Benzoic acid, 4-hydroxy-, C18-22-alkyl esters

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

13 - 20 Oct 2005

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP-Guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

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)

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

The Department of Health of the Government of the United Kingdom

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

IUPAC Name:

68411-27-8

Details on test material:

- Name of test material (as cited in study report): C12 - C15 alkyl benzoate
- Substance type: UVCB
- Physical state: clear colourless liquid
- Analytical purity: > 99%
- Purity test date: 10 Aug 2005
- Lot/batch No.: ES65530820
- Expiration date of the lot/batch: 19 May 2007
- Storage condition of test material: at room temperature in the dark

Method

Target gene:

his operon

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbitone/β-naphthoflavone

Test concentrations with justification for top dose:

Preliminary toxicity test: 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate with and without metabolic activation
Experiments 1 and 2: 50, 150, 500, 1500, 5000 µg/plate with and without metabolic activation

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: The test substance was immiscible in both sterile distilled water and DMSO at 50 mg/mL, but was fully miscible in acetone at the same concentration in solubility checks performed in-house.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG): 2, 3 and 5 µg/plate for WP2 uvrA, TA 100 and TA 1535, respectively; 9-aminoacridine (9AA): 80 µg/plate for TA 1537; 4-nitroquinoline-1-oxide (4NQO): 0.2 µg/plate for TA 98

Remarks:

without metabolic activation

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (2AA): 1, 2 and 10 µg/plate for TA 100, TA 1535/TA 1537 and WP2 uvrA, respectively; benzo(a)pyrene (BP): 5 µg/plate for TA 98

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation

DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: triplicates each in two independent experiments

NUMBER OF CELLS EVALUATED:

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; inspection of bacterial background lawn

Evaluation criteria:

There are several criteria for determining a positive result, such as a dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolic activation. Biological relevance of the results is considered first, statistical methods, as recommended by the UKEMS (Kirkland, 1989) can also be used as an aid to evaluation, however statistical significance is the only determining factor for a positive response.
A test material is considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments give clear positive or negative results, in some instances the data generated prohibits a definite judgement about the test material activity. Results of this type are reported as equivocal.

Reference
Kirkland D.J. (Ed) (1989). Statistical Evaluation of Mutagenicity Test Data. UKEMS Subcommittee on Guidelines for Mutagenicity Testing, Report – Part III, Cambridge University Press.

Statistics:

Mean values and standard deviation were calculated.

Results and discussion

Test results

Test results 1

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: The test material was immiscible in both distilled water and dimethyl sulphoxide at 50 mg/mL but was fully miscible in acetone at the same concentration in solubility checks performed in-house at the test facility. Acetone was therefore selected as the vehicle of choice.
- Precipitation: A precipitate (oily in appearance) was observed at and above 1500 µg/plate, this did not prevent the scoring of revertant colonies.
- Other confounding effects: No biologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any concentration of the test material, either with or without metabolic activation. A small increase in revertant colony frequency was observed in tester strain TA 100, with S9-mix only, at 50 µg/plate in Experiment 1. However, this increase was not reproduced in Experiment 2 and was considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the revertant counts at 50 µg/plate were well within the in-house range of the tester strain and the fold increase was only 1.32 times the concurrent vehicle control.

RANGE-FINDING/SCREENING STUDIES:
In the preliminary toxicity test, the test material was non-toxic to the strains of bacteria used (TA 100 and WP2 uvrA) (see Table 1). The test material formulation and S9-mix used in this experiment were both shown to be sterile.

COMPARISON WITH HISTORICAL CONTROL DATA:
The revertant counts for the negative, vehicle and positive controls were within the historical in-house ranges for the two foregoing years.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level. The test material was, therefore, tested up to the maximum recommended concentration of 5000 µg/plate.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1. Preliminary toxicity test

 

With (+) or without (-) S9-mix	Strain	Concentration (µg/plate)
		0	0.15	0.5	1.5	5	15	50	150	500	1500	5000
-	TA 100	169	133	133	141	148	126	159	163	150	145P	150P
+	TA 100	102	81	86	77	84	78	98	98	78	90P	89P
-	WP2 uvrA	26	27	21	24	20	24	21	17	18	21P	26P
+	WP2 uvrA	37	24	34	29	29	36	30	30	37	29P	38P

 

P: Precipitate

 

Table 2. Spontaneous mutation rates (concurrent negative controls)

 

	Base-pair substitution type			Frameshift type
	TA 100	TA 1535	WP2 uvrA	TA 98	TA1537
Experiment 1
Number of revertants per plate	99	16	30	18	10
	117	14	32	18	10
	85	12	22	21	13
Mean	100	14	28	19	11
Experiment 2
Number of revertants per plate	166	23	29	26	9
	151	26	24	21	13
	110	24	26	26	14
Mean	142	24	26	24	12

 

 

Table 3. Results of Experiment 1

 

With ( ± ) or without (-) S9-mix	Test substance concentration (µg/plate)	Number of revertants (mean number of colonies per plate ± standard deviation)
		Base-pair substitution type			Frameshift type
		TA 100	TA 1535	WP2 uvrA	TA 98	TA1537
-	0	76 90 108 91 ± 16.0	17 16 18 17 ± 1.0	28 22 23 24 ± 3.2	19 14 15 16 ± 2.6	7 5 11 8 ± 3.1
-	50	91 90 88 90 ± 1.5	10 27 20 19 ± 8.5	22 15 18 18 ± 3.5	16 26 23 22 ± 5.1	9 6 9 8 ± 1.7
-	150	103 117 88 103 ± 14.5	14 21 25 20 ± 5.6	17 16 10 14 ± 3.8	22 18 13 18 ± 4.5	5 5 10 7 ± 2.9
-	500	88 96 103 96 ± 7.5	14 33 26 24 ± 9.6	24 21 20 22 ± 2.1	13 26 21 20 ± 6.6	6 5 6 6 ± 0.6
-	1500	96P 103P 94P 98 ± 4.7	29P 23P 23P 25 ± 3.5	22P 16P 20P 19 ± 3.1	13P 14P 10P 12 ± 2.1	5P 11P 9P 8 ± 3.1
-	5000	105P 115P 101P 107 ± 7.2	26P 20P 23P 23 ± 3.0	14P 21P 10P 15 ± 5.6	14P 17P 9P 13 ± 4.0	8P 8P 15P 10 ± 4.0
-	ENNG (3 µg/plate)	539 666 561 589 ± 67.9	-	-	-	-
-	ENNG (5 µg/plate)	-	332 407 401 380 ± 41.7	-	-	-
-	ENNG (2 µg/plate)	-	-	904 901 924 910 ± 12.5	-	-
-	4NQO (0.2 µg/plate)	-	-	-	373 361 318 351 ± 28.9	-
-	9AA (80 µg/plate)	-	-	-	-	2002 1543 1548 1698 ± 263.6
±	0	86 72 81 80 ± 7.1	14 9 13 12 ± 2.6	C 40 28 34 ± 8.5	22 24 20 22 ± 2.0	11 6 10 9 ± 2.6
±	50	94 126 95 105 ± 18.2*	10 10 14 11 ± 2.3	21 25 23 23 ± 2.0	15 22 22 20 ± 4.0	8 8 9 8 ± 0.6
±	150	89 94 102 95 ± 6.6	15 16 14 15 ± 1.0	21 27 28 25 ± 3.8	9 23 23 18 ± 8.1	10 9 9 9 ± 0.6
±	500	91 100 91 94 ± 5.2	15 14 19 16 ± 2.6	27 29 30 29 ± 1.5	21 13 18 17 ± 4.0	C 5 10 8 ± 3.5
±	1500	89P 79P 97P 88 ± 9.0	8P 15P 17P 13 ± 4.7	22P 31P 30P 28 ± 4.9	22P 26P 23P 24 ± 2.1	6P 7P 9P 7 ± 1.5
±	5000	77P 94P 100P 90 ± 11.9	16P 11P 8P 12 ± 4.0	28P 30P 32P 30 ± 2.0	30P 22P 18P 23 ± 6.1	7P 7P 7P 7 ± 0.0
±	2AA (1 µg/plate)	1944 1744 1988 1892 ± 130.0	-	-	-	-
±	2AA (2 µg/plate)	-	213 184 275 224 ± 46.5	-	-	-
±	2AA (10 µg/plate)	-	-	997 960 1040 999 ± 40.0	-	-
±	BP (5 µg/plate)	-	-	-	212 267 251 243 ± 28.3	-
±	2AA (2 µg/plate)	-	-	-	-	174 207 234 205 ± 30.0

 

ENNG: N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO: 4-nitroquinoline-1-oxide

9AA: 9-aminoacridine

2AA: 2-aminoanthracene

BP: benzo(a)pyrene

P: Precipitate

C: Contaminated

* p ≤ 0.05

 

 

Table 4. Results of Experiment 2

 

With ( ± ) or without (-) S9-mix	Test substance concentration (µg/plate)	Number of revertants (mean number of colonies per plate ± standard deviation)
		Base-pair substitution type			Frameshift type
		TA 100	TA 1535	WP2 uvrA	TA 98	TA1537
-	0	100 128 152 127 ± 26.0	30 38 39 36 ± 4.9	29 31 22 27 ± 4.7	18 19 15 17 ± 2.1	8 15 17 13 ± 4.7
-	50	111 146 154 137 ± 22.9	32 26 35 31 ± 4.6	18 19 18 18 ± 0.6	15 14 19 16 ± 2.6	9 11 10 10 ± 1.0
-	150	123 C 127 125 ± 2.8	32 41 35 36 ± 4.6	20 21 22 21 ± 1.0	18 26 26 23 ± 4.6	7 13 9 10 ± 3.1
-	500	125 112 102 113 ± 11.5	34 37 38 36 ± 2.1	33 18 17 23 ± 9.0	26 24 16 22 ± 5.3	14 18 17 16 ± 2.1
-	1500	116P 108P 95P 106 ± 10.6	33P 31P 33P 32 ± 1.2	18P 20P 11P 16 ± 4.7	22P 17P 19P 19 ± 2.5	8P 13P 11P 11 ± 2.5
-	5000	117P 104P 104P 108 ± 7.5	29P 40P 27P 32 ± 7.0	17P 22P 14P 18 ± 4.0	16P 17P 19P 17 ± 1.5	13P 19P 22P 18 ± 4.6
-	ENNG (3 µg/plate)	496 514 562 524 ± 34.1	-	-	-	-
-	ENNG (5 µg/plate)	-	247 330 335 304 ± 49.4	-	-	-
-	ENNG (2 µg/plate)	-	-	730 660 609 666 ± 60.7	-	-
-	4NQO (0.2 µg/plate)	-	-	-	235 200 195 210 ± 21.8	-
-	9AA (80 µg/plate)	-	-	-	-	897 735 757 796 ± 87.9
±	0	158 102 113 124 ± 29.7	14 16 18 16 ± 2.0	20 27 33 27 ± 6.5	22 17 33 24 ± 8.2	17 16 14 16 ± 1.5
±	50	101 104 103 103 ± 1.5	15 17 10 14 ± 3.6	31 18 23 24 ± 6.6	29 17 26 24 ± 6.2	7 8 16 10 ± 4.9
±	150	94 94 111 100 ± 9.8	18 14 24 19 ± 5.0	30 17 25 24 ± 6.6	18 16 20 18 ± 2.0	13 15 15 14 ± 1.2
±	500	86 112 103 100 ± 13.2	16 14 6 12 ± 5.3	24 29 20 24 ± 4.5	20 21 25 22 ± 2.6	13 18 8 13 ± 5.0
±	1500	96P 108P 94P 99 ± 7.6	14P 15P 18P 16 ± 2.1	23P 21P 29P 24 ± 4.2	17P 17P 29P 21 ± 6.9	7P 15P 15P 12 ± 4.6
±	5000	100P 89P 125P 105 ± 18.4	16P 16P 13P 15 ± 1.7	25P 34P 36P 32 ± 5.9	20P 17P 21P 19 ± 2.1	11P 17P 15P 14 ± 3.1
±	2AA (1 µg/plate)	1676 1724 1418 1606 ± 164.6	-	-	-	-
±	2AA (2 µg/plate)	-	262 264 332 286 ± 39.8	-	-	-
±	2AA (10 µg/plate)	-	-	497 484 463 481 ± 17.2	-	-
±	BP (5 µg/plate)	-	-	-	202 235 254 230 ± 26.3	-
±	2AA (2 µg/plate)	-	-	-	-	254 377 254 295 ± 71.0

 

ENNG: N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO: 4-nitroquinoline-1-oxide

9AA: 9-aminoacridine

2AA: 2-aminoanthracene

BP: benzo(a)pyrene

P: Precipitate

C: Contaminated

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative