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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test substance was found to cause reverse mutations in bacteria.

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Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 January 1999 to 25 February 1999
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:
yes
Remarks:
only 4 tester strains tested; no tester strain to detect cross-linking mutagens was included
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
yes
Remarks:
only 4 tester strains tested; no tester strain to detect cross-linking mutagens was included
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
- Identification: FAT 93460/A
- Source and lot/batch No.of test material: EN-No.: 037098 A7
- Expiration date of the lot/batch: November, 2003

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability of the test substance in the solvent/vehicle: at least 24 hours in water, PEG 400, saline, FCA/NaCl-solution, and CMC-solution

OTHER SPECIFICS:
- Aggregate state at room temperature: Solid
- Colour: Yellow
- Purity: 23 % active ingredient
Target gene:
Histidine requiring genes of Salmonella strains
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
CELLS USED
- Type and source of cells: The bacterial strains TA 1535, TA 98, and TA 100 were obtained from Ames (University of California, 94720 Berkeley, U.S.A.). The bacterial strain TA 1537 was obtained from BASF (D-67063 Ludwigshafen).

The strain cultures were stored as stock cultures in ampoules with nutrient broth + 5 % DMSO (MERCK, D-64293 Darmstadt) in liquid nitrogen.
Precultures; From the thawed ampoules of the strains 0.5 ml bacterial suspension was transferred into 250 mL Erlenmeyer flasks containing 20 ml nutrient medium. A solution of 20 µL ampicillin (25 µg/ml) was added to the strains TA 98, and TA 100.
This nutrient medium contains per litre:
8 g Merck Nutrient Broth (MERCK, D-64293 Darmstadt)
5 g NaCl (MERCK, D-64293 Darmstadt)
The bacterial cultures were incubated in a shaking water bath for 8 hours at 37° C.
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9 : The S9 liver microsomal fraction was obtained from the livers of 8 - 12 weeks old male rats, strain Wistar Hanlbm (BRL, CH-4414 Füllinsdorf; weight approx. 220 - 320 g)
- method of preparation of S9 mix: Rats received daily applications of 80 mg/kg b.w. Phenobarbital i.p. dissolved in deionised water (Desitin; D-22335 Hamburg) and ß-Naphthoflavone orally dissolved in corn oil (Aldrich, D-89555 Steinheim) on three subsequent days. The livers were prepared 24 hours after the last treatment. After decapitation of the anaesthetised animals, the livers of the animals were removed, washed in 150 mM KCl and homogenised. The homogenate was diluted 1+3 in KCl and centrifuged at 9,000 g for 10 minutes at 4 °C. A stock of the supernatant containing the microsomes was frozen in ampoules and stored at -80 °C. Small numbers of the ampoules are kept at -20 °C for up to one week before use. The protein content was determined using an analysis kit of Bio-Rad Laboratories, D-80939 München (Bio-Rad protein assay, Catalogue No. 5000006). The protein concentration in the S9 preparation was 28.3 mg/ml (lot no. 271198) in the preexperiment and in experiment I, and 50.4 mg/ml (lot no. 181298) in experiment I and II.

- concentration or volume of S9 mix and S9 in the final culture medium: Before the experiment an appropriate quantity of S9 supernatant was thawed and mixed with S9 co-factor solution. The amount of S9 supernatant was 15 % v/v in the cultures. The composition of the co-factor solution was chosen to yield the following concentrations in the S9 mix:
8mM MgCl2
33 mM KCl
5mM Glucose-6-phosphate
5mM NADP
in 100 mM sodium-ortho-phosphate-buffer, pH 7.4.
During the experiment the S9 mix was stored in an ice bath. The S9 mix preparation was performed according to Ames et al.
Test concentrations with justification for top dose:
31; 92; 307; 920; 2300; and 4600 μg/plate (active ingredient)
Vehicle / solvent:
DMSO (purity >99 %, MERCK, D-64293 Darmstadt)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Without metabolism: TA 1535 and TA 100
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylene-diamine, 4-NOPD
Remarks:
Without metabolism: TA 1537 and TA 98
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene, 2-AA
Remarks:
With metabolic activation: TA 1535, TA 1537, TA 98, TA 100
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration Triplicate
- Number of independent experiments : Two

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium; Plate incorporation assay.

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: The bacterial cultures were incubated in a shaking water bath for 8 hours at 37° C.

FOR GENE MUTATION:
- Method used: Agar

METHODS FOR MEASUREMENT OF CYTOTOXICITY - Method: Background growth inhibition.

METHODS FOR MEASUREMENTS OF GENOTOXICIY - A test article is considered mutagenic if the number of reversions is at least twice the spontaneous reversion rate in strains TA 98, and TA 100 or thrice in strains TA 1535 and TA 1537 (3, 4). Also, a dose-dependent and reproducible increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of the test article regardless whether the highest dose induced the criteria described above or not.
Evaluation criteria:
A test article is considered positive if either a dose related increase in the number of revertants or a biologically relevant increase for at least one test concentration is induced. A test article producing neither a dose related increase in the number of revertants nor a biologically relevant positive response at any one of the test points is considered nonmutagenic in this system. A biologically relevant response is described as follows: A test article is considered mutagenic if the number of reversions is at least twice the spontaneous reversion rate in strains TA 98, and TA 100 or thrice in strains TA 1535 and TA 1537. Also, a dose-dependent and reproducible increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of the test article regardless whether the highest dose induced the criteria described above or not.
Statistics:
A statistical analysis of the data is not required.
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES: Test article concentration per plate µg: 3, 9, 31, 92, 307, 920, 2300, 4600.
The plates with the test article showed normal background growth up to 4600 µg/plate in strain TA 98 and TA 100.

- No toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation.

- The plates incubated with the test article showed normal background growth up to 4600 µg/plate with and without S9 mix in all strains used.

- Substantial and dose dependent increases in revertant colony numbers were observed following treatment with FAT 93460/A in all strains with and without metabolic activation in experiment I and II. The required threshold of twice (strains TA 98 and TA 100) and thrice (TA 1535 and TA 1537) the number of the corresponding solvent control was reached or exceeded at the following concentrations:

Strain

Experiment I (μg/plate)

Experiment II (μg/plate)

without S9 mix

with S9 mix

without S9 mix

with S9 mix

TA 1535

>307

> 307

>307

>307

TA 1537

>307

> 920

>307

> 92

TA 98

>920

> 31

> 92

> 31

TA 100

>920

>2300

>920

>920

Positive control substances showed a distinct increase in induced revertant colonies, thereby validating the test.

Conclusions:
FAT 93460/A is considered to be mutagenic in this Salmonella typhimurium reverse mutation assay.
Executive summary:

A study was performed according to OECD test guideline 471 and EU Method B.13/14 to investigate the potential of FAT 93460/A to induce gene mutations according to the plate incorporation test using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test article was tested at the following concentrations: 31; 92; 307; 920; 2300; and 4600 μg/plate (active ingredient). No toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation. The plates incubated with the test article showed normal background growth up to 4600 μg/plate with and without S9 mix in all strains used. Substantial and dose dependent increases in revertant colony numbers were observed following treatment with FAT 93460/A in all strains with and without metabolic activation in experiment I and II. The required threshold of twice (strains TA 98 and TA 100) and trice (TA 1535 and TA 1537) the number of the corresponding solvent control was reached or exceeded at the concentrations. Based on the findings of the study, it can be concluded that the test article induced gene mutations by base pair changes and frameshifts in the genome of the strains TA 1535, TA 1537, TA 98, and TA 100. Therefore, FAT 93460/A is considered to be mutagenic in this Salmonella typhimurium reverse mutation assay.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Test start date: 15 November 1995; Test end date: 16 January 1996; Study completion date: 21 March 1996
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
92/69/EEC (Cytogenetics, metaphase analysis)
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
Identification: FAT 40543/A
Description: Red powder
Batch: TVRN 196-200
Purity: 90 %
Test substance storage: At room temperature in dark
Stability under storage conditions: Stable
Expiry date: November 01, 2000
Species / strain / cell type:
Chinese hamster lung (CHL/IU)
Details on mammalian cell type (if applicable):
Chinese hamster lung (CHL) cells, Source: European Collection of Animal Cell Cultures, Division of Biologies, PHLS Centre for Applied Microbiology and Research. Porton Down, Salisbury SP4 OJG, United Kingdom. AGT(Average Generation Time) = 12.5 h (November 1995)
Metabolic activation:
with and without
Metabolic activation system:
Preparation of S9-homogenate:
Rat liver microsomal enzymes were routinely prepared from adult male Wistar rats, which were obtained from Charles River Wiga, Sulzfeld, Germany. The animals were housed at NOTOX in a special room under standard laboratory conditions, as described in the SOP's. The rats were injected
intraperitoneally with a solution (20 % w/v) of Aroclor 1254 (500 mg/kg body weight) in corn oil. Five days later, they were killed by decapitation; (they were denied access to food for at least 12 h preceding sacrifice). The livers of the rats were removed aseptically, and washed in cold (0 °C) sterile 0.1 M sodium phosphate buffer (pH 7.4) containing 0.1 mM Disodium-EDTA. Subsequently the livers were minced in a blender and homogenized in 3 volumes of phosphate buffer with a Potter homogenizer. The homogenate was centrifuged for 15 min at 9000 g. The supernatant (S9) was transferred into sterile ampules, which were stored in liquid nitrogen (-196 °C).

Preparation of S9-roix
S9-mix was prepared immediately before use and kept on ice. S9-mix contained per ml: 1.02 mg MgCl2 6H2O; 2.46 mg KCl; 1.7 mg glucose-6-phosphate; 3.4 mg NADP; 4 µmol HEPES and 0.5 ml S9. The above solution was filter (0.22 µm)-sterilized. To 0.5 ml S9-mix components 0.5 ml S9-fraction (batch 95-7) was added (50 % (v/v) S9-fraction). Metabolic activation was achieved by addding 0.2 ml liver S9-mix to each ml cell culture.
Test concentrations with justification for top dose:
Concentration range in the main test (with metabolic activation): 3, 10, 33, 100 µg/ml
Concentration range in the main test (without metabolic activation): 1, 3, 10, 33, 56 µg/ml
Vehicle / solvent:
Dimethylsulfoxide
Untreated negative controls:
yes
Remarks:
solvent control
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
without metabolic activation
Untreated negative controls:
yes
Remarks:
solvent control
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:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Duplicate.
- Number of independent experiments : Two

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 3 x 10E4 and 1 x 10E4 cells/dish were seeded for a 24 h and 48 h fixation period respectively

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 24h and 48 h.

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor (cytogenetic assays): Colchicine (1 µg/mL)
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): Fixed cells were dropped onto previously cleaned slides which were immersed for 24 hours in a 1:1 mixture of 96% (v/v) ethanol/ether and cleaned with a tissue. The slides were marked with the NOTOX study identification number and group number. Two slides were prepared per culture. Slides were allowed
to dry and thereafter stained for 10-30 min with 5% (v/v) Giemsa solution in tap water. Thereafter slides were rinsed in tap water and allowed to dry. The dry slides were cleared by dipping them in xylene before they were embedded in MicroMount and mounted with a coverslip.
- Criteria for scoring chromosome aberrations (selection of analysable cells and aberration identification): Aall slides were randomly coded before examination of chromosome aberrations and scored. An adhesive label with NOTOX study identification number and code were stuck over the marked slide. At least 100 metaphase chromosome spreads per culture were examined by light microscopy for chromosome aberrations. Only metaphases containing 25 ± 2 chromosomes were analysed. The number of cells with aberrations and the number of aberrations were calculated
- Determination of polyploidy: Yes
- Determination of endoreplication Yes:

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Mitotic index (MI).

METHODS FOR MEASUREMENTS OF GENOTOXICIY : Chromosome abberations.
Evaluation criteria:
A chromosome aberration test was considered acceptable if it met the following criterion:
The positive control substances should produce a statistically significant (Chi-square test, P < 0.05) increase in the number of cells with chromosome aberrations.
A test substance was considered positive (clastogenic) in the chromosome aberration test if:
a) It induced a dose-related statistically significant (Chi-square test, P < 0.05) increase in the number of cells with chromosome aberrations.
b) A statistically significant increase in the frequencies of aberrations was observed in the absence of a clear dose-response relationship, but the results were reproducible in an independently repeated experiment.
A test substance was considered negative (not clastogenic) in the chromosome aberration test if:
a) None of the tested concentrations induced a statistically significant (Chi-square test, P < 0.05) increase in the number of cells with chromosome aberrations.
The preceding criteria were not absolute and other modifying factors might enter into the final evaluation decision.
Statistics:
The incidence of aberrant cells (cells with one or more chromosome aberrations, inclusive or exclusive gaps) for each treatment group was compared to that of the solvent control using Chi-square statistics.
If P is small (P< 0.05) the hypothesis that the incidence of cells with chromosome aberrations is the same for both the treated and the solvent control group is rejected and the number of aberrant cells in the test group is considered to be significantly different from the control group at the 95 % confidence level.
Key result
Species / strain:
Chinese hamster lung (CHL/IU)
Metabolic activation:
with
Genotoxicity:
ambiguous
Remarks:
only at 33 µg/ml (24h) increase of chromosome abberations
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
(at 100 µg/ml precipitation)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
Chinese hamster lung (CHL/IU)
Metabolic activation:
without
Genotoxicity:
positive
Remarks:
as of 10 µg/ml (24h) and as of 1 µg/ml (48h)
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
(>= 33 µg/ml)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
Chinese hamster lung (CHL/IU)
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
(at 100 µg/ml precipitation)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
Chinese hamster lung (CHL/IU)
Metabolic activation:
without
Genotoxicity:
positive
Remarks:
as of 10 µg/ml (24h) and as of 3 µg/ml (48h)
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
(>= 10 µg/ml)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Observations:
Reproducible, statistically significant (P<0.001), increases in the number of chromosomal aberrations (excluding gaps) were observed in the absence of S9 in experiments 1 (≥1 µg/ml) and 2 (≥5.6 µg/ml).

In the presence of S9, a statistically significant (P<0.05) increase in chromosomal aberrations (excluding gaps) was observed only in experiment 1 at the 24 h fixation, at 33 micrograms/ml.

