

2110±84.9

10.0

Solvent control: A. dest.

Positive controls: 4-nitro-o-phenylene-diamine (TA 98, TA 1537); sodium azide (TA 100, TA 1535); methylmethanesulfonate (TA 102)

Mutation Factor = mean revertants (test item)/mean revertants (solvent control)

 

Tab. 2: Experiment 1 (plate incorporation) with metabolic activation - Number of revertants per plate (mean of 3 plates ± standard deviation)

Conc. [µg/plate]

TA 98

Mutation Factor

TA 100

Mutation Factor

TA 1535

Mutation Factor

TA 1537

Mutation Factor

TA 102

Mutation Factor

Solvent control

34±7.6

1.0

113±13.5

1.0

7±2.5

1.0

8±3.8

1.0

296±16.1

1.0

31.6

29±6.0

0.9

130±20.8

1.1

8±1.2

1.0

6±4.2

0.8

278±25.1

0.9

100

26±1.2

0.8

111±5.9

1.0

5±2.1

0.7

7±1.7

0.9

255±14.8

0.9

316

30±3.0

0.9

113±4.6

1.0

9±1.5

1.2

8±1.0

1.0

281±18.5

0.9

1000

20±7.2

0.6

110±2.6

1.0

6±2.3

0.8

9±2.3

1.2

250±28.2

0.8

2500

25±1.7

0.7

101±6.0

0.9

8±1.5

1.0

8±0.6

1.1

213±8.7

0.7

5000

16±2.5

0.5

108±4.0

1.0

10±3.1

1.4

6±4.6

0.7

216±20.2

0.7

Positive control

2247±270.3

66.8

1768±89.8

15.6

56±7.2

7.6

263±40.9

34.4

944±42.7

3.2

Solvent control: A. dest.

Positive controls: 2-aminoanthracene (all tester strains)

Mutation Factor = mean revertants (test item)/mean revertants (solvent control)

 

Tab. 3: Experiment 2 (preincubation) without metabolic activation - Number of revertants per plate (mean of 3 plates ± standard deviation)

Conc. [µg/plate]

TA 98

Mutation Factor

TA 100

Mutation Factor

TA 1535

Mutation Factor

TA 1537

Mutation Factor

TA 102

Mutation Factor

Solvent control

17±1.2

1.0

84±11.6

1.0

6±3.1

1.0

8±3.5

1.0

171±13.5

1.0

31.6

23±3.8

1.3

104±5.7

1.2

9±1.7

1.6

9±2.6

1.1

183±21.6

1.1

100

23±3.0

1.3

82±5.0

1.0

6±3.1

1.1

9±1.0

1.1

188±10.5

1.1

316

21±4.7

1.2

87±9.5

1.0

9±1.5

1.5

8±4.6

1.0

162±16.1

0.9

1000

22±8.4

1.3

81±4.0

1.0

6±1.5

1.1

10±3.6

1.2

163±19.6

1.0

2500

18±7.8

1.1

71±9.7

0.8

7±0.6

1.3

7±4.9

1.1

184±6.8

1.1

5000

22±4.0

1.3

73±20.0

0.9

7±0.0

1.2

6±3.2

0.7

180±20.1

1.1

Positive control

390±33.5

22.5

1256±99.0

15.0

932±74.1

164.4

103±14.3

12.4

1811±82.9

10.6

Solvent control: A. dest.

Positive controls: 4-nitro-o-phenylene-diamine (TA 98, TA 1537); sodium azide (TA 100, TA 1535); methylmethanesulfonate (TA 102)

Mutation Factor = mean revertants (test item)/mean revertants (solvent control)

 

Tab. 4: Experiment 2 (preincubation) with metabolic activation - Number of revertants per plate (mean of 3 plates ± standard deviation)

Conc. [µg/plate]

TA 98

Mutation Factor

TA 100

Mutation Factor

TA 1535

Mutation Factor

TA 1537

Mutation Factor

TA 102

Mutation Factor

Solvent control

37±10.1

1.0

104±7.8

1.0

6±1.2

1.0

9±0.6

1.0

256±30.9

1.0

31.6

42±7.5

1.1

101±16.6

1.0

11±2.1

1.8

9۬.3

1.1

277±25.5

1.1

100

41±13.1

1.1

84±8.5

0.8

9±2.1

1.5

9±3.8

1.1

277±11.2

1.1

316

31±6.7

0.9

102±20.5

1.0

7±1.5

1.2

8±4.4

0.9

270±19.5

1.1

1000

38±11.4

1.0

101±8.1

1.0

7±2.6

1.1

8±4.0

0.9

262±15.8

1.0

2500

30±9.3

0.8

86±13.7

0.8

7±2.5

1.1

6±5.9

0.7

225±1.7

0.9

5000

27±2.1

0.7

78±4.6

0.8

7±3.6

1.1

8±2.5

1.0

227±16.2

0.9

Positive control

2095±255.2

79.2

1933±303.9

18.6

81±18.1

12.8

190±40.9

21.9

612±102.0

2.4

Solvent control: A. dest.

Positive controls: 2-aminoanthracene (all tester strains)

Mutation Factor = mean revertants (test item)/mean revertants (solvent control)

Applicant's summary and conclusion

Conclusions:
Interpretation of results: negative