2-[2,4-bis(tert-pentyl)phenoxy]-N-(3,5-dichloro-4-ethyl-2-hydroxyphenyl)butyramide

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:

September 2018

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2-[2,4-bis(tert-pentyl)phenoxy]-N-(3,5-dichloro-4-ethyl-2-hydroxyphenyl)butyramide

EC Number:

300-634-0

EC Name:

2-[2,4-bis(tert-pentyl)phenoxy]-N-(3,5-dichloro-4-ethyl-2-hydroxyphenyl)butyramide

Cas Number:

93951-12-3

Molecular formula:

C28H39Cl2NO3

IUPAC Name:

2-[2,4-bis(tert-pentyl)phenoxy]-N-(3,5-dichloro-4-ethyl-2-hydroxyphenyl)butyramide

Test material form:

solid

Details on test material:

Batch 44029
colour : White to off-white
retest date : 01/06/2019

Specific details on test material used for the study:

Stored ate room temperature (15-25°C)- keep away from light - non hygroscopic

Method

Target gene:

strains histidine-dependent and tryptophane-dependent

Species / strain

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

E Coli WP2 (uvr A-) pKM101

Metabolic activation:

with and without

Metabolic activation system:

microsomal fraction of rat liver

Test concentrations with justification for top dose:

5000, 1500, 500, 150 and 50 µg/plate

Vehicle / solvent:

DMSO

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

9-aminoacridine

2-nitrofluorene

sodium azide

other: cis-platinum (II) Diammine Dichloride and 2-anthramine

Details on test system and experimental conditions:

Histidine and tryptophane requirements with cultures in presence and in absence of L-histidine and L-triptophane for Salmonella typhimurium and Escherichia coli strains respectively.
Loss of cell wall LPS (rfa mutation) measuring crystal violet inhibition for Salmonella typhimurium strains.
Ampicillin resistance for the strains which have the pKM 101 plasmide.
deltauvr B mutation i.e. U.V.B sensitivity for Salmonella typhimurium and deltauvr A mutation i.e. U.V.A sensitivity for Escherichia coli.
Spontaneous revertant rate.

Evaluation criteria:

Ensure that the criteria of validity of the study are well respected namely :
 the bacteriostatic activity of the highest concentration tested shall be equal to or less than 75 %,
 the spontaneous reversion rate of the absolute negative control shall comply with the historical values of the laboratory,
 the spontaneous reversion rate of the solvent shall not be statistically different from absolute negative control,
 the mean number of revertant colonies obtained for each strain and the corresponding positive control, with and/or without metabolic activation shall comply with the historical values of the laboratory.
 Negative and positive values should not show significant difference with the historical values of the laboratory (± 2 standard deviations).

Results and discussion

Test results

Test results 1

Key result

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

Key result

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:

valid

Positive controls validity:

valid

Test results 3

Key result

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:

valid

Positive controls validity:

valid

Test results 4

Key result

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

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

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

Additional information on results:

No deviation

Any other information on results incl. tables

Mutation assay interpretation

There is no significant difference between the number of spontaneous reversions, the number of reversions obtained for the positive controls (without and with metabolic activation), and the mean of corresponding experimental historical values obtained in the laboratory.

There is no evidence of any increase in the number of revertant colonies in the presence of the test item stock solution and dilutions (5 000, 1 500, 500, 150 and 50 μg/plate) without and with metabolic activation in (Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 and in Escherichia coli WP2(uvrA-) (pKM 101).

One can observe in presence of the highest doses tested the presence of precipitates no hindering the scoring.

Results are confirmed in an independent experiment.

Applicant's summary and conclusion

Conclusions:

Doses (5 000, 1 500, 500, 150 and 50 μg/plate) prepared from solutions of the test item 80ACQ 2-[2,4-bis(tert-pentyl)phenoxy]-N-(3,5-dichloro-4-ethyl-2-hydroxyphenyl)butyramide BATCH: 44029 (LEMI code: LM-18/0226) provided by LA MESTA, do not induce any mutagenic change in Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 and in Escherichia coli WP2 (uvrA¯) (pKM 101) without, or with metabolic activation, according to the OECD Guidelines n° 471.

Executive summary:

Solutions obtained from test item, have been tested for their capacity to induce reverse mutation in four Salmonella typhimurium strains and one Escherichia coli WP2(uvr A¯)(pKM101) strain. This study was performed in the absence and presence of metabolic activation. Two independent assays were carried out

For assay n° 1, various concentrations were put in contact with the strains in the absence and presence of a metabolic activation system (S9-mix 10% (v/v)).

For assay n° 2, various concentrations were put in contact with the strains in the absence of metabolic activation and with pre-incubation in the presence of metabolic activation system (S9-mix 10% (v/v)).

For the two assays, negative and positive controls were carried out in parallel. Positive controls induced a significant increase in the number of revertant colonies compared to negative controls. There is no significant difference between the number of spontaneous reversions, the number of reversions obtained in the positive controls (without and with metabolic activation), and the mean of corresponding experimental “historical” values obtained in the laboratory.

These results validate the two tests.

There is no evidence of any increase in the number of revertant colonies in the presence of the various concentrations of the test item (5 000, 1 500, 500, 150 and 50 μg/plate), without and with metabolic activation in Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 and in Escherichia coli WP2(uvrA¯) (pKM 101).