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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Based on the Ames assay, Chromosomal aberration and HPRT study test substance is considered as non-mutagenic.

Link to relevant study records

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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
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: TZ2382-BB-503C81
- Expiration date of the lot/batch: 28-01-2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature
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 4h treatment; MEM + 10 % FBS 20h 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
Conclusions:
FAT 40224/H is considered to be non-mutagenic in the HPRT locus using V 79 cells of the Chinese Hamster.
Executive summary:

The test item FAT 40224/H was assessed for its potential to induce gene mutations at the HPRT locus using V 79 cells of the Chinese hamster. The main experiments were carried out without and with metabolic activation. The experiments with metabolic activation were performed by including liver microsomes and NADP for efficient detection of a wide variety of carcinogens requiring metabolic activation. The selection of the concentrations used in the main experiments was based on data from the pre-experiments according to the OECD guideline 476. In experiment 1 2500 µg/mL (with and without metabolic activation) was selected as the highest concentration. In experiment II 800 µg/mL (without metabolic activation) and 3000 µg/mL (with metabolic activation) were selected as the highest concentrations. 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 was performed as 20 h long time exposure assay (without metabolic activation). The pH-value detected with the test item was within the physiological range. The test item was investigated at the following concentrations:


Experiment I


without metabolic activation:


S, 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, 50, 100, 200, 400, 600 and 800 µg/mL


and with metabolic activation:


100, 316, 1000, 1250, 1 500, 2000, 2400, 2800 and 3000 µg/mL


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 10cells). 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/ 10cells 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/ 10cells and in the range of 5.22 to 43.12 mutants/10cells with the test item, respectively. The highest mutation rate (compared to the negative control values) of 1.30 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 BIOSERVICF. No dose-response relationship could be observed. The mutation frequencies found in the groups treated with the tcst 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 10cells 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 544 mutants per 10cells). 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/10cells 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.


Based on the study results, FAT 40224/H is considered to be non-mutagenic in the HPRT locus using V 79 cells of the Chinese Hamster.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12 October 2020 to 29 November 2020
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
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:
BOP 01-19 (MC-58955700)
- Expiration date of the lot/batch:
May 14th, 2024

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
Deep Frozen (-10 to -25 °C)
Species / strain / cell type:
other: human peripheral blood lymphocytes
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced rat liver S9 mix
Test concentrations with justification for top dose:
Experiments 1 & 2 (presence and absence of metabolic activation with 3-hour exposure)

a) 375 b) 750 and c) 1500 µg/mL (factor of 2)

Experiment 3 (absence of metabolic activation with 22-hour exposure)

a) 125 b) 250 and c) 500 µg/mL (factor of 2)
Vehicle / solvent:
Sterile water
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
Presence of metabolic activation; 14 µg/mL
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
Absence of metabolic activation 600 µg/mL
Details on test system and experimental conditions:
Test System:
Test approaches currently accepted under the OECD and ICH guidance for the assessment of mammalian cell clastogenicity involve the use of human peripheral blood lymphocytes for the analysis of chromosome aberrations in mutagen tests.
Human blood cultures were established and used as the test system.

Justification for the Selection of Test System:
Studies on exposed individuals and on cultured cells have shown that the human peripheral blood lymphocyte is an extremely sensitive indicator of both in vivo and in vitro clastogen induced chromosome structural changes and these changes in chromosome structure offer readily scored morphological evidence of damage to the genetic material.

Human peripheral blood lymphocytes are useful in in vitro cytogenetic testing because of easy availability of large numbers of human cells, their distribution throughout the body, circulation in all the tissues and a proportion of them are long-lived and they maintain a stable karyotype.

A proportion of the lymphocytes can be stimulated by mitogens to undergo mitosis in culture, they are easy to culture and thus provide a ready source of dividing cells for the scoring of chromosome aberrations.
Source of the Test System:
Blood was collected from individual male donor of age 33 years for preliminary cytotoxicity test. Blood was collected from individual male donor of age 26 years for chromosomal aberration test. Donors were non-smokers, not undergoing any drug treatment and not suspected of exposure to high levels of radiation or hazardous chemicals or any viral infection.
Evaluation criteria:
EVALUATION AND INTERPRETATION OF RESULTS
When all the validity criteria are fulfilled:
1.    A test chemical is considered to be clearly positive if, in any of the experimental conditions examined:
·      At least one of the test concentrations exhibits a statistically significant increase compared with the concurrent vehicle control.
·      The increase is dose-dependent when evaluated with an appropriate trend test.
·      Any of the results are outside the distribution of the historical vehicle control data.
 
2.    A test chemical is considered to be clearly negative if, in all experimental conditions examined:
·      None of the test concentrations exhibits a statistically significant increase compared with the concurrent vehicle control.
·      There is no concentration-related increase when evaluated with an appropriate trend test.
·      All results are inside the distribution of the historical vehicle control data.
 
3.    The results will be considered equivocal if they do not meet the criteria specified for a positive or negative response.
4.    An increase in the number of polyploidy cells may indicate that the test item has the potential to inhibit mitotic processes and to induce numerical chromosome aberrations. An increase in the number of cells with endore duplicated chromosomes may indicate that the test item has the potential to inhibit cell cycle progression.
Statistics:
The statistical analysis of the experimental data was carried out using validated SYSTAT Statistical package ver.12.0. Data were analysed for proportions of aberrant metaphases in each sample, excluding gaps as aberrations. Pooled data from each test concentration and the positive control were compared with the vehicle control using Fischer exact test. All analysis and comparisons were evaluated at 5 % (p<0.05) level.
Species / strain:
lymphocytes: human peripheral blood lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Human peripheral blood lymphocytes has been used

Results:


S9 Characterization


a)        Sterility Check


The S9 homogenate was found to be sterile.


b)        Metabolic Activation


The S9 homogenate was found to be active.


c)        Protein Content


The protein content of the S9 homogenate was34.724mg/mL.


 


Stability of Test Item in the Vehicle and Analytical Verification of Dosing Formulations


FAT 40224/I TE was also found to be stable in the vehicle for 24 hours at room temperature, at the fortification levels of 50µg/mL and 200000µg/mL. The concentration analysis results of chromosomal aberration test indicated a mean % agreement of 106.9 and 105 % of their claimed target concentrations of 12500 and 150000µg/mL, respectively, (with a corresponding RSD of 0.324 and 0.2857 %, respectively) confirming that the concentration of the test item was within the acceptable limits (±10 % of claimed concentrations and an RSD of10 %).


No test item was detected in the vehicle control.


 


Preliminary Solubility Test


Test item was soluble in SW at 50 mg/mL.


 


Preliminary Cytotoxicity Test and Justification for the Selection of Test Concentrations


At the end of the 3-hour exposure period, FAT 40224/I TE did not cause precipitation in the test solution at any of the test doses, both in the presence and absence of metabolic activation, at the end of the 3-hour exposure period. In the presence of metabolic activation, at the end of 3-hour exposure to the test item, the pH of the test medium ranged between 7.23 and 7.29 with a pH value of 7.27 in the SW control. In the absence of metabolic activation, the pH ranged between 7.21 and 7.29 with a pH value of 7.25 in the SW control. At the end of 3-hour exposure, osmolality of the test medium at the highest test item treatment condition (2000µg/mL) was 0.289 and 0.299 OSMOL/kg in the presence and absence of metabolic activation, respectively. The corresponding osmolality in the SW control was 0.290 and 0.292 OSMOL/kg in the presence and absence of metabolic activation, respectively. FAT 40224/I exhibited the required level of cytotoxicity (reduction in the mitotic index by 45±5% of the concurrent vehicle control) at 1000µg/mL both in the presence and absence of metabolic activation with 3 hour exposure and at 500 µg/mL with 22-hour exposure. Based on these observations, in the chromosomal aberration assay, a top concentration of 1500 µg/mL will be tested in the 3-hour exposure, in the presence and absence of metabolic activation and in the absence of metabolic activation and 500 µg/mL with 22-hour exposure. Test item showed 65 % and 63 % cytotoxicity at 2000 µg/mL, respectively in the presence and absence of metabolic activation at 3-hour exposure, compared to the concurrent vehicle control. Test item exhibited 64 % and 88 % toxicity at 1000 and 2000 µg/mL, respectively at 22-hour exposure, compared to the vehicle control


 


Chromosomal Aberration Assay


Experiment 1


At the highest concentration tested (1500µg/mL), the mitotic inhibition was 49% compared to the vehicle control. The incidence of aberrations in the vehicle control was within the range of the in-house historical control data. There was no statistically significant increase in the number of aberrant metaphases in any of the test concentrations when compared to the vehicle control. No incidences of polyploidy and endoreduplication were seen. The positive control, cyclophosphamide monohydrate caused a statistically significant (p<0.05) increase in the aberrant metaphases excluding gaps.


 


Experiment 2


At the highest concentration tested (1500µg/mL), the mitotic inhibition was 50% compared to the vehicle control. The incidence of aberrations in the vehicle control was within the range of the in-house historical control data. There was no statistically significant increase in the number of aberrant metaphases in any of the test concentrations when compared to the vehicle control. There were no incidences of polyploidy and endoreduplication.


 


Experiment 3


At the highest concentration tested (500 µg/mL), the mitotic inhibition was 50% compared to the vehicle control. The incidence of aberrations in the vehicle control was within the range of the in-house historical control data. There was no statistically significant increase in the number of aberrant metaphases in any of the test concentrations when compared to the vehicle control. No incidences of polyploidy and endoreduplication were seen. The positive control, Ethyl methanesulfonate caused a statistically significant (p<0.05) increase in the aberrant metaphases excluding gaps.

Conclusions:
FAT 40224/I is not clastogenic under the conditions of testing employed.
Executive summary:

The clastogenic potential of FAT 40224/I to induce chromosomal aberrations in mammalian cells was evaluated using cultured human peripheral blood lymphocytes. The test was carried out according to OECD guideline 473. Human lymphocytes in whole blood culture, stimulated to divide by addition of Phytohaemagglutinin (PHA) 48 hours prior to treatment, were exposed to the test item in the presence and absence of an exogenous metabolic activation system (S9 fraction prepared from Aroclor 1254 induced rat liver). The study consisted of a preliminary cytotoxicity test and a chromosomal aberration assay. Chromosomal aberration assay consisted of three independent experiments: Experiments 1 and 2 in the presence and absence of metabolic activation system with 3-hourexposure, respectively, and Experiment 3 in the absence of metabolic activation system with 22-hourexposure. The test item was also found to be stable in the vehicle for 24 hours at room temperature, at the fortification levels of 50 µg/mL and 200000 µg/mL. In a preliminary cytotoxicity test, blood cells were exposed to FAT 40224/I at the concentrations of 62.5, 125, 250, 500, 1000 and 2000 µg/mL along with a Sterile Water (SW) control. At the end of the 3-hour exposure period, FAT 40224/I did not cause precipitation in the test solution at any of the test doses, both in the presence and absence of metabolic activation, at the end of the 3-hour exposure period. FAT 40224/I exhibited the required level of cytotoxicity (reduction in the mitotic index by 45±5 % of the concurrent vehicle control) at 1000 µg/mL both in the presence and absence of metabolic activation with 3 hour exposure and at 500 µg/mL with 22-hour exposure. Test item showed 65 % and 63 % cytotoxicity at 2000 µg/mL, respectively in the presence and absence of metabolic activation at 3-hour exposure, compared to the concurrent vehicle control. Test item exhibited 64 % and 88 % toxicity at 1000 and 2000 µg/mL, respectively, at 22-hour exposure, compared to the vehicle control. Based on these observations, in the chromosomal aberration assay, a top concentration of 1500 µg/mL was tested in the 3-hour exposure, in the presence and absence of metabolic activation and in the absence of metabolic activation and 500 µg/mL with 22-hour exposure. In the chromosomal aberration assay, blood cultures were exposed to the test item in duplicate at the concentrations of 375, 750 and 1500µg/mL in Experiments 1& 2 (presence and absence of metabolic activation with 3-hour test item exposure) and 125, 250 and 500 µg/mL in Experiment 3 (absence of metabolic activation with 22-hour test item exposure). In a similar way, concurrent vehicle control (SW) and the positive controls (cyclophosphamide monohydrate in the presence of metabolic activation and ethyl methanesulfonate in the absence of metabolic activation) were tested in duplicate cultures. In each case, the cells in the C-metaphase were harvested at approximately 22 hours after the start of the treatment from the vehicle control, test item concentrations and the positive control groups. At the highest concentration tested, the reduction in mitotic index was 49, 50 and 50% in Experiments 1, 2 and 3, respectively, compared to the vehicle control. A total of 300 metaphases from duplicate cultures from each of the controls and three treatment levels were evaluated for chromosomal aberrations. The data from the treatment groups and the positive controls were statistically compared with the vehicle control. There were no statistically significant increase in the incidence of structurally aberrant metaphases, either at 3 hours in the presence and absence of metabolic activation and 22 hours exposure, in the absence of metabolic activation in any of the tested concentrations. Under identical conditions, the respective positive control substances produced statistically significant (p<0.05) increases in aberrant metaphases confirming the sensitivity of the test system and the activity of the S9 mix.


The results of the concentration analysis of the dose formulation samples were within the acceptable specification range and confirmed that the targeted top concentration level was achieved and the results support the validity of the study conclusion. The study indicated that, under the conditions of this study, FAT 40224/I was not clastogenic in human peripheral blood lymphocytes up to the respective highest concentration of 125 µg/mL.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20 November 2020 to 23 November 2020
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 0119 (MC-58955700)
- Expiration date of the lot/batch:
May 14th,2024

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
Deep Frozen (-10 to -25 °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,(828) 264-9099,USA), will be used as the metabolic activation system. The S9 homogenate will be 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 test: 50, 100, 200, 400, 800, 1600, 3200 and 5000 µg/plate
Mutation assay: 50, 158, 500, 1581 and 5000 µg/plate
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
congo red
Remarks:
In Presence of Metabolic Activation:TA98, TA100, TA1535, TA1537, WP2uvrA (pKM101)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
In Absence of Metabolic Activation: TA 98
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
In Absence of Metabolic Activation: TA100, TA1535
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
In Absence of Metabolic Activation: TA1537
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
In Absence of Metabolic Activation: WP2uvrA (pKM101)
Details on test system and experimental conditions:
Test System:
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).

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 2 ºC to 8ºC. The master plates prepared on 8 February and 21 May 2018 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.
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.
Species / strain:
bacteria, other: TA98, TA100, TA1535, TA1537, WP2uvrA
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:
Both the Salmonella typhimurium and Escherichia coli tester strains were found to be reliable and responsive to the different genotypic characterization tests like the amino acid requirement, rfa mutation, uvr mutation and the
R-factor plasmids. Similarly, the spontaneous revertant counts of the vehicle control groups of these tester strains were in the ranges of the test facility’s historical control data.

The positive controls produced a more than 3-fold increase in the mean numbers of revertant colonies when compared to the respective vehicle controls, demonstrating the sensitivity of the assay procedure.

The test item at doses up to 5000 µg/plate did not cause a two fold increase in the mean numbers of revertant colonies in the strains TA98, TA100 and WP2uvrA (pKM101) or three fold increase in the mean numbers of revertant colonies in the strains TA1535 and TA1537 either in the presence or absence of the metabolic activation system when compared to the respective vehicle control plates.
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.


 


Stability of Test Item in the Vehicle and Analytical Verification of Dosing Formulations


FAT 40224/I was found to be stable and re-suspendable in Milli-Q water for 24 hours at room temperature at the concentrations of 50 and 200000 mg/mL. The results of concentration analysis of FAT 40224/I dose formulations from the initial mutation assay indicated that the mean of percentage agreement with claimed concentration were 102.4 and 107.0 % (with an RSD of 0.5012 and 0.2804 %, respectively) of their respective claimed concentrations of 500 and 50000 µg/mL, confirming that the concentration of the test item was within the acceptable limit No test item was detected in the vehicle control.


The results of concentration analysis of FAT 40224/I TE dose formulations from the confirmatory mutation assay indicated that the mean of percentage agreement with claimed concentration were 99.77 and 108.6 % (with an RSD of 0.1968 and 0.4776 %, respectively) of their respective claimed concentrations of 500 and 50000 µg/mL, confirming that the concentration of the test item was within the acceptable limits. No test item was detected in the vehicle control.


These results from the initial and confirmatory mutation assays indicate that the top dose of 5000 µg/plate level was achieved and support the validity of the study conclusion.


 


Preliminary Solubility Test for selection of vehicle


The test item was soluble in sterile water at 50 mg/mL, on sonication.


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.


 


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


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 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


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 109CFU/mL for the initial toxicity-mutation as well as the confirmatory mutation assay.


 


Initial Mutation Assay


The mean numbers of revertant colonies/plate in the SW control was comparable to the in-house spontaneous revertant counts for all the tester strains.


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 mean numbers of revertant colonies/plate in the SW control was comparable to the in-house spontaneous revertant counts for all the tester strains.


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.


 


DISCUSSION


Both the Salmonella typhimurium and Escherichia coli tester strains were found to be reliable and responsive to the different genotypic characterization tests like the amino acid requirement, rfa mutation, uvr mutation and the R-factor plasmids. Similarly, the spontaneous revertant counts of the vehicle control groups of these tester strains were in the ranges of the test facility’s historical control data. 


The positive controls produced a more than 3-fold increase in the mean numbers of revertant colonies when compared to the respective vehicle controls, demonstrating the sensitivity of the assay procedure. 


The test item at doses up to 5000 µg/plate did not cause a two fold increase in the mean numbers of revertant colonies in the strains TA98, TA100 and WP2uvrA (pKM101) or three foldincrease in the mean numbers of revertant colonies in the strains TA1535 and TA1537 either in the presence or absence of the metabolic activation system when compared to the respective vehicle control plates.

Conclusions:
FAT 40224/I was not mutagenic in the bacterial reverse mutation test up to the top dose of 5000 µg/plate under the conditions of testing employed.
Executive summary:

The test item, FAT 40224/I 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. FAT 40224/I formed a free-flowing suspension in sterile water (SW) at 50 mg/mL. The test item was found stable in Milli-Q water for 24 hours at room temperature, at the fortification levels of 50 and 200000 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 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 40224/I 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 40224/I 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 40224/I 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

Based on the micronucleus assay test substance is considered as non-mutagenic.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
06 March 1987
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:
not specified
Qualifier:
according to guideline
Guideline:
other: EEC Directive 79/831, Annex V, Part B, Paragraph 4.3.1 for the micronucleus test
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
mouse
Strain:
other: OF-1 albino mice
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: IFFA-CREDO, L'Arbresle, France
- Weight at study initiation: 25g
- Housing: Animals were housed 5 of the same sex per cage in Makrolon type III cages.
- Diet: Aliment Rats-Souris Charles River, produced by U.A.R., Villemoisson/Orge, France, ad libitum
- Water: ad libitum
- Quarantaine period: 1 week at Battelle
Route of administration:
oral: gavage
Vehicle:
Vehicle: distilled water
The test substance was dissolved in distilled water to obtain a stock solution of 250 mg/mL.
Details on exposure:
Intragastric intubation: using 0.5 mL of a solution at 250 mg/mL per 25 g body weight.
Frequency of treatment:
single treatment
Post exposure period:
20, 44, 68 hours
Dose / conc.:
5 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
Thio-TEPA (N, N', N"-triethylenethiophosphoramide), reference N° 509-227, made by Lederle Laboratories Ltd.
- Doses / concentrations: 20 mg/kg
Tissues and cell types examined:
normochromatic and polychromatic erythrocytes
Details of tissue and slide preparation:
DETAILS OF SLIDE PREPARATION:
After sacrifice of the animals the femurs were taken and broken open at one end. Bone marrow cells were suspended in foetal calf serum using a small syringe, and the cells were centrifuged at 120 x g for 5 minutes. The supernatant was removed with a Pasteur pipette, cells were spread on a microscope slide and the smears allowed to dry in air. The following day smears were stained with Giemsa (1:6 in water), dried and mounted with a coverslip.

METHOD OF ANALYSIS:
Two types of erythrocytes were observed in the bone marrow smears: normochromatic (mature red blood cells about to pass into the blood stream) and polychromatic (immature red blood cells). The latter are stained blue by Giemsa for around 24 h after the expulsion of the erythroblast nucleus: the staining is probably due to traces of RNA remaining in these cells. The proportion of polychromatic erythrocytes containing one or more micronuclei was compared with the total number of polychromatic erythrocytes, and statistical comparisons were made between these ratios for the different groups. A minimum of 500 polychromatic erythrocytes were counted per smear (two smears per animal). In each smear an evaluation was made of the number of nucleated cells and the two types of erythrocytes (normochromatic and polychromatic) were counted up to a total of 2000 erythrocytes per animal. This was done in order to gain information on the mode of action of the test compound in bone marrow cells, and also to identify possible artifacts. All slides were given coded labels and were microscopically analysed without knowledge of their treatment groups.
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF DEFINITIVE STUDY
At low magnification of the microscope no noticeable differences in bone marrow nucleated cells were observed between animals treated with FAT 40'224/C and negative controls. In the positive control group (Thio-TEPA) decreased numbers of nucleated bone marrow cells were noted. There was no statistically significant increase in the number of micronucleated polychromatic erythrocytes in animals exposed to 5000 mg/kg of FAT 40'224/C compared to negative control animals. In animals treated with Thio-TEPA there was a statistically significant increased number of micronucleated cells. The ratio of polychromatic to normochromatic erythrocytes was markedly decreased in mice treated with Thio-TEPA. There was no statistically significant difference in this ratio between animals treated with FAT 40224/C and the negative controls.
Conclusions:
FAT 40224 is not considered to be mutagenic in the micronucleus test.
Executive summary:

In a GLP-compliant micronucleus test, tested according to OECD guideline 474, 5 non-consanguinous OF-1 albino mice originating from an SPF colony per sex per treatment group were treated once by oral gavage with the test substance 5000 mg/kg bw dissolved in distilled water followed by a 20, 44, 68 hour exposure period. In a preliminary range finding study the concentration of 5000 mg/kg bw was chosen for the main-test. A positive control (Thio-TEPA) administered at a concentration of 20 mg/kg bw showed pronounced evidence of mutagenicity 44 h after administration. No mutagenic effect was observed in bone marrow smears taken 20, 44 and 68 h after administration of the test substance. Based on the study results, FAT 40226/C is not considered to be mutagenic in the micronucleus test.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Mode of Action Analysis / Human Relevance Framework

The test substance is considered as non-mutagenic.

Additional information

Genetic toxicity in vitro, Ames test:

In a key study with FAT 40224/I was tested for its mutagenic potential in the Prival modification of 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. The bacterial tester strains were exposed to the test item in the presence and absence of hamster uninduced liver S9. The results of the study showed that the test substance 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. In conclusion, FAT 40224/I was not mutagenic in this Bacterial Reverse Mutation Assay up to 5000 µg/plate concentration.

 

In two independent GLP compliant Ames test studies, performed according to OECD guideline 471, 4 Salmonella typhimurium strains (TA 1535, TA 1537, TA 98, and TA 100) were used to test the mutagenic potential of the test substance (20, 80, 320, 1280 and 5120 µg/plate), both with and without metabolic activation system. The test substance did not show any mutagenic activity in any strain.

 

Genetic toxicity in vitro, Chromosome aberration test:

In a chromosomal aberration key study, the clastogenic potential of FAT 40224/I TE to induce chromosomal aberrations in mammalian cells was evaluated using cultured human peripheral blood lymphocytes. The test was carried out according to OECD guideline 473. At the highest concentration tested, the reduction in mitotic index was 49, 50 and 50% in Experiments 1, 2 and 3, respectively, compared to the vehicle control. A total of 300 metaphases from duplicate cultures from each of the controls and three treatment levels were evaluated for chromosomal aberrations. The data from the treatment groups and the positive controls were statistically compared with the vehicle control. There were no statistically significant increase in the incidence of structurally aberrant metaphases, either at 3 hours in the presence and absence of metabolic activation and 22 hours exposure, in the absence of metabolic activation in any of the tested concentrations. Under identical conditions, the respective positive control substances produced statistically significant (p<0.05) increases in aberrant metaphases confirming the sensitivity of the test system and the activity of the S9 mix. The study indicated that, under the conditions of this study, FAT 40224/I TE was not clastogenic in human peripheral blood lymphocytes up to the respective highest concentration of 125 µg/ml.

In another supporting study tested according to OECD guideline 743, V79 cells of the Chinese Hamster were exposed to the test substance with and without metabolic activation (CCR 1987). Different exposure times (up to 28 hours) and dosages (up to 3.00 mg/mL) were chosen in this study. 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 of the cells with 3.00 mg/mL reduced clearly the plating efficiency of the V79 cells. Also the mitotic index was reduced with the highest concentrations in the presence and absence of S9 mix. There were relevant enhancements of cells with structural aberrations after treatment with the test article at fixation intervals 7 h and 28 h with metabolic activation by S9 mix. Appropriate reference mutagens were used as positive controls and showed distinct increases of 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 induced structural chromosome aberrations as determined by the chromosomal aberration test in the V79 Chinese Hamster cell line. Therefore, the test substance is considered to be mutagenic in this chromosomal aberration assay.

 

Genetic toxicity in vitro, HPRT mutation assay:

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 20h long time exposure assay. The following concentrations were used. Experiment I without metabolic activation: 5, 10, 25, 50, 100, 250, 500, 1000, 2000 and 2500µg/mL; Experiment I with metabolic activation: 5, 10, 25, 50, 100, 250, 500, 1000, and 2500µg/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.

Genetic toxicity in vivo, micronucleus test:

In a GLP-compliant micronucleus test, tested according to OECD guideline 474, five albino mice originating from an SPF colony per sex per treatment group were treated once by oral gavage with the test substance 5000 mg/kg bw dissolved in distilled water followed by a 20, 44, 68 hour exposure period. In a preliminary range finding study the concentration of 5000 mg/kg bw was chosen for the main-test. A positive control (Thio-TEPA) administered at a concentration of 20 mg/kg bw showed pronounced evidence of mutagenicity 44 h after administration. No mutagenic effect was observed in bone marrow smears taken 20, 44 and 68 h after administration of the test substance.

 

Justification for classification or non-classification

Based on the findings of the genetic toxicity studies, the test substance does not considered to be classified according to the EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.