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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 based on grouping of substances (category approach)
Adequacy of study:
weight of evidence
Study period:
18 Oct - 29 Oct 2001
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP - Guideline study. According to the ECHA guidance document "Practical guide 6: How to report read-across and categories (March 2010)", the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report Date:
2004

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
Details on test material:
- Name of test material (as cited in study report): only trade name given
- Chemical name: acetic acid esters of monoglyceride of fully hydrogenated castor oil
- Substance type: clear liquid
- Analytical purity: no data
- Lot/batch No.: 10102
- Expiration date of the lot/batch: 26-04-2002
- Item No.: 175540
- Storage condition of test material: at room temperature, protected from light

Method

Target gene:
his operon
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Details on mammalian cell type (if applicable):
- Type and identity of media:
Liquid growth medium (broth): Oxoid Nutrient Broth No. 2 (2.5% (w/v) in distilled water
Selective agar plates: Vogel-Bonner medium composed of bacto agar (1.5%), D-glucose (2%), MgSO4 x 7H20 (0.02%), citric acid (0.2%), K2HPO4 (1%), NaNH4HPO4 x 4 H2O (0.35%) in distilled water (percentages correspond to w/v)
The components were autoclaved separately and mixed afterwards.
Top-agar: bacto agar (0.6%), NaCl (0.5%) in distilled water (percentages correspond to w/v), supplemented with 0.05 mM histidine and 0.05 mM biotin before use
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)
Test concentrations with justification for top dose:
50, 160, 500, 1600 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: - S9: sodium azide (SA, 1 µg/plate) for TA 100 and TA 1535; 2-nitrofluorene (2NF, 1 µg/plate) for TA 98 and TA 1537; cumene hydroperoxide (CHP, 100 µg/plate for plate incorporation and 25 µg/plate for preincubation) for TA 102;
Positive control substance:
other: + S9: 2-aminoanthracene (2-AA, 4 µg/plate for TA 102 or 2 µg/plate for remaining strains)
Details on test system and experimental conditions:
METHOD OF APPLICATION:
1. in agar (plate incorporation)

DURATION
- Exposure duration: 72 h

2. preincubation

DURATION
- Preincubation period: 1 h
- Exposure duration: 72 h

NUMBER OF REPLICATIONS: 3 replications each in 2 independent experiments including 1 plate incorporation and 1 preincubation test


DETERMINATION OF CYTOTOXICITY
- Method: other: thinning of the background lawn of non-revertant bacteria, appearance of micro-colonies and/or reduction in the number of revertant colonies on the test plates comparised to the solvent control plates

Evaluation criteria:
The test was considered to be valid when the following criteria were met:
1. negative and positive control data were consistent and within the range of historical control data (see table 4)
2. positive controls revealed a marked increase over the concurrent solvent controls
3. the evaluation was not restricted by loss of plates (e.g. through contamination)

The test material may be considered mutagenic in this test system if all of the following criteria were met:
1. dose-related increases in the number of revertants at one or more test points
2. reproducible increases in revertants between replicate plates
3. statistically significance in the increases of revertants
4. increases count more than twice the corresponding solvent control values

The test material may be considered non-mutagenic in this test system when no increases in the number of revertants is observed which exceed 1.5 times the solvent control values at any test point. Sporadically ocurring statistically significanct increases in the number of revertants which were not dose-related will usually be considered incidental and not relevant for the evaluation.

Increases between 1.5 and 2 fold compared to the respective solvent controls meeting the other criteria for a positive result were considered to demonstrate weak mutagenicity.
Statistics:
Statistical analyses were performed with the SAS (R) procedures version 8.1 (SAS Institute Inc., Cary, North Carolina 27513, USA).
In detail, the number of revertant colonies at each treatment test point were compared to the corresponding solvent control values using the Analysis of Variance test. Statistically significant differences were further evaluated via Dunnett´s test to determine the statistical significance of increases and decreases in the number of revertant colonies for each set of triplicates.

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:
other: no but tested up to limit concentrations (a small reduction in the number of revertants was induced in TA 100 (5000 µg: -15% (preincubation assay, +S9) and TA 1535 (160 µg: -28%; 1600 µg: -46%; 5000 µg: -30% (plate incorporation assay, + S9))
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
The test item did not induce toxicity within the conducted preliminary toxicity test as evaluated by large reductions in the number of revertants or poor growth of the background lawn of non-revertant bacteria either in the absence or presence of S9 mix (see table 1).

COMPARISON WITH HISTORICAL CONTROL DATA:
The solvent and positive control values were acceptable and compatible with the historical control values (slight increases have been determined in number of revertants for TA 102 in the plate incorporation (-S9: 470 ± 6 vs 409) and preincubation test (+S9: 460 ± 4 vs 432) and for TA 1535 in the plate incorporation assay after treatment with sodium azide (-S9: 1063 ± 83 vs 908), see table 4).

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 the preliminary toxicity test (plate incorporation)

 

 

 

With or without S9-Mix

Test substance concentration

Mean number of revertant colonies per plate

(μg/plate)

(average of 3 plates ± Standard Deviation)

 

Frameshift type

 

TA98

0

36.0 ± 1.7

50

30.3 ± 3.1

160

28.7 ± 5.1

500

33.0 ± 6.0

1600

34.3 ± 2.9

5000

34.3 ± 7.0

Positive controls, –S9

Name

2NF

Concentrations (μg/plate)

1

Mean No. of colonies/plate (average of 3 ± SD)

499.7 ± 88.3

+

0

48 ± 3

+

50

48.3 ± 5.5

+

160

43.3 ± 2.9

+

500

40.0 ± 6.6

+

1600

40.0 ± 1.0

+

5000

39.7 ± 6.1

Positive controls, +S9

Name

2AA

Concentrations (μg/plate)

2

Mean No. of colonies/plate (average of 3 ± SD)

682 ± 26.5

2NF = 2 Nitrofluorene

 

 

2AA = 2-Aminoanthracene

 

 

No statistical analysis was performed.

 

 

 

Table 2. Test results of main test 1 (plate incorporation)

 

 

 

 

 

 

 

 

 

 

With or without S9-Mix

Test substance concentration

Mean number of revertant colonies per plate

(μg/plate)

(average of 3 plates ± Standard deviation)

 

Base-pair substitution type

Frameshift type

 

TA 100

TA1535

TA102

TA98

TA1537

0

210.3 ± 7.5

14.7 ± 1.5

469.7 ± 6.1

30.7 ± 7.6

14.0 ± 2.0

50

210.7 ± 7.8

15.7 ± 2.5

474.0 ± 3.0

33.3 ± 7.2

17.7 ± 2.5

160

206.0 ± 5.3

15.0 ± 1.0

480.0 ± 6.6

27.7 ± 0.6

18.0 ± 2.0

500

205.7 ± 3.8

16.3 ± 2.1

478.7 ± 5.7

27.0 ± 2.6

15.0 ± 2.0

1600

208.7 ± 9.3

15.0 ± 3.6

479.0 ± 6.2

26.0 ± 5.0

16.3 ± 2.3

5000

208.3 ± 2.9

12.3 ± 1.5

483.7 ± 5.5

32.7 ± 3.1

15.7 ± 2.5

Positive controls, –S9

Name

SA

SA

CHP

2NF

2NF

Concentrations (μg/plate)

1

1

100

1

1

Mean No. of colonies/plate (average of 3 ± SD)

1136.7 ± 60.8

1063 ± 82.5

968.3 ± 78.4

749.7 ± 100.5

202.0 ± 7.9

+

0

213.7 ± 3.8

20.3 ± 2.9

405.3 ± 1.5

43.3 ± 2.1

16.7 ± 1.5

+

50

213.3 ± 3.1

19.0 ± 1.0

399.7 ± 6.8

41.0 ± 2.0

20.3 ± 0.6

+

160

215.7 ± 3.8

14.7* ± 1.5

401.3 ± 10.2

44.0 ± 2.0

19.7 ± 0.6

+

500

202.0 ± 5.3

17.0 ± 2.6

405.0 ± 11.5

40.3 ± 2.5

18.7 ± 0.6

+

1600

208.0 ± 11.1

11.0 ± 1.7

406.7 ± 9.7

42.0 ± 3.6

18.7 ± 2.1

+

5000

216.7 ± 4.2

14.3* ± 3.1

405.7 ± 4.7

43.0 ± 1.7

19.0 ± 1.7

Positive controls, +S9

Name

2AA

2AA

2AA

2AA

2AA

Concentrations (μg/plate)

2

2

4

2

2

Mean No. of colonies/plate (average of 3 ± SD)

838.0 ± 71.4

276.0 ± 80.0

1030.3 ± 102.6

597.0 ± 25.6

366.7 ± 34.0

SA = sodium azide

 

 

 

 

 

CHP = cumene hydroperoxide

 

 

 

 

 

2NF = 2-nitrofluorene

 

 

 

 

 

2AA = 2-Aminoanthracene

 

 

 

 

 

* = statistically significant at 5% level

 

 

 

 

* * = statistically significant at 1% level

 

 

 

 

Otherwise not statistically significant at 5% level (positive controls were not included)

 

 

Table 3. Test results of main test 2 (preincubation)

 

 

 

 

 

 

 

 

 

 

With or without S9-Mix

Test substance concentration

Mean number of revertant colonies per plate

(μg/plate)

(average of 3 plates ± Standard deviation)

 

Base-pair substitution type

Frameshift type

 

TA 100

TA1535

TA102

TA98

TA1537

0

214.7 ± 4.0

18.0 ± 3.0

403.7 ± 3.1

36.0 ± 1.7

15.7 ± 1.2

50

213.3 ± 6.4

17.3± 0.6

405.0 ± 5.6

30.3 ± 3.1

18.3 ± 0.6

160

213.7 ± 6.0

18.3 ± 3.5

407.7 ± 2.3

28.7 ± 5.1

18.3 ± 1.5

500

213.0 ± 1.7

21.3 ± 4.2

403.7 ± 1.5

33.0 ± 6.0

19.0 ± 1.0

1600

211.0 ± 11.5

21.3 ± 3.2

406.7 ± 4.9

34.3 ± 2.9

18.3 ± 2.1

5000

212.3 ± 6.5

17.0 ± 1.7

411.3 ± 13.7

34.3 ± 7.0

17.0 ± 1.0

Positive controls, –S9

Name

SA

SA

CHP

2NF

2NF

Concentrations (μg/plate)

1

1

25

1

1

Mean No. of colonies/plate (average of 3 ± SD)

1103.0 ± 25.5

596.3 ± 25.5

1158.0 ± 33.2

499.7 ± 88.3

338.7± 22.7

+

0

203.7 ± 7.5

11.7 ± 1.2

459.7 ± 4.2

48.0 ± 3.0

18.7 ± 1.2

+

50

206.0 ± 12.2

17.0 ± 1.0

460.0 ± 2.6

48.3 ± 5.5

20.0 ± 1.0

+

160

213.7 ± 4.7

16.3 ± 4.2

456.7 ± 4.5

43.3 ± 2.9

19.0 ± 1.0

+

500

207.7 ± 6.8

15.7 ± 2.1

455.3 ± 3.8

40.0 ± 6.6

18.0 ± 1.7

+

1600

188.7 ± 3.1

15.0 ± 4.0

458.3 ± 2.5

40.0 ± 1.0

19.0 ± 1.0

+

5000

173.3** ± 9.5

17.7 ± 2.3

459.7 ± 4.2

39.7 ± 6.1

19.0 ± 1.0

Positive controls, +S9

Name

2AA

2AA

2AA

2AA

2AA

Concentrations (μg/plate)

2

2

4

2

2

Mean No. of colonies/plate (average of 3 ± SD)

574.7 ± 30.2

231.7 ± 18.9

1140.7 ± 98.8

682.0 ± 26.5

210.3 ± 16.0

SA = sodium azide

 

 

 

 

 

CH = cumene hydroperoxide

 

 

 

 

 

2NF = 2-nitrofluorene

 

 

 

 

 

2AA = 2-Aminoanthracene

 

 

 

 

 

* = statistically significant at 5% level

 

 

 

 

* * = statistically significant at 1% level

 

 

 

 

Otherwise not statistically significant at 5% level (positive controls were not included)

 

 

 

Table 4. Historical control values

 

 

 

 

 

 

 

 

 

 

 

 

 

Strain

Treatment (µg/mL)

S9 mix

Number of revertant colonies/plate

Number of plates

Mean

Standard Deviation

Minimum

Maximum

 

TA 102

solvent

-

305

40

249

409

114

 

solvent

+

321

52

224

432

117

 

CHP (25)

-

1262

346

836

2487

54

 

CHP (100)

-

1252

258

903

2000

48

 

2AA (4)

+

1076

197

500

1522

105

TA 100

solvent

-

144

33

91

221

162

 

solvent

+

143

35

90

234

168

 

SA (1)

-

1006

196

578

1460

150

 

2AA (2)

+

1007

362

123

2163

156

TA 98

solvent

-

51

16

19

82

159

 

solvent

+

61

17

21

97

162

 

2NF (1)

-

658

247

216

1189

147

 

2AA (2)

+

851

284

411

1622

150

TA 1537

solvent

-

15

5

5

28

111

 

solvent

+

19

7

7

43

117

 

2NF (1)

-

357

154

121

919

99

 

2AA (2)

+

301

202

67

1121

105

TA 1535

solvent

-

20

8

7

38

117

 

solvent

+

18

7

7

42

123

 

SA (1)

-

541

169

206

908

105

 

2AA (2)

+

272

129

110

648

111

SA = sodium azide

 

CHP = cumene hydroperoxide

 

2NF = 2-nitrofluorene

 

2AA = 2-Aminoanthracene

 

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative