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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:
weight of evidence
Study period:
19 July 2017 - 28 August 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

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
Qualifier:
according to guideline
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Version / remarks:
cf. Sofuni, 1993
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Tetrachloro-μ-hydroxy(μ-methacrylato-O:O')dichromium
EC Number:
239-147-2
EC Name:
Tetrachloro-μ-hydroxy(μ-methacrylato-O:O')dichromium
Cas Number:
15096-41-0
Molecular formula:
C10H23Cl4Cr2O5
IUPAC Name:
tetrachloro-μ-hydroxy(μ-methacrylato-O:O')dichromium
Test material form:
liquid

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
ß-Naphthoflavone/Phenobarbital induced rat liver S9
Test concentrations with justification for top dose:
5, 15.8, 50, 158, 500, 1580 and 5000 µg/plate
top dose: maximum recommended concentration according to OECD guideline 471
Vehicle / solvent:
- Vehicle/solvent used: ultrapure water
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
sodium azide
other: daunomycin, 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 2 days

NUMBER OF REPLICATIONS: 3 (two independent experiments)

DETERMINATION OF CYTOTOXICITY
- Method: counting numbers of revertant colonies
Evaluation criteria:
A test material was to be defined as positive or mutagenic in this assay if
- the assay is considered valid and
- a biologically relevant increase in the mean number of revertants above a threshold of 2¬fold (TA 98, TA 100, WP2 uvrA) or 3-fold (TA 1535, TA 1537) as compared to the concurrent negative controls is observed
- an increase exceeding the threshold at only one concentration is considered as biologically meaningful if reproduced in a second independent experiment - a concentration-dependent increase is considered biologically meaningful if the threshold is exceeded at more than one concentration

A test material is defined as negative or non-mutagenic in this assay if
- the assay is considered valid and
- none of the above mentioned criteria are met

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: at 5000 µg/plate, only at the beginning of the experiment

Any other information on results incl. tables

Table 1: Summary 1st Series

Metabolic Activation

Test Material

Concentr. [µg/plate]

Revertants per plate (Mean ± SD)

TA 98

TA 100

TA 1535

TA 1537

WP2 uvrA

Without Activation

H2O

 

25 ± 6

125 ± 9

17 ± 3

8 ± 2

29 ± 6

Test item

5.00

26 ± 5

128 ± 9

22 ± 5

7 ± 4

34 ± 5

15.8

36 ± 9

139 ± 19

18 ± 1

10 ± 5

37 ± 6

50.0

22 ± 3

137 ± 14

15 ± 3

9 ± 3

36 ± 5

158

20 ± 6

134 ± 21

18 ± 6

8 ± 1

41 ± 5

500

25 ± 4

131 ± 14

20 ± 2

9 ± 4

39 ± 6

1580

27 ± 4

145 ± 9

19 ± 6

12 ± 4

33 ± 1

5000

22 ± 7B

171 ± 13B

22 ± 4B

6 ± 1B

40 ± 8B

DAUN

1.00

226 ± 27

 

 

 

 

NaN3

2.00

 

1725 ± 52

906 ± 14

 

 

9-AA

50.0

 

 

 

1235 ± 401

 

NQO

2.00

 

 

 

 

1975 ± 85

With Activation

H2O

 

41 ± 5

138 ± 6

19 ± 3

10 ± 4

35 ± 6

Test item

5.00

28 ± 1C

143 ± 21

19 ± 3

11 ± 3

43 ± 3

15.8

29 ± 10

149 ± 9

18 ± 15

10 ± 0

33 ± 3

50.0

35 ± 5

136 ± 14

14 ± 7

9 ± 6

49 ± 4

158

31 ± 4

152 ± 6

18 ± 4

9 ± 3

36 ± 6

500

35 ± 5

192 ± 19

16 ± 4

8 ± 3

37 ± 1

1580

31 ± 8

170 ± 22

20 ± 1

4 ± 2

47 ± 4

5000

25 ± 4B

146 ± 19B

15 ± 1B

4 ± 3B

33 ± 2B

2-AA

2.00

969 ± 226

1720 ± 57

 

 

 

2-AA

5.00

 

 

153 ± 28

351 ± 18

 

2-AA

10.0

 

 

 

 

377 ± 34

 Key to Positive Controls

NaN3    Sodium azide

2-AA     2-Aminoanthracene

9-AA     9-Aminoacridine

DAUN   Daunomycin

NQO     4-Nitroquinoline-N-oxide

Key to Plate Postfix Codes

B          Precipitation at beginning of experiment

C          Contaminated

 

Table 1: Summary 2nd Series

Metabolic Activation

Test Material

Concentr. [µg/plate]

Revertants per plate (Mean ± SD)

TA 98

TA 100

TA 1535

TA 1537

WP2 uvrA

Without Activation

H2O

 

