Sodium long chain alkyl salicylate

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:

08 September 2014 to 22 September 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

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:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Benzoic acid, 2-hydroxy-, mono-C14-18-alkyl derivs., sodium salts

Molecular formula:

Na(C21-25H33-41O3)

IUPAC Name:

Benzoic acid, 2-hydroxy-, mono-C14-18-alkyl derivs., sodium salts

Test material form:

other: sticky solid

Details on test material:

- Appearance: Green-black solid
- Storage conditions of test material: At room temperature in the dark

Method

Target gene:

S. typhimurium: Histidine locus
E. coli: Tryptophan locus

Species / strain

Species / strain 1

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

- Type and identity of media: Enriched nutrient broth (Oxoid LTD, Hampshire, England)
- Properly maintained: Yes. The strains are regularly checked to confirm their histidine-requirement, crystal violet sensitivity, ampicillin resistance (TA98 and TA100), UV-sensitivity and the number of spontaneous revertants. Stock cultures were stored in liquid nitrogen (-196 °C).

Additional strain / cell type characteristics:

other: Additional mutations: rfa: deep rough; gal: mutation in the galactose metabolism; chl: mutation in nitrate reductase; bio: defective biotin synthesis; uvrB: loss of the excision repair system; and TA98 and TA100 possessed the R-factor = plasmid pKM101.

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

- Type and identity of media: Enriched nutrient broth (Oxoid LTD, Hampshire, England)
- Properly maintained: Yes. The strain is regularly checked to confirm the tryptophan-requirement, UV-sensitivity and the number of spontaneous revertants. Stock cultures were stored in liquid nitrogen (-196 °C).

Additional strain / cell type characteristics:

other: Additional mutations: rfa: deep rough (defective lipopolysaccharide cellcoat); gal: mutation in the galactose metabolism; chl: mutation in nitrate reductase; bio: defective biotin synthesis; and uvrB.

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

- Preliminary test: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate (TA100 and WP2uvrA)
- Main study Experiment 1 (without metabolic activation): 5.4, 17, 52, 164, 512 and 1600 µg/plate (TA1535, TA1537 and TA98)
- Main study Experiment 1 (with metabolic activation): 52, 164, 512, 1600, 3300 and 5000 µg/plate (TA1535, TA1537 and TA98)
- Main study Experiment 2 (without metabolic activation): 5.4, 17, 52, 164, 512 and 1600 µg/plate (TA1535, TA1537, TA98 and TA100)
- Main study Experiment 2 (with metabolic activation): 52, 164, 512, 1600, 3300 and 5000 µg/plate (TA1535, TA98 and TA100)
- Main study Experiment 2 (with metabolic activation): 52, 164, 512, 900, 1600 and 3300 µg/plate (TA1537)
- Main study Experiment 2 (with and without metabolic activation): 52, 164, 512, 1600 and 5000 µg/plate (WP2uvrA)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Ethanol
The stock solution was treated with ultrasonic waves to obtain a homogeneous suspension and heated to 66 °C. Test material concentrations were used within 1.5 hours of preparation.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: ICR-191; 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)
Top agar in top agar tubes was melted by heating to 45 °C. The following solutions were successively added to 3 mL molten top agar: 0.1 mL of a fresh bacterial culture (10¿ cells/mL) of one of the tester strains, 0.1 mL of a dilution of the test material in ethanol and either 0.5 mL S9-mix (in case of activation assays) or 0.5 mL 0.1 M phosphate buffer (in case of non-activation assays). The ingredients were vortex mixed and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were inverted and incubated in the dark at 37.0 ± 1.0 °C.

DURATION
- Exposure duration: 48 ± 4 h

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: Assessment of background lawn

Evaluation criteria:

A test material is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is not greater than 2 times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is not greater than 3 times the concurrent vehicle control.
b) The negative response should be reproducible in at least one a follow-up experiment.

A test material is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is greater than 2 times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is greater than 3 times the concurrent vehicle control.
b) In case a follow up experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.

Statistics:

No formal hypothesis testing was done.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535

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:

S. typhimurium TA 1537

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 3

Species / strain:

S. typhimurium TA 98

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 4

Species / strain:

S. typhimurium 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 5

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

DOSE RANGE FINDING TEST/FIRST MUTATION EXPERIMENT
- Precipitate: Precipitation of the test material on the plates was not observed at the start or at the end of the incubation period in any tester strain.
- Toxicity: In the tester strain WP2uvrA, no reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants were observed. The reduction of the bacterial background lawn and the reduction in the number of revertants in the other tester strains are presented in Table 1.
- Mutagenicity: No increase in the number of revertants was observed upon treatment with the test material under all conditions tested.

MAIN STUDY EXPERIMENT 2
- Precipitate: Precipitation of the test material on the plates was not observed at the start or at the end of the incubation period.
- Toxicity: In the tester strain WP2uvrA, no reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants were observed. The reduction of the bacterial background lawn and the reduction in the number of revertants in the other tester strains are presented in Table 2.
- Mutagenicity: In the second mutation assay, no increase in the number of revertants was observed upon treatment with the test material under all conditions tested.

DISCUSSION
All bacterial strains showed negative responses over the entire dose range, i.e. no significant dose-related increase in the number of revertants in two experiments.
The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Toxicity of the Test Material in the Dose Range Finding Test/First Experiment (Reduction of the bacterial background lawn and in the number of revertant colonies)

Strain	Without S9-mix			With S9-mix
	Dose (µg/plate)	Bacterial background lawn	Revertant colonies	Dose (µg/plate)	Bacterial background lawn	Revertant colonies
TA100	512 1600 5000	Slight Extreme Extreme	Moderate Microcolonies Microcolonies	5000	Extreme	Microcolonies
TA1535	512 1600	Slight Extreme	No reduction Microcolonies	3300 5000	Slight Extreme	Extreme Microcolonies
TA1537	512 1600	Moderate Extreme	Extreme Microcolonies	1600 3300 5000	Normal Extreme Extreme	Extreme Microcolonies Microcolonies
TA98	512 1600	Slight Extreme	No reduction Microcolonies	3300 5000	Normal Extreme	Extreme Microcolonies

 

Table 2: Toxicity of the Test Material in the Second Experiment (Reduction of the bacterial background lawn and in the number of revertant colonies)

Strain	Without S9-mix			With S9-mix
	Dose (µg/plate)	Bacterial background lawn	Revertant colonies	Dose (µg/plate)	Bacterial background lawn	Revertant colonies
TA100	512 1600	Slight Extreme	Extreme Microcolonies	3300 5000	Normal Slight	Moderate Extreme
TA1535	1600	Extreme	Microcolonies	5000	Normal	No reduction
TA1537	512 1600	Moderate Absent	Extreme Complete	1600 3300	Normal Normal	Extreme Extreme
TA98	512 1600	Slight Extreme	No reduction Microcolonies	5000	Normal	Extreme

 

Key to Toxicity Tables

Bacterial background lawn evaluation

- Normal: Distinguished by a healthy microcolony lawn

- Slightly reduced: Distinguished by a slight thinning of the microcolony lawn

- Moderately reduced: Distinguished by a moderate thinning of the microcolony lawn

- Extremely reduced: Distinguished by an extreme thinning of the microcolony lawn and an increase in the size of the microcolonies compared to the solvent control plate

- Absent: Distinguished by a complete lack of any microcolony background lawn

 

Evaluation of the reduction in the number of revertants

- A reduction of 21 to 40 %: slight reduction

- A reduction of 41 to 60 %: moderate reduction

- A reduction of 61 to 99 %: extreme reduction

If the size of the microcolonies was increased to small colonies due to an extremely reduced background lawn the reduction is evaluated as microcolonies. If no revertant colonies are observed on the plates the reduction is evaluated as a complete lack of revertants.

Any mean plate count equal to the minimal value of the historical control data range should be considered not toxic.

Table 3: Summary of Experiment 1

+/- 5 % S9 Mix	Concentration (µg/plate)	Mean number of colonies/plate
		Base-pair Substitution Type			Frameshift Type
		TA100	TA1535	WP2uvrA	TA98	TA1537
-	Solvent 1.7 5.4 17 52 164 512 1600 5000	106 125 123 119 98 87 47 MC MC	15 - 17 17 24 13 11 MC -	30 29 28 29 31 27 33 28 22	15 - 20 17 25 17 11 MC -	9 - 9 8 6 4 0 MC -
+	Solvent 1.7 5.4 17 52 164 512 1600 3300 5000	117 115 125 121 130 139 117 89 - MC	18 - - - 17 14 19 9 2 MC	39 37 34 37 44 28 41 42 - 31	35 - - - 50 42 28 18 8 MC	13 - - - 9 10 4 1 MC MC
Positive Controls
-	Name	MMS	SA	4NQO	NF	ICR-191
	Concentration (µg/plate)	650	5	10	10	2.5
	Mean no. colonies/plate	884	282	1561	1028	648
+	Name	2AA	2AA	2AA	2AA	2AA
	Concentration (µg/plate)	1	2.5	15	1	2.5
	Mean no. colonies/plate	1369	644	296	775	437

MC = Microcolonies and an extremely reduced background lawn were seen

MMS = Methylmethanesulfonate

SA = Sodium azide

4NQO = 4-Nitroquinoline-N-oxide

NF = 2-Nitrofluorene

2AA = 2-aminoanthracene

 

Table 4: Summary of Experiment 2

+/- 10 % S9 Mix	Concentration (µg/plate)	Mean number of colonies/plate
		Base-pair Substitution Type			Frameshift Type
		TA100	TA1535	WP2uvrA	TA98	TA1537
-	Solvent 5.4 17 52 164 512 1600 5000	105 88 106 105 80 40 MC -	8 8 8 8 6 9 MC -	25 - - 26 27 25 23 28	17 17 12 21 15 8 MC -	6 6 7 6 5 2 0 -
+	Solvent 52 164 512 900 1600 3300 5000	104 95 126 121 - 102 61 8	10 9 12 11 - 9 6 3	38 40 42 38 - 38 - 35	38 32 38 41 - 23 11 2	11 14 12 10 6 3 1 -
Positive Controls
-	Name	MMS	SA	4NQO	NF	ICR-191
	Concentration (µg/plate)	650	5	10	10	2.5
	Mean no. colonies/plate	768	701	1777	965	860
+	Name	2AA	2AA	2AA	2AA	2AA
	Concentration (µg/plate)	2	2.5	15	1	5
	Mean no. colonies/plate	1013	221	310	611	454

MMS = Methylmethanesulfonate

SA = Sodium azide

4NQO = 4-Nitroquinoline-N-oxide

NF = 2-Nitrofluorene

2AA = 2-aminoanthracene

Applicant's summary and conclusion

Conclusions:

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

Under the conditions of this study, the test material was determined to be non-mutagenic in both the presence and absence of metabolic activation.

Executive summary:

The mutagenic activity of the test material was evaluated in a bacterial reverse mutation assay conducted in accordance with the standardised guidelines OECD 471 and EU Method B.13/14 under GLP conditions.

The test material was tested with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and with a tryptophan-requiring strain of Escherichia coli (WP2uvrA). The test was performed in two independent experiments in the presence and absence of S9-mix (rat liver S9-mix induced by Aroclor 1254).

In the dose range finding test, the test material was tested up to concentrations of 5000 µg/plate in the absence and presence of S9-mix in the strains TA100 and WP2uvrA. The test material did not precipitate on the plates at this dose level. In the tester strain TA100, toxicity was observed at dose levels of 512 µg/plate and above in the absence of S9-mix and at 5000 µg/plate in the presence of S9-mix. In the tester strain WP2uvrA, no toxicity was observed at any of the dose levels tested.

Based on the results of the dose range finding test, the test material was tested in the first mutation assay at a concentration range of 5.4 to 1600 µg/plate in the absence of S9-mix and at 52 to 5000 µg/plate in the presence of 5 % (v/v) S9-mix in tester strains TA1535, TA1537 and TA98. Toxicity was observed in all three tester strains.

In the second mutation assay, the test material was tested up to concentrations of 1600 µg/plate in the absence of S9-mix in tester strains TA1535, TA1537, TA98 and TA100 and up to 3330 µg/plate (TA1537) and 5000 µg/plate (TA1535, TA98, TA100) in the presence of 10 % (v/v) S9-mix. The test material was tested up to concentrations of 5000 µg/plate in the absence and presence of S9-mix in the tester strain WP2uvrA. Toxicity was observed in all tester strains, except in the tester strain WP2uvrA in both the absence and presence of S9-mix and in the tester strain TA1535 in the presence of S9-mix.

The test material did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four Salmonella tester strains and in the number of revertant (Trp+) colonies in the Escherichia tester strain both in the absence and presence of S9-metabolic activation. These results were confirmed in a follow-up experiment.

In this study, the negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Under the conditions of this study, the test material was determined to be non-mutagenic in both the presence and absence of metabolic activation.