Registration Dossier

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:
Experimental Starting Date (first day of data collection): 17 August 2018; Experimental Start Date (first day test substance administered to test system): 21 August 2018; Experimental Completion Date: 13 September 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
OECD Guideline 471 (Genetic Toxicology: Bacterial Reverse Mutation Test), Ninth Addendum to the OECD Guidelines for the Testing of Chemicals, adopted July 21, 1997.
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: crystal powder
Details on test material:
- Synonym (Trade Name): ADDITIVE® 9735
- CAS Number: 102-40-9
- Molecular Formula: C10H14O4
- Molecular Weight: 198 g/mol
- Appearance: White crystal powder
- Expiration date: 30 August 2019
- Purity: 100%

Method

Target gene:
Tester strains TA98 and TA1537 are reverted from histidine dependence (auxotrophy) to histidine independence (prototrophy) by frameshift mutagens. Tester strain TA1535 is reverted by mutagens that cause basepair substitutions. Tester strain TA100 is reverted by mutagens that cause both frameshift and basepair substitution mutations. Specificity of the reversion mechanism in E. coli is sensitive to basepair substitution mutations, rather than frameshift mutations (Green and Muriel, 1976).
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
not applicable
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
not applicable
Additional strain / cell type characteristics:
not applicable
Cytokinesis block (if used):
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9 was used as the metabolic activation system
Test concentrations with justification for top dose:
- In the intitial toxicity mutation assay, the dose levels tested were 1.50 / 5.00 / 15.0 / 50.0 / 150 / 500 / 1500 and 5000 µg per plate.
In the intial toxicity mutation assay, neither precipitate nor toxicity was observed. No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. Based upon these results, the maximum dose tested in the confirmatory mutagenicity assay was 5000 µg per plate.

- In the confirmatory mutagenicity assay, the dose levels tested were 15.0, 50.0, 150, 500, 1500 and 5000 µg per plate.

Vehicle / solvent:
The vehicle used to deliver the test item to the test system was DMSO (Dimethyl sulfoxide).
DMSO was the vehicle of choice based on the solubility of the test substance and compatibility with the target cells.
Controlsopen allclose all
Untreated negative controls:
other: vehicle control
Remarks:
DMSO
Negative solvent / vehicle controls:
yes
Remarks:
DMSO used to prepare positive control
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Untreated negative controls:
other: vehicle control
Remarks:
DMSO
Negative solvent / vehicle controls:
yes
Remarks:
DMSO used to prepare positive control
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Untreated negative controls:
other: vehicle control
Remarks:
DMSO
Negative solvent / vehicle controls:
yes
Remarks:
Sterile water used to prepare positive control
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Untreated negative controls:
other: vehicle control
Remarks:
DMSO
Negative solvent / vehicle controls:
yes
Remarks:
DMSO used to prepare positive control
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Untreated negative controls:
other: vehicle control
Remarks:
DMSO
Negative solvent / vehicle controls:
yes
Remarks:
DMSO used to prepare positive control
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: In agar (plate incorporation)
- Cell density at seeding (if applicable): to ensure that appropriate numbers of bacteria are plated, tester strain culture titers must be greater than or equal to 0.3x10E9 cells/mL

DURATION
- Exposure duration: 48 to 72 hours

NUMBER OF REPLICATIONS: 2 in the initial toxicity-mutation assay; 3 in the confirmatory mutagenicity assay.

NUMBER OF CELLS Analyzed/Culture: 1.0 to 2.8 x10E8 cells per plate.

DETERMINATION OF CYTOTOXICITY
- Method: other: Counting of revertant colony numbers and evaluation of the condition of the bacterial background lawn.
Rationale for test conditions:
not specified
Evaluation criteria:
Evaluation of Test Results
For each replicate plating, the mean and standard deviation of the number of revertants per plate were calculated and are reported.
For the test substance to be evaluated positive, it must cause a dose-related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of test substance as specified below:
Strains TA1535 and TA1537
Data sets were judged positive if the increase in mean revertants at the peak of the dose response was equal to or greater than 3.0-times the mean vehicle control value and above the corresponding acceptable vehicle control range.

Strains TA98, TA100 and WP2 uvrA
Data sets were judged positive if the increase in mean revertants at the peak of the dose response was equal to or greater than 2.0-times the mean vehicle control value and above the corresponding acceptable vehicle control range.
An equivocal response is a biologically relevant increase in a revertant count that partially meets the criteria for evaluation as positive. This could be a dose-responsive increase that does not achieve the respective threshold cited above or a non-dose responsive increase that is equal to or greater than the respective threshold cited. A response was evaluated as negative if it was neither positive nor equivocal.
Statistics:
According to the test guidelines, the biological relevance of the results is the criterion for the interpretation of the results, and a statistical evaluation of the results is not regarded as necessary.

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
Remarks:
DMSO
Untreated negative controls validity:
valid
Remarks:
solvent for positive control preparation
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
Remarks:
DMSO
Untreated negative controls validity:
valid
Remarks:
solvent for positive control preparation
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
Remarks:
DMSO
Untreated negative controls validity:
valid
Remarks:
solvent for positive control preparation
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
Remarks:
DMSO
Untreated negative controls validity:
valid
Remarks:
solvent for positive controp preparation
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:
cytotoxicity
Remarks:
Toxicity as reduction in revertant count was observed at 5000 µg per plate with tester strain WP2 uvrA in the absence of S9 activation
Vehicle controls validity:
valid
Remarks:
DMSO
Untreated negative controls validity:
valid
Remarks:
solvent for positive control preparation
Positive controls validity:
valid
Additional information on results:
Initial toxicity-Mutation assay:
- Neither precipitate not toxicity was observed.
- No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation.


Confirmatory mutagenicity Assay:
- Based upon the results of the initial toxicity-mutation assay, the dose levels selected for the confirmatory mutagenicity assay were 15.0, 50.0, 150, 500, 1500 and 5000 µg per plate.
- No precipitate was observed.
- Toxicity as reduction in revertant count was observed at 5000 µg per plate with tester strain WP2 uvrA in the absence of S9 activation.
- No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation.

Any other information on results incl. tables

Historical control data:

 

Historical Negative and Positive Control Values

2016

revertants per plate

Strain

Control

Activation

None

Rat Liver

Mean

SD

Min

Max

95% CL

Mean

SD

Min

Max

95% CL

TA98

Neg

15

5

6

34

5-25

22

6

8

42

10-34

Pos

198

174

36

1826

 

287

159

47

1916

 

TA100

Neg

90

12

60

146

66-114

94

14

63

181

66-122

Pos

629

159

186

1383

 

620

294

192

3483

 

TA1535

Neg

12

4

3

31

4-20

12

4

3

26

4-20

Pos

541

164

34

1082

 

150

122

27

1114

 

TA1537

Neg

8

3

1

21

2-14

9

3

2

23

3-15

Pos

368

227

21

1791

 

91

90

17

951

 

WP2uvrA

Neg

24

7

7

44

10-38

27

7

8

51

13-41

Pos

336

119

25

876

 

300

111

41

1059

 

SD=standard deviation; Min=minimum value; Max=maximum value; 95% CL = Mean ±2 SD (but not less than zero); Neg=negative control (including but not limited to deionized water, dimethyl sulfoxide, ethanol and acetone); Pos=positive control

Metabolic

Activation

Test

Substance

Dose Level

(µg/plate)

 

Revertant Colony Counts (Mean ±SD) (B1: Initial Toxicity-Mutation Assay)

 

 

 

 

 

 

 

 

 

 

 

 

 

TA98

TA100

TA1535

TA1537

WP2uvrA

 

 

 

 

 

 

 

 

 

Without Activation

DMSO

50.0 µL/plate

 

16 ± 2

87 ± 19

11 ± 1

7 ± 1

29 ± 2

Test Substance

1.50

 

11 ± 3

104 ± 25

11 ± 6

6 ± 5

29 ± 7

 

5.00

 

18 ± 6

97 ± 23

9 ± 0

8 ± 4

32 ± 3

 

15.0

 

12 ± 1

105 ± 0

10 ± 1

11 ± 5

36 ± 4

 

50.0

 

19 ± 3

105 ± 21

10 ± 6

8 ± 2

32 ± 8

 

150

 

10 ± 0

88 ± 1

10 ± 4

4 ± 2

25 ± 0

 

500

 

11 ± 4

96 ± 5

7 ± 3

9 ± 6

37 ± 10

 

1500

 

11 ± 4

100 ± 6

15 ± 2

4 ± 1

26 ± 0

 

5000

 

16 ± 3

97 ± 6

14 ± 1

5 ± 0

21 ± 4

2NF

1.00

 

65 ± 14

 

 

 

 

SA

1.00

 

 

712 ± 28

646 ± 6

 

 

9AAD

75.0

 

 

 

 

657 ± 49

 

MMS

1000

 

 

 

 

 

386 ± 17

With Activation

DMSO

50.0 µL/plate

 

19 ± 1

104 ± 11

10 ± 1

5 ± 0

24 ± 0

Test Substance

1.50

 

23 ± 1

106 ± 11

9 ± 0

7 ± 1

39 ± 6

 

5.00

 

22 ± 0

105 ± 6

10 ± 1

6 ± 1

27 ± 7

 

15.0

 

18 ± 4

106 ± 1

14 ± 1

7 ± 1

28 ± 2

 

50.0

 

23 ± 2

119 ± 15

10 ± 1

8 ± 2

31 ± 1

 

150

 

22 ± 1

108 ± 23

13 ± 0

11 ± 0

29 ± 6

 

500

 

20 ± 5

97 ± 6

10 ± 1

7 ± 0

28 ± 4

 

1500

 

21 ± 2

103 ± 13

11 ± 4

7 ± 1

31 ± 8

 

5000

 

19 ± 7

113 ± 8

12 ± 1

4 ± 4

25 ± 2

2AA

1.00

 

