Benzenepropanoic acid, 3,5-bis(1,1-dimethylethyl)-4-hydroxy-, 2-ethylhexyl ester

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:

10 Nov 2017 - 24 Nov 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

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)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Benzenepropanoic acid, 3,5-bis(1,1-dimethylethyl)-4-hydroxy-, 2-ethylhexyl ester

EC Number:

807-747-9

Cas Number:

144429-84-5

Molecular formula:

C25H42O3

IUPAC Name:

Benzenepropanoic acid, 3,5-bis(1,1-dimethylethyl)-4-hydroxy-, 2-ethylhexyl ester

Test material form:

liquid

Method

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

Metabolic activation system:

liver S9 mix from induced rats

Test concentrations with justification for top dose:

0; 33; 100; 333; 1000; 2500 and 5000 μg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
Due to the insolubility of the test substance in water, DMSO was used as vehicle, which had been demonstrated to be suitable in bacterial reverse mutation tests and for which historical control data are available.

The test substance was weighed and topped up with the chosen vehicle to achieve the required concentration of the stock solution. To achieve a clear solution of the test substance in the vehicle, the test substance preparation was shaken thoroughly. The further concentrations were diluted from the stock solution according to the planned doses. All test substance formulations were prepared immediately before administration.

Controls

Untreated negative controls:

yes

Remarks:

sterility control

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: see below

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicates
- Number of independent experiments: two

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): After incubation at 37°C for 48 – 72 hours in the dark, the bacterial colonies (his+ revertants) were counted.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
Toxicity detected by a
• decrease in the number of revertants (factor ≤ 0.6)
• clearing or diminution of the background lawn (= reduced his- or trp- background growth)


POSITIVE CONTROLS
The following positive controls were used to check the mutability of the bacteria and the activity of the S9 mix:
With S9 mix
• 2-aminoanthracene (2-AA)
- 2.5 μg/plate, dissolved in DMSO
- strains: TA 1535, TA 100, TA 1537, TA 98
- 60 μg/plate, dissolved in DMSO
- strain: Escherichia coli WP2 uvrA

Without S9 mix
• N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)
- 5 μg/plate, dissolved in DMSO
- strains: TA 1535, TA 100
• 4-nitro-o-phenylenediamine (NOPD)
- 10 μg/plate, dissolved in DMSO
- strain: TA 98
• 9-aminoacridine (AAC)
- 100 μg/plate, dissolved in DMSO
- strain: TA 1537
• 4-nitroquinoline-N-oxide (4-NQO)
- 5 μg/plate, dissolved in DMSO
- strain: E. coli WP2 uvrA

Evaluation criteria:

The test substance was considered positive in this assay if the following criteria were met:
• A dose-related and reproducible increase in the number of revertant colonies, i.e. at least doubling (bacteria strains with high spontaneous mutation rate, like TA 98, TA 100 and E.coli WP2 uvrA) or tripling (bacteria strains with low spontaneous mutation rate, like TA 1535 and TA 1537) of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.
A test substance was generally considered non-mutagenic in this test if:
• The number of revertants for all tester strains were within the range of the historical negative control data under all experimental conditions in at least two experiments carried out independently of each other.

Results and discussion

Test results

Species / strain:

other: all strains used

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

A weak bacteriotox effect (slight decrease in the number of his+ revertants) was observed only with tester strain TA 1535 with S9 mix at a concentration of 5000 μg/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: Test substance precipitation was found from about 1000 μg/plate onward both with and without S9 mix.

Ames test:
- Signs of toxicity : A weak bacteriotox effect (slight decrease in the number of his+ revertants) was observed only with tester strain TA 1535 with S9 mix at a concentration of 5000 μg/plate. In the preincaubation assay no bacteriotoxic effect (reduced his- or trp- background growth, decrease in the number of his+ or trp+ revertants) was observed up to the highest concentration

Any other information on results incl. tables

EXPERIMENTAL RESULTS

Standard Plate Test:

	Mean revertants per plate
Strain	TA 1535		TA 100		TA 1537		TA 98		WP2uvrA
Dose (µg/plate)	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
DMSO	9.3	8.7	106.7	120.3	8.3	7.3	19.7	23.0	24.0	22.0
33	10.3	10.7	105.7	102.3	7.0	7.3	14.3	22.0	22.0	21.0
100	13.0	9.7	98.7	119.0	6.3	5.3	17.7	24.3	22.7	26.7
333	9.0	11.0	95.0	116.3	9.7	11.7	16.3	25.7	24.7	24.7
1000	9.7	12.0	100.3	107.0	10.3	8.7	16.3	23.7	21.7	26.7
2500	8.0	7.0	106.3	104.0	8.3	10.7	14.3	28.7	24.7	25.3
5000	10.3	4.3	103.3	98.7	10.3	9.7	15.7	23.7	23.7	23.3
positive control	4514.0	209.3	3176.7	1659.7	763.3	85.3	922.0	854.3	740.0	86.7

Preincubation Test

	Mean revertants per plate
Strain	TA 1535		TA 100		TA 1537		TA 98		WP2uvrA
Dose (µg/plate)	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
DMSO	9.0	10.3	98.7	109.7	7.0	9.3	15.7	21.7	22.0	22.3
33	12.3	8.0	105.0	102.7	9.7	9.7	18.7	19.7	20.7	17.0
100	10.0	9.3	98.7	109.0	9.7	7.3	13.3	23.7	19.3	25.0
333	8.0	11.0	110.0	97.7	9.7	8.7	13.0	25.0	18.3	33.7
1000	7.7	8.7	97.7	107.7	8.7	7.3	18.3	29.3	16.7	23.7
2600	10.7	12.3	81.7	80.7	10.3	9.7	15.0	22.0	22.3	22.3
5200	8.7	9.0	103.7	97.0	8.3	13.3	16.7	30.0	19.0	24.0
positive control	3321.3	227.0	2361.3	1677.3	842.7	85.0	1057.0	872.0	238.3	88.7

Applicant's summary and conclusion

Conclusions:

Under the experimental conditions of this study, the test substance XPDL 920 is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation.

Executive summary:

The test substance was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of bacterial strains Salmonella typhimurium TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA in a reverse mutation assay. The dose range was 33 μg - 5000 μg/plate in standard plate test (SPT) and preincubation test (PIT) both with and without metabolic activation (liver S9 mix from induced rats). Precipitation of the test substance was found from about 1000 μg/plate onward both with and without S9 mix. A weak bacteriotoxic effect was observed only in the SPT with tester strain TA 1535 after the addition of S9 mix at a concentration of 5000 μg/plate. A relevant increase in the number of his+ or trp+ revertants (factor ≥ 2: TA 100, TA 98 and E.coli WP2 uvrA or factor ≥ 3: TA 1535 and TA 1537) was not observed in the standard plate test or in the preincubation test without S9 mix or after the addition of a metabolizing system. Under the experimental conditions of this study, the test substance XPDL 920 is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation.