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Genetic toxicity: in vitro

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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:
2012 - 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
Alcohols, C16-18 (even numbered, C18-unsatd.), ethoxylated, and alcohols C20-22 (even numbered), sulfates, ammonium salts
EC Number:
938-445-6
Molecular formula:
C16H37NO4S - C34H72NO12S
IUPAC Name:
Alcohols, C16-18 (even numbered, C18-unsatd.), ethoxylated, and alcohols C20-22 (even numbered), sulfates, ammonium salts
Test material form:
solid: compact
Details on test material:
- Name of test material (as cited in study report): DE07_2012_001_PLSW
- Physical state: solid
- Lot/Batch No. PU20740016, PU20740019
- Expiration date of lot/batch: 14/03/2014
- Storage condition of test materilal: common room light, room temperature
Specific details on test material used for the study:
- Name of test material (as cited in study report): DE07_2012_001_PLSW
- Substance type: Alkylsulfate/alkylethersulfate
- Chemical name: Alcohols, C16-18 (even numbered, C18-unsatd.), ethoxylated, and alcohols C20-22 (even numbered), sulfates, ammonium salts
- CAS: n/a
- Physical state: ivory paste-like solid at 20 °C
- Batch No.: PU20740016, PU20740019
- Purity: 100 % (UVCB)
- Storage condition of test material: Room temperature, protected from light
- Stability: stable under test conditions

Method

Target gene:
his- / his+ reversions
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
S9-mix (rat liver, induced with phenobarbital and 5,6-benzoflavone)
Test concentrations with justification for top dose:
Dose-finding study:
3.16, 10.0, 31.6, 100, 316, 2500, 5000 μg/plate (TA 98, TA 100)
Doses: 3.16, 10.0, 31.6, 100, 316, 2500, 5000 μg/plate

Main study:
-S9 mix: 0, 31.6, 100, 316, 1000, 2500, 5000 μg/plate (all strains)
+S9 mix: 0, 31.6, 100, 316, 1000, 2500, 5000 μg/plate (all strains)

The test item was tested in a pre-experiment eith the following concentrations:
3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate

The test item concentration to be applied in the main experiments were choosen according to the results of the pre-experiment. 5000 µg/plate was selected as the maximum concentration. The concentration range covered two logarithmic decades. Three independent experiments were performed with the following concentrations:
3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate
Vehicle / solvent:
dist. Water
Controls
Untreated negative controls:
yes
Remarks:
dist. water
Negative solvent / vehicle controls:
yes
Remarks:
dist. water
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-Nitro-o-phenylenediamine, 2-aminoanthracene
Details on test system and experimental conditions:
Procedures:
Pre-incubation method
Plate incorporation method

For each strain and dose level, including controls, three plates were used.

Number of replicates: 3

Positive controls:
-S9 mix; Sodium azide (TA 100, TA 1535), 4-nitro-o-phenylene-diamine (TA 98, TA 1537), methylmethanesulfonate (TA 102)
+S9 mix; 2-Aminoanthracene (for all strains)
Evaluation criteria:
To be considered positive if:
- a clear and dose related increase in the number of revertants accours and/or
- a biologically relevant positive response for at least one of the dose groups occurs in at least one tester strain with or without metabolic activation.

A biologically relevant increase is described as follows;

- if in tester strains TA 98, TA 100 and TA 102 the number of reversions is at least twice as high
- if in tester strains TA 1535 and TA 1537 the number of reversions is at least three times higher than the reversion rate of the solvent control.
Statistics:
no data

Results and discussion

Test results
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Remarks:
a slight toxic effect oberved in tester strain TA 1535 at a concentration of 5000 µg/plate (+ S9 mix)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
No biological relevant increases in revertant colony numbers of any of the five strains were observed in the test with either the non-activation method (-S9 mix) or the activation method (+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 Revertants per plate (mean of 3 plates, data of experiment 1 of 3 shown)

Concentration µg/plate

TA 98

TA100

TA1535

TA1537

TA102

- S9

+ S9

- S9

+ S9

- S9

+ S9

- S9

+ S9

- S9

+ S9

Negative control

25

32

106

128

5

6

7

9

309

177

31.6

19

33

108

113

9

11

9

12

239

220

100.0

14

30

116

119

8

6

14

14

256

191

316

22

30

125

122

8

5

7

11

252

182

1000

20

24

127

125

8

5

9

7

297

158

2500

27

28

115

117

12

5

11

13

268

207

5000

22

22

114

105

17

4

17

11

341

193

4-NOPD/NaN3 (10µg)/MMS ()* 382  - 711 387 30  - 1784
2-AA (2.5 µg)**  -  2500  -  2004  -  66  -  210  - 379 

* positive control with 4-Nitro-o-phenylene-diamine (4-NOPD, 10 µg (TA 98) and 40 µg (TA 1537)), sodium azide (NaN3, 10 µg (TA 100, TA 1535)) or Methylmethansulfonate (MMS, 1µL (TA 102))

** positive control with 2-Aminoanthracene (2-AA, 2.5 µg (TA 98, TA 100, TA 1535, TA 1537) and 10 µg (TA 102))

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
The test material was considered to be non-mutagenic to Salmonella typhimurium under the conditions of the test.
Executive summary:

The test item did not increase the reverse mutation rate in histidine dependent bacterial strains of Salmonella typhimurium in the presence or absence of metabolic activation at concentrations up to and including 5000 µg/plate. It is concluded that the test substance is negative for mutagenicity in bacteria under the conditions of the test.