Benzoic acid, 2-hydroxy-, C14-18-alkyl derivs.

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 September 2014 to 29 January 2015

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:

2015

Report date:

2015

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-,C14-18 alkyl derivs.

EC Number:

931-472-4

Cas Number:

182700-89-6

IUPAC Name:

Benzoic acid, 2-hydroxy-,C14-18 alkyl derivs.

Test material form:

liquid: viscous

Details on test material:

- Appearance: Clear reddish-brown viscous liquid
- 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:

- Experiment 1: 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate (all tester strains)
- Experiment 2 (without metabolic activation): 17, 52, 164, 512, 878, 1600 and 2800 µg/plate (all strains)
- Experiment 2 (with metabolic activation): 17, 52, 164, 512, 1600, 2800 and 5000 µg/plate (all strains)
- Experiments 3 and 4 (without metabolic activation): 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate (TA98 only)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

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 DMSO 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 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 vehicle 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 vehicle 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

Remarks:

In TA98 in Experiment 1 (- S9-mix) an up to 4.9-fold increase in the number of revertant colonies was observed at two intermediate dose levels but could not be replicated. This increase is considered to be incidental and not biologically relevant.

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:

FIRST MUTATION EXPERIMENT
- Precipitate: Heavy precipitation of the test material on the plates was observed at the start and at the end of the incubation period at the concentration of 5000 µg/plate. The concurrent control plates with only the test material showed small round-off test material droplets (quite similar to microcolonies) at the highest dose level of 5000 µg/plate. Therefore the highest dose level was judged as not countable due to heavy precipitate.
- Toxicity: To determine the toxicity of the test material, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were examined. The reduction of the bacterial background lawn and the reduction in the number of revertants are presented in Table 1.
- Mutagenicity: In tester strain TA98, the test material induced an up to 4.9-fold increase in the number of revertant colonies compared to the solvent control in the absence of S9-mix. This increase was observed at two intermediate dose levels (52 and 164 µg/plate). In the other tester strains, no increase in the number of revertants was observed upon treatment with the test material.

SECOND MUTATION ASSAY
- Precipitate: Precipitation of the test material on the plates was observed at the start of the incubation period at concentrations of 2800 and 5000 µg/plate in the presence of S9-mix. At the end of the incubation period precipitate was observed at concentrations of 2800 µg/plate in the absence of S9-mix and at 2800 and 5000 µg/plate in the presence of S9-mix in all tester strains. In the tester strain TA1537 in the absence of S9-mix, precipitate was already observed at the concentration of 1600 µg/plate. The concurrent control plates with only the test material showed small round-off test material droplets (quite similar to microcolonies) at the highest dose level of 5000 µg/plate. Therefore the highest dose level was judged as not countable due to heavy precipitate.
- Toxicity: The reduction of the bacterial background lawn and the reduction in the number of revertants are presented in Table 2.
- Mutagenicity: No increase in the number of revertants was observed upon treatment with the test material under all conditions tested.

ADDITIONAL MUTATION ASSAY
To verify the mutagenic response observed in the first mutation experiment in the tester strain TA98 in the absence of S9-mix, two additional experiments (experiments 3 and 4) were performed.
- Precipitate: Precipitation of the test material on the plates was observed at the start and at the end of the incubation period at the concentration of 5000 µg/plate.
- Toxicity: An extreme thinning of the micro-colony lawn and an increase in the size of the microcolonies compared to the solvent control plate was observed in the third mutation experiment at test material concentrations of 512 and 1600 µg/plate and in the fourth experiment at 512 µg/plate. A complete lack of any micro-colony background lawn was observed in the third mutation experiment at the test material concentration of 5000 µg/plate and in the fourth experiment at 1600 and 5000 µg/plate.
- Mutagenicity: No increase in the number of revertants was observed upon treatment with the test material under all conditions tested.

DISCUSSION
In the tester strain TA98 in the absence of S9-mix, the test material induced an up to 4.9-fold increase in the number of revertant colonies compared to the solvent control. However, this increase was observed only at two intermediate dose levels (52 and 164 µg/plate). Furthermore, this increase could not be repeated in three repeat experiments. Therefore, this increase is considered to be an incidental finding not biologically relevant.
All other bacterial strains showed negative responses over the entire dose range, i.e. no significant dose-related increase in the number of revertants in two independently repeated experiments.
The negative and strain-specific positive control values were within the laboratory background 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 in the First Mutation Experiment (Reduction of the bacterial background lawn and in the number of revertant colonies)

Strain	Without S-9 Mix			With S9-Mix
	Dose (µg/plate)	Bacterial Background Lawn	Revertant Colonies	Dose (µg/plate)	Bacterial Background Lawn	Revertant Colonies
TA1535	512 1600 5000	Slight Extreme Absent	No reduction Microcolonies ND	5000	Normal	ND
TA1537	512 1600 5000	Slight Extreme Absent	Moderate Microcolonies ND	1600 5000	Normal Normal	Extreme ND
TA98	512 1600 5000	Slight Extreme Absent	Reduction* Microcolonies ND	5000	Normal	ND
TA100	52 to 164 512 1600 5000	Normal Slight Extreme Absent	Extreme Extreme Microcolonies ND	1600 5000	Normal Normal	Moderate ND
WP2uvrA	5000	Normal	ND	5000	Normal	ND

*Reduction in the number of revertant colonies, but not less than the minimal value of the historical control data range.

ND = Due to heavy precipitate, no revertant colonies could be determined

 

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

Strain	Without S-9 Mix			With S9-Mix
	Dose (µg/plate)	Bacterial Background Lawn	Revertant Colonies	Dose (µg/plate)	Bacterial Background Lawn	Revertant Colonies
TA1535	512 878 1600 2800	Slight Slight Extreme Extreme	Moderate No reduction Microcolonies Microcolonies	5000	Normal	ND
TA1537	164 512 878 1600 2800	Slight Mod to ex* Extreme Extreme Extreme	No reduction Ex or micro** Microcolonies Microcolonies Microcolonies	2800 5000	Normal Normal	Extreme ND
TA98	164 512 878 1600 2800	Slight Moderate Moderate Extreme Extreme	No reduction No reduction Extreme Microcolonies Microcolonies	5000	Normal	ND
TA100	512 878 1600 2800	Slight Moderate Extreme Extreme	Moderate Extreme Microcolonies Microcolonies	5000	Normal	ND
WP2uvrA	2800	Normal	No reduction	5000	Normal	ND

*Moderate to extreme

**Extreme or microcolonies

ND = Due to heavy precipitate, no revertant colonies could be determined

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 5.4 17 52 164 512 1600 5000	88 112 92 16 15 34 MC HP	20 19 20 12 13 15 MC HP	29 28 25 23 24 19 24 HP	16 11 20 78 54 8 MC HP	5 8 10 5 4 2 MC HP
+	Solvent 5.4 17 52 164 512 1600 5000	103 87 90 83 84 76 56 HP	11 11 10 10 12 14 10 HP	30 34 34 26 21 32 31 HP	26 29 29 27 25 18 14 HP	12 7 9 8 8 4 2 HP
Positive Controls
-	Name	MMS	SA	4NQO	NF	ICR-191
	Concentration (µg/plate)	650	5	10	10	2.5
	Mean no. colonies/plate	549	728	1087	699	729
+	Name	2AA	2AA	2AA	2AA	2AA
	Concentration (µg/plate)	1	2.5	15	1	2.5
	Mean no. colonies/plate	806	174	158	756	320

MC = Microcolonies and an extremely reduced background lawn were seen

HP = Heavy precipitate. The number of revertant colonies could not be determined

MMS = Methylmethanesulfonate

SA = Sodium azide

4NQO = 4-Nitroquinoline-N-oxide

NF = 2-Nitrofluorene

2AA = 2-aminoanthracene

 

Table 2: 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 17 52 164 512 878 1600 2800	103 95 94 74 49 29 MC MC	11 15 13 13 5 7 MC MC	38 26 34 20 27 26 22 17	8 14 15 13 8 2 MC MC	5 8 6 7 1 MC MC MC
+	Solvent 17 52 164 512 1600 2800 5000	98 103 98 92 87 89 71 HP	14 12 12 12 9 10 9 HP	38 34 40 41 43 39 29 HP	21 21 24 20 18 16 22 HP	10 6 9 7 13 3 2 HP
Positive Controls
-	Name	MMS	SA	4NQO	NF	ICR-191
	Concentration (µg/plate)	650	5	10	10	2.5
	Mean no. colonies/plate	821	639	1285	933	844
+	Name	2AA	2AA	2AA	2AA	2AA
	Concentration (µg/plate)	2	2.5	15	1	5
	Mean no. colonies/plate	1360	190	309	500	387

MC = Microcolonies and an extremely reduced background lawn were seen

HP = Heavy precipitate. The number of revertant colonies could not be determined

MMS = Methylmethanesulfonate

SA = Sodium azide

4NQO = 4-Nitroquinoline-N-oxide

NF = 2-Nitrofluorene

2AA = 2-aminoanthracene

 

Table 5: Summary of Experiments 3 and 4

+/- S9 Mix	Concentration (µg/plate)	Experiment 3	Experiment 4
		TA98	TA98
-	Solvent 17 52 164 512 878 1600 2800	19 18 13 18 17 MC MC/HP HP	12 15 16 12 15 MC MC MC/HP
	NF 10	984	1224

MC = Microcolonies and an extremely reduced background lawn were seen

HP = Heavy precipitate. The number of revertant colonies could not be determined

NF = 2-Nitrofluorene

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). To obtain more information about the possible mutagenicity of the test material, additional experiments were performed with the tester strain TA98 in the absence of S9-mix.

In the first mutation assay, the test material was tested up to the concentration of 5000 µg/plate in DMSO in the absence and presence of 5 % (v/v) S9-mix. The test material precipitated heavily on the plates at the dose level of 5000 µg/plate and due to this heavy precipitate, the number of revertants at this dose level could not be determined. Cytotoxicity, as evidenced by a decrease in the number of revertants and/or reduction of the bacterial background lawn, was observed in the tester strains TA1535, TA1537, TA98 and TA100 in the absence of S9-mix and in TA1537 and TA100 in the presence of S9-mix. In tester strain TA98, the test material induced an up to 4.9-fold increase in the number of revertant colonies compared to the solvent control in the absence of S9-mix. This increase was observed at two intermediate dose levels (52 and 164 µg/plate). In the other tester strains, no increase in the number of revertants was observed upon treatment with the test material.

In the second mutation assay, the test material was tested up to the concentration of 2800 µg/plate in the absence of S9-mix and up to 5000 µg/plate in the presence of 10 % (v/v) S9-mix. The test material precipitated on the plates at dose levels of 2800 µg/plate and above. In the tester strain TA1537 in the absence of S9-mix, precipitate was already observed at the concentration of 1600 µg/plate. Due to the heavy precipitate on the plates at the test material concentration of 5000 µg/plate, the number of revertants at this dose level could not be determined. Cytotoxicity was observed in the tester strains TA1535, TA1537, TA98 and TA100 in the absence of S9-mix and in TA1537 in the presence of S9-mix. No increase in the number of revertants was observed upon treatment with the test material under all conditions tested.

To verify the mutagenic response observed in the first mutation experiment in the tester strain TA98 in the absence of S9-mix, two additional experiments (experiments 3 and 4) were performed. The test material was tested up to the concentration of 5000 µg/plate in the absence of S9-mix. The test material precipitated on the plates at the dose level of 5000 µg/plate. Cytotoxicity was observed in the third mutation experiment at dose levels of 164 µg/plate and above and in the fourth mutation experiment at dose levels of 512 µg/plate and above. No increase in the number of revertants was observed upon treatment with the test material under all conditions tested

In the tester strain TA98 in the absence of S9-mix in the first experiment, the test material induced an up to 4.9-fold increase in the number of revertant colonies compared to the solvent control. However, this increase was observed only at two intermediate dose levels (52 and 164 µg/plate). Furthermore, this increase could not be repeated in three repeat experiments. Therefore, this increase is considered to be an incidental finding and not biologically relevant.

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

Based on the results of this study it is concluded that the test substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.