JAUNE REACTIF 181

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Genetic Toxicity In vitro: Ames test

In a GLP-compliant study, FAT 40400/F was tested for its mutagenic potential in the bacterial reverse mutation assay according to OECD guideline 471. 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 homogenate. FAT 40400/F was soluble in sterile water (SW) at 50 mg/mL. In a preliminary toxicity test conducted for the selection of test doses for the mutation assay, the test item did not precipitate on the basal agar plates at any of the tested doses. The test item did not cause 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. In the mutation assay the bacterial tester strains were exposed to FAT 40400/F 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 (SW) 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 40400/F 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. Based on the study results, FAT 40400/F 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.

In another supporting study (GLP-compliant), performed according to EU-Method B.14, 5 Salmonella typhimurium strains (TA 1535, TA 1537, TA 1538, TA 98, and TA 100) were used to the test the mutagenic potential of the test substance (10, 100, 333.3, 1000, 5000 µg per plate), both with and without metabolic activation (CCR 1990). No cytotoxic effects were observed in the test groups with and without metabolic activation in both experiments. Up to the highest investigated dose, neither a significant and reproducible increase of the number of revertants was found in any strain as compared to the solvent control nor a concentration-dependent enhancement of the revertant number exists. The presence of liver microsomal activation did not influence these findings. The test substance did not show any mutagenic activity in any strain. Therefore, it was concluded that the test substance did not show any mutagenic activity in any strain.

Genetic Toxicity In vitro: Chromosome Aberration

In a GLP-compliant chromosome aberration test, tested according to OECD guideline 473, Chinese hamster lung fibroblast (V79) were exposed to the test substance with and without metabolic activation (CCR 1990). Different exposure times (up to 28 hours) and dosage (up to 5 mg/mL) were chosen in this study. The mitotic index was reduced after treatment with the highest concentrations (with and without S9 mix) indicating that the test substance had cytotoxic properties. There was no relevant increase in cells with structural aberrations after treatment with the test article at any fixation interval either without or with metabolic activation by S9 mix. Therefore, it was stated that the test substance did not induce structural chromosome aberrations in the V79 Chinese hamster cell line.

In vitro Mammalian Cell Gene Mutation Test (HPRT-Locus) in Chinese Hamster V79 Cells:

No studies on the mutagenicity in mammalian cells of FAT40400 were available. However, a HPRT gene mutation test is available for the structural analogue FAT 40426.

In a GLP-compliant mammalian cell gene mutation assay, tested according to OECD guideline 476, Chinese hamster V79 cells were exposed to the test substance with and without metabolic activation and the potential to induce mutations at the HPRT locus was assessed (BSL Bioservice 2013). The selection of the concentrations was based on data from the pre-experiments. Experiment I with and without metabolic activation and experiment II with metabolic activation were performed as a 4h short-term exposure assay. Experiment II without metabolic activation was performed as 20 h long-term exposure assay. The following concentrations were used. Experiment I without metabolic activation: 5, 10, 25, 50, 100, 250, 500, 1000, 2000 and 2500 µg/mL; Experiment I with metabolic activation: 5, 10, 25, 50, 100, 250, 500, 1000, and 2500 µg/mL; Experiment II without metabolic activation: 10, 25, 50, 100, 200, 400, 600 and 800µg/mL; Experiment II with metabolic activation: 100, 316, 1000, 1250, 1500, 2000, 2400, 2800, and 3000µg/mL. No precipitation was noted in the experiments. Biologically relevant growth inhibition was observed in experiment I and II with and without metabolic activation. In experiment I without metabolic activation the relative growth was 16.5 % for the highest concentration (2500 µg/mL) evaluated. The highest biologically relevant concentration evaluated with metabolic activation was 2500 µg/mL with a relative growth of 17.4 %. In experiment II without metabolic activation the relative growth was 12.2 % for the highest concentration (800 µg/mL) evaluated. The highest concentration evaluated with metabolic activation was 3000 µg/mL with a relative growth of 21.8 %. In both experiments no biologically relevant increase of mutants was found after treatment with the test item (with and without metabolic activation). No dose-response relationship was observed. The positive controls showed distinct biologically relevant effects in mutation frequency. In conclusion, the test substance is considered to be non-mutagenic in the HPRT locus using V79 cells of the Chinese hamster.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 August 1990 to 19 November 1990

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Qualifier:

according to guideline

Guideline:

other: Revised Chemical Substance Law (1987) according to the notification of December 9, 1986 by EA, Environmental Agency (No. 700); MHW, Ministry of Health and Welfare (No. 1039) and MITI, Ministry of International Trade and Industry (No. 1014), Japan.

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: BG 3247/TV 6
- Expiration date of the lot/batch: March 1995

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under storage conditions: Pure: 5 years; In solvent: in water, methanol, acetone, DMSO and DMF > 72 hours
- Stability under test conditions:

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Metabolic activation system:

aroclor 1254 induced rat liver S9 mix

Test concentrations with justification for top dose:

Evaluated test concentrations: with and without S9 mix: 7 hours: 5.0 mg/mL, 18 hours: 0.5, 4.0, 5.0 mg/mL, 28 hours: 5.0 mg/mL

Vehicle / solvent:

culture medium without fetal calf serum

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation: Two days old logarithmically growing stock cultures more than 50 % confluent were trypsinised and a single cell suspension was prepared. The cells were seeded into Quadriperm dishes ( Heraeus, D-6450 Hanau, F.R.G. ) which contained microscopic slides (2 chambers per dish and test group). In each chamber 5E4 - 1E5 cells were seeded with regard to preparation time. The medium was MEM + 10 % FCS.
- Exposure: After 48 hours (7 hours, 28 hours preparation interval) and 55 hours (18 hours preparation interval) the medium was replaced with serum-free medium containing the test article, either without S9 mix or with 20 µL/mL S9 mix. After 4 hours this medium was replaced with normal medium after rinsing twice with 'saline G'.
- Fixation: 5, 15.5 and 25.5 hours after the start of the treatment colcemid was added (0.2 µg/mL culture medium) to the cultures. 2 hours (7 hours
interval) or 2.5 hours later, (18 hours and 28 hours interval) the cells were treated on the slides in the chambers with hypotonic solution (0.4 % KCl) for 20 minutes at 37 °C. After incubation in the hypotonic solution the cells were fixed with 3 + 1 absolute methanol + glacial acetic acid. All two slides per group were prepared. After fixation the cells were stained with Giemsa (Merck, D-6100 Darmstadt, F.R.G.).

NUMBER OF CELLS EVALUATED:
- At least 100 well spread metaphases per slide were scored for cytogenetic damage on coded slides. Only metaphases with characteristic chromosome number of 22 ± 1 were included in the analysis.

DETERMINATION OF CYTOTOXICITY
- To describe a cytotoxic effect the mitotic index (% cells in mitosis) was determined.

Evaluation criteria:

A test article is classified as mutagenic if it induces either a significant dose-related increase in the number of structural chromosomal aberrations or a significant positive response for at least one of the test points. A test article producing neither a significant dose-related increase in the number of structural chromosomal aberrations nor a significant and reproducible positive response at any one of the test points is considered non-mutagenic in this system.

Statistics:

chi-square test

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

- Additional information on genotoxicity:
Both, in the absence and presence of S9 mix the test article did not increase significantly the frequency of cells with aberrations at any fixation interval. The aberration rates of the cells after treatment with the test article (0.5 % - 4.0 %) were in or near to the range of the control values : 0.0 % - 2.0 %. At fixation interval 18 hours in the absence of S9 mix in one sample treated with 4.0 mg/mL the aberration rate (4.0 %) was slightly increased as compared to the control value (2.0 %). This effect was observed only in one of the duplicate cultures. As this value falls within our historical data range: 0.0 % - 4.0 % the observed effect is not regarded as biologically relevant. Also, the occurrence of two exchanges is restricted to this sample and do not influence in this case the evaluation of the test article. Therefore, a dose-depended increase in the exchange rate (1 % with 4.0 mg; 1.5 % with 5.0 mg/mL) can not be evaluated unequivocally. In addition, the exchange rate found in the sample treated with 5.0 mg/mL is near to our historical exchange control range. 0.0 % - 1.0 %.

- Additional information on cytotoxicity:
In the pre-experiment on toxicity (colony forming ability) in the presence of S9 mix after treatment with 5.0 mg/mL the colony forming ability was distinctly reduced; in the absence of S9 mix only a slight reduction was observed. Higher concentrations were not applied. Also, in the main experiment the mitotic index was reduced in the high dose range (1.0 - 5.0 mg/mL) at fixation intervals 7 and 28 hours both in the presence and absence of S9 mix. At fixation interval 18 hours, only in the presence of S9 mix some indications of toxicity were observed (using the mitotic index as indicator).

Remarks on result:

other: all strains/cell types tested

Conclusions:

FAT 40400/A is considered to be not clastogenic in this chromosomal aberration test.

Executive summary:

In a GLP-compliant chromosome aberration test, tested according to OECD guideline 473, chinese hamster lung fibroblast (V79) were exposed to the test substance with and without metabolic activation.

The test article FAT 40400/A was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro. Preparation of chromosomes was done 7 h (high dose), 18 h (low, medium and high dose), and 28 h (high dose) after start of treatment with the test article. The treatment interval was 4 h. In each experimental group two parallel cultures were used. Per culture 100 metaphases were scored for structural chromosomal aberrations.

The following dose levels were evaluated:

without S9 mix:

7 h: 5.0 mg/ml

18 h: 0.5; 4.0; 5.0 mg/ml

28 h: 5.0 mg/ml

with S9 mix:

7 h: 5.0 mg/ml

18 h: 0.5; 4.0; 5.0 mg/ml

28 h : 5.0 mg/ml

 

The concentration range of the test article applied had been determined in a pre-experiment using the plating efficiency assay as indicator for toxicity response. Treatment with 5.0 mg/ml reduced distinctly the plating efficiency of the V79 cells in the presence of S9 mix; in the absence of S9 mix only a slight reduction was observed. Also, toxic effects were observed in the main experiment using the mitotic index as indicator for toxicity. Higher concentrations than 5.0 mg/ml were not applied. There was no relevant increase in cells with structural aberrations after treatment with the test article at any fixation interval either without or with metabolic activation by S9 mix. Appropriate reference mutagens were used as positive controls and showed distinct increases in cells with structural chromosome aberrations. In conclusion, it can be stated that in the study described and under the experimental conditions reported, the test article did not induce structural chromosome aberrations as determined by the chromosomal aberration test in the V79 Chinese hamster cell line. Therefore, FAT 40400/A is considered to be non-clastigenic in this chromosomal aberration test.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 November 2012 to 14 March 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

(Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Germany)

Type of assay:

mammalian cell gene mutation assay

Target gene:

hypoxanthine-guanine-phosphoribosyl-transferase (HPRT)

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

-Type and identity of media: MEM
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

Liver S9 of Wistar Phenobarbital and ß-Naphthoflavone-induced rat liver S9 mix

Test concentrations with justification for top dose:

Pre-experiment for experiment I (without metabolic activation):
5, 10, 25, 50, 100, 250, 500, 1000, 2500 and 5000 µg/mL
Pre-experiment for experiment II (only without metabolic activation, 20 h long-term exposure assay):
50, 100, 250, 500, 1000, 2000, 3000 and 5000 µg/mL
Experiment I
without metabolic activation: 5, 10, 25, 50, 100, 250, 500, 1000, 2000 and 2500 µg/mL
and with metabolic activation: 5, 10, 25, 50, 100, 250, 500, 1000 and 2500 µg/mL
Experiment II
without metabolic activation: 10, 25, 50, 100, 200, 400, 600 and 800 µg/mL
and with metabolic activation: 100, 316, 1000, 1250, 1500, 2000, 2400, 2800 and 3000 µg/mL

Vehicle / solvent:

Vehicle (Solvent) used: cell culture medium (MEM + 0 % FBS 4 h treatment; MEM + 10 % FBS 20 h treatment). The test item was dissolved under stirring in cell culture medium and diluted prior to treatment

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation; 300 µg/mL

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

with metabolic activation; 1 and 1.5 µg/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: dissolved in medium
DURATION: 4 h (short-term exposure), 20 h (long-term exposure)
Expression time (cells in growth medium): 5 days
Selection time (if incubation with selection agent): about one week

SELECTION AGENT ( mutation assay) 11 µg/mL 6-thioguanine (TG)
NUMBER OF REPLICATIONS: two separate experiments (I+II) with single exposure; 5 individual flasks were seeded and evaluated
NUMBER OF CELLS EVALUATED: 400000 cells per flask
DETERMINATION OF CYTOTOXICITY: Method: relative growth

Evaluation criteria:

A test is considered to be negative if there is no biologically relevant increase in the number of mutants.
There are several criteria for determining a positive result:
-a reproducible three times higher mutation frequency than the solvent control for at least one of the concentrations;
-a concentration related increase of the mutation frequency; such an evaluation may be considered also in the case that a three-fold increase of
the mutant frequency is not observed;
-if there is by chance a low spontaneous mutation rate in the corresponding negative and solvent controls a concentration related increase of the mutations within their range has to be discussed.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Experiment I without S9: ≥ 500 μg/mL; experiment I with S9: ≥ 100 μg/mL; Experiment II without S9: ≥ 400 μg/mL; Experiment II with S9:≥ 1000 μg/mL

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Precipitation:

No precipitation of the test item was noted in the experiments.

Toxicity:

A biologically relevant growth inhibition (reduction of relative growth below 70 %) was observed after the treatment with the test item in experiment I and II with and without metabolic activation.

In experiment I without metabolic activation the relative growth was 16.5 % for the highest concentration (2500 µg/mL) evaluated. The highest biologically relevant concentration evaluated with metabolic activation was 2500 µg/mL with a relative growth of 17.4 %.

In experiment II without metabolic activation the relative growth was 12.2 % for the highest concentration (800 µg/mL) evaluated, The highest concentration evaluated with metabolic activation was 3000 µg/mL with a relative growth of 21.8 %, which is slightly higher than the postulated 10-20 % survival. Due to the fact that in both experiments with metabolic activation no hint at mutagenicity was found, this deficiency is considered to be not biologically relevant,

 

Mutagenicity:

In experiment I without metabolic activation all mutant values of the negative controls and test item concentrations found were within the historical control data of the test facility BSL BIOSERVICE (about 5-43 mutants per 10⁶ cells). No dose-response relationship could be observed. The mutation frequencies found in the groups treated with the test item did not show a biologically relevant increase as compared to the negative controls. Mutation frequencies with the negative control were found to be 41.47 and 32.60 mutants/10⁶ cells and in the range of 20.69 to 40.74 mutants/10⁶ cells with the test item, respectively. The highest mutation rate (compared to the negative control values) of 1.10 was found at a concentration of 1000 µg/mL with a relative growth of 45.6 %. With metabolic activation all mutant values of the negative controls and test item concentrations found were within the historical control data of the test facility BSL BIOSERVICE (about 5-44 mutants per 10⁶ cells). No dose-response relationship could be observed. The mutation frequencies found in the groups treated with the test item did not show a biologically relevant increase as compared to the negative controls. Mutation frequencies with the negative control were found to be 29.75 and 36.36 mutants/10⁶ cells and in the range of 5.22 to 43.12 mutants/10⁶ cells with the test item, respectively. The highest mutation rate (compared to the negative control values) of 1.10 was found at a concentration of 25 µg/mL with a relative growth of 82.1 %.

In experiment II without metabolic activation all mutant values of the negative controls and test item concentrations found were within the historical control data of the test facility BSL BIOSERVICE (about 5-43 mutants per 10⁶ cells), No dose-response relationship could be observed. The mutation frequencies found in the groups treated with the test item did not show a biologically relevant increase as compared to the negative controls.

Mutation frequencies with the negative control were found to be 38.18 and 19.92 mutants/10⁶ cells and in the range of 13.16 to 41.84 mutants/10⁶ cells with the test item, respectively. The highest mutation rate (compared to the negative control values) of 1.44 was found at a concentration of 50 µg/mL with a relative growth of 100.2 %.

In experiment II with metabolic activation all mutant values of the negative controls and test item concentrations found were within the historical control data of the test facility BSL BIOSERVICE (about 5-44 mutants per 10⁶ cells). No dose-response relationship could be observed. The mutation frequencies found in the groups treated with the test item did not show a biologically relevant increase as compared to the negative controls.

Mutation frequencies with the negative control were found to be 36.45 and 24.39 mutants/10⁶ cells and in the range of 9.26 to 36.89 mutants/10⁶ cells with the test item, respectively. The highest mutation rate (compared to the negative control values) of 1.21 was found at a concentration of 2000 µg/mL with a relative growth of 35.5 %, DMBA (1.0 and 1.5 µg/mL) and EMS (300 µg/mL) were used as positive controls and showed distinct and biologically relevant effects in mutation frequency.

Conclusions:

FAT 40224/H is considered to be non-mutagenic in the HPRT locus using V79 cells of the Chinese Hamster.

Executive summary:

The test item FAT 40224/H was assessed for its potential to induce mutations at the HPRT locus using V79 cells of the Chinese Hamster. The study was carried out according to OECD guideline 476 and EU method B 17.

The selection of the concentrations was based on data from the pre-experiments. Experiment I with and without metabolic activation and experiment II with metabolic activation were performed as a 4 h short-term exposure assay. Experiment II without metabolic activation was performed as 20 h long time exposure assay,

The test item was investigated at the following concentrations:

Experiment I

without metabolic activation:

5, 10, 25, 50, 100, 250, 500, 1000, 2000 and 2500 µg/mL

and with metabolic activation:

5, 10, 25, 50, 100, 250, 500, 1000 and 2500 µg/mL

Experiment II

without metabolic activation:

10, 25, 50, 100, 200, 400, 600 and 800 µg/mL

and with metabolic activation:

100, 316, 1000, 1250, 1500, 2000, 2400, 2800 and 3000 µg/mL

No precipitation of the test item was noted in the experiments.

Biologically relevant growth inhibition was observed in experiment I and II with and without metabolic activation. In experiment I without metabolic activation the relative growth was 16.5 % for the highest concentration (2500 µg/mL) evaluated. The highest biologically relevant concentration evaluated with metabolic activation was 2500 µg/mL with a relative growth of 17.4 %. In experiment II without metabolic activation the relative growth was 12.2 % for the highest concentration (800 µg/mL) evaluated. The highest concentration evaluated with metabolic activation was 3000 µg/mL with a relative growth of 21.8 %.

In both experiments no biologically relevant increase of mutants was found after treatment with the test item (with and without metabolic activation). No dose-response relationship was observed.

DMBA and EMS were used as positive controls and showed distinct and biologically relevant effects in mutation frequency.

In conclusion, in the described mutagenicity test under the experimental conditions reported, the test item FAT 40224/H is considered to be non-mutagenic in the HPRT locus using V79 cells of the Chinese Hamster. 

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 November, 2020 to 18 March, 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:

SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material:
BOP 01-20 (MC-66870600)
- Expiration date of the lot/batch:
June, 8th 2025
- Test item: FAT 40400/F TE
- Physical appearance: Red powder, solid at 20 ºC

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
Deep Frozen (-10 to -20 °C),

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:

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.

