Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental Starting Date (first day of data collection): 17 August 2018 and Experimental Start Date (first day test substance administered to test system): 21 August 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:
21 July 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:
liquid
Details on test material:
Name: XTJ-785, experimental
Lot No.: 9570-2-6738
CAS No.: Not listed
Purity: >92% C1214 alcohol, propoxylated, aminated, ethyoxylated
Expiry Date: No date established

Method

Target gene:
The tester strains used were the Salmonella typhimurium histidine auxotrophs TA98, TA100, TA1535 and TA1537 as described by Ames et al. (1975) and Escherichia coli WP2 uvrA as described by Green and Muriel (1976).
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 preliminary toxicity assay, the dose levels tested were 1.50, 5.00, 15.0, 50.0, 150, 500, 1500 and 5000 µg per plate. The top dose is determined per the guidelines and solubility of the test substance. Based upon these results, the maximum dose tested in the confirmatory mutagenicity assay was 500 or 1500 µg per plate. In the confirmatory mutagenicity assay, the dose levels tested were 1.50, 5.00, 15.0, 50.0, 150 and 500 µg per plate in the absence of S9 activation and 5.00, 15.0, 50.0, 150, 500 and 1500 µg per plate in the presence of S9 activation.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO (Dimethyl sulfoxide)
- Justification for choice of solvent/vehicle: DMSO was the vehicle of choice based on the solubility of the test substance and compatibility with the target cells. The test substance formed a clear solution in DMSO at a concentration of approximately 500 mg/mL in the solubility test conducted at BioReliance.

- Suplier: Sigma-Aldrich
- Lot number: SHBH9867
- Purity: 99.92%
- Expiration date: Aug 2021
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
vehicle control
Negative solvent / vehicle controls:
yes
Remarks:
solvent used to prepare positive control (DMSO)
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
2-aminoanthracene: positive control for testing with S9 metabolic activation.
Untreated negative controls:
yes
Remarks:
vehicle control
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
2-nitrofluorene: positive control for testing of TA98 without metabolic activation.
Untreated negative controls:
yes
Remarks:
vehicle control
Negative solvent / vehicle controls:
yes
Remarks:
sterile water
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
sodium azide: positive control for testing of TA100 and TA1535 without metabolic activation.
Untreated negative controls:
yes
Remarks:
vehicle control
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
9-aminoacridine: positive control for testing of TA1537 without metabolic activation.
Untreated negative controls:
yes
Remarks:
vehicle control
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
methylmethanesulfonate: positive control for testing of WP2 uvra without metabolic activation.
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 preliminary toxicity assay; 3 in the mutagenicity assay.

NUMBER OF CELLS EVALUATED: 1.6 to 3.9 x10E8 cells per plate.

DETERMINATION OF CYTOTOXICITY
- Method: The condition of the bacterial background lawn was evaluated for evidence of test substance toxicity by using a dissecting microscope. Precipitate was evaluated after the incubation period by visual examination without magnification. Toxicity and degree of precipitation were scored relative to the vehicle control plate using the codes shown in the following table. As appropriate, colonies were enumerated either by hand or by machine.

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:
cytotoxicity
Remarks:
toxicity observed at >= 50µg per plate w/o metabolic activation and toxicty observed at >= 500 µg per plate with metabolic activation.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
toxicity observed at >= 150µg per plate w/o metabolic activation and toxicty observed at >= 500 µg per plate with metabolic activation.
Vehicle controls validity:
valid
Untreated negative controls validity:
not valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
toxicity observed at >= 150µg per plate w/o metabolic activation and toxicty observed at >= 500 µg per plate with metabolic activation.
Vehicle controls validity:
valid
Untreated negative controls validity:
not valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
toxicity observed at >= 150µg per plate w/o metabolic activation and toxicty observed at >= 500 µg per plate with metabolic activation.
Vehicle controls validity:
valid
Untreated negative controls validity:
not valid
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 observed at >= 500µg per plate w/o metabolic activation and toxicty observed at 1500 µg per plate with metabolic activation.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other: precipitate was observed at 1500 µg per plate.

Any other information on results incl. tables

Sterility Results

No contaminant colonies were observed on the sterility plates for the vehicle control, the test substance dilutions or the S9 and Sham mixes.

Tester Strain Titer Results

Experiment

Tester Strain

TA98

TA100

TA1535

TA1537

WP2uvrA

Titer Value (x 109cells per mL)

B1

1.2

1.0

1.6

1.3

2.8

B2

1.3

1.1

1.5

1.3

2.7

Initial Toxicity-Mutation Assay

The maximum dose of 5000 µg per plate was achieved using a concentration of 100 mg/mL and a 50.0 µL plating aliquot. Precipitate was observed beginning at 1500 µg per plate with all conditions. Toxicity was observed as indicated in the following table:

Tester Strains

Without metabolic activation

(µg per plate)

With metabolic activation

(µg per plate)

Toxicity

Toxicity

TA98

≥ 150

≥ 500

TA100

≥ 150

≥ 500

TA1535

≥ 50.0

≥ 500

TA1537

≥ 150

≥ 500

WP2uvrA

≥ 500

≥ 1500

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 1.50, 5.00, 15.0, 50.0, 150 and 500 µg per plate in the absence of S9 activation and 5.00, 15.0, 50.0, 150, 500 and 1500 µg per plate in the presence of S9 activation.

Precipitate was observed at 1500 µg per plate with all tester strains in the presence of S9 activation. Toxicity was observed as indicated in the following table:

Tester Strains

Without metabolic activation

(µg per plate)

With metabolic activation

(µg per plate)

Toxicity

Toxicity

TA98

≥ 150

≥ 500

TA100

≥ 150

≥ 500

TA1535

≥ 50.0

≥ 500

TA1537

≥ 150

≥ 500

WP2uvrA

≥ 500

1500

No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation.

 

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

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, Alcohols, C12-14 alkyl ethers, propoxylated, aminated, ethoxylated 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, Alcohols, C12-14 alkyl ethers, propoxylated, aminated, ethoxylated, was tested to evaluate its mutagenic potential by measuring its ability to induce reverse mutations at selected loci of several strains ofSalmonella typhimuriumand at the tryptophan locus ofEscherichia colistrain 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. Precipitate was observed beginning at 1500 µg per plate with all conditions. Toxicity was observed beginning at 50.0, 150, 500 or 1500 µg per plate with all conditions. 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 500 or 1500 µg per plate.

In the confirmatory mutagenicity assay, the dose levels tested were 1.50, 5.00, 15.0, 50.0, 150 and 500 µg per plate in the absence of S9 activation and 5.00, 15.0, 50.0, 150, 500 and 1500 µg per plate in the presence of S9 activation. Precipitate was observed at 1500 µg per plate with all tester strains in the presence of S9 activation. Toxicity was observed beginning at 50.0, 150, 500 or 1500 µg per plate with all conditions. No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation.

These results indicate Alcohols, C12-14 alkyl ethers, propoxylated, aminated, ethoxylated was negative for the ability to induce reverse mutations at selected loci of several strains ofSalmonella typhimuriumand at the tryptophan locus ofEscherichia colistrain WP2 uvrA in the presence and absence of an exogenous metabolic activation system.