232 ± 15

 

85 ± 1

 

 

2AA

2.00

 

 

840 ± 24

 

33 ± 0

 

2AA

15.0

 

 

 

 

 

302 ± 17

 

 

 

 

 

 

 

 

 

Key to Positive Controls

 

 

 

SA

2AA

9AAD

2NF

MMS

sodium azide

2-aminoanthracene

9-Aminoacridine

2-nitrofluorene

methyl methanesulfonate

 

 

Test Substance:HER (Resorcinol bis-(2-hydroxyethyl) ether, 2-[3-(2-hydroxyethoxy)phenoxy]ethanol)

Metabolic

Activation

Test

Substance

Dose Level

(µg/plate)

 

Revertant Colony Counts (Mean ±SD) (B2: Confirmatory Mutagenicity Assay)

 

 

 

 

 

 

 

 

 

 

 

 

 

TA98

TA100

TA1535

TA1537

WP2uvrA

 

 

 

 

 

 

 

 

 

Without Activation

DMSO

50.0 µL/plate

 

15 ± 3

95 ± 18

14 ± 3

7 ± 1

29 ± 4

Test Substance

15.0

 

13 ± 4

95 ± 12

17 ± 2

6 ± 2

24 ± 6

 

50.0

 

15 ± 5

88 ± 11

14 ± 4

5 ± 1

30 ± 7

 

150

 

16 ± 2

85 ± 15

15 ± 2

5 ± 0

29 ± 6

 

500

 

14 ± 1

83 ± 8

15 ± 3

6 ± 3

20 ± 6

 

1500

 

11 ± 3

75 ± 12

12 ± 1

6 ± 2

21 ± 5

 

5000

 

15 ± 1

81 ± 5

11 ± 3

5 ± 2

13 ± 3

2NF

1.00

 

64 ± 13

 

 

 

 

SA

1.00

 

 

718 ± 73

736 ± 70

 

 

9AAD

75.0

 

 

 

 

444 ± 54

 

MMS

1000

 

 

 

 

 

348 ± 21

 

 

 

 

 

 

 

 

 

With Activation

DMSO

50.0 µL/plate

 

19 ± 3

106 ± 6

8 ± 2

6 ± 2

24 ± 0

Test Substance

15.0

 

27 ± 4

107 ± 16

10 ± 0

9 ± 6

30 ± 2

 

50.0

 

22 ± 1

111 ± 17

16 ± 2

8 ± 2

25 ± 10

 

150

 

21 ± 3

108 ± 4

8 ± 3

6 ± 0

26 ± 1

 

500

 

24 ± 1

102 ± 13

9 ± 4

9 ± 2

29 ± 9

 

1500

 

14 ± 1

99 ± 23

6 ± 2

6 ± 2

25 ± 3

 

5000

 

18 ± 4

96 ± 18

10 ± 2

5 ± 2

20 ± 6

2AA

1.00

 

247 ± 10

 

87 ± 8

 

 

2AA

2.00

 

 

661 ± 40

 

50 ± 13

 

2AA

15.0

 

 

 

 

 

236 ± 27

 

 

 

 

 

 

 

 

 

Key to Positive Controls

 

 

 

SA

2AA

9AAD

2NF

MMS

sodium azide

2-aminoanthracene

9-Aminoacridine

2-nitrofluorene

methyl methanesulfonate

 

 

  Test Substance:HER (Resorcinol bis-(2-hydroxyethyl) ether, 2-[3-(2-hydroxyethoxy)phenoxy]ethanol)

Applicant's summary and conclusion

Conclusions:
All criteria for a valid study were met as described in the protocol. The results of the Bacterial Reverse Mutation Assay indicate that, under the conditions of this study, the test item did not cause a positive mutagenic response with any of the tester strains in either the presence or absence of Aroclor induced rat liver S9.
Executive summary:

The test substance, the test item, was tested to evaluate its mutagenic potential by measuring its ability to induce reverse mutations at selected loci of several strains of Salmonella typhimurium and at the tryptophan locus of Escherichia colis train WP2 uvrA in the presence and absence of an exogenous metabolic activation system. Dimethyl sulfoxide (DMSO) was used as the vehicle

In the initial toxicity-mutation assay, the dose levels tested were 1.50, 5.00, 15.0, 50.0, 150, 500, 1500 and 5000 µg per plate. Neither precipitate nor toxicity was observed. No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. Based upon these results, the maximum dose tested in the confirmatory mutagenicity assay was 5000 µg per plate.

In the confirmatory mutagenicity assay, the dose levels tested were 15.0, 50.0, 150, 500, 1500 and 5000 µg per plate. No precipitate was observed. Toxicity as reduction in revertant count was observed at 5000 µg per plate with tester strain WP2 uvrA in the absence of S9 activation. No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation.

These results indicate that the test item was negative for the ability to induce reverse mutations at selected loci of several strains of Salmonella typhimurium and at the tryptophan locus ofEscherichia colistrain WP2 uvrA in the presence and absence of an exogenous metabolic activation system.