Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20 March 2017 - 13 April 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:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21 July 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
31 May 2008
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Remarks:
paste
Details on test material:
Name as cited in report: DDDA TEA salt
Physical appearance: beige to pale yellow paste
Storage conditions: at room temperature
Specific details on test material used for the study:
Purity correction factor: 1.25
Solubility in vehicle: not indicated
Stability in vehicle: not indicated
Test substance is irritant or corrosive

Method

Target gene:
- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene
Species / strainopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9-mix induced by Aroclor 1254 (5 and 10%)
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9-mix induced by Aroclor 1254 (5 and 10%)
Test concentrations with justification for top dose:
Dose-range finding test (TA100 and WP2 uvrA): 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate (reported as part of experiment 1)
Experiment 1 (TA1535, TA1537 and TA98): 52, 164, 512, 1600 and 5000 μg/plate
Experiment 2 (all tester strains): 492, 878, 1568, 2800 and 5000 μg/plate

The top dose used is according to OECD guideline 471.
Vehicle:
- Vehicle used: dimethyl sulfoxide
- Justification for choice of solvent/vehicle: the vehicle has been accepted and approved by authorities and international guidelines
Controlsopen allclose all
Negative controls:
yes
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Without S9
Negative controls:
yes
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: ICR-191
Remarks:
Without S9
Negative controls:
yes
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
Without S9
Negative controls:
yes
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
Without S9
Negative controls:
yes
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
Without S9
Positive control substance:
other: 2-aminoanthracene
Remarks:
With 5 and 10% S9
Negative controls:
yes
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
With 5% S9
Negative controls:
yes
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
With 5 and 10% S9
Negative controls:
yes
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
With 5% S9
Negative controls:
yes
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
With 10% S9
Negative controls:
yes
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
with 5 and 10% S9
Negative controls:
yes
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-acetylaminofluorene
Remarks:
with 10% S9
Details on test system and conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: no
- Exposure duration: 48 ± 4 h (in the dark at 37.0 ± 1.0 °C)

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain. Two independent experiments were conducted: in the first experiment 5% S9 was used and in the second experiment 10% S9 was used.

METHODS OF SLIDE PREPARATION: Top agar in top agar tubes was melted by heating to 45 ± 2°C. Fresh bacterial culture, dilution of the test item in DMSO and either S9-mix or 0.1 M phosphate buffer were added to the molten top agar. The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate.

DETERMINATION OF CYTOTOXICITY
- Method: the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies.

OTHER EXAMINATIONS:
- The presence of precipitation of the test compound on the plates was determined.
Evaluation criteria:
DECISION CRITERIA:
A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is not greater than two times the concurrent vehicle control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three times the concurrent vehicle control.
b) The negative response should be reproducible in at least one follow-up experiment.

A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is greater than two times the concurrent vehicle control, or the total number of revertants in tester strains TA1535, TA1537, TA98 is greater than three times the concurrent vehicle control.
b) In case a follow up experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.

In addition to the criteria stated above, any increase in the total number of revertants should be evaluated for its biological relevance including a comparison of the results with the historical control data range.

ACCEPTABILITY CRITERIA:
a) The vehicle control and positive control plates from each tester strain (with or without S9-mix) must exhibit a characteristic number of revertant colonies when compared against relevant historical control data generated at Charles River Den Bosch.
b) The selected dose-range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.
c) No more than 5% of the plates are lost through contamination or some other unforeseen event. If the results are considered invalid due to contamination, the experiment will be repeated.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
At all concentrations tested.
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity:
no, but tested up to limit concentrations
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no, but tested up to limit concentrations
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no, but tested up to limit concentrations
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no, but tested up to limit concentrations
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no, but tested up to limit concentrations
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: no precipitation of the test substance on the plates was observed at the start or at the end of the incubation periods for both experiments.

RANGE-FINDING/SCREENING STUDIES: a range-finding test was conducted as part of the first experiment in tester strains TA100 and WP2 uvr A. No cytotoxicity or mutagenicity was observed.

- Cytotoxicity: in the first experiment, cytotoxicity was only observed in tester strain TA98 in the absence of S9-mix; in all other tester strain, no reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants were observed. In the second mutation assay, there was no reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants at any of the concentrations tested in all tester strains in the absence and presence of S9-mix.
- In strains TA1535 (absence of S9-mix) and TA1537 (absence and presence of S9-mix), fluctuations in the number of revertant colonies below the laboratory historical control data range were observed. However, since no dose-relationship was observed, these reductions
are not considered to be caused by toxicity of the test item. It is more likely these reductions are caused by an incidental fluctuation in the number of revertant colonies.

HISTORICAL CONTROL DATA: see table 1 and 2 in 'any other information on results'.
- The negative control values were within the laboratory historical control data ranges, except the responses for TA1535 and TA1537 in the absence of S9-mix in the first experiment. However since the mean number of revertant colonies showed a characteristic number of revertant colonies (4 and 2 revertant colonies in the tester strains TA1535 and TA1537, respectively) when compared against relevant historical control data (5 and 3 revertant colonies in the tester strains TA1535 and TA1537, respectively), the validity of the test was considered to be not affected.
- The strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Any other information on results incl. tables

Table 1 Historical data of the solvent control

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

TA1535

TA1537

TA98

TA100

WP2uvrA

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

S9-mix

-

+

-

+

-

+

-

+

-

+

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Range

5 - 36

3 - 32

3 – 23

3 – 23

8 - 41

9 - 52

66 - 156

65 - 154

10 – 56

9 - 69

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Mean

12

12

6

8

16

23

100

100

25

31

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

SD

5

4

3

4

5

7

15

16

6

7

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

n

1865

1862

1740

1715

1852

1912

1853

1877

1571

1583

 

 

 

 

 

 

 

 

 

 

 

 

 

 

SD = Standard deviation

n = Number of observations

Historical control data from experiments performed between November 2014 and November 2016.

Table 2 Historical data of the positive control items

 

TA1535

TA1537

TA98

TA100

WP2uvrA

S9-mix

-

+

-

+

-

+

-

+

-

+

Range

125 - 1381

78 - 1058

55 – 1311

55 – 1051

410 – 1995

250 - 1907

554 – 1848

408 - 2651

112 – 1951

85 - 1359

Mean

828

218

686

376

1270

883

892

1352

1165

388

SD

151

109

320

142

338

340

174

342

488

152

n

1875

1829

1560

1716

1766

1851

1820

1857

1506

1557

SD = Standard deviation

n = Number of observations

Historical control data from experiments performed between November 2014 and November 2016.

Applicant's summary and conclusion

Conclusions:
In an AMES test, performed according to OECD guideline 471 and GLP principles, DDDA TEA salt was found not to be mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay, with or without metabolic activation.