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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:
key study
Study period:
13 Nov - 22 Nov 2007
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:
2007
Report date:
2007

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Ministerium für Umwelt und Naturschutz, Landwirtschaft und Verbraucherschutz des Landes Nordrhein-Westfalen, Düsseldorf, Germany
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
97593-30-1 (C12)
IUPAC Name:
97593-30-1 (C12)
Details on test material:
- Name of test material (as cited in study report): only trade name given
- Chemical name: acetylated glyceride-mixture
- Physical state: clear yellowish liquid
- Analytical purity: 100%
- Purity test date: 15 Oct 2007
- Lot/batch No.: CH70920C
- Expiration date of the lot/batch: 20 Sep 2008
- Stability under test conditions: The batch used was analytically examined for identity prior to study initiation and was approved for use for the test period. A stability test in the solvent did not reveal significant degradation of the active ingredient.
- Storage condition of test material: at room temperature

Method

Target gene:
his operon
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
S. typhimurium TA 102
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
Test concentrations with justification for top dose:
50, 158, 500, 1581 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO (0.1 mL/plate)
- Justification for choice of solvent/vehicle: The solvent used was chosen out of the following solvents, in the order given: water, DMSO, methanol, ethanol, acetone, ethylene glycol dimethylether (EGDE), and DMF according to information given by the sponsor. The order of these solvents is based on their bacteriotoxic effects in preincubation experiments.
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 (µg/plate, strain): sodium azide (10, TA1535); Nitrofurantoin (0.2, TA100); 4-nitro-1,2-phenylene diamine (0.5 and 10, TA98 and TA1537, respectively); Mitomycin C (0.2, TA102 [plate incorporation]); cumene hydroperoxide (50, TA102 [preincubation])
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: +S9 (µg/plate, strain): 2-aminoanthracene (3, all strains)
Details on test system and experimental conditions:
METHOD OF APPLICATION:
First experiment: in agar (plate incorporation)
Second experiment: preincubation

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

NUMBER OF REPLICATIONS: 3 in each experiment (plate incorporation and preincubation)

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency and/or reduction in background growth
Evaluation criteria:
A reproducible and dose-related increase in mutant counts of at least one strain is considered to be a positive result. For TA 1535, TA 100 and TA 98 this increase should be about twice that of negative controls, whereas for TA 1537, at least a threefold increase should be reached. For TA 102 an increase of about 100 mutants should be reached. Otherwise, the result is evaluated as negative. However, these guidelines may be overruled by good scientific judgement.
In case of questionable results, investigations should continue, possibly with modifications, until a final evaluation is possible.
Statistics:
Mean values and standard deviation were calculated.

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:
cytotoxicity
Remarks:
TA1535, TA98 and TA100: at 1581 µg/plate and above (with S9); TA1537: at 500 µg/plate and above (with S9)
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 1581 µg/plate and above (with S9)
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: At 1581 µg/plate, the substance started to precipitate.

COMPARISON WITH HISTORICAL CONTROL DATA:
The negative controls (DMSO) were within the expected range and the positive controls showed sufficient effects when compared to the testing laboratory's historical negative and positive control data presented in the study report for the preceding 10 years.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Cytotoxic effects, as assessed by a reduction in the number of revertants per plate and/or reduction in background lawn, were observed in the plate incorporation assay in:

TA1535 at 5000 µg/plate with metabolic activation
TA100 at 1581 µg/plate and above with metabolic activation
TA1537 at 1581 µg/plate and above with metabolic activation

In the preincubation assay, cytotoxic effects were observed in:

TA1535 at 1581 µg/plate and above with metabolic activation
TA100 at 1581 µg/plate and above with metabolic activation
TA1537 at 500 µg/plate and above with metabolic activation, and at 5000 µg/plate without metabolic activation
TA98 at 1581 µg/plate and above with metabolic activation
TA102 at 1581 µg/plate and above with metabolic activation
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 first experiment (plate incorporation).

 

With or without S9-Mix

Test substance concentration

(μg/plate)

Mean number of revertant colonies per plate

(average of 3 plates ± Standard deviation)

Base-pair substitution type

Frameshift type

 

 

TA 100

TA1535

TA102

TA98

TA1537

DMSO

130 ± 19

11 ± 3

263 ± 5

28 ± 6

7 ± 2

50

147 ± 12

8 ± 2

255 ± 6

21 ± 6

9 ± 1

158

159 ± 14

11 ± 4

215 ± 35

19 ± 5

7 ± 2

500

141 ± 15

10 ± 2

235 ± 9

26 ± 2

6 ± 1

1581

142 ± 14P

11 ± 1P

253 ± 28P

24 ± 7P

5 ± 2P

5000

143 ± 20P

9 ± 3P

231 ± 16P

23 ± 9P

5 ± 2P

Positive controls, –S9

Name

NF

Na-azide

MMC

4-NPDA

4-NPDA

Concentrations (μg/plate)

0.2

10

0.2

0.5

10

Mean No. of colonies/plate

(average of 3 ± SD)

387 ± 16

527 ± 24

517 ± 16

137 ± 5

88 ± 8

+

DMSO

184 ± 4

12 ± 2

340 ± 39

35 ± 8

12 ± 3

+

50

187 ± 6

12 ± 4

333 ± 13

31 ± 6

12 ± 4

+

158

198 ± 20

11 ± 3

348 ± 8

33 ± 7

10 ± 1

+

500

176 ± 8

13 ± 5

311 ± 8

30 ± 6

10 ± 1

+

1581

156 ± 12P*

10 ± 5P

322 ± 45P

24 ± 5P

6 ± 2P*

+

5000

151 ± 8P*

7 ± 3P*

300 ± 27P

27 ± 11P

4 ± 1P*

Positive controls, +S9

Name

2AA

2AA

2AA

2AA

2AA

Concentrations (μg/plate)

3

3

3

3

3

Mean No. of colonies/plate

(average of 3 ± SD)

2434 ± 260

149 ± 26

847 ± 80

1340 ± 110

446 ± 46

 

NF = nitrofurantoin

Na-azide = sodium azide

MMC = mitomycin C

4-NPDA = 4-nitro-1,2-phenylene diamine

2AA = 2-aminoanthracene

P = precipitate

* = cytotoxicity

 

 

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

 

With or without S9-Mix

Test substance concentration

(μg/plate)

Mean number of revertant colonies per plate

(average of 3 plates ± Standard deviation)

Base-pair substitution type

Frameshift type

 

 

TA 100

TA1535

TA102

TA98

TA1537

DMSO

131 ± 5

12 ± 2

266 ± 4

16 ± 3

8 ± 1

50

112 ± 16

9 ± 1

268 ± 6

17 ± 2

6 ± 1

158

113 ± 17

9 ± 1

266 ± 13

13 ± 4

6 ± 1

500

111 ± 12

8 ± 1

263 ± 12

15 ± 4

7 ± 3

1581

104 ± 14

8 ± 2

222 ± 28

16 ± 7

6 ± 2

5000

99 ± 3P

7 ± 1P

254 ± 16P

12 ± 5P

0 ± 0BP

Positive controls, –S9

Name

NF

Na-azide

Cumene

4-NPDA

4-NPDA

Concentrations (μg/plate)

0.2

10

0.2

0.5

10

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

560 ± 17

701 ± 27

503 ± 27

137 ± 5

102 ± 6

+

DMSO

199 ± 6

12 ± 0

354 ± 16

27 ± 0

8 ± 2

+

50

211 ± 9

11 ± 3

320 ± 22

25 ± 5

10 ± 1

+

158

197 ± 33

12 ± 3

334 ± 31

22 ± 4

6 ± 1

+

500

100 ± 14

9 ± 2

267 ± 21

16 ± 3

5 ± 2

+

1581

73 ± 5B

7 ± 2

178 ± 5B

10 ± 4B

1 ± 1BP

+

5000

71 ± 14BP

3 ± 2BP

156 ± 22BP

1 ± 2BP

0 ± 0BP

Positive controls, +S9

Name

2AA

2AA

2AA

2AA

2AA

Concentrations (μg/plate)

3

3

3

3

3

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

2021 ± 235

176 ± 19

594 ± 47

1719 ± 168

178 ± 36

 

NF = nitrofurantoin

Na-azide = sodium azide

Cumene = cumene hydroperoxide

4-NPDA = 4-nitro-1,2-phenylene diamine

2AA = 2-aminoanthracene

B = background lawn reduced

P = precipitate

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative