Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

FAT 40000 was found to be not mutagenic in the Salmonella reverse mutation assays. The read across substance, FAT 40277, was not mutagenic in the bacterial reverse mutation assays and the mammalian cell gene mutation assay in vitro. FAT 40277 was found to be clastogenic in a chromosome aberration assay in vitro.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1978
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Justification for type of information:
None
Qualifier:
according to
Guideline:
other: Ames et al, 1973
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
no tester strain to detect cross-linking mutagens has been included
Principles of method if other than guideline:
None
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
None
Target gene:
None
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
S9-mix from rat liver
Test concentrations with justification for top dose:
25, 7 5, 225, 675, and 2025 µg / 0 .1 ml
Vehicle / solvent:
phosphate buffer
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
Without metabolic activation - for TA 1535
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
Without metabolic activation - for TA 1537
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: daunoblastin
Remarks:
Without metabolic activation - for TA 98
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
without metabolic activation - for TA 100 strain
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with metabolic acyivation - for TA 1535 strain
Details on test system and experimental conditions:
None
Rationale for test conditions:
None
Evaluation criteria:
When the colonies had been counted, the arithmetic mean was calculated. The test substance was considered to be non-mutagenic if the colony count in relation to the negative control was not doubled at any concentration.
Statistics:
None
Key result
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 1538, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
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:
None
Remarks on result:
other: all strains/cell types tested

None

Conclusions:
FAT 40000/B was determined to be non-mutagenic in the bacterial reverse mutation assay.
Executive summary:

A supporting study was performed wherein preparation FAT 40000/B was tested for mutagenic effects on histidine-auxotrophic mutants of Salmonella typhimurium. The investigations were performed with the following concentrations of the trial substance with and without microsomal activation: 25, 75, 225, 675 and 2025 µg/0.1 ml. Since considerable fluctuations in the number of back-mutants produced were observed in the experiments with Strain TA 100, the experiments were repeated, in accordance with the usual practice in the laboratory. These tests permit the detection of point mutations in bacteria induced by chemical substances. Any mutagenic effects of the substances are demonstrable on comparison of the numbers of bacteria in the treated and control cultures that have undergone back-mutation to histidine-prototrophism. To ensure that mutagenic effects of metabolites of the test substances formed in mammals would also be detected, experiments were performed in which the cultures were additionally treated with an activation mixture (rat liver microsomes and co-factors). In the experiments performed with and without microsomal activation, comparison of the numbers of back-mutant colonies in the controls and the cultures treated with the various concentrations of FAT 40000/B revealed no marked deviations. No evidence of the induction of point mutations by FAT 40000/B or by the metabolites of the substance formed as a result of microsomal activation was detectable in the strains of S. typhimurium used in these experiments.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1990
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
None
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
no tester strain to detect cross-linking mutagens has been included
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Principles of method if other than guideline:
None
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
None
Target gene:
None
Species / strain / cell type:
other: S. typhimurium TA 1535, TA 1537, TA 1538, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
S9-mix from rat liver
Test concentrations with justification for top dose:
10.0, 33.3, 100.0, 333.3, 1000.0 and 5000.0 ug/plate
Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Without metabolic activation - for TA 1535, TA 100 strains
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 metabolic activation - for TA 1537, TA 1538, TA 98 strains
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 - for TA 1535, TA 1537, TA 1538, TA 98, TA 100 strains
Details on test system and experimental conditions:
None
Rationale for test conditions:
None
Evaluation criteria:
The generally accepted conditions for the evaluation of the results are: corresponding background growth on both negative control and test plates normal range of spontaneous reversion rates.
Statistics:
None
Key result
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 1538, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
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:
None
Remarks on result:
other: all strains/cell types tested

None

Conclusions:
FAT 40000/G was determined to be non-mutagenic in the bacterial reverse mutation assay.
Executive summary:

A key study was performed to investigate the potential of FAT 40000/G to induce gene mutations according to the plate incorporation test using the Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100. The assay was performed in two independent experiments, using identical procedures, 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 : 10.0, 33.3, 100.0, 333.3, 1000.0; and 5000.0 ug/plate A slight toxic effect, evidenced by a reduction in the number of revertants, occurred only in strain TA 98 at 5000.0 ug/plate in the presence of S9 mix in experiment I. The plates incubated with the test article showed normal background growth up to 5000.0 ug/plate with and without S9 mix in all strains used. No substantial increases in revertant colony numbers of any of the five tester strains were observed following treatment with FAT 40000/G at any dose level, either in the presence or absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of significance. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies. In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test article did not induce point mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, FAT 40000/G is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1988
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Metabolic activation system:
aroclor 1254 induced rat liver S9 mix
Test concentrations with justification for top dose:
Treatment was performed with the following concentrations:
- 7 hours: 100, 200, 300, 400 µg/mL
- 18 hours: 10, 50, 100, 200, 300, 400 µg/mL
- 28 hours: 100, 100, 300, 400 µg/mL

The following dose levels were evaluated:
- 7 hours: 400 µg/mL
- 18 hours: 10, 200, 400 µg/mL
- 28 hours: 400 µg/mL
Vehicle / solvent:
culture medium without fetal calf serum
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation: Two days old logarithmically growing stock cultures more than 50 % confluent were trypsinised and a single cell suspension was prepared. The cells were seeded into Quadriperm dishes ( Heraeus, D-6450 Hanau, F.R.G. ) which contained microscopic slides (4 chambers per dish and test group). In each chamber 5E4 - 1E5 cells were seeded with regard to preparation time. The medium was MEM + 10 % FCS.
- Exposure: After 48 h (7 h, 28 h preparation interval) and 55 h (18 h preparation interval) the medium was replaced with serum-free medium containing the test article, either without S9 mix or with 20 µL/ml S9 mix. After 4h this medium was replaced with normal medium after rinsing twice. All incubations were done at 37°C in a humidified atmosphere with 5 % CO2.
- Fixation: 5, 15.5 and 25.5 h after the start of the treatment colcemid was added (0.2 µg/ml culture medium) to the cultures. 2.0 h (7 h interval) or 2.5 h later, (18 h and 28 h interval). the cells were treated on the slides in the chambers with hypotonic solution (0.4 % KCl) for 20 min at 37°C. After incubation in the hypotonic solution the cells are fixed with 3:1 absolute methanol:glacial acetic acid. All four slides per group were prepared. After fixation the cells were stained with aceto-orcein.

NUMBER OF REPLICATIONS: 4

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

DETERMINATION OF CYTOTOXICITY
- To describe a cytotoxic effect the mitotic index (% cells in mitosis) was determined.
Evaluation criteria:
The test article will be classified as mutagenic if it induces a significantly increased aberration rate with at least one of the concentrations tested as compared with the negative control. A test article which produce no significant positive response at any test point will be regarded as non-mutagenic in this assay.
Statistics:
nonparametric Mann-Whitney test
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
ADDITIONAL INFORMATION ON GENOTOXICITY:
In the presence of S9 mix the test article did not increase the frequency of cells with aberrations at any fixation interval. The aberration rates of the cells after treatment with the test article (1.00 % - 3.25 %) are in or very near to the range of the control values: 0.50 % - 2.50 %. In the absence of S9 mix the test article increased the structural aberration rate after treatment with 400 µg/mL at fixation interval 28 h. Additionally, 3.75 % of the cells were carrying exchanges as compared to 0.00 % in the corresponding solvent control.

RANGE-FINDING/SCREENING STUDIES:
In the pre-experiment on toxicity (colony forming ability) in the absence and presence of S9 mix with concentration of 400 µg/mL the colony forming ability was clearly reduced as compared to the solvent controls.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the main experiment the mitotic index was reduced after treatment with the highest dose level in the absence and presence of S9 mix, indicating that FAT 40'277/B had cytotoxic properties.
Remarks on result:
other: all strains/cell types tested
Conclusions:
The test substance is considered to be clastogenic in this chromosomal aberration test.
Executive summary:

In a GLP-compliant chromosome aberration test, tested according to OECD guideline 473, chinese hamster lung fibroblast (V79) were exposed to the test substance with and without metabolic activation. Different preparation intervals (up to 28 hours) and dosage (up to 400 µg/mL) were chosen in this study. The mitotic index was reduced after treatment with the highest concentrations in the presence and absence of S9 mix indicating that the test substance had cytotoxic properties. In the presence of S9 mix the test article did not increase the frequency of cells with aberrations at any fixation interval, but in the absence of S9 mix the test article increased the structural aberration rate after treatment with 400 µg/mL at fixation interval 28 hours. Based on the results the test substance is considered to be clastogenic in this chromosomal aberration test.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-12-12 to 2013-03-07
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
(Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Germany)
Type of assay:
mammalian cell gene mutation assay
Target gene:
hypoxanthine-guanine-phosphoribosyl-transferase (HPRT)
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
-Type and identity of media: MEM
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
Liver S9 of Wistar Phenobarbital and ß-Naphthoflavone-induced rat liver S9 mix (pre-experiment) and Liver S9 of Sprague Dawley Phenobarbital and ß-Naphthoflavone-induced rat liver S9 mix (main experiments)
Test concentrations with justification for top dose:
Pre-experiment for experiment I (with and without metabolic activation):
5, 10, 25, 50, 100, 250, 500, 1000, 2500, 5000 µg/mL
Experiment I
without metabolic activation: 10, 25, 50, 100, 200, 400, 600, 800, 1000 and 1200 µg/mL
and with metabolic activation: 1.0, 2.5, 5, 10, 25, 50, 100 and 125 µg/mL
Experiment II
without metabolic activation: 5, 10, 25, 50, 100, 250, 500, 1000 and 2000 µg/mL
and with metabolic activation: 1.00, 3.16, 10, 20, 40, 60, 80, 100 and 120 µg/mL
Vehicle / solvent:
Vehicle (Solvent) used:
For the pre-experiment the test item was dissolved in cell culture medium (MEM + 0% FBS 4h treatment; MEM + 10% FBS 20h treatment).
For the main experiments a stock solution of the test item in Aqua ad injectabilia were prepared (tenfold) and processed by sterile filtration. The dilution series was prepared in Aqua ad injectabilia. 10% of the dilution series and/or Aqua ad injectabilia were added to cell culture medium prior to treatment (resulting in the designated concentrations of the test item).
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without metabolic activation; 300 µg/mL
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 Aqua ad inj. / 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: ≥ 400 μg/mL; experiment I with S9: ≥ 50 μg/mL; Experiment II without S9: ≥ 500 μg/mL; Experiment II with S9:≥ 40 μg/mL
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
In the described in vitro cell gene mutagenicity test under the experimental conditions reported, the test item FAT 40277/D TE is considered to be non-mutagenic in the HPRT locus using V79 cells of the Chinese Hamster.
Executive summary:

In a mammalian cell gene mutation assay (HPRT locus),V79 cells culturedin vitro were exposed to FAT 40277/D TE at concentrations of

- 10, 25, 50, 100, 200, 400, 600, 800, 1000 and 1200 µg/mL (without metabolic activation, Experiment I)

- 1.0, 2.5, 5, 10, 25, 50, 100 and 125 µg/mL (with metabolic activation, Experiment I)

- 5, 10, 25, 50, 100, 250, 500, 1000 and 2000 µg/mL (without metabolic activation, Experiment II)

- 1.00, 3.16, 10, 20, 40, 60, 80, 100 and 120 µg/mL (with metabolic activation, Experiment II).

FAT 40277/D TE was tested up to cytotoxic concentrations.

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 14.0% for 1200 µg/mL evaluated. The highest biologically relevant concentration evaluated with metabolic activation was 125 µg/mL with a relative growth of 14.3%. In experiment II without metabolic activation the relative growth was 13.9% for the highest concentration (2000 µg/mL) evaluated. The highest concentration evaluated with metabolic activation was 120 µg/mL with a relative growth of 10.6%.

In experiment I without metabolic activation the highest mutation rate (compared to the solvent control values) of 2.13 was found at a concentration of 1200 µg/mL with a relative growth of 14.0%.

In experiment I with metabolic activation the highest mutation rate (compared to the solvent control values) of 3.07 was found at a concentration of 100 µg/mL with a relative growth of 20.7%.
In experiment II without metabolic activation the highest mutation rate (compared to the solvent control values) of 2.14 was found at a concentration of 10 µg/mL with a relative growth of 109.2%.
In experiment II with metabolic activation the highest mutation rate (compared to the solvent control values) of 1.74 was found at a concentration of 10 µg/mL with a relative growth of 79.1%.

The observed elevated number of mutant colonies and the mutation factor of 3.07 in experiment I (with metabolic activation) at a concentration of 100 µg/mL could not be reproduced in experiment II (with metabolic activation). At the same concentration (100 µg/mL) only 32.43 mutants per 106cells were observed, resulting in a mutation factor of 1.34. Due to this finding, the absence of a dose-response relationship as well as due to the overall integrity of the data the observed increased mutant values resulting in an elevated mutation factor in experiment I (with metabolic activation) is considered to be not biologically relevant.

The positive controls did induce the appropriate response. 

There was no evidence of a concentration related positive response of induced mutant colonies over background.

This study is classified as acceptable.  This study satisfies the requirement for Test Guideline OPPTS 870.5300, OECD 476 forin vitromutagenicity (mammalian forward gene mutation) data.

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

Genetic toxicity in vivo

Description of key information

FAT 40277 did not induce micronuclei in the micronucleus assay, hence considered to be not clastogenic.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1988
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Zentralinstitut für Versuchstierzucht D-3000 Hannover, F.R.G.
- Age at start of Acclimatization: minimum 10 weeks
- Weight at start of treatment: approximately 30 g
- Fasting period before study: approximately 18 hours
- Housing: Individually in Makrolon type-1 cages with wire mesh top (EBECO, D-4620 Castrop-Rauxel, F.R.G.) with granulated soft wood bedding (ALTROMIN, D-4937 Lage/Lippe, F.R.G.)
- Diet: pelleted standard diet (ALTROMIN, D-4937 Lage/Lippe, F.R.G.)
- Water: tap water, ad libitum (Südhessische Gas- und Wasser AG, D-6100 Darmstadt)
- Acclimation period: minimum 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 3
- Humidity (%): relative humidity not regulated
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: aqua dest
- Justification for choice of solvent/vehicle: The vehicle was chosen to its non-toxicity for the animals.
- Concentration of test material in vehicle: 3000 mg/kg bw
- Amount of vehicle: 20 mL/kg bw
Frequency of treatment:
Single treatment
Post exposure period:
24, 48, and 72 hours
Remarks:
Doses / Concentrations:
3000 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
6
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide;
- Route of administration: oral (gavage)
- Doses: 30 mg/kg bw
- Volume administrated: 10 mL/kg bw
Tissues and cell types examined:
Normochromatic and polychromatic erythrocytes
Details of tissue and slide preparation:
DETAILS OF SLIDE PREPARATION:
The animals were sacrificed by cervical dislocation. The femora were removed, the epiphyses were cut off and the marrow was flushed out with 2.0 mL fetal calf serum, using a 5 mL syringe, into 1 mL fetal calf serum. The cell suspension was centrifuged at 1,000 rpm for 5 minutes and the supernatant was discarded. A small drop of the resuspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Gruenwald/Giemsa. Cover slips were mounted with EUKITT (KINDLER, D-7800 Freiburg F.R.G.). At least one slide was made from each bone marrow sample.

METHOD OF ANALYSIS:
Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives. 1000 polychromatic erythrocytes (PCE) were analysed per animal for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined in same sample and expressed in normochromatic erythrocytes per 1000 PCEs. The analysis was performed with coded slides. Five animals per sex and group were evaluated as described. The remaining animal of each test group was evaluated in case an animal had died in its test group spontaneously or due to gavage error.
Evaluation criteria:
The frequencies of micronucleated PCEs in the groups treated with the test article are compared with their corresponding negative controls. Positive responses are those in which an increase of micronucleated PCEs can be confirmed statistically significant at the five per cent level (p < 0.05). However, both biological and statistical significance should be considered together in the evaluation.
Statistics:
Statistical significance at the five per cent level (p < 0.05) was evaluated by means of the non-parametric Mann-Whitney test.
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
in vivo
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- In a pre-experiment 2 animals (1 male, 1 female) received orally a single dose of 1000, 1500, 2000, or 3000 mg/kg bw. FAT 40'277/B dissolved in aqua dest. The volume administered was 20 mL/kg bw. Animals in the 1000 and 1500 mg/kg bw dose group did not express toxic reactions. The males in the 2000 mg/kg bw group expressed toxic reactions including reduction of spontaneous activity and apathy. The females however expressed no toxic reactions. Animals of the 3000 mg/kg bw dose group showed reduction of spontaneous activity, eyelid closure and apathy. The mean number of normochromatic erythrocytes was not increased after treatment with the test article as compared to the mean values of NCEs of the corresponding negative controls indicating that FAT 40'277/B had no cytotoxic properties.

RESULTS OF DEFINITIVE STUDY
- In comparison with the corresponding negative controls there was no substantial enhancement in the frequency of the detected micronuclei at any preparation interval after application of the test article. Biometric analysis proved no significant difference between control and test article groups.
- 30 mg/kg b.w. cyclophosphamide administered per os was used as positive control which showed a distinct increase of induced micronuleus frequency.
Conclusions:
The test substance did not induce micronuclei in bone marrow cells of the mouse.
Executive summary:

In a GLP-compliant erythrocyte micronucleus test, tested according to OECD guideline 474, 6 NMRI mice per sex were treated once by oral gavage with the test substance (3000 mg/kg bw) dissolved in water followed by a 24, 48 and 72 hours post exposure period. In pre-experiments 3000 mg/kg bw was estimated to be the maximum tolerated dose. Based on the finding that the mean number of normochromatic erythrocytes was not increased after treatment with the test article as compared to the negative controls, that the test substance had no cytotoxic properties. In comparison with the corresponding negative controls there was no enhancement in the frequency of the detected micronuclei at any preparation interval after application of the test article and therefore it could be concluded that the test substance did not induce micronuclei in this study.

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

Additional information

The genetic toxicity database of FAT 40000 consists of two bacterial reverse mutation assays. However, to complete data requirement at this tonnage level as well as genetic toxicity assessment, a study investigating mutagenic potential in mammalian cells and a study investigating clastogenic potential in mammalian cells is required. The genetic toxicity database of read across substance FAT 40277 consists of three bacterial reverse mutation assays, an in vitro mammalian cell gene mutation assay, an in vitro chromosomal aberration assay and a micronucleus assay. Hence the studies with read across substance were used to complete the genetic toxicity assessment of the target substance.

Bacterial reverse mutation assays with FAT 40000

FAT 40000 was evaluated for mutagenic potential in two Salmonella reverse mutation assays (1990 and 1978). The assays were performed with and without metabolic activation systems. No substantial increases in revertant colony numbers or tendency of higher mutation rates with increasing concentrations was observed. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies. Hence it was concluded, that in these assays the target substance did not induce mutations in the bacterial cells.

Bacterial reverse mutation assays with FAT 40277 (read across)

Three reverse mutation assays were performed to test the mutagenic potential of FAT 40277 (Battelle 1987, CCR 1987, RCC 2005). All three assays concluded that the test substance did not induce gene mutations in the genome of the strains used. Therefore, FAT 40277 is considered to be non-mutagenic in bacterial cells.

In vitro mammalian cell gene mutation assay with FAT 40277 (read across)

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: 10, 25, 50, 100, 200, 400, 600, 800, 1000 and 1200 µg/mL; Experiment I with metabolic activation: 1.0, 2.5, 5, 10, 25, 50, 100, and 125 µg/mL; Experiment II without metabolic activation: 5, 10, 25, 50, 100, 250, 500, 1000, and 2000 µg/mL; Experiment II with metabolic activation: 1.0, 3.16, 10, 20, 40, 60, 80, 100 and 120 µg/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 14.0% for the highest concentration (1200 µg/mL) evaluated. The highest biologically relevant concentration evaluated with metabolic activation was 125 µg/mL with a relative growth of 14.3%. In experiment II without metabolic activation the relative growth was 13.9% for the highest concentration (2000 µg/mL) evaluated. The highest concentration evaluated with metabolic activation was 120µg/mL with a relative growth of 10.6%. 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 Chines hamster.

In vitro chromosomal aberration assay with FAT 40277 (read across)

In a GLP-compliant chromosome aberration test, tested according to OECD Guideline 473, Chinese hamster lung fibroblast (V79) were exposed to the FAT 40277 with and without metabolic activation (CCR 1988). Different preparation intervals (up to 28 hours) and dosage (up to 400 µg/mL) were chosen in this study. The mitotic index was reduced after treatment with the highest concentrations in the presence and absence of S9 mix indicating that the test substance had cytotoxic properties. In the presence of S9 mix the test article did not increase the frequency of cells with aberrations at any fixation interval, but in the absence of S9 mix the test article increased the structural aberration rate after treatment with 400 µg/mL at fixation interval 28 hours. Based on the results the test substance is considered to be clastogenic in this chromosomal aberration test.

Micronucleus assay with FAT 40277 (read across)

In a GLP-compliant erythrocyte micronucleus test, tested according to OECD Guideline 474, 6 NMRI mice per sex were treated once by oral gavage with the test substance (3000 mg/kg bw) dissolved in water followed by a 24, 48 and 72 hours post exposure period (CCR 1988). In pre-experiments 3000 mg/kg bw was estimated to be the maximum tolerated dose. Based on the finding that the mean number of normochromatic erythrocytes was not increased after treatment with the test article as compared to the negative controls, that the test substance had no cytotoxic properties. In comparison with the corresponding negative controls there was no enhancement in the frequency of the detected micronuclei at any preparation interval after application of the test article and therefore it could be concluded that the test substance did not induce micronuclei in this study.

As discussed above, FAT 40000 was found to be not mutagenic in the Salmonella reverse mutation assays. The read across substance, FAT 40277, was not mutagenic in the bacterial reverse mutation assays and the mammalian cell gene mutation assay in vitro. FAT 40277 was found to be clastogenic in a chromosome aberration assay in vitro. However, it did not induce micronuclei in the micronucleus assay, hence considered to be not clastogenic. Therefore, based on the negative results in the bacterial reverse mutation assays with the target and source substance, in vitro mammalian cell gene mutation assay with the source substance as well as micronucleus assay with the source substance, both FAT 40000 and FAT 40277 are considered to be devoid of mutagenic as well as clastogenic potential, and hence to be not genotoxic.

Justification for classification or non-classification

Based on the above information, the substance does not warrant classification for genotoxicity as per the CLP (Regulation 1272/2008) criteria.