Benzoic acid, C12-15-alkyl esters

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames Test (OECD TG 471, S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 ): 50 - 5000 µg/plate: negative
MNT in vitro (OECD TG 487, primary human lymphocytes): not clastogenic up to 5 µL/mL
MLA in vitro (OECD TG 490, mouse lymphoma L5178Y cells): 0.0195 -5 μL/mL not mutagenic

Link to relevant study records

Reference

Reference 1

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.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Batch FPIL1807384

Target gene:

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

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.

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.

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

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

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

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

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

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

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.

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Target gene:

Species Rat
Strain Sprague Dawley
Tissue Liver
Inducing Agents Phenobarbital – 5,6-Benzoflavone

Species / strain / cell type:

lymphocytes:

Metabolic activation:

with and without

Test concentrations with justification for top dose:

Solutions of the test item, as received, were prepared immediately before use in ethanol (see justification in chapter 5.1). Solutions were prepared on a volume/volume basis with correction for the displacement due to the volume of the test item. For the continuous treatment, where the total amount of organic solvent used for both the test item and Cytochalasin B should not exceed 1% (v/v), at the maximum dose level of 5 μL/mL, the test item was added to the treatment mixture as supplied. Concentrations were expressed in terms of material as received. All test item solutions were used within 30 minutes from the initial formulation. All dose levels in this report are expressed to three significant figures.

Vehicle / solvent:

Ethanol

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

colchicine

cyclophosphamide

Statistics:

The statistical significance of the recorded number of cells bearing micronuclei together with the results of linear trend analysis are shown in Table 4.

The incidence of micronucleated cells of the negative controls fell within the distribution range of our historical control values.

Adequate cell proliferation was observed in negative control cultures and the appropriate number of doses and cells was analysed.

Treatment with the positive control items, Cyclophosphamide and Colchicine, gave marked responses that were compatible with those generated in the historical control database and produced a statistically significant increase in the incidence of micronucleated cells, compared with the concurrent negative control, indicating the correct functioning of the test system.

Based on these results, the study was accepted as valid.

After treatment with the test item, no statistically significant increase in the incidence of micronucleated cells was seen at any dose level, in any treatment series. No concentration related increase of cells bearing micronuclei was observed in any treatment series.

For all treatment series the incidence of micronucleated cells was within the normal distribution of the historical control data reported in Appendix 1 with the exception of the intermediate dose level selected for scoring in the absence of S9 (short exposure), where, the incidence (0.75%) was slightly higher than the Upper Confidence Limit (0.63%). The result however was due to the erratic finding observed for one replicate culture and considered to be attributable to a chance event.

On the basis of the above mentioned results and in accordance with the criteria for outcome of the study, the test item was not considered to induce micronuclei in human lymphocytes after in vitro treatment.

Key result

Species / strain:

lymphocytes: batches of human whole blood, were pooled

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

No remarkable cytotoxicity was observed at the two highest dose levels of 5.00 and 3.33 μL/mL. Mild to not remarkable cyotoxicity was observed over the remaining dose range.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks:

Marked increases in the incidence of micronucleated cells were observed following treatments with the positive controls Cyclophosphamide and Colchicine, indicating the correct functioning of the test system.

Solubility test

The test item was found to be miscible with ethanol at 500 μL/mL. Miscibility was assessed by adding 0.2 mL of test item to 0.2 mL of ethanol. This solvent was selected since it is compatible with the survival of cells and the S9 metabolic activity. An aliquot of this stock solution added to culture medium in the ratio 1:200 gave a slightly opaque solution with micelles in suspension. Since for the continuous treatment (Main Assay 1), the test item had to be added as supplied to reach the maximum concentration of 5.00 μL/mL (the upper limit to testing indicated in the Study Protocol for UVCB substances), an additional solubility trial was performed where 25 μL of the test item were

added to 4975 μL of culture medium. An opaque medium with micelles in suspension was observed.

These results permitted a maximum concentration of 5.00 μL/mL to be used in the study. In Main Assay 1, following treatment with the test item, a slight opacity of the medium was observed at the end of the three hour exposure in the absence and presence of S9 metabolism over the whole dose range. A slight dose-related opacity of the medium was also observed at the end of the continuous exposure over the whole dose range. For the repeated treatment (Main Assay 2), a dose related turbidity was observed at the end of the continuous exposure from 0.381 μL/mL onwards.

Osmolality and pH results

Following treatment with the test item, no remarkable variation of pH and osmolality over the concurrent controls was observed in the absence or presence of S9 metabolism.

Assay results

The number of binucleated cells with micronuclei is presented.

Following treatment with the test item, no remarkable increase in the incidence of micronucleated cells over the concurrent solvent control was observed in the absence or presence of S9 metabolism, at any concentration, in any treatment series.

Marked increases in the incidence of micronucleated cells were observed following treatments with the positive controls Cyclophosphamide and Colchicine, indicating the correct functioning of the test system.

Conclusions:

It is concluded that C12-15 alkyl benzoates does not induce micronuclei in human lymphocytes after in vitro treatment, under the reported experimental conditions.

Executive summary:

The test item C12-15 alkyl benzoates was assayed for the ability to induce micronuclei in human lymphocytes, following in vitro treatment in the absence and presence of S9 metabolic activation.

Three treatment series were included in the study. A short treatment, where the cells were treated for 3 hours, was performed in the absence and presence of S9 metabolism. The harvest time of approximately 32 hours, corresponding to approximately two cell cycle lengths, was used. A long term (continuous) treatment was also performed only in the absence of S9 metabolism, until harvest at 31 hours.

Based on solubility data, the maximum practicable concentration of the test item in the final treatment medium was 5.00 μL/mL. Since the test item is not of defined composition (i.e. UVCB), this concentration is the upper limit to testing as indicated in the Study Protocol.

For the 3-hour treatment series, both in the absence and presence of S9 metabolism, the test item was assayed at the maximum concentration of 5.00 μL/mL and at the following dose levels: 3.33, 2.22, 1.48, 0.988, 0.658, 0.439, 0.293 and 0.195 μL/mL. For the continuous treatment in the absence of S9 metabolism, the additional dose level of 0.130 μL/mL was employed (Main Assay 1). All treatments were performed using the test item as a solution in ethanol with the exception of the continuous treatment in the absence of S9 at the highest dose level where, in order to reach the maximum concentration of 5.00 μL/mL, the test item was used as supplied.

Since for the continuous treatment, possibly due to the chemical and physical properties of the test item, an unexpected cytoxicity profile was observed, it was considered appropriate to repeat the treatment by slightly modifying the procedures of treatment and incubation of the cultures (Main Assay 2) in order to improve the miscibility of the test item. The test item was assayed as a solution in ethanol by using a maximum dose level of 2.50 μL/mL and a larger space interval in order to cover a wider range of concentrations. Dose levels of 2.50, 1.56, 0.977, 0.610, 0.381, 0.238, 0.149, 0.0931, 0.0582, 0.0364, 0.0227, 0.0142 and 0.00888 μL/mL were tested.

Each treatment series included appropriate negative controls (untreated and solvent vehicle controls). In addition, positive controls were included for the short termand the long term treatment series (Cyclophosphamide and Colchicine, respectively).

Two cell cultures were prepared at each test point.

The actin polymerisation inhibitor Cytochalasin B was added prior to the targeted mitosis to allow the selective analysis of micronucleus frequency in binucleated cells. The cytokinesisblock proliferation index CBPI was calculated in order to evaluate cytotoxicity.

Dose levels for the scoring of micronuclei were selected with the aim to evaluate the test item concentrations at adequate levels of cytotoxicity and considering turbidity or test item precipitation at the end of the exposure.

Based on the results obtained, the following concentrations were selected for the scoring of micronuclei:

Assay No.       S9       Treatment time       Harvest time       Dose level                     Cytotoxicity

              (hours)        (hours)              (μL/mL)                      (%)

1 - 3 32 5.00, 3.33 and 2.22 6, 15 and 12

+                                                                             6, 0 and 9

2                   - 31                         31        0.610, 0.238 and 0.0931       32, 29 and 19

One thousand binucleated cells per culture were scored to assess the frequency of micronucleated cells.

Following treatment with the test item, no statistically significant increase in the incidence of micronucleated cells over the control value was observed at any dose level, in any treatment series. All the results obtained were inside the distribution of historical control data and no dose effect relationship was indicated by a linear trend analysis.

Statistically significant increases in the incidence of micronucleated cells were observed following treatments with the positive controls Cyclophosphamide and Colchicine, indicating the correct functioning of the test system.

It is concluded that C12-15 alkyl benzoates does not induce micronuclei in human lymphocytes after in vitro treatment, under the reported experimental conditions.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Principles of method if other than guideline:

The mutation assay method used in this study is based on the identification of L5178Y colonies which have become resistant to a toxic thymidine analogue trifluorothymidine (TFT). This analogue can be metabolised by the enzyme thymidine kinase (TK) into nucleosides, which are used in nucleic acid synthesis resulting in the death of TK-competent cells.

TK-deficient cells, which are presumed to arise through mutations in the TK gene, cannot metabolise trifluorothymidine and thus survive and grow in its presence.

In the L5178Y mouse lymphoma cells, the gene which codes for the TK enzyme is located on chromosome 11. Cells which are heterozygous at the TK locus (TK+/−) may undergo a single step forward mutation to the TK−/− genotype in which little or no TK activity remains.

The cells used, L5178Y TK+/−, are derived from one of the two clones originated from a thymic tumour induced in a DBA/2 mouse by methylcholanthrene. The use of the TK mutation system in L5178Y mouse lymphoma cells has been well characterised and validated (D. Clive et al., 1979) and is accepted by most of the regulatory authorities.

The mouse lymphoma assay often produces a bimodal size distribution of TFT resistant colonies designated as small or large. It has been evaluated that point mutations and deletions within the active allele (intragenic event) produce large colonies. Small colonies result in part from lesions that affect not only the active TK allele but also a flanking gene whose expression modulates the growth rate of cells.

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Specific details on test material used for the study:

Batch no. FPIL1807384
Purity 100%
CAS no. 68411-27-8
Date of expiry 03May 2022
Appearance clear colourless liquid
Storage conditions +18°C (+14°C ÷ +23°C)
RTC number 15951

Species / strain / cell type:

mammalian cell line, other:

Details on mammalian cell type (if applicable):

Species Rat
Strain Sprague Dawley
Tissue Liver

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Test concentrations with justification for top dose:

Based on solubility data (RTC Study No. A3309), the maximum practicable concentration of the test item in the final treatment medium was 5 μL/mL using ethanol as solvent. Since the test item is not of defined composition (i.e. UVCB), this concentration is the upper limit to testing as indicated in the Study Protocol. On the basis of the solubility result, a preliminary cytotoxicity assay was performed both in the absence and presence of S9 metabolic activation, using the maximum dose level of 5 μL/mL and a wide range of lower dose levels: 2.50, 1.25, 0.625, 0.313, 0.156, 0.0781, 0.0391 and 0.0195 μg/mL.

Vehicle / solvent:

Ethanol

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

methylmethanesulfonate

Key result

Species / strain:

mammalian cell line, other: Species Rat Strain Sprague Dawley Tissue Liver

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Remarks:

The mutant frequencies in the solvent control cultures fell within the normal range

Positive controls validity:

valid

Remarks:

Marked increases were obtained with the positive control treatments indicating the correct functioning of the assay system.

In the presence of S9 metabolism and using the long treatment in the absence of S9 metabolic activation, adequate levels of cytotoxicity, covering a range from the maximum to slight or no toxicity, were observed. Using the short treatment time in the absence of S9 metabolism, moderate toxicity was noticed at higher concentrations, however opacity of treatment medium was observed, indicating the adequate selection of dose levels. No relevant increases in mutant frequencies were observed following treatment with the test item, in the absence or presence of S9 metabolism, in any experiment.

Conclusions:

It is concluded that C12-15 alkyl benzoates does not induce mutation at the TK locus of L5178Y mouse lymphoma cells in vitro in the absence or presence of S9 metabolic activation, under the reported experimental conditions.
RTC

Executive summary:

The test item C12-15 alkyl benzoates was examined for mutagenic activity by assaying for the induction of 5 trifluorothymidine resistant mutants in mouse lymphoma L5178Y cells after in vitro treatment, in the absence and presence of S9 metabolic activation, using a fluctuation method.

Based on solubility data (RTC Study No. A3309), the maximum practicable concentration of the test item in the final treatment medium was 5 μL/mL using ethanol as solvent. Since the test item is not of defined composition (i.e. UVCB), this concentration is the upper limit to testing as indicated in the Study Protocol. On the basis of the solubility result, a preliminary cytotoxicity assay was performed both in the absence and presence of S9 metabolic activation, using the maximum dose level of 5 μL/mL and a wide range of lower dose levels: 2.50, 1.25, 0.625, 0.313, 0.156, 0.0781, 0.0391 and 0.0195 μg/mL.

A U-shaped profile of toxicity was observed in all treatment series probably related to the formation of test item micelles detectable by the end of treatment time as opacity of the culture medium or as the presence of micelles visible to the unaided eye. In the absence of S9 metabolic activation, using the 3 hour treatment time, no relevant toxicity was observed at the lowest concentration tested (0.0195 μg/mL), slight reduction of Relative Survival (RS) was noted at 0.0391 μL/mL; while mild toxicity was seen at 0.0781 and 0.156 μL/mL, reducing RS to 43 and 46%, respectively. Moderate to marked toxicity was observed between 0.313 and 2.50 μL/mL; while no relevant toxicity was seen at the highest dose level of 5.00 μL/mL, where formation of test item micelles was noted. Using the 24 hour treatment time, no toxicity was observed at the lowest dose level, while treatment at 0.0391 and 0.0781 μL/mL yielded a reduction of RS to 42 and 28% of the concurrent negative control value, respectively. No cells survived treatment from 0.156 μL/mL to 2.50 μL/mL; while no relevant toxicity was seen at the highest dose level. Following treatment in the presence of S9 metabolic activation, using the short treatment time (3 hours), no toxicity was noted at the three lowest concentrations, while mild to moderate toxicity was observed at the two intermediate dose levels of 0.156 and 0.313 μL/mL, with a reduction of RS to 45 and 38% of the negative control, respectively. Slight toxicity (RS=65%) was observed at 0.625 μL/mL; while no relevant reduction of RS was seen at the three highest concentrations tested.

Two independent assays for mutation at the TK locus were performed. Based on the results obtained in the preliminary trial, the dose levels described in the following table were selected in order to achieve the appropriate toxicity of 10-20% relative total growth (RTG).

Assay No	S9	Treatment time (hrs)	Dose level (μL/mL)
1	-	3	0.625, 0.313, 0.156, 0.0781, 0.0.039 and 0.0195
1	+	3	0.500, 0.313, 0.195, 0.122 and 0.0763
2	-	24	0.120, 0.0750, 0.0469, 0.0293, 0.0183 and 0.0114

In the presence of S9 metabolism and using the long treatment in the absence of S9 metabolic activation, adequate levels of cytotoxicity, covering a range from the maximum to slight or no toxicity, were observed. Using the short treatment time in the absence of S9 metabolism, moderate toxicity was noticed at higher concentrations, however opacity of treatment medium was observed, indicating the adequate selection of dose levels. No relevant increases in mutant frequencies were observed following treatment with the test item, in the absence or presence of S9 metabolism, in any experiment.

Negative and positive control treatments were included in each mutation experiment in the absence and presence of S9 metabolism. The mutant frequencies in the solvent control cultures fell within the normal range. Marked increases were obtained with the positive control treatments indicating the correct functioning of the assay system.

It is concluded that C12-15 alkyl benzoates does not induce mutation at the TK locus of L5178Y mouse lymphoma cells in vitro in the absence or presence of S9 metabolic activation, under the reported experimental conditions.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Bacterial reverse mutation assay

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 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.

 

A further Ames Mutagenicity assay was conducted to determine whether a test article solution of C12 -15 Alkyl Benzoate, Lot #34449, would cause mutagenic changes in histidine-dependent Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and TA1538 in the presence and absence of S-9 metabolic activation. The methodology of Ames et al (1975) was followed but modified to use a test article solution.

The test article solution of C12 -15 Alkyl Benzoate, Lot #34449, (prepared by the sponsor) was found to be non-inhibitory to growth of the tester strains.

Under the conditions of this mutagenicity assay, the test article solution of C12-15 Alkyl Benzoate was not considered mutagenic to Salmonella typhimurium tester strains TA98, TA100, TA1535, TA1537 and TA1538.

 

Mammalian cell gene mutation assay

The test item C12-15 alkyl benzoates was examined for mutagenic activity by assaying for the induction of 5 trifluorothymidine resistant mutants in mouse lymphoma L5178Y cells after in vitro treatment, in the absence and presence of S9 metabolic activation, using a fluctuation method.

Based on solubility data (RTC Study No. A3309), the maximum practicable concentration of the test item in the final treatment medium was 5 μL/mL using ethanol as solvent. Since the test item is not of defined composition (i.e. UVCB), this concentration is the upper limit to testing as indicated in the Study Protocol. On the basis of the solubility result, a preliminary cytotoxicity assay was performed both in the absence and presence of S9 metabolic activation, using the maximum dose level of 5 μL/mL and a wide range of lower dose levels: 2.50, 1.25, 0.625, 0.313, 0.156, 0.0781, 0.0391 and 0.0195 μg/mL.

A U-shaped profile of toxicity was observed in all treatment series probably related to the formation of test item micelles detectable by the end of treatment time as opacity of the culture medium or as the presence of micelles visible to the unaided eye. In the absence of S9 metabolic activation, using the 3 hour treatment time, no relevant toxicity was observed at the lowest concentration tested (0.0195 μg/mL), slight reduction of Relative Survival (RS) was noted at 0.0391 μL/mL; while mild toxicity was seen at 0.0781 and 0.156 μL/mL, reducing RS to 43 and 46%, respectively. Moderate to marked toxicity was observed between 0.313 and 2.50 μL/mL; while no relevant toxicity was seen at the highest dose level of 5.00 μL/mL, where formation of test item micelles was noted. Using the 24 hour treatment time, no toxicity was observed at the lowest dose level, while treatment at 0.0391 and 0.0781 μL/mL yielded a reduction of RS to 42 and 28% of the concurrent negative control value, respectively. No cells survived treatment from 0.156 μL/mL to 2.50 μL/mL; while no relevant toxicity was seen at the highest dose level. Following treatment in the presence of S9 metabolic activation, using the short treatment time (3 hours), no toxicity was noted at the three lowest concentrations, while mild to moderate toxicity was observed at the two intermediate dose levels of 0.156 and 0.313 μL/mL, with a reduction of RS to 45 and 38% of the negative control, respectively. Slight toxicity (RS=65%) was observed at 0.625 μL/mL; while no relevant reduction of RS was seen at the three highest concentrations tested.

Two independent assays for mutation at the TK locus were performed.

In the presence of S9 metabolism and using the long treatment in the absence of S9 metabolic activation, adequate levels of cytotoxicity, covering a range from the maximum to slight or no toxicity, were observed. Using the short treatment time in the absence of S9 metabolism, moderate toxicity was noticed at higher concentrations, however opacity of treatment medium was observed, indicating the adequate selection of dose levels. No relevant increases in mutant frequencies were observed following treatment with the test item, in the absence or presence of S9 metabolism, in any experiment.

Negative and positive control treatments were included in each mutation experiment in the absence and presence of S9 metabolism. The mutant frequencies in the solvent control cultures fell within the normal range. Marked increases were obtained with the positive control treatments indicating the correct functioning of the assay system.

It is concluded that C12-15 alkyl benzoates does not induce mutation at the TK locus of L5178Y mouse lymphoma cells in vitro in the absence or presence of S9 metabolic activation, under the reported experimental conditions.

 

Micronucleus assay

The test item C12-15 alkyl benzoates was assayed for the ability to induce micronuclei in human lymphocytes, following in vitro treatment in the absence and presence of S9 metabolic activation.

Three treatment series were included in the study. A short treatment, where the cells were treated for 3 hours, was performed in the absence and presence of S9 metabolism. The harvest time of approximately 32 hours, corresponding to approximately two cell cycle lengths, was used. A long term (continuous) treatment was also performed only in the absence of S9 metabolism, until harvest at 31 hours.

Based on solubility data, the maximum practicable concentration of the test item in the final treatment medium was 5.00 μL/mL. Since the test item is not of defined composition (i.e. UVCB), this concentration is the upper limit to testing as indicated in the Study Protocol.

For the 3-hour treatment series, both in the absence and presence of S9 metabolism, the test item was assayed at the maximum concentration of 5.00 μL/mL and at the following dose levels: 3.33, 2.22, 1.48, 0.988, 0.658, 0.439, 0.293 and 0.195 μL/mL. For the continuous treatment in the absence of S9 metabolism, the additional dose level of 0.130 μL/mL was employed (Main Assay 1). All treatments were performed using the test item as a solution in ethanol with the exception of the continuous treatment in the absence of S9 at the highest dose level where, in order to reach the maximum concentration of 5.00 μL/mL, the test item was used as supplied.

Since for the continuous treatment, possibly due to the chemical and physical properties of the test item, an unexpected cytotoxicity profile was observed, it was considered appropriate to repeat the treatment by slightly modifying the procedures of treatment and incubation of the cultures (Main Assay 2) in order to improve the miscibility of the test item. The test item was assayed as a solution in ethanol by using a maximum dose level of 2.50 μL/mL and a larger space interval in order to cover a wider range of concentrations. Dose levels of 2.50, 1.56, 0.977, 0.610, 0.381, 0.238, 0.149, 0.0931, 0.0582, 0.0364, 0.0227, 0.0142 and 0.00888 μL/mL were tested.

Each treatment series included appropriate negative controls (untreated and solvent vehicle controls). In addition, positive controls were included for the short term and the long term treatment series (Cyclophosphamide and Colchicine, respectively).

Two cell cultures were prepared at each test point.

The actin polymerisation inhibitor Cytochalasin B was added prior to the targeted mitosis to allow the selective analysis of micronucleus frequency in binucleated cells. The cytokinesis block proliferation index CBPI was calculated in order to evaluate cytotoxicity.

Dose levels for the scoring of micronuclei were selected with the aim to evaluate the test item concentrations at adequate levels of cytotoxicity and considering turbidity or test item precipitation at the end of the exposure.

One thousand binucleated cells per culture were scored to assess the frequency of micronucleated cells.

Following treatment with the test item, no statistically significant increase in the incidence of micronucleated cells over the control value was observed at any dose level, in any treatment series. All the results obtained were inside the distribution of historical control data and no dose effect relationship was indicated by a linear trend analysis.

Statistically significant increases in the incidence of micronucleated cells were observed following treatments with the positive controls Cyclophosphamide and Colchicine, indicating the correct functioning of the test system.

It is concluded that C12-15 alkyl benzoates does not induce micronuclei in human lymphocytes after in vitro treatment, under the reported experimental conditions.

Justification for classification or non-classification

Based on the available data, the substance is not classified as mutagenic according to regulation (EC) 1272/2008.