2-[4-(4-Alkoxyphenyl)-(pyridin-2-yl)-heteromonocyclyl]phenol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

FAT 75637 was found to be non-mutagenic in Ames test.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Study initiation date - 03 December 2020; Experiment start date - 03 December 2020; Experiment completion date - 21 December 2020; Study completion date - 25 February 2021.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Test Item: FAT 75637/B TE
Physical Appearance: Light yellow powder
Purity: 99.4 % all organic constituents; 95.0 % main constituent
Batch No: AT-0063765400
Manufactured Date: 21st April 2020
Expiry Date: May 27th, 2025

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):

Salmonella typhimurium:
Health Protection Agency, National Collection of Type Cultures (NCTC) 61, Colindale Avenue, London NW9 5EQ, Great Britain

Escherichia coli: The National Collection of Industrial and Marine Bacteria Ltd. (NCIMB), Ferguson Building, Craibstone Estate, Bucksburn Aberdeen, AB21 9YA, Scotland, U.K.

Stock cultures of tester strains were stored in Oxoid nutrient broth No. 2 in the
test facility as frozen permanents in liquid nitrogen. Laboratory stocks were
maintained on respective minimal glucose agar plates as master plates of each
strain, for a maximum period of 2 months and refrigerated at 2 ºC to 8 ºC. The master plates prepared on 09 October 2020 and 30 November 2020 were used in the study.
The growth requirements and the genetic identity of strains like histidine or
tryptophan requirement, sensitivity to UV radiation, resistance of strains TA98, TA100 and WP2uvrA (pKM101) to ampicillin and rfa mutation of Salmonella typhimurium strains were checked along with the range of spontaneous revertants after preparation of the master plates.

Metabolic activation:

with and without

Metabolic activation system:

Preparation of S9 Homogenate and Activation Mixture
Hamster rat liver S9 homogenate (procured from Molecular Toxicology, Inc 157, Industrial Park Dr. Boone, NC 28607,[phone],USA), was used as the metabolic activation system.
The S9 homogenate was thawed immediately before use and mixed with the co-factor solution containing 4 mM NADP, 20 mM Glucose-6-phosphate, 8 mM MgCl2 ,2mM FMN, 2mM NADH,2.8 units of Glucose-6-phosphate dehydrogenase per mL and 33 mM KCl in PBS to achieve a final concentration of 30 % S9 (v/v) in the activation mixture.

S9 Mix
The co-factors solution was prepared by dissolving the following in 8.75 and 39.4 mL of cold PBS for the preliminary test and mutation assay, respectively. This solution was filter sterilized using a 0.2 μm disposable syringe filter.

Co-factor Preliminary toxicity test Mutation assay
NADP (4 mM) 78.73 196.86
Glucose-6-phosphate (20 mM) 85.05 382.7
Magnesium chloride (8 mM) 9.52 79.84
Potassium chloride (33 mM) 30.75 42.84
NADH(2mM) 17.74 53.82
FMN(2mM) 11.96 138.4
Glucose-6-phosphate dehydrogenase 2.8 units/mL 175 μL 788 μL

S9 mix was prepared by mixing 3.75 and 16.9 mL of the S9 homogenate with 8.75 and 39.4 mL of the co-factors solution for the preliminary toxicity and mutation assay, respectively, kept in an ice bath.

Test concentrations with justification for top dose:

Based on the observations of the preliminary toxicity test, the following five test doses were selected for testing in the mutation assay:
50, 158, 500, 1581 and 5000 μg/plate.

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

congo red

Remarks:

With metabolic activation - S. typhimurium TA 98, TA 100, TA1535, TA 1537; E. coli - WP2 uvrA (pKM101)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

Without metabolic activation - TA 98

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Without metabolic activation - TA 100 and TA 1535

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

Without metabolic activation - TA 1537

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

Without metabolic activation - WP2 uvrA (pKM101)

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Triplicate
- Number of independent experiments: Two

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium; in agar (plate incorporation) in initial mutation assay; preincubation - in confirmatory assay.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Background growth inhibition

METHODS FOR MEASUREMENTS OF GENOTOXICIY
: Dose related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of the test item either in the presence or absence of the metabolic activation system.

Evaluation criteria:

EVALUATION AND INTERPRETATION OF RESULTS
To determine a positive result, there should be a dose related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of the test item either in the presence or absence of the metabolic activation system. The test will be judged positive, if the increase in mean revertants at the peak of the dose response is equal to or greater than 2 times the mean vehicle control value for strains TA98, TA100 and WP2uvrA (pKM101) or equal to or greater than 3 times the mean vehicle control value for strains TA1535 and TA1537.
An equivocal response is a biologically relevant increase in a revertant count that partially meets the criteria for evaluation as positive. This could be a dose responsive increase that does not achieve the respective threshold cited above or a non-dose responsive increase that is equal to or greater than the respective threshold cited. A response will be evaluated as negative, if it is neither positive nor equivocal.

Key result

Species / strain:

other: S. typhimurium TA98, TA100, TA1535, TA1537 and E.coli WP2uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

RESULTS

Genotypic Characterization

Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 demonstrated the requirement of histidine amino acid for their growth. Escherichia coli strain WP2uvrA (pKM101) demonstrated the requirement of tryptophan amino acid for its growth. Ampicillin resistance was demonstrated by the strains TA98, TA100 and WP2uvrA (pKM101) which carry R-factor plasmids. The presence of characteristic mutations like the rfa mutation was demonstrated by all the Salmonella typhimurium strains by their sensitivity to crystal violet. The uvrA mutation in the Escherichia coli strain and the uvrB mutation in the Salmonella typhimurium strains were demonstrated through their sensitivity to ultraviolet light. Finally, all these tester strains produced spontaneous revertant colonies which were within the frequency ranges of the test facility’s historical control data.

Preliminary Solubility Test for selection of vehicle

The test item was insoluble in sterile water and formed free flowing suspension in DMSO at 50 mg/mL, on sonication. DMSO is one of the vehicles compatible with this test system. Hence, based on the results of the solubility test, DMSO was selected as the vehicle of choice in the mutation assay.

Preliminary Toxicity Test and Justification for the Selection of Target Top Dose

The test item did not cause precipitation on the basal agar plates up to 3200 μg/plate. Test item exhibited non-interfering precipitate at 5000 μg/plate at any of the tested doses. The test item did not show toxicity to the tester strain at any of the tested doses as the intensity of the bacterial background lawn as well as the mean number of revertant colonies were comparable to the vehicle control plates, both in the presence and absence of metabolic activation. Based on these observations, it was decided to test the OECD 471- recommended top dose of 5000 μg/plate in the mutation assay.

Mutation Assay

Viable Counts of the Overnight Tester Strains

Viable counts of all the tester strains were within the required range of 1-2 x 10E9 CFU/mL for the initial toxicity-mutation as well as the confirmatory mutation assay.

Initial Mutation Assay

The test item did not cause precipitation up to 1581 μg/plate. However non-interfering precipitate was observed at 5000 μg/plate on the basal agar plates. The test item did not show toxicity to any of the tester strains up to the top dose of 5000 μg/plate, as the intensity of the bacterial background lawn and the mean number of revertant colonies was comparable to the vehicle control plates both in the presence and absence of metabolic activation. The tested doses showed no positive mutagenic increase in the mean number of revertant colonies for any of the tester strains when compared to the respective vehicle control plates, either in the presence or absence of metabolic activation. Positive control chemicals tested simultaneously produced a more than 3-fold increase in the mean numbers of revertant colonies for all the strains when compared to the respective vehicle control plates. No toxicity was observed in the positive controls as the intensity of the bacterial background lawn of all the tester strains was comparable to that of the respective vehicle control plates.

Confirmatory Mutation Assay

The test item did not cause precipitation up to 1581 μg/plate. However non-interfering precipitate was observed at 5000 μg/plate on the basal agar plates. The test item did not show toxicity to any of the tester strains up to the top dose of 5000 μg/plate, as the intensity of the bacterial background lawn and the mean number of revertant colonies was comparable to the vehicle control plates both in the presence and absence of metabolic activation. The tested doses showed no positive mutagenic increase in the mean number of revertant colonies for any of the tester strains when compared to the respective vehicle control plates, either in the presence or absence of metabolic activation. Positive control chemicals tested simultaneously produced a more than 3-fold increase in the mean numbers of revertant colonies for all the strains when compared to the respective vehicle control plates. No toxicity was observed in the positive controls as the intensity of the bacterial background lawn of all the tester strains was comparable to that of the respective vehicle control plates.

Conclusions:

FAT 75637/B was not mutagenic in this bacterial reverse mutation test up to the OECD 471-recommended top dose of 5000 µg/plate under the conditions of testing employed.

Executive summary:

The test item, FAT 75637/B was tested for its mutagenic potential in the bacterial reverse mutation assay in a study conducted according to OECD test guideline 471 in a GLP certified laboratory. The study was conducted using TA98, TA100, TA1535 and TA1537 strains of Salmonella typhimurium and WP2uvrA (pKM101) strain of Escherichia coli in three phases, a preliminary toxicity test, an initial mutation assay and a confirmatory mutation assay. The bacterial tester strains were exposed to the test item in the presence and absence of hamster uninduced liver S9. FAT 75637/B formed a free-flowing suspension in DMSO at 50 mg/mL. In a preliminary toxicity test for the selection of test doses for the mutation assay, the test item did not precipitate on the basal agar plates up to 3200 µg/plate. However, at 5000 µg/plate, test item showed non interfering precipitate, both in the presence and absence of metabolic activation system. The test item did not cause toxicity to the strain at any of the tested doses as the intensity of the bacterial background lawn as well as the mean number of revertant colonies were comparable to the vehicle control plates. Based on these observations, the OECD 471-recommended top dose of 5000 µg/plate was tested in the mutation assay. In the mutation assay the bacterial tester strains were exposed to FAT 75637/B in triplicate at 50, 158, 500, 1581 and 5000 µg/plate. The initial mutation assay was conducted using the direct plate incorporation mode of exposure whereas, the confirmatory mutation assay was carried out using the pre-incubation mode of exposure. The vehicle control (DMSO) and the appropriate positive controls were tested simultaneously. The mean and standard deviation of numbers of revertant colonies were calculated for each test dose and the controls for all the tester strains. The results of the study from both the initial and confirmatory mutation assays showed that, FAT 75637/B did not show any positive mutagenic increase at any of the tested doses either in the presence or in the absence of metabolic activation. Under identical test conditions, there was a more than 3-fold increase in the mean numbers of revertant colonies in the positive controls, demonstrating the sensitivity of the assay procedure used.  The study indicated that FAT 75637/B was not mutagenic in this Bacterial Reverse Mutation Assay up to the OECD 471-recommended top dose of 5000 µg/plate, under the conditions of testing employed.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Ames test

The test item, FAT 75637/B was tested for its mutagenic potential in the bacterial reverse mutation assay in a study conducted according to OECD test guideline 471 in a GLP-certified laboratory. The study was conducted using TA98, TA100, TA1535 and TA1537 strains of Salmonella typhimurium and WP2uvrA (pKM101) strain of Escherichia coli in three phases, a preliminary toxicity test, an initial mutation assay and a confirmatory mutation assay. The bacterial tester strains were exposed to the test item in the presence and absence of hamster uninduced liver S9. FAT 75637/B formed a free-flowing suspension in DMSO at 50 mg/mL. In a preliminary toxicity test for the selection of test doses for the mutation assay, the test item did not precipitate on the basal agar plates up to 3200 µg/plate. However, at 5000 µg/plate, test item showed non interfering precipitate, both in the presence and absence of metabolic activation system. The test item did not cause toxicity to the strain at any of the tested doses as the intensity of the bacterial background lawn as well as the mean number of revertant colonies were comparable to the vehicle control plates. Based on these observations, the OECD 471-recommended top dose of 5000 µg/plate was tested in the mutation assay. In the mutation assay the bacterial tester strains were exposed to FAT 75637/B in triplicate at 50, 158, 500, 1581 and 5000 µg/plate. The initial mutation assay was conducted using the direct plate incorporation mode of exposure whereas, the confirmatory mutation assay was carried out using the pre-incubation mode of exposure. The vehicle control (DMSO) and the appropriate positive controls were tested simultaneously. The mean and standard deviation of numbers of revertant colonies were calculated for each test dose and the controls for all the tester strains. The results of the study from both the initial and confirmatory mutation assays showed that, FAT 75637/B did not show any positive mutagenic increase at any of the tested doses either in the presence or in the absence of metabolic activation. Under identical test conditions, there was a more than 3-fold increase in the mean numbers of revertant colonies in the positive controls, demonstrating the sensitivity of the assay procedure used. The study indicated that FAT 75637/B was not mutagenic in this Bacterial Reverse Mutation Assay up to the OECD 471-recommended top dose of 5000 µg/plate, under the conditions of testing employed.

Justification for classification or non-classification

Based on the available result of the bacterial reverse mutation assay, the substance is not to be classified for mutagenicity as per EU CLP [Regulation (EC) No 1272/2008].