Benzoic acid, C12-15-alkyl esters

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:

June 2018 - April 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

This is a Klimisch 1 OECD 471 guideline study conducted on Benzoic acid, C12-15-alkyl esters in accordance with GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Benzoic acid, C12-15-alkyl esters

EC Number:

270-112-4

EC Name:

Benzoic acid, C12-15-alkyl esters

Cas Number:

68411-27-8

Molecular formula:

C19-22 H30-36 O2

IUPAC Name:

Benzoic acid, C12-15-alkyl esters

Specific details on test material used for the study:

Batch FPIL1807384

Method

Target gene:

Histidine (in S. Tryhimurium); Tryptophan (in E-coli)

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Vehicle / solvent:

Solvents used in this study were Ethanol, Dimethylsulfoxide and sterile water. The test item was used as a solution in ethanol.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: 2-aminoanthracene

Details on test system and experimental conditions:

Preliminary toxicity test – Conducted in order to select the concentrations of the test item to be used in the Main Assays. In this test a wide range of dose levels of the test item, set at half-log intervals, were used. Treatments were performed both in the absence and presence of S9 metabolism using the plate incorporation method; a single plate was used at each test point and positive controls were not included. Toxicity was assessed on the basis of a decline in the number of spontaneous revertants, a thinning of the background lawn or a microcolony formation.

Main Assays – Two Main Assays were performed including negative and positive controls in the absence and presence of an S9 metabolising system. Three replicate plates were used at each test point. In addition, plates were prepared to check the sterility of the test item solutions and the S9 mix and dilutions of the bacterial cultures were plated on nutrient agar plates to establish the number of bacteria in the cultures.

Plate-incorporation method - The components of the assay (the tester strain bacteria, the test item and S9 mix or phosphate buffer) were added to molten overlay agar and vortexed. The mixture was then poured onto the surface of a minimal medium agar plate and allowed to solidify prior to incubation.

The overlay mixture was composed as follows:
Overlay agar (held at 45°C) 2.0mL
Test or control item solution 0.1mL
S9 mix or phosphate buffer (pH 7.4, 0.1 M) 0.5mL
Bacterial suspension 0.1mL

Pre-incubation method - The components were added in turn to an empty test-tube:
Bacterial suspension 0.1mL
Ethanol or test item solution 0.01mL
or control item solution 0.05mL
S9 mix or phosphate buffer (pH 7.4, 0.1 M) 0.5mL

The incubate was vortexed and placed at 37°C for 30 minutes. Two mL of overlay agar was then added and the mixture vortexed again and poured onto the surface of a minimal medium agar plate and allowed to solidify.

Incubation and scoring - The prepared plates were inverted and incubated for approximately 72 hours at 37°C. After this period of incubation, plates were immediately scored by counting the number of revertant colonies on each plate.

Evaluation criteria:

The assay was considered valid if the following criteria were met:
1. Mean plate counts for untreated and positive control plates should fall within 2 standard deviations of the current historical mean values.
2. The estimated numbers of viable bacteria/plate should fall in the range of 100 – 500 million for each strain.
3. No more than 5% of the plates should be lost through contamination or other unforeseen event.

For the test item to be considered mutagenic, two-fold (or more) increases in mean revertant numbers must be observed at two consecutive dose levels or at the highest practicable dose level only. In addition, there must be evidence of a dose-response relationship showing increasing numbers of mutant colonies with increasing dose levels.

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:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

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:

no 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:

no 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:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

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:

valid

Positive controls validity:

valid

Any other information on results incl. tables

Plate Incorporation method – TA1535
Dose Level (μL/pl)	WITHOUT METABOLIC ACTIVATION					WITH METABOLIC ACTIVATION
	Plate Counts			Mean	S.E.	Plate Counts			Mean	S.E.
Untreated	13	15	18	15	1.5	14	16	16	15	0.7
0.00	20	22	16	19	1.8	13	13	15	14	0.7
0.313	14	13	13	13	0.3	18	14	15	16	1.2
0.625	15	14	15	15	0.3	21	15	20	19	1.9
1.25	18	22	20	20	1.2	17	16	22	18	1.9
2.50	15	18	18	17P	1.0	22	20	15	19P	2.1
5.00	13	16	15	15P	0.9	20	20	24	21P	1.3
Positive and Negative Control Plates
Treatment	Plate Counts			Mean	S.E.	Plate Counts			Mean	S.E.
Untreated	13	15	18	15	1.5
Sodium Azide	586	549	538	558	14.5
DMSO						13	14	16	14	0.9
2-Aminoanthracene						140	158	139	146	6.2

 

Plate Incorporation method – TA1537
Dose Level (μL/pl)	WITHOUT METABOLIC ACTIVATION					WITH METABOLIC ACTIVATION
	Plate Counts			Mean	S.E.	Plate Counts			Mean	S.E.
Untreated	14	20	19	18	1.9	26	19	23	23	2.0
0.00	22	24	17	21	2.1	26	26	26	26	0.0
0.313	14	19	16	16	1.5	16	15	13	15	0.9
0.625	18	16	13	16	1.5	16	15	14	15	0.6
1.25	16	15	14	15	0.6	14	15	13	14	0.6
2.50	13	18	20	17P	2.1	14	14	13P	14	0.3
5.00	14	14	13	14P	0.3	14	12	15P	14	0.9
Positive and Negative Control Plates
Treatment	Plate Counts			Mean	S.E.	Plate Counts			Mean	S.E.
DMSO	13	15	20	16	2.1
9-Aminoacridine	222	171	164	186	18.3
DMSO						18	17	30	18	0.9
2-Aminoanthracene						94	93	119	102	8.5

Plate Incorporation method –WP2uvrA
Dose Level (μL/pl)	WITHOUT METABOLIC ACTIVATION					WITH METABOLIC ACTIVATION
	Plate Counts			Mean	S.E.	Plate Counts			Mean	S.E.
Untreated	31	25	31	29	2.0	27	29	28	28	0.6
0.00	28	31	29	29	0.9	27	31	25	28	1.8
0.313	28	22	29	26	2.2	34	31	35	33	1.2
0.625	24	31	29	28	2.1	29	29	33	30	1.3
1.25	26	26	26	26	0.0	32	33	27	31	1.9
2.50	26	31	29	29P	1.5	29	28	27	28P	0.6
5.00	28	33	30	30P	1.5	27	27	29	28P	0.7
Positive and Negative Control Plates
Treatment	Plate Counts			Mean	S.E.	Plate Counts			Mean	S.E.
Untreated	31	25	31	29	2.0
MMS	167	173	197	179	9.2
DMSO						27	28	28	28	0.3
2-Aminoanthracene						191	215	223	210	9.6

 

Plate Incorporation method – TA98
Dose Level (μL/pl)	WITHOUT METABOLIC ACTIVATION					WITH METABOLIC ACTIVATION
	Plate Counts			Mean	S.E.	Plate Counts			Mean	S.E.
Untreated	27	26	26	26	0.3	31	32	31	31	0.3
0.00	36	38	37	37	0.6	31	29	31	30	0.7
0.313	27	30	31	29	1.2	30	33	30	31	1.0
0.625	28	31	30	30	0.9	38	33	30	34	2.3
1.25	28	30	30	29	0.7	35	36	38	36	0.9
2.50	30	35	33	33P	1.5	38	42	40	40P	1.2
5.00	26	25	31	27P	1.9	35	35	37	36P	0.7
Positive and Negative Control Plates
Treatment	Plate Counts			Mean	S.E.	Plate Counts			Mean	S.E.
DMSO	30	28	30	29	0.7
2-Nitrofluorene	116	106	139	120	9.8
DMSO						30	32	30	31	0.7
2-Aminoanthracene						368	388	395	384	8.1

  

Plate Incorporation method – TA100
Dose Level (μL/pl)	WITHOUT METABOLIC ACTIVATION					WITH METABOLIC ACTIVATION
	Plate Counts			Mean	S.E.	Plate Counts			Mean	S.E.
Untreated	128	126	126	127	0.7	124	126	128	126	1.2
0.00	106	112	109	109	1.7	113	119	101	111	5.3
0.313	118	124	119	120	1.9	117	119	112	116	2.1
0.625	119	106	114	113	3.8	123	134	126	128	3.3
1.25	130	125	115	123	4.4	114	115	112	114	0.9
2.50	111	116	110	112P	1.9	130	115	120	122P	4.4
5.00	112	107	110	110P	1.5	128	126	124	126P	1.2
Positive and Negative Control Plates
Treatment	Plate Counts			Mean	S.E.	Plate Counts			Mean	S.E.
Untreated	128	126	126	127	0.7
Sodium Azide	561	529	449	513	33.3
DMSO						167	143	150	153	7.1
2-Aminoanthracene						740	872	891	834	47.5

 

Pre-incubation method – TA1535
Dose Level (μL/pl)	WITHOUT METABOLIC ACTIVATION					WITH METABOLIC ACTIVATION
	Plate Counts			Mean	S.E.	Plate Counts			Mean	S.E.
Untreated	17	17	14	16	1.0	13	14	15	14	0.6
0.00	17	19	19	18	0.7	14	15	13	14	0.6
0.313	13	17	15	15	1.2	13	13	15	14	0.7
0.625	19	13	13	15	2.0	15	13	12	13	0.9
1.25	13	17	13	14	1.3	13	16	17	15	1.2
2.50	13	14	13	13	0.3	16	13	15	15	0.9
5.00	19	16	14	16P	1.5	15	13	17	15P	1.2
Positive and Negative Control Plates
Treatment	Plate Counts			Mean	S.E.	Plate Counts			Mean	S.E.
Untreated	17	17	14	16	1.0
Sodium Azide	415	436	389	413	13.6
DMSO						13	13	17	14	1.3
2-Aminoanthracene						102	95	114	104	5.5

 

Pre-incubation method – TA1537
Dose Level (μL/pl)	WITHOUT METABOLIC ACTIVATION					WITH METABOLIC ACTIVATION
	Plate Counts			Mean	S.E.	Plate Counts			Mean	S.E.
Untreated	14	14	15	14	0.3	24	24	23	24	0.3
0.00	16	16	18	17	0.7	23	23	19	22	1.3
0.313	14	15	17	15	0.9	16	16	14	15	0.7
0.625	16	13	13	14	1.0	18	13	18	16	1.7
1.25	15	13	12	13	0.9	14	14	18	15	1.3
2.50	13	15	13	14	0.7	17	20	17	18	1.0
5.00	14	17	16	16P	0.9	18	20	21	20P	0.9
Positive and Negative Control Plates
Treatment	Plate Counts			Mean	S.E.	Plate Counts			Mean	S.E.
DMSO	20	15	15	17	1.7
9-Aminoacridine	280	169	194	214	33.6
DMSO						23	22	24	23	0.6
2-Aminoanthracene						85	91	87	88	1.8

 

Pre-incubation method –WP2uvrA
Dose Level (μL/pl)	WITHOUT METABOLIC ACTIVATION					WITH METABOLIC ACTIVATION
	Plate Counts			Mean	S.E.	Plate Counts			Mean	S.E.
Untreated	30	35	30	32	1.7	32	38	36	35	1.8
0.00	36	31	33	33	1.5	31	33	32	32	0.6
0.313	28	25	25	26	1.0	39	39	40	39	0.3
0.625	33	30	27	30	1.7	39	39	38	39	0.3
1.25	30	29	32	30	0.9	39	30	30	33	3.0
2.50	28	30	36	31	2.4	36	28	33	32	2.3
5.00	33	26	28	29P	2.1	30	30	36	32P	2.0
Positive and Negative Control Plates
Treatment	Plate Counts			Mean	S.E.	Plate Counts			Mean	S.E.
Untreated	30	35	30	32	1.7
MMS	139	121	145	135	7.2
DMSO						34	34	30	33	1.3
2-Aminoanthracene						212	197	212	207	5.0

 

Pre-incubation method – TA98
Dose Level (μL/pl)	WITHOUT METABOLIC ACTIVATION					WITH METABOLIC ACTIVATION
	Plate Counts			Mean	S.E.	Plate Counts			Mean	S.E.
Untreated	33	31	33	32	0.7	42	37	47	42	2.9
0.00	37	30	35	34	2.1	38	43	45	42	2.1
0.313	34	34	30	33	1.3	44	40	31	38	3.8
0.625	38	35	36	36	0.9	42	42	38	41	1.3
1.25	30	28	27	28	0.9	48	38	40	42	3.1
2.50	31	26	30	29	1.5	43	36	39	39	2.0
5.00	36	30	25	30P	3.2	39	41	45	42P	1.8
Positive and Negative Control Plates
Treatment	Plate Counts			Mean	S.E.	Plate Counts			Mean	S.E.
DMSO	32	29	29	30	1.0
2-Nitrofluorene	175	164	170	170	3.2
DMSO						38	44	43	42	1.9
2-Aminoanthracene						614	702	665	660	25.5

 

Pre-incubation method – TA100
Dose Level (μL/pl)	WITHOUT METABOLIC ACTIVATION					WITH METABOLIC ACTIVATION
	Plate Counts			Mean	S.E.	Plate Counts			Mean	S.E.
Untreated	159	153	155	156	1.8	166	165	171	167	1.9
0.00	162	163	174	166	3.8	166	142	148	152	7.2
0.313	159	164	157	160	2.1	168	170	164	167	1.8
0.625	144	158	165	156	6.2	154	150	168	157	5.5
1.25	143	123	150	139	8.1	144	159	171	158	7.8
2.50	140	139	151	143	3.8	163	158	170	164	3.5
5.00	166	158	150	158P	4.6	152	165	166	161P	4.5
Positive and Negative Control Plates
Treatment	Plate Counts			Mean	S.E.	Plate Counts			Mean	S.E.
Untreated	159	153	155	156	1.8
Sodium Azide	479	421	400	433	23.6
DMSO						161	164	154	160	3.0
2-Aminoanthracene						1488	1215	1281	1328	82.2

Applicant's summary and conclusion

Conclusions:

Benzoic acid, C12-15-alkyl esters, CAS No. 68411-27-8, EC No. 270-112-4, was found to be non-mutagenic in the OECD 471, Bacterial Reverse Phase Mutation study, as it did not induce mutation in four histidine-requiring strains of Salmonella typhimurium (TA 98, TA100, TA1535 and TA1537) and one tryptophan-requiring strain of Escherichia coli (WP2 uvrA) in the absence and presence of a rat liver metabolic activation system (S-9).

Executive summary:

The mutagenic potential of Benzoic acid, C12-15-alkyl esters was evaluated in a study a conducted according to OECD guideline 471. The study was assigned a reliability score of 1 in accordance with the criteria for assessing data quality set forth by Klimisch et al. (1997).

 

The study was accepted as valid as the following criteria were met. The results show that mean plate counts for untreated and positive control plates fell within the normal distribution range based on historical control data. The estimated numbers of viable bacteria/plate fell in the range of 100 - 500 million for each strain. No plates were lost through contamination or cracking.

 

The results show that Benzoic acid, C12-15-alkyl esters did not induce two-fold increases in the number of revertant colonies in the plate incorporation or pre-incubation assay, at any dose level, in any tester strain, in the absence or presence of S9 metabolism.

 

It was therefore concluded that Benzoic acid, C12-15-alkyl esters does not induce reverse mutation in Salmonella typhimurium or Escherichia coli in the absence or presence of S9 metabolism, under the reported experimental conditions.