Reaction products of disodium 3,5-diