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:
01 - 11 June 2015
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The study was conducted according to the appropriate OECD test guideline and in compliance with GLP. The study is considered to be an addendum to a previous study (880075, 1988) where no E. coli strain was tested. Therefore, the test material was only tested in the E. coli strain.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
(1997)
Deviations:
yes
Remarks:
The study was only conducted in one tester strain and is considered to be an addendum to a previous genetic toxicity study
GLP compliance:
yes (incl. certificate)
Remarks:
Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht Rheinland Pfalz
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: solid
Details on test material:
- Name of test material (as cited in study report): sodium dodecyl sulphate
- Physical state: white solid
- Analytical purity: 97.8%
- Composition of test material, percentage of components: 97.8 area-%; water: 0.91%
- Lot/batch No.: 0012897906
- Stability under test conditions: stability of the test substance under storage conditions is guaranteed until 23 Nov 2016
- Storage condition of test material: room temperature

Method

Target gene:
trp operon
Species / strain
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
- Type and identity of media:
soft agar (overlay agar), which consists of 100 mL agar (0.8% (w/v) agar + 0.6% (w/v) NaCl) and 10 mL amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM tryptophan).
- Properly maintained: yes
Additional strain / cell type characteristics:
other: Escherichia coli WP2 uvrA (deficiency of excision repair), (trp-)
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented postmitochondrial fraction (S9-mix), prepared from the livers of rats treated with phenobarbital and β-naphthoflavone.
Test concentrations with justification for top dose:
Experiment I: Plate incorporation
- 33, 100, 333, 1000, 2600 and 5200 μg/plate (with and without metabolic activation)

Experiment II: Pre-incubation
- 33, 100, 333, 1000, 2600 and 5200 μg/plate (with and without metabolic activation)
Vehicle / solvent:
- Vehicle used: water
- Justification for choice of solvent/vehicle: Due to the good solubility of the test substance in water, water was used as vehicle.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: + S9-mix: 2-aminoanthracene (2-AA): 60 µg/plate (in DMSO); -S9-mix: 4-nitroquinoline-N-oxide (4-NQO): 5 µg/plate (in DMSO)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); pre-incubation

DURATION
- Pre-incubation period: 20 min
- Expression time (cells in growth medium): 48 - 72 h

NUMBER OF REPLICATIONS: 3 test plates per dose or per control

DETERMINATION OF CYTOTOXICITY
- Method: decrease in the number of revertants (factor ≤ 0.6), clearing or diminution of the background lawn
Evaluation criteria:
Individual plate counts, the mean number of revertant colonies per plate and the standard deviations were given for all dose groups as well as for the positive and negative (vehicle) controls in all experiments. In general, six doses of the test substance are tested with a maximum of 5 mg/plate, and triplicate plating is used for all test groups at least in the 1st Experiment. Dose selection and evaluation as well as the number of plates used in repeat studies or further experiments are based on the findings of the 1st Experiment.
Statistics:
For each replicate plating, the mean and standard deviation of the number of revertants per plate were calculated and are reported.

Results and discussion

Test results
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
≥ 2600 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
COMPARISON WITH HISTORICAL CONTROL DATA:
In this study with and without S9 mix, the number of revertant colonies in the negative controls was within the range of the historical negative control data for the tester strain. In addition, the positive control substances both with and without S9 mix induced a significant increase in the number of revertant colonies within the range of the historical positive control data.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
A cytotoxic effect (reduced trp (-) background growth, decrease in the number of trp (+) revertants) was observed in the standard plate test at a concentration of 5200 μg/plate. In the pre-incubation assay cytotoxicity (reduced trp (-) background growth, decrease in the number of trp (+) revertants) was observed depending on the test conditions from about 2600 μg/plate onward. Furthermore, no test substance precipitation was found with and without S9 mix.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Results Experiment I (plate incorporation):

With or without S9-Mix

Test substance concentration

(μg/plate)

Mean number of revertant colonies per plate (average of 3 plates ± Standard deviation)

Base-pair substitution type

WP2 uvr A

0

19.3 ± 2.3

33

16.3 ± 4.2

100

17.0 ± 4.4

333

17.7 ± 5.1

1000

20.3 ± 2.3

2600

13.7 ± 0.6

5200 B

4.7 ± 0.6

Positive controls, –S9-Mix

Name

4-NQO

Concentrations (μg/plate)

5

Mean No. of colonies/plate

(average of 3 ± SD)

1019 ± 7.5

+

0

22.7 ± 2.3

+

33

23.7 ± 6.0

+

100

20.7 ± 5.9

+

333

23.7 ± 4.0

+

1000

20.7 ± 0.6

+

2600

22.0 ± 2.6

+

5200 B

6.0 ± 1.7

Positive controls, +S9-Mix

Name

2-AA

Concentrations (μg/plate)

60

Mean No. of colonies/plate

(average of 3 ± SD)

96 ± 2.6

4-NQO: 4-nitroquinoline-N-oxide

2-AA: 2-aminoanthracene

B: Reduced background growth

Table 2: Results Experiment II (pre-incubation):

With or without S9-Mix

Test substance concentration

(μg/plate)

Mean number of revertant colonies per plate (average of 3 plates ± Standard deviation)

Base-pair substitution type

WP2 uvr A

0

21.0 ± 7.5

33

20.0 ± 3.5

100

18.0 ± 4.6

333

18.3 ± 4.7

1000

19.3 ± 6.4

2600 B

8.3 ± 1.2

5200 B

6.3 ± 3.1

Positive controls, –S9-Mix

Name

4-NQO

Concentrations (μg/plate)

5

Mean No. of colonies/plate

(average of 3 ± SD)

374 ± 23.3

+

0

19.7 ± 2.5

+

33

26.3 ± 1.5

+

100

28.0 ± 4.0

+

333

24.0 ± 4.0

+

1000

20.0 ± 1.0

+

2600

14.0 ± 6.0

+

5200 B

4.3 ± 3.2

Positive controls, +S9-Mix

Name

2-AA

Concentrations (μg/plate)

60

Mean No. of colonies/plate

(average of 3 ± SD)

92 ± 0

4-NQO: 4-nitroquinoline-N-oxide

2-AA: 2-aminoanthracene

B: Reduced background growth

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative