Fatty acids, C8-14 (even numbered) methyl-2-sulfoethyl esters, sodium 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:

19 January 2011 to 10 February 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Refer to read-across justification document for SCMI in section 13

Cross-reference

Reason / purpose for cross-reference:

assessment report

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

(1997)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

(2008)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Sodium 2-(dodecanoyloxy)propane-1-sulfonate

EC Number:

700-150-3

Cas Number:

156572-81-5

Molecular formula:

C15H29NaO5S

IUPAC Name:

Sodium 2-(dodecanoyloxy)propane-1-sulfonate

Details on test material:

- Name of test material (as cited in study report): Sodoium lauroyl methyl isethionate, CAS No 156572-81-5
- Physical state: Off white solid
- Analytical purity: >80%
- Impurities (identity and concentrations):
- Purity test date: Not stated
- Lot/batch No.: Not supplied
- Expiration date of the lot/batch: 15 December 2011
- Stability under test conditions: Assumed stable for 4h

Method

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Additional strain / cell type characteristics:

not applicable

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

phenobarbiotone/¿-naphthoflavone induced S9

Vehicle / solvent:

Sterile, distilled water

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Sterile, distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

-S9 Migrated to IUCLID6: WP2uvrA; TA100; TA1535

Controls 2

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

-S9 Migrated to IUCLID6: TA1537

Controls 3

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

-S9 Migrated to IUCLID6: TA98

Controls 4

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene: TA100; TA1537; TA1537; WPsuvrA

Remarks:

+S9

Controls 5

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

+S9 Migrated to IUCLID6: TA98

Details on test system and experimental conditions:

Preliminary toxicity test:
Conducted to determine the toxicity of the test article. Concentrations tested were in the range of 0 ¿ 5 mg/plate. The test was performed by mixing bacterial culture (TA100 or WP2uvrA), molten trace histidine or typtophan supplemented top agar, test material formulation and S9 or PBS and overlaying onto sterile plates of Vogel-Bonner minimal agar.  Sterility of the test item was assessed (test article formulation alone added to agar (as described above). Following 48h incubation at 37°C plates were assessed for numbers of revertant colonies sing a Domino colony counter and examined for effects on the growth of the background lawn.
 
Initial toxicity-mutation assay:
Plate incorporation method used (i.e. soft agar, bacteria, TA and S9 / buffer mixed prior to immediate incorporation on to agar plate) test followed the directions of Ames et al.,(1973, 1975). Vehicle and positive controls and seven dose levels of the test material were plated in triplicate/dose. All strains listed above were tested in the presence and absence of S9 mix. 
 
Plates were incubated for approximately 48 hours at 37°C.
 
Confirmatory mutagenicity assay:
Pre-incubation method used (i.e. bacterial strain, test article formulation and S9 or phosphate buffer were incubated for 20 minutes at 37°C). following incubation the bacterial/test article formulation etc. mix was plated on to minimal agar. Vehicle and positive controls and seven dose levels of the test material were plated in triplicate/dose, dosing being adjusted based on the results of the initial experiment. . Again plates were incubated for at 48 hours at 37°C. Means and standard deviations were calculated for the both the initial and confirmatory test.

Evaluation criteria:

The test material was considered positive: dose related increase in mutant frequency over the dose range tested, reproducible increase at 1 or more concentrations, biological relevance against in-house historical control ranges, fold increase greater than 2 times the concurrent solvent control for any tester strain.
 
Toxicity was assessed by reduction in the background lawn; dose-dependent reduction in the mutant count/plate (concurrent vehicle control).

Statistics:

Statistical analysis was performed as described by UKEMS (Mahonet al., 1989)

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

INITIAL TOXICITY-MUTATION ASSAY:

Six doses of the test material ranging from 5 to 5000 ug/plate±S9 or 15 to 5000 ug/plate±S9 were evaluated in the plate incorporation test inSalmonellaandE.coli strains, respectively.. No precipitation was observed up to the limit dose, 5000 ug/plate. Toxicity was observed in all strains at 500 ¿g/plate and above.

 

Table 1: Bacterial mutation assay, summary of results¿ Initial (plate incorporation) test

Dose (ug/plate)	0	5	15	50	150	500	1500	5000	Positive control
REVERTANTS/PLATE
TA1535 ¿S9	24±2.1	24±1.5	20±1.2	25±0.6	22±2.6	25±5.2S	17±3.1S	6±2.1V	921±56.0
TA1535 +S9	16±1.5	13±2.0	16±1.5	13±1.7	17±4.4	17±2.1	13±4.9S	9±2.5V	264±31.5
TA100 ¿S9	111±1.7	110±9.1	115±13.1	112±8.2	109±6.4	93±5.8S	46±16.8S	18±6.2V	379±46.8
TA100 +S9	99±6.5	99±9.5	112±6.5	106±16.1	105±20.5	82±8.0	53±3.8S	14±6.4V	1195±5.5
TA1537 ¿S9	14±3.2	14±1.2	14±1.2	17±1.2	17±3.6	13±1.2	6±3.2S	0±0.0T	798±200.3
TA1537 +S9	15±2.6	14±2.1	16±.7	15±2.3	13±1.5	15±2.6	3±2.1S	0±0.0T	208±4.6
TA98 ¿S9	29±3.5	30±1.2	32±3.8	28±2.1	27±3.1	30±1.0	7±2.6	3±1.5	214±15.7
TA98 +S9	31±5.9	33±1.0	25±1.0	31±2.6	31±6.1	2±1.7	12±6.5S	3±2.0V	208±10.5
WP2uvrA ¿S9	35±1.5	N/T	32±3.8	28±2.1	27±3.1	30±1.0	7±2.6	3±1.5	214±15.7
WP2uvrA +S9	44±5.2	N/T	37±7.9	39±3.5	39±4.0	38±1.5	33±4.4	21±9.0	332±18.0

 

CONFIRMATORY MUTAGENICITY ASSAY:

Again no precipitation was observed in any of the strains tested. Cytotoxicity was observed in allSalmonellastrains in the absence and presence of S9.

 

Table 2A: Bacterial mutation assay, summary of results¿ confirmatory (pre-incubation) test ¿ TA1535 & TA100 ¿S9

Dose (ug/plate)	0	1.5	5	15	50	150	500	1500	Positive control
REVERTANTS/PLATE
TA1535 -S9	25±0.6	20±1.0	25±7.1	26±2.9	23±8.4	26±0.6S	12±2.1V	0±0.0V	708±82.1
TA100 -S9	93±10.5	91±4.2	95±7.2	97±7.2	103±8.5	101±3.6S	83±22.6V	44±5.9V	1799±198.6

 

Table 2B: Bacterial mutation assay, summary of results¿ confirmatory (pre-incubation) test ¿ TA1537, TA98 & WP2uvrA¿S9

Dose (ug/plate)	0	5	15	50	150	500	1500	5000	Positive control
REVERTANTS/PLATE
TA1537 -S9	9±2.9	8±0.6	10±1.0	7±4.0	6±2.3	6±1.7S	0±0.0T	0±0.0V	2079±280.0
TA98 -S9	26±4.4	31±3.1	22±1.7	15±1.5	16±1.7	9±1.5S	5±3.2V	0±0.0V	187±15.5
WP2uvrA -S9	26±4.5	N/T	31±4.6	25±3.5	28±1.0	26±3.2	27±3.5	15±3.8	976±62.4

 

Table 2C: Bacterial mutation assay, summary of results¿ confirmatory (pre-incubation) test All strains +S9

Dose (ug/plate)	0	5	15	50	150	500	1500	5000	Positive control
REVERTANTS/PLATE
TA1535 +S9	16±7.09	12±1.7	15±3.2	14±0.6	15±1.2	8±0.6S	7±1.2V	0±0.0V	258±16.8
TA100 +S9	99±8.4	75±11.0	81±9.1	87±2.5	87±5.3	51±4.9S	40±0.6V	0±0.0V	938±63.0
TA1537 +S9	11±2.1	12±2.5	11±1.5	13±1.5	6±1.2	7±1.0S	2±1.0	0±0.0V	221±51.2
TA98 +S9	30±6.7	34±4.0	31±0.6	27±2.0	31±8.4	12±4.0	9±1.7V	0±0.0V	231±17.3
WP2uvrA +S9	31±7.8	N/T	28±5.5	31±22.6	29±9.6	38±0.6	36±8.3	16±0.6	353±17.1

 

N/T      Not treated

S         Sparse background lawn

T         Toxic, no background lawn

V         Very weak background lawn

 

The test material was tested up to either the maximum recommended dose level (5000 ¿g/plate) or the toxic limit, depending on the bacterial strain type. No significant increases in the frequency of revertant colonies were recorded for any of the strains of bacteria, at any dose level either with or without metabolic activation or exposure method.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

Based on the results from this study,sodium lauroyl methyl isethionatewas not mutagenic in the bacterial strains tested, either in the presence or absence of metabolic activation when tested up either 5000 ug/plate (the maximum dose in accordance with regulatory guidelines) or cytotoxic concentrations.

Executive summary:

In a reverse gene mutation assay in bacteria,Salmonella typhimuriumstrains TA98, TA100, TA1535, and TA1537 andEscherichiacoli WP2uvrAwere exposed to sodium lauroyl methyl isethionate formulated in sterile, distilled water. The assay was performed in two phases, using the plate incorporation and the pre-incubation method.

 

In an initial test, the plate incorporation method was used with dose levels of 5 to 5000 ug/plate (depending on strain) in the presence and absence of S9 activation. In the confirmatory test, the pre-incubation method was used. The same dose range was used.  No precipitation was observed in any strain tested. The test material was tested up to either the maximum recommended dose level (5000 ¿g/plate) or the toxic limit, depending on the bacterial strain type. No significant increases in the frequency of revertant colonies were recorded for any of the strains of bacteria, at any dose level either with or without metabolic activation or exposure method.

 

Based on the results from this study,sodium lauroyl methyl isethionatewas not mutagenic in the bacterial strains tested, either in the presence or absence of metabolic activation when tested up either 5000 ug/plate (the maximum dose in accordance with regulatory guidelines) or cytotoxic concentrations.