Test concentrations with justification for top dose:

Preliminary Toxicity Test: 50, 100, 200, 400, 800, 1600, 3200 and 5000 µg/plate.
Based on the observations of the preliminary toxicity test, the following five test doses were selected (with approximately half-log dose interval) for testing in the mutation assay:
50, 158, 500, 1581 and 5000 µg/plate.

Vehicle / solvent:

Sterile water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

congo red

Remarks:

WIth metabolic activation: TA98, TA100, TA1535, TA1537 and WP2 uvrA (pKM101)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

WIthout metabolic activation: TA100 and TA1535

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

WIthout metabolic activation: TA98

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

WIthout metabolic activation: TA1537

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-Nitroquinoline-1-Oxide

Remarks:

Without metabolic activation: WP2 uvrA (pKM101)

Details on test system and experimental conditions:

Test System:
Following strains of bacteria accepted under the OECD guideline for the assessment of point gene mutation were used:
Histidine auxotrophic strains of Salmonella typhimurium: TA98, TA100, TA1535 and TA1537.
Tryptophan auxotrophic strain of Escherichia coli: WP2uvrA (pKM101).

Justification for the Selection of Test System:
Each S. typhimurium tester strain contains, in addition to a mutation in the histidine operon, additional mutations that enhance sensitivity to some mutagens. The rfa mutation results in a cell wall deficiency that increases the permeability of the cell to certain classes of chemicals such as those containing large ring systems that would otherwise be excluded. The deletion in the uvrB gene results in a deficient DNA excision repair system. Tester strains TA98 and TA100 also contain the pKM101 plasmid (carrying the R factor). It has been suggested that the plasmid increases sensitivity to mutagens by modifying an existing bacterial DNA repair polymerase complex involved with the mismatch repair process. TA98 and TA1537 are reverted from histidine dependence (auxotrophy) to histidine independence (prototrophy) by frameshift mutagens. TA100 is reverted by both frameshift and base substitution mutagens and TA1535 is reverted only by mutagens that cause base substitutions. The E. coli tester strain has an AT base pair at the critical mutation site within the trpE gene (Wilcox et al., 1990). Tester strain WP2uvrA (pKM101) has a deletion in the uvrA gene resulting in a deficient DNA excision repair system. Tryptophan revertants can arise due to a base change at the originally mutated site or by a base change elsewhere in the chromosome causing the original mutation to be suppressed. Thus, the specificity of the reversion mechanism is sensitive to base pair substitution mutations (Green and Muriel, 1976).

Source of the Test System:
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.

Storage of Test System
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 2bºC to 8ºC. The master plates prepared on 05 October 2020 were used in the study.

Test Medium and Solutions
VB agar
Soft agar
Soft agar containing 0.5 mM histidine and biotin
Soft agar containing 5 µg/mL tryptophan
Nutrient agar
Oxoid Nutrient broth No. 2
PBS (pH 7.4)

Evaluation criteria:

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:

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

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

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

True negative controls validity:

not examined

Positive controls validity:

valid

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

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

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

True negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: All strains has been used

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 soluble in sterile water at 50 mg/mL. Water is one of the vehicles compatible with this test system. Hence, based on the results of the solubility test, SW was selected as the vehicle of choice in the mutation assay.

 

Mutation Assay

Initial mutation assay was conducted using the direct plate incorporation procedure and the confirmatory mutation assay was conducted using a pre-incubation procedure.

 

Viable Counts of the Overnight Tester Strains

Viable counts of all the tester strains were within the required range of 1-2 x 10⁹ CFU/mL for the initial toxicity-mutation as well as the confirmatory mutation assay.

 

Initial Mutation Assay

The test item did not cause precipitation on the basal agar plates at any of the tested doses. 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 on the basal agar plates at any of the tested doses. 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 40400/F was not mutagenic in the bacterial reverse mutation test up to 5000 µg/plate dose.

Executive summary:

FAT 40400/F was tested for its mutagenic potential in the bacterial reverse mutation assay according to OECD guideline 471. 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 homogenate. FAT 40400/F TE was soluble in sterile water (SW) at 50 mg/mL. In a preliminary toxicity test conducted for the selection of test doses for the mutation assay, the test item did not precipitate on the basal agar plates at any of the tested doses. The test item did not cause 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. 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 40400/F TE 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 (SW) 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 40400/F 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. Based on the study results, FAT 40400/F 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

Description of key information

Genetic Toxicity In vivo: Erythrocyte micronucleus test

In a GLP-compliant erythrocyte micronucleus test, tested according to OECD guideline 474, 6 NMRI mice per sex were treated once by oral gavage with the test substance(5000 mg/kg bw) dissolved in 1 % CMC followed by a 24, 48 and 72 hours post exposure period (CCR 1990). In a pre-experiment 5000 mg/kg bw was estimated to be the maximum tolerated dose. The finding that the mean number of normochromatic erythrocytes was not increased after treatment with the test article as compared to the negative controls, indicated that the test substancehad no cytotoxic properties. In comparison with the corresponding negative controls there was no enhancement in the frequency of the detected micronuclei at any preparation interval after application of the test article and therefore, it could be concluded that the test substance did not induce micronuclei in this study.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 April 1990 to 04 July 1990

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Qualifier:

according to guideline

Guideline:

EPA OTS 798.5395 (In Vivo Mammalian Cytogenics Tests: Erythrocyte Micronucleus Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material:
BG 3247/TV 6
- Expiration date of the lot/batch:
March 1995

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
room temperature
- Stability under storage conditions:
Pure: stable; In vehicle: stable in water, methanol, DMSO, DMF, acetone >72 hours

Species:

mouse

Strain:

NMRI

Details on species / strain selection:

The mouse is an animal which has been used for many years 'as suitable experimental animal in cytogenetic investigations. There are many data available from such investigations which may be helpful in the interpretation of results from the micronucleus test. In addition, the mouse is an experimental animal in many physiological, pharmacological and toxicological studies. Data from such experiments also may be useful for the design and the performance of the micronucleus test

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: BRL Tierfarm Füllinsdorf, CH-4414 Füllinsdorf/Basel, Switzerland
- Age at start of acclimatization: minimum 10 weeks
- Weight at study initiation: approximately 30 g
- Fasting period before study: 18 hours
- Housing: Individually in Makrolon type-1 cages with wire mesh top (EHRET GmbH, D-7830 Emmendingen) with granulated soft wood bedding (ALTROMIN, D-4937 Lage/Lippe, F.R.G.)
- Diet: pelleted standard diet (ALTROMIN, D-4937 Lage/Lippe, F.R.G.)
- Water: tap water, ad libitum (Gemeindewerke, D-6101 Roßdorf, F.R.G.)
- Acclimation period: minimum 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 +/- 3
- Humidity (%): 30-70 %
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: carboxymethylcellulose-suspension (1%)
- Justification for choice of solvent/vehicle: The vehicle was chosen to its relative non-toxicity for the animals.
- Concentration of test material in vehicle: 5000 mg/kg bw
- Amount of vehicle (if gavage or dermal): 20 mL/kg bw

Frequency of treatment:

Single treatment

Post exposure period:

24, 48, and 72 hours

Dose / conc.:

5 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

6

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide;
- Route of administration: orally
- Doses: 40 mg/kg bw
- Volume administrated: 10 mL/kg bw
- Vehicle: physiological saline

Tissues and cell types examined:

Normochromatic and polychromatic erythrocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
A preliminary study on acute toxicity was performed with the same strain and under identical conditions as in the mutagenicity study. The maximum tolerated dose level was determined to be the dose that caused toxic reactions without having major effects on survival within 72 hours.

DETAILS OF SLIDE PREPARATION:
The animals were sacrificed by cervical dislocation. The femora were removed, the epiphyses were cut off and the marrow was flushed out with fetal calf serum, using a 5 mL syringe. The cell suspension was centrifuged at 1,500 rpm for 5 minutes and the supernatant was discarded. A small drop of the resuspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Grunwald (Merck, D-6100 Darmstadt F.R.G.)/Giemsa (Gurr, BDH Limited Poole, Great Britain). Cover slips were mounted with EUKITT (KINDLER, D-7800 Freiburg F.R.G.). At least one slide was made from each bone marrow sample.

METHOD OF ANALYSIS:
Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives. 1,000 polychromatic erythrocytes (PCE) were analysed per animal for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and expressed in normochromatic erythrocytes per 1000 PCEs. The analysis was performed with coded slides. Five animals per sex and group were evaluated as described. The remaining animal of each test group was evaluated in case an animal had died in its test group spontaneously or due to gavage error.

Evaluation criteria:

A test article is classified as mutagenic if it induces either a statistically significant dose-related increase in the number of micronucleated polychromatic erythrocytes or a reproducible statistically significant positive response for at least one of the test points. A test article producing neither a statistically significant dose-related increase in the number of micronucleated polychromatic erythrocytes nor a statistically significant and reproducible positive response at any one of the test points is considered non-mutagenic in this system.

Statistics:

Statistical significance at the five per cent level (p <0.05) was evaluated by means of the non-parametric Mann-Whitney test.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

In vivo, but no cytotoxicity was observed.

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- In a pre-experiment 4 animals (2 males, 2 females) received orally a single dose of 5000 mg/kg b.w., FAT 40400/A suspended in 1 % CMC. The volume administered was 20 mL/kg b.w. One male animal expressed eyelid closure and one male animal showed a reduction of spontaneous activity.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei: In comparison with the corresponding negative controls there was no substantial enhancement in the frequency of the detected micronuclei at any preparation interval after application of the test article. The mean values of micronuclei observed after treatment with FAT 40'400/A were in the same range as compared to the negative control groups.
- Ratio of PCE/NCE: The mean number of normochromatic erythrocytes was not increased after treatment with the test article as compared to the mean values of NCEs of the corresponding negative controls, indicating that FAT 40400/A had no cytotoxic properties.
- 40 mg/kg b.w. cyclophosphamide administered per os was used as positive control which showed a distinct increase of induced micronucleus frequency.

Conclusions:

FAT 40400/A did not induce micronuclei in bone marrow cells of the mouse.

Executive summary:

A study was performed to investigate the potential of FAT 40400/A to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse according to OECD guideline 474. The test article was formulated in carboxymethylcellulose suspension (1 %). This suspending agent was used as negative control. The volume administered orally was 20 ml/kg b.w. 24 h, 48 h and 72 h after a single application of the test article the bone marrow cells were collected for micronuclei analysis. Ten animals (5 males, 5 females) per test group were evaluated for the occurrence of micronuclei. 1000 polychromatic erythrocytes (PCE) per animal were scored for micronuclei. To describe a cytotoxic effect due to the treatment with the test article the ratio between polychromatic and normochromatic erythrocytes (NCE) was determined in the same sample and reported as the number of NCE per 1000 PCE. The 5000 mg/kg b.w. dose level of the test article was investigated at 24 h, 48 h, and 72 h preparation interval. In a pre-experiment this dose level was estimated to be the maximum attainable dose.

After treatment with the test article the ratio between PCEs and NCEs was not affected as compared to the corresponding negative controls thus indicating no cytotoxic effects. In comparison to the corresponding negative controls there was no substantial enhancement in the frequency of the detected micronuclei at any preparation interval after application of the test article. An appropriate reference mutagen was used as positive control which showed a distinct increase of induced micronucleus frequency. In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test article did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse. Therefore, FAT 40'400/A is considered to be non-mutagenic in this micronucleus assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

Based on the available genotoxicity studies, the test substance does not need to be classified for genotoxicity according to the EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008