The average doubling time of the cells used was reported to be 12.5 h. An additional exposure time of 45 hours in the absence of metabolic activation was used.

Conclusions:
FAT 40543/A, was shown to be clastogenic in this study, in particular without metabolic activation. With metabolic activation the clastogenicity was almost completely eliminated.
Executive summary:

This report describes the effect of FAT 40543/A on induction of chromosome aberrations in cultured Chinese hamster lung (CHL) cells in the presence and absence of a metabolic activation system (Aroclor-1254 induced rat liver S9-mix). This experiment was conducted in accordance with OECD test guideline 473 and EEC Directive 67/548/EEC B.10. In the absence of S9-mix, in the first experiment, FAT 40543/A was tested up to 33 µg/ml for a 24 h fixation time and up to 10 µg/ml for a 48 h fixation time and in the second experiment FAT 40543/A was tested up to 10 µg/ml for a 24 h fixation time, and up to 5.6 µg/ml for a 48 h fixation time. In the presence of S9-mix FAT 40543/A was tested up to 100 µg/ml for a 24 h and 48 h fixation period in both experiments. In the absence of S9-mix, in experiment 1, at the 24 h fixation period concentrations of 10 and 33 µg/ml and at the 48 h fixation period concentrations of 1, 3 and 10 µg/ml induced statistically and biologically significant increases in the number of cells with chromosome aberrations. In experiment 2 at the 24 h fixation period a concentration of 10 µg/ml induced a statistically significant increase in the number of cells with chromosome aberrations when gaps were excluded. At the 48 h fixation period a concentration of 3 µg/ml induced a statistically significant increase in the number of cells with chromosome aberrations when gaps were excluded and a concentration of 5.6 µg/ml induced a statistically and biologically significant increase in the number of cells with chromosome aberrations when gaps were included and excluded. It is concluded that the test substance is clastogenic in the absence of S9-mix. In the presence of S9-mix, in experiment 1, at the 24 h fixation period a concentration of 33 µg/ml induced a statistically significant increase in the number of cells with chromosome aberrations. None of the other tested concentrations induced a statistically or biologically significant increase in the number of cells with chromosome aberrations. Therefore, it is concluded that clastogenicity of FAT 40543/A is almost completely eliminated by the presence of S9-mix. The positive control chemicals (MMC-C and CP) both produced statistically significant increases in the frequency of aberrant cells. It was therefore concluded that the test conditions were optimal and that the metabolic activation system (S9-mix) functioned properly. It is concluded that the test substance is clastogenic in Chinese hamster lung (CHL) cells under the experimental conditions described in this report.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Study completion date: 26 March 1996
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
GLP compliance:
yes
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes
Specific details on test material used for the study:
Identification: FAT 40543/A
Description: Red powder
Batch: TVRN 196-200
Purity: 90 %
Test substance storage: At room temperature in dark
Stability under storage conditions: Stable
Expiry date: November 01, 2000
Target gene:
The objective of this study was to evaluate the test substance for its ability to induce forward mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in V79 Chinese hamster cells. The assay was conducted in the absence and presence of a metabolic system (S9-mix).
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
V79 Chinese hamster cells were cultured in F10 complete culture medium. Cultures were incubated in a humid atmosphere containing 5% CO2 in air at 37°C. Cell density was preferably kept below 8 x 10^6 per 150 sq.cm.
Metabolic activation:
with and without
Metabolic activation system:
Aroclor-induced rat liver S9

Preparation of S9-homogenate
Rat liver microsomal enzymes were routinely prepared from adult male Wistar rats, which were obtained from Charles River Wiga, Sulzfeld, Germany. Switzerland. The animals were housed at NOTOX in a special room under standard laboratory conditions, as described in the SOP's. The rats were injected intraperitoneally with a solution (20 % w/v) of Aroclor 1254 (500 mg/kg body weight) in corn oil. Five days later, they were killed by decapitation; (they were denied access to food for at least 12 hours preceding sacrifice). The livers of the rats were removed aseptically, and washed in
cold (0°C) sterile 0.1 M sodium phosphate buffer (pH 7.4) containing 0.1 mM Na2-EDTA. Subsequently the livers were minced in a blender and homogenized in 3 volumes of phosphate buffer with a Potter homogenizer. The homogenate was centrifuged for 15 min at 9000 g. The supernatant (S9) was transferred into sterile ampules, which were stored in liquid nitrogen (-196 °C).

Preparation of S9-mix
S9-mix was prepared immediately before use and kept on ice. S9-mix contained per ml: 1.02 mg MgCl2.6H20; 2.46 mg KCl; 1.7 mg glucose-6-phosphate; 3.4 mg NADP; 4 µmol HEPES. The above solution was filter (0.22 µm)-sterilized. To 0.5 ml S9-mix components 0.5 ml S9-fraction (batch 95-8) was added (50% (v/v) S9-fraction). Metabolic activation was achieved by adding 0.2 mL liver S9-mix to each mL cell suspension. The concentration of the S9-fraction in the exposition medium was 8 % (v/v).
Test concentrations with justification for top dose:
Concentration range in the main test (with metabolic activation): 3, 10, 33 and 100 µg/mL
Concentration range in the main test (without metabolic activation): 3, 10, 13, 18 and 24.5 µg/mL
Vehicle / solvent:
Dimethylsulfoxide
The test substance was suspended or dissolved in dimethylsulphoxide of spectroscopic quality (Merck). Test substance concentrations were prepared directly prior to use. The final concentration of the solvent in the culture medium amounted to 0.8% (v/v).
Untreated negative controls:
yes
Remarks:
solvent control
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without metabolic activation (S9-mix)
Untreated negative controls:
yes
Remarks:
solvent control
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-dimethylnitrosamine
Remarks:
with metabolic activation (S9-mix)
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Single
- Number of independent experiments : Two

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 6 x 10E6
- Test substance added in medium; Cell cultures were exposed for 3 hours to FAT 40543/A in exposition medium. The cell cultures were placed in 30 ml centrifuge tubes on a roller mixer at 37 °C.

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 3 hours

FOR GENE MUTATION:
- Expression time: 7 days
- Selection time (if incubation with a selective agent): 7 days
- Fixation time (start of exposure up to fixation or harvest of cells): 7 days
- If a selective agent is used (e.g., 6-thioguanine or trifluorothymidine), indicate its identity, its concentration and, duration and period of cell exposure.: Selective medium consisted of F10 complete culture medium containing 5 µg/ml 6-thioguanine (6-TG; Fluka).
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: For determination of the MF a total number of 106 cells were seeded in 9 cm petri dishes (10^5 cells/dish) containing 10 ml selective medium. The petridishes for CE3 and MF were incubated at 37 °C in humified air with 5 % CO2 for 7 days. Colonies were fixed with methanol, stained with 1% methylene blue and counted with the naked eye or with the Artek colony counter. The mutant frequency was expressed as the number of mutants per 10^5 surviving cells.


METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Cloning efficiency.

METHODS FOR MEASUREMENTS OF GENOTOXICIY : Increase in mutant frequency.
Evaluation criteria:
ACCEPTABILITY OF ASSAY
A mutation assay was considered acceptable if it met the following criteria:
a) The absolute colony forming efficiency of the solvent controls was >50 %.
b) At least three of the four doses of the test substance had an acceptable number of surviving cells (10E6) analysed for expression of the HPRT mutation.
c) The spontaneous mutant frequency in the untreated or solvent-treated control was <5 per 10E5 survivors.
d) The positive control substances induced significant (at least 3-fold) increases in the mutant frequencies.
Statistics:
DATA EVALUATION AND STATISTICAL PROCEDURES
No formal hypothesis testing was done. A test substance was considered positive (mutagenic) in the mutation assay if:
a) The test substance induced a mutant frequency that was at least 3 times higher than the spontaneous mutant frequency of the untreated or solvent-treated control.
b) The results showed a dose response relationship and were reproducible in an independently repeated test.
A test substance was considered negative (not mutagenic) in the mutation assay if:
a) None of the tested concentrations showed a mutant frequency at least 3 times that of the solvent control.
b) The results were confirmed in an independently repeated test.
The preceding criteria were not absolute and other modifying factors might enter into the final evaluation decision.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
as of 100 µg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
other: as specified above
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
as of 18 µg/ml
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
EVALUATION OF THE RESULTS
The spontaneous mutant frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range ({0.3- and 5.1E+5 (mean 2.0E+5) in the absence of S9-mix} and {0.8- and 5.4E+5 (mean 2.0E+5) in the presence of S9-mix}; for n=29 and 32, respectively). With the exception of the solvent control in the presence of S9, in the second experiment, which was 0.6E+5, it is just outside the historical control data range. Mutant frequencies induced by positive control chemicals were increased by 42- to 32-fold for EMS, in the first and second experiment, respectively, and by 19- to 21-fold for DMN, in the first and second experiment, respectively. It was therefore concluded that the test conditions were optimal and that the metabolic activation system (S9-mix) functioned properly. In the first experiment, in the absence of S9 metabolic activation FAT40543/A induced a three-fold increase in the mutant frequency at the HPRT-locus at a test substance concentration of 24.5 µg/mL. However, this increase was just three-fold, this increase was in the absence of a dose-response relationship and this increase was within the historical control data range. In the second experiment, in the absence of S9 metabolic activation FAT40543/A induced a two-fold increase in the mutant frequency at the HPRT-locus at the test substance concentrations of 10 and 13 µg/mL. However, this increase was not three-fold as stated in the "Data evaluation", this increase was in the absence of a dose-response relationship and this increase was within the historical control data range. Therefore, these increases were considered not biologically relevant and FAT40543/A is considered not mutagenic in the absence of S9-mix. In the presence of S9 metabolic activation in the first experiment FAT40543/A induced a two-fold increase in the mutant frequency at the HPRT-locus at the precipitating concentration of 100 µg/mL. However, this increase was not three-fold as stated in the "Data evaluation", this increase was in the absence of a dose-response relationship and this increase was within the historical control data range. Moreover, the increase was observed in only one experiment. Therefore, this increase was considered not biologically relevant and FAT40543/A is considered not mutagenic in the presence of S9-mix.
Conclusions:
FAT 40543/A was found to be not mutagenic in the V79/HPRT mutation test system under the experimental conditions described in this report.
Executive summary:

This report describes the effects of FAT 40543/A, on the induction of forward mutations at the HPRT-locus in V79 Chinese hamster cells in the presence and absence of S9-mix. This study was conducted in accordance with OECD test guideline 476 in a GLP-certified laboratory. FAT 40543/A was tested from 3 to 24.5 µg/mL in the absence of S9-mix and from 3 to 100 µg/mL in the presence of 8 % (v/v) S9-fraction, in the first and second experiment, respectively. In the absence of S9-mix, at these dose levels appropriate cytotoxicity was observed. FAT 40543/A did not induce a dose-related increase in the mutant frequency at the HPRT-locus in the absence and presence of S9-mix, in two independently repeated experiments. Mutant frequencies induced by positive control chemicals were increased by 42- and 32-fold for EMS, in the first and second experiment, respectively, and by 19- and 21-fold for DMN, in the first and second experiment, respectively. It was therefore concluded that the test conditions, both in the absence and presence of S9-mix, were appropriate and that the metabolic activation system (S9-mix) functioned properly. It is concluded that FAT 40543/A is not mutagenic in the mutation test with V79 Chinese hamster cells under the experimental conditions described in this report.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

The test substance did not cause clastogenic effects in a micronucleus assay.

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:
Test start date: March 27, 2001; Test end date: 24 April 2001; Study completion date: July 17, 2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
July 21, 1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
May 19, 2000
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
micronucleus assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Test material identified as: FAT 93529/A
- Source and batch No.of test material: 1-5 / 2000 laborgetrocknet
- Expiration date of the batch: March 2006

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability of the test substance in the solvent/vehicle: 24 hours in water

OTHER SPECIFICS:
- Aggregate state at room temperature: solid
- Colour: yellow
- Purity: technical purity
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
- Strain: NMRI
- Source: RCC Ltd., Biotechnology and Animal Breeding Division; CH-4414 Füllinsdorf
- Number of animals: 72 (36 males/36 females)
- Initial age at start of acclimatization: 8-10 weeks
- Acclimatization: minimum 5 days
- Initial body weight at start of treatment: males mean value 33.6 g (SD ± 2.3 g); females mean value 25.5 g (SD ± 2.3 g)

Husbandry
The animals were kept conventionally. The experiment was conducted under standard laboratory conditions.
- Housing: single
- Cage Type: Makrolon Type I, with wire mesh top (EHRET GmbH, D-79302 Emmendingen)
- Bedding: Granulated soft wood bedding (ALTROMIN, D-32791 Lage/Lippe)
- Feed: Pelleted standard diet, ad libitum (ALTROMIN 1324, D-32791 Lage/Lippe)
- Water: Tap water, ad libitum, (Gemeindewerke, D-64380 Roßdorf)
- Environment: temperature 21 ± 4°C
- Relative humidity 30-70%
- Artificial light: 6.00 a.m. - 6.00 p.m.
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: Corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: On the day of the experiment, the test article was formulated in corn oil.

- All animals received a single standard volume of 20 mL/kg bw orally.
Duration of treatment / exposure:
The animals received the test article, the vehicle or the positive control substance once.
Frequency of treatment:
Single treatment
Post exposure period:
Sampling of the bone marrow was done 24 and 48 hours after treatment, respectively.
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Vehicle
Dose / conc.:
500 mg/kg bw/day (nominal)
Remarks:
24 hour interval only
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
24 hour interval only
Dose / conc.:
2 000 mg/kg bw/day (nominal)
Remarks:
24 and 48 hour interval
No. of animals per sex per dose:
6 male and 6 female rats
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide;
- Justification for choice of positive control(s): Recommended by the guidelines
- Route of administration: Oral
- Doses : 40 mg/kg b.w.
- Frequency: Once
Tissues and cell types examined:
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 syringe. The cell suspension was centrifuged at 1500 rpm (390 x g) for 10 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-Griinwald (MERCK, D-64293 Darmstadt)/Giemsa (Gurr, BDH Limited Poole, Great Britain). Cover slips were mounted with EUKITT (KINDLER, D-79110 Freiburg). At least one slide was made from each bone marrow sample.
Details of tissue and slide preparation:
Analysis of Cells:
Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives. At least 2000 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 2000 the PCEs. The analysis was performed with coded slides. Five animals per sex and group were evaluated.
Evaluation criteria:
- A test article is classified as mutagenic if it induces either a dose-related increase or a clear increase in the number of micronucleated polychromatic erythrocytes in a single dose group.
- A test item that fails to produce a biological relevant increase in the number of micronucleated polychromatic erythrocytes is considered non-mutagenic in this system..
- Statistical methods (nonparametric Mann-Whitney test (8)) will be used as an aid in evaluating the results.
- However, the primary point of consideratrion is the biological relevance of the results.
Statistics:
Non parametric Mann-Whitney test
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: A single dose of 2000 mg/kg bw was used
- Clinical signs of toxicity in test animals: The treated animals expressed no toxic reactions. The urine color was orange at 2-4 hours after treatment, while 6 hours after treatment, it was yellow orange.
- Rationale for exposure: Highest (limit dose) was used as recommended by the guideline.


RESULTS OF DEFINITIVE STUDY
- The mean number of normochromatic erythrocytes was not increased after treatment with the test item as compared to the mean value of NCEs of the vehicle control indicating that FAT 93'529/A had no cytotoxic properties in the bone marrow. However, the urine colour of the treated animals had taken on the colour of the test item indicating the systemic distribution of the test item and its bioavailability in the target organ.

- In comparison to the corresponding vehicle controls there was no statistically significant or biologically relevant enhancement in the frequency of the detected micronuclei at any preparation interval and dose level after administration of the test item. The mean values of micronuclei observed after treatment with FAT 93'529/A were below or near to the value of the vehicle control group.

- 40 mg/kg b.w. cyclophosphamide administered orally was used as positive control which showed a statistically significant increase of induced micronucleus frequency.
Conclusions:
The test substance did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse.
Executive summary:

The test substance FAT 93529/A was assessed in the micronucleus assay for its potential to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse as per the methodology described in OECD Guideline 474 and EU Method B.12. The test substance was formulated in corn oil. Corn oil was used as vehicle control. The volume administered orally was 20 mL/kg bw. 24 h and 48 h after a single administration of the test substance 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. At least 2000 polychromatic erythrocytes (PCE) per animal were scored for micronuclei. To describe a cytotoxic effect due to the treatment with the test item the ratio between polychromatic and normochromatic erythrocytes (NCE) was determined in the same sample and reported as the number of NCEs per 2000 PCEs. The following dose levels of the test substance were investigated: 24 h preparation interval: 500, 1000 and 2000 mg/kg bw, 48 h preparation interval: 2000 mg/kg bw. After treatment with the test item the number of NCEs was not substantially increased as compared to the mean value of NCEs of the vehicle control thus indicating that FAT 93529/A had no cytotoxic effectiveness in the bone marrow. However, the urine of the treated animals had taken over the colour of the test item indicating the systemic distribution and the bioavailability of the test item in the target organ. In comparison to the corresponding vehicle controls there was no biologically relevant or statistically significant enhancement in the frequency of the detected micronuclei at any preparation interval after administration of the test item and with any dose level used. 40 mg/kg b.w. cyclophosphamide administered orally was used as positive control which showed a substantial increase of induced micronucleus frequency. In conclusion, it can be stated, that the test substance did not induce micronuclei as determined by the micronucleus test in the bone marrow cells of the mouse.

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Genetic toxicity (in vitro): bacetrial reverse mutation assay

A study was performed to investigate the potential of FAT 93460/A to induce gene mutations according to the plate incorporation test using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test article was tested at the following concentrations: 31; 92; 307; 920; 2300; and 4600 μg/plate (active ingredient). No toxic effects, evident as a reduction in the number of revenants, occurred in the test groups with and without metabolic activation. The plates incubated with the test article showed normal background growth up to 4600 μg/plate with and without S9 mix in all strains used. Substantial and dose dependent increases in revenant colony numbers were observed following treatment with FAT 93460/A in all strains with and without metabolic activation in experiment I and II. The required threshold of twice (strains TA 98 and TA 100) and trice (TA 1535 and TA 1537) the number of the corresponding solvent control was reached or exceeded at the concentrations. Based on the findings of the study, it can be concluded that the test article induced gene mutations by base pair changes and frameshifts in the genome of the strains TA 1535, TA 1537, TA 98, and TA 100. Therefore, FAT 93460/A is considered to be mutagenic in this Salmonella typhimurium reverse mutation assay.

Genetic toxicity (in vivo): Micronucleus assay

The test substance FAT 93529/A was assessed in the micronucleus assay for its potential to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse as per the methodology described in OECD Guideline 474 and EU Method B.12. The test substance was formulated in corn oil. Corn oil was used as vehicle control. The volume administered orally was 20 mL/kg bw. 24 h and 48 h after a single administration of the test substance 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. At least 2000 polychromatic erythrocytes (PCE) per animal were scored for micronuclei. To describe a cytotoxic effect due to the treatment with the test item the ratio between polychromatic and normochromatic erythrocytes (NCE) was determined in the same sample and reported as the number of NCEs per 2000 PCEs. The following dose levels of the test substance were investigated: 24 h preparation interval: 500, 1000 and 2000 mg/kg bw, 48 h preparation interval: 2000 mg/kg bw. After treatment with the test item the number of NCEs was not substantially increased as compared to the mean value of NCEs of the vehicle control thus indicating that FAT 93529/A had no cytotoxic effectiveness in the bone marrow. However, the urine of the treated animals had taken over the colour of the test item indicating the systemic distribution and the bioavailability of the test item in the target organ. In comparison to the corresponding vehicle controls there was no biologically relevant or statistically significant enhancement in the frequency of the detected micronuclei at any preparation interval after administration of the test item and with any dose level used. 40 mg/kg b.w. cyclophosphamide administered orally was used as positive control which showed a substantial increase of induced micronucleus frequency. In conclusion, it can be stated, that the test substance did not induce micronuclei as determined by the micronucleus test in the bone marrow cells of the mouse. Based on the above data, it can be concluded that the substance induced reverse mutations in bacteria, while it had no clastogenic effect in an in vivo micronucleus assay.

Data from the read across substance (FAT 40543)

In vitro chromosome aberrations in chinese hamster lung cells

The effect of FAT 40543/A on the induction of chromosome aberrations in cultured Chinese hamster lung (CHL) cells in the presence and absence of a metabolic activation system (Aroclor-1254 induced rat liver S9-mix) was investigated. In the absence of S9-mix, in the first experiment, FAT 40543/A was tested up to 33 µg/ml for a 24 h fixation time and up to 10 µg/ml for a 48 h fixation time and in the second experiment FAT 40543/A was tested up to 10 µg/ml for a 24 h fixation time, and up to 5.6 µg/ml for a 48 h fixation time. In the presence of S9-mix FAT 40543/A was tested up to 100 µg/ml for a 24 h and 48 h fixation period in both experiments. In the absence of S9-mix, in experiment 1, at the 24 h fixation period concentrations of 10 and 33 µg/ml and at the 48 h fixation period concentrations of 1, 3 and 10 µg/ml induced statistically and biologically significant increases in the number of cells with chromosome aberrations. In experiment 2 at the 24 h fixation period a concentration of 10 µg/ml induced a statistically significant increase in the number of cells with chromosome aberrations when gaps were excluded. At the 48 h fixation period a concentration of 3 µg/ml induced a statistically significant increase in the number of cells with chromosome aberrations when gaps were excluded and a concentration of 5.6 µg/ml induced a statistically and biologically significant increase in the number of cells with chromosome aberrations when gaps were included and excluded. It is concluded that the test substance is clastogenic in the absence of S9-mix.

 

In vitro mammalian cell gene mutation test with V79 Chinese hamster cells

This report describes the effects of FAT 40543/A, a read across substance to FAT 93460/D, on the induction of forward mutations at the HPRT-locus in V79 Chinese hamster cells in the presence and absence of S9-mix. This study was conducted in accordance with OECD test guideline 476 in a GLP certified laboratory. FAT 40543/A was tested from 3 to 24.5 µg/mL in the absence of S9-mix and from 3 to 100 µg/mL in the presence of 8 % (v/v) S9-fraction, in the first and second experiment, respectively. In the absence of S9-mix, at these dose levels appropriate cytotoxicity was observed. FAT 40543/A did not induce a dose-related increase in the mutant frequency at the HPRT-locus in the absence and presence of S9-mix, in two independently repeated experiments. Mutant frequencies induced by positive control chemicals were increased by 42- and 32-fold for EMS, in the first and second experiment respectively, and by 19- and 21-fold for DMN, in the first and second experiment, respectively. It was therefore concluded that the test conditions, both in the absence and presence of S9-mix, were appropriate and that the metabolic activation system (S9-mix) functioned properly. It is concluded that FAT 40543/A is not mutagenic in the mutation test with V79 Chinese hamster cells under the experimental conditions described in this report. The source substance FAT 40543 was found to be mutagenic in bacterial cells, however, it did not induce mutagenicity in mammalian cells in an in vitro mammalian cell gene mutation test with V79 Chinese hamster cells. Similarly, it was clastogenic in an in vitro chromosomal aberration assay, however, the clastogenic effect was not reproduced in an in vivo micronucleus assay. Hence, it can be concluded that FAT 40543 is not genotoxic. Hence, using the read across principles with the current substance, it can be concluded that the substance is not genotoxic.

Justification for classification or non-classification

Based on the available database of genetic toxicity studies, the substance does not warrant classification according to CLP (Regulation 1272/2008) criteria.