Alcohols, C16-18 (even numbered, C18-unsatd.), ethoxylated, and alcohols C20-22 (even numbered), sulfates, ammonium salts

Genetic toxicity: in vitro

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 guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

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

Test material information

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.