31 ± 11

125 ± 6

20 ± 5

8 ± 3

44 ± 6

Test item

158

22 ± 5

127 ± 3

29 ± 5

8 ± 1

49 ± 4

500

37 ± 4

152 ± 7

21 ± 4

7 ± 2

33 ± 4

1580

24 ± 7

125 ± 14

27 ± 5

8 ± 3

47 ± 9

2810

24 ± 2

135 ± 17

21 ± 6

8 ± 1

50 ± 6

5000

20 ± 3B

138 ± 11B

26 ± 11B

7 ± 4B

51 ± 11B

DAUN

1.00

265 ± 4

 

 

 

 

NaN3

2.00

 

1721 ± 43

989 ± 2

 

 

9-AA

50.0

 

 

 

804 ± 190

 

NQO

2.00

 

 

 

 

1988 ± 166

With Activation

H2O

 

40 ± 3

122 ± 12

17 ± 4

11 ± 5

47 ± 11

Test item

158

30 ± 5

140 ± 17

17 ± 5

9 ± 3

51 ± 9

500

39 ± 9

157 ± 15

19 ± 5

11 ± 3

47 ± 10

1580

35 ± 6

154 ± 12

15 ± 8

11 ± 5

44 ± 5

2810

25 ± 3

142 ± 12

11 ± 6

4 ± 2

41 ± 4

5000

29 ± 3B

128 ± 16B

11 ± 8B

8 ± 2B

43 ± 4B

2-AA

2.00

349 ± 55

781 ± 57

 

 

 

2-AA

5.00

 

 

162 ± 21

349 ± 59

 

2-AA

10.0

 

 

 

 

309 ± 7

 Key to Positive Controls

NaN3    Sodium azide

2-AA     2-Aminoanthracene

9-AA     9-Aminoacridine

DAUN   Daunomycin

NQO     4-Nitroquinoline-N-oxide

Key to Plate Postfix Codes

B           Precipitation at beginning of experiment

 

 

Table 3: Historical Data

Negative Controls

Strain

TA 98

TA 100

TA 1535

TA 1537

WP2 uvrA

S9 Mix

Without

With

Without

With

Without

With

Without

With

Without

With

Compound

Solvent

Solvent

Solvent

Solvent

Solvent

Solvent

Solvent

Solvent

Solvent

Solvent

Total Plates

234

234

229

234

170

170

190

190

190

189

Number of Values

45

45

44

45

29

29

34

34

34

34

Minimum

19

20

94

90

15

6

4

6

22

28

Maximum

52

58

152

151

38

28

11

15

39

45

Mean

38

44

120

125

25

21

8

9

30

37

Standard Deviation

7.3

7.6

12.4

12.5

5.8

4.2

1.6

1.9

5.2

4.4

Positive Controls

Strain

TA 98

TA 100

TA 1535

TA 1537

WP2 uvrA

S9 Mix

Without

With

Without

With

Without

With

Without

With

Without

With

Compound

DAUN

2-AA

NaN3

2-AA

NaN3

2-AA

9-AA

2-AA

NQO

2-AA

Total Plates

117

117

115

117

85

85

95

95

95

95

Number of Values

45

45

44

45

29

29

34

34

34

34

Minimum

84

112

821

437

351

43

247

88

872

187

Maximum

779

3015

2376

3429

2149

758

1485

705

2275

696

Mean

285

816

1524

1411

869

186

673

299

1704

375

Standard Deviation

155.6

532.0

238.7

736.6

279.1

126.1

291.1

174.9

337.2

140.5

 

Applicant's summary and conclusion

Conclusions:
The test substance was considered to be non-mutagenic with and without metabolic activation in bacteria.
Executive summary:

The present study was conducted to investigate the test material for its mutagenic potential in a bacterial reverse gene mutation assay in the absence and presence of a rat liver metabolizing system (S9 mix).

The investigations for the mutagenic potential of the test item were performed using Salmonella typhimurium tester strains TA 98, TA 100, TA 1535 and TA 1537 and Escherichia coli WP2 uvrA. The plate incorporation test with and without addition of liver S9 mix from rats pretreated with (3-Naphthoflavone/Phenobarbital was used. In this study, two experimental series were performed. The S9 mix used contained 10% S9 in the 1st and 20% S9 in the 2nd series, respectively.

Solvent and positive control treatments were included for all strains. The mean numbers of revertant colonies were all within acceptable ranges for solvent control treatments, or were clearly elevated by positive control treatments, thus, showing the expected reversion properties of all strains and good metabolic activity of the S9 mix used.

Following treatment of all bacteria tester strains with the test item in the absence and presence of S9 mix, no relevant increases in revertant numbers were observed. It was concluded that with and without addition of S9 mix as the exogenous metabolizing system, the test item was not mutagenic under the experimental conditions described.