Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

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:
18 Aug - 12 Sep 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Mainz, Germany
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Details on test material:
- Name of test material (as cited in study report): trade name given
- Substance type: white, crystalline powder
- Analytical purity: 99.5%
- Solubility: water: < 0.1 g/L
- Lot/batch No.: 1592ZG-076
- Expiration date of the lot/batch: Aug 2015
- Storage condition of test material: at room temperature 20 ± 5 °C

Method

Target gene:
his operon (for S. typhimurium strains)
Species / strain
Species / strain / cell type:
S. typhimurium, other: TA97a, TA98, TA100, TA102 and TA1535
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254 (500 mg/kg bw i.p.)
Test concentrations with justification for top dose:
First experiment: 50, 150, 500, 1500 and 5000 µg/plate with and without metabolic activation.
Second experiment: 78, 156, 313, 625, 1250, 2500 and 5000 µg/plate with and without metabolic activation.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
benzo(a)pyrene
other: 4-Nitro-1,2-phenylene diamine (4-NOPD); 2-Amino-Anthracene (2-AA)
Remarks:
+S9: 2-AA (1 µg/plate, TA100, TA102, TA1535, TA97a); B[a]P (20 µg/plate, TA98); -S9: sodium azide (1 µg/plate, TA100); 4-NOPD (20 µg/plate, TA102, TA97a, TA98, TA1535)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) (experiment 1); preincubation (experiment 2)

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

NUMBER OF REPLICATIONS: quadruplicates each in 2 independent experiment

DETERMINATION OF CYTOTOXICITY
- Method: reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn
Evaluation criteria:
The increase factor f(I) of revertant induction (mean revertants divided by mean spontaneous revertants) and the absolute number of revertants (“Rev. abs.”, mean revertants less mean spontaneous revertants) were also calculated.
A test item is considered as a mutagen if a significant, reproducible increase of revertant colonies per plate (increase factor ≥ 2) in at least one strain can be observed. A concentration-related increase over the range tested can also be taken as a sign of mutagenic activity.
Statistics:
Mean values and standard deviation were calculated.

Results and discussion

Test results
Species / strain:
S. typhimurium, other: TA97a, TA98, TA100, TA102 and TA1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
but tested up to 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: As the test item was tested as suspension precipitation were observed on all plates in all tested concentrations.

COMPARISON WITH HISTORICAL CONTROL DATA: All positive control values were within the range of the historical data. The most values of the spontaneous revertants of the solvent control ethanol which lie outside the range of the historical data are in the range of the literature data. Therefore, the study is concidered valid.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1. Test results of experiment 1 (plate incorporation).

With or without S9 mix

Test substance concentration

(μg/plate)

Mean number of revertant colonies per plate

(average of 4 plates ± Standard deviation)

Base-pair substitution type

Frameshift type

TA 100

TA102

TA1535

TA97a

TA98

-

0 (EtOH)

121 ± 17.1

248 ± 22.3

12 ± 1.0

99 ± 11.3

14 ± 1.0

-

50

115 ± 22.7

209 ± 8.9

13 ± 2.5

105 ± 4.5

13 ± 0.8

-

150

107 ± 12.0

208 ± 28.3

13 ± 3.2

112 ± 5.0

14 ± 0.6

-

500

109 ± 9.2

247 ± 42.0

14 ± 1.9

133 ± 9.1

10 ± 2.1

-

1500

115 ± 14.4

244 ± 47.7

15 ± 1.0

132 ± 1.8

13 ± 2.2

-

5000

119 ± 18.6

304 ± 102.8

15 ± 2.1

141 ± 31.0

15 ± 1.5

Positive controls,
-S9 mix

Name

NaN3

4-NOPD

NaN3

4-NOPD

4-NOPD

Concentrations

(μg/plate)

1

20

1

20

20

Mean No. of colonies/plate

(average of 4 ± SD)

412 ± 30.1

748 ± 13.1

103 ± 14.5

525 ± 45.8

384 ± 54.0

+

0 (EtOH)

103 ± 9.7

229 ± 32.1

15 ± 1.0

125 ± 4.8

17 ± 3.0

+

50

116 ± 22.5

235 ± 16.4

13 ± 1.7

117 ± 5.7

13 ± 2.2

+

150

136 ± 4.1

238 ± 40.6

15 ± 2.4

113 ± 4.1

13 ± 3.1

+

500

126 ± 7.5

218 ± 19.1

13 ± 2.1

130 ± 16.4

14 ± 0.8

+

1500

125 ± 22.6

219 ± 14.1

14 ± 3.4

145 ± 15.2

13 ± 4.1

+

5000

107 ± 21.4

268 ± 36.5

14 ± 3.2

133 ± 9.0

14 ± 4.0

Positive controls, +S9 mix

Name

2-AA

2-AA

2-AA

2-AA

B[a]P

Concentrations

(μg/plate)

1

1

1

1

20

Mean No. of colonies/plate

(average of 4 ± SD)

492 ± 93.9

689 ± 80.2

107 ± 3.8

578 ± 34.3

99 ± 8.7

EtOH: Ethanol

NaN3: Sodium azide

4-NOPD: 4-nitro-o-phenylene-diamine

2-AA: 2-aminoanthracene

B[a]P: Benzo-a-pyrene

Table 2. Test results of experiment 2 (preincubation).

With or without S9 mix

Test substance concentration

(μg/plate)

Mean number of revertant colonies per plate

(average of 4 plates ± Standard deviation)

Base-pair substitution type

Frameshift type

TA 100

TA102

TA1535

TA97a

TA98

-

0 (EtOH)

112 ± 5.2

199 ± 11.3

10 ± 0.8

101 ± 13.0

10 ± 2.1

-

78

135 ± 4.8

299 ± 59.9

13 ± 3.3

106 ± 13.7

10 ± 0.0

-

156

134 ± 4.9

324 ± 22.9

12 ± 1.5

108 ± 14.1

11 ± 3.2

-

312

122 ± 20.0

279 ± 76.5

14 ± 2.9

104 ± 10.0

10 ± 2.6

-

625

109 ± 16.1

275 ± 22.0

11 ± 1.0

121 ± 3.6

13 ± 2.9

-

1250

107 ± 7.9

230 ± 23.2

11 ± 4.0

119 ± 3.1

12 ± 2.2

-

2500

104 ± 9.5

240 ± 35.0

9 ± 2.2

114 ± 2.1

19 ± 2.1

-

5000

110 ± 20.0

248 ± 40.9

9 ± 1.9

113 ± 4.4

12 ± 2.4

Positive controls,
-S9 mix

Name

NaN3

4-NOPD

NaN3

4-NOPD

4-NOPD

Concentrations

(μg/plate)

1

20

1

20

20

Mean No. of colonies/plate

(average of 4 ± SD)

563 ± 69.2

949 ± 113.2

114 ± 15.6

547 ± 76.3

111 ± 13.6

-

0 (EtOH)

99 ± 16.9

187 ± 5.7

9 ± 2.2

115 ± 2.7

17 ± 2.6

-

78

126 ± 14.2

327 ± 34.8

15 ± 4.0

126.± 7.3

12 ± 3.3

-

156

114 ± 21.1

285 ± 41.1

15 ± 2.6

115 ± 1.8

14 ± 5.1

-

312

115 ± 21.2

160 ± 13.5

12 ± 2.2

117 ± 6.6

13 ± 2.5

-

625

134 ± 9.3

318 ± 50.8

15 ± 2.2

109 ± 14.0

12 ± 2.4

-

1250

97 ± 6.9

295 ± 53.8

12 ± 3.6

113 ± 14.4

10 ± 0.5

-

2500

120 ± 13.4

349 ± 30.2

9 ± 2.2

115 ± 1.9

14 ± 2.4

-

5000

106 ± 17.1

237 ± 18.0

11 ± 2.4

119 ± 2.8

12 ± 1.3

Positive controls, +S9 mix

Name

2-AA

2-AA

2-AA

2-AA

B[a]P

Concentrations

(μg/plate)

1

1

1

1

20

Mean No. of colonies/plate

(average of 4 ± SD)

539 ± 113.6

1018 ± 101.2

99 ± 11.8

556 ± 72.2

114 ± 16.8

EtOH: Ethanol

NaN3: Sodium azide

4-NOPD: 4-nitro-o-phenylene-diamine

2-AA: 2-aminoanthracene

B[a]P: Benzo-a-pyrene

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative