Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

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:
18 July 2012-13 December 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study performed under GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report date:
2012

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Acetic acid, 2-sulfo-, mono-C12-14(even numbered)-alkyl esters, sodium salt
EC Number:
939-512-2
Cas Number:
85681-55-6
IUPAC Name:
Acetic acid, 2-sulfo-, mono-C12-14(even numbered)-alkyl esters, sodium salt
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): Lathanol LAL coarse
- Physical state: powder
- Analytical purity: 100% (under REACH)
- Lot/batch No.: 7472548
- Expiration date of the lot/batch: 19 December 2013
- Stability under test conditions: stable
- Storage condition of test material: Controlled room temperature (15-25 °C, < 70 % RH)

Method

Target gene:
Histidine and Tryptophan
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:
S-9 rat liver induced with phenobarbital/β-naphthoflavone
Test concentrations with justification for top dose:
Range-finding test (TA 98, TA100): 5000; 2500; 1000; 316; 100; 31.6 and 10 μg/plate
Main tests: 5000; 1581; 500; 158.1; 50; 15.81, 5 and 1.581 μg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: distilled water
- Justification for choice of solvent/vehicle: Substance is soluble in water
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
methylmethanesulfonate
other: 4-nitro-1,2-phenylenediamine; 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) Experiment 1; preincubation Experiment 2

DURATION
- Preincubation period: 20 minutes
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: Triplicate plates per duplicate experiment

DETERMINATION OF CYTOTOXICITY
- Method: evaluation of background lawn of bacteria
Evaluation criteria:
Criteria for a Positive Response:
A test item is considered mutagenic if:
- a dose–related increase in the number of revertants occurs and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.
An increase is considered biologically relevant if:
- the number of reversions is more than two times higher than the reversion rate of the negative (solvent) control in Salmonella typhimurium TA98, TA100 and Escherichia coli WP2 uvrA bacterial strains;
- the number of reversions is more than three times higher than the reversion rate of the negative (solvent) control in Salmonella typhimurium TA1535 and TA1537 bacterial strains.

According to the OECD guidelines, statistical method may be used as an aid in evaluating the test results. However, statistical significance should not be the only determining factor for a positive response.

Criteria for a Negative Response:
A test article is considered non-mutagenic if it produces neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation.
Statistics:
Not applicable.

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: None
- Effects of osmolality: None
- Evaporation from medium: None
- Water solubility: Soluble
- Precipitation: None
- Other confounding effects:

RANGE-FINDING/SCREENING STUDIES: Range-finder experiment performed to set the dose levels for the two main experiments.

COMPARISON WITH HISTORICAL CONTROL DATA: Yes, all control data within the historical ranges.

ADDITIONAL INFORMATION ON CYTOTOXICITY: Cytotoxicity varied slightly between bacterial strains; more toxic to strains TA1535 and TA1537.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Summary Table of the Initial Mutation Test

 

Concentrations
(µg/plate)

Mean
values of revertants / Mutation factor (MF)

Salmonella typhimuriumtester strains

Escherichia coli

TA98

TA100

TA1535

TA1537

WP2uvrA

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

Untreated control

Mean

24.7

24.0

92.7

111.7

8.0

10.0

7.7

6.3

25.7

35.0

Distilled water control (50µL)

Mean

-

-

82.0

-

4.7

-

-

-

29.3

-

DMSO
control

Mean

23.3

24.3

-

118.7

-

7.3

5.3

6.0

-

27.0

Distilled water control (200µL)

Mean

25.3

28.7

82.0

102.3

8.7

8.3

9.0

8.3

30.0

32.7

5000

Mean

0.0

0.0

0.0

61.0

0.0

0.0

3.3

5.0

21.7

26.0

1581

Mean

0.0

3.0

12.7

60.7

0.0

4.0

2.0

1.0

27.7

31.3

500

Mean

8.0

18.7

38.0

115.3

2.0

8.0

4.0

11.3

30.3

41.7

158.1

Mean

13.7

17.0

73.3

123.3

8.7

7.3

9.0

10.7

27.3

36.7

50

Mean

20.0

17.7

112.0

125.7

6.7

9.0

8.0

8.3

25.7

43.7

15.81

Mean

17.7

20.7

111.0

120.3

8.0

10.0

6.7

5.7

38.7

38.7

5

Mean

17.0

24.3

107.7

113.7

10.3

16.7

10.0

11.0

31.3

36.0

1.581

Mean

20.3

23.3

118.0

122.7

7.7

10.3

7.3

6.7

30.0

38.3

NPD (4µg)

Mean

298.3

-

-

-

-

-

-

-

-

-

2AA (2µg)

Mean

-

2370.7

-

2386.7

-

198.0

-

203.3

-

-

2AA (50µg)

Mean

-

-

-

-

-

-

-

-

-

215.3

SAZ (2µg)

Mean

-

-

1292.0

-

1189.3

-

-

-

-

-

9AA (50µg)

Mean

-

-

-

-

-

-

394.7

-

-

-

MMS (2µL)

Mean

-

-

-

-

-

-

-

-

1048.0

-

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

In conclusion, the test item Lathanol LAL coarse had no mutagenic activity in the applied bacterium tester strains under the test conditions used in this study.
Executive summary:

The test item Lathanol LAL coarse was tested for potential mutagenic activity using the Bacterial Reverse Mutation Assay. The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537) and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2uvrA) in the presence and absence of a post mitochondrial supernatant (S9 fraction) prepared from the livers of phenobarbital/b-naphthoflavone-induced rats. The study included a Preliminary Solubility Test, a Preliminary Range Finding Test (Informatory Toxicity Test), an Initial Mutation Test (Plate Incorporation Method) and a Confirmatory Mutation Test (Pre-Incubation Method). Based on the results of the Solubility Test, the test item was formulated in Distilled water. Concentrations of 5000; 2500; 1000; 316; 100; 31.6 and 10µg/plate were examined in the Range Finding Test. Based on the results of the Range Finding Test, the test item concentrations in theInitial Mutation Test were 5000, 1581, 500, 158.1, 50, 15.81, 5 and 1.581 μg test item/plate. Examined concentrations in the Confirmatory Mutation Test were 5000, 1581, 500, 158.1, 50, 15.81, 5, 1.581 and 0.5μg test item/plate. In the Initial Mutation Test and Confirmatory Mutation Test, none of the observed revertant colony numbers were above the respective biological threshold value. There were no consistent dose-related trends and no indication of any treatment effect. In all test item treated groups, the numbers of revertant colonies were below the threshold of biological relevance when compared with the solvent controls, were within the historical control range and were within the range of normal biological variability of the test system.Inhibitory, cytotoxic effect of the test item was observed in the Initial Mutation Test in all examined bacterial strains without metabolic activation, and in all Salmonella typhimurium strains with metabolic activation. Similar inhibitory, cytotoxic effect of the test item was observed in the Confirmatory Mutation Test in all Salmonella typhimurium strains with and without metabolic activation. The mean values of revertant colonies of the solvent control plates were within the historical control range, the reference mutagens induced the expected increase in the number of revertant colonies, the viability of the bacterial cells was checked by a plating experiment in each test. At least five analyzable concentrations were presented in all strains of the main tests.The tests were considered to be valid.

 

The reported data of this mutagenicity assay show that under the experimental conditions applied the test item did not induce gene mutations by base pair changes or frameshift mutations in the genome of the strains used. In conclusion, the test item Lathanol LAL coarse had no mutagenic activity in the bacterium tester strains under the test conditions used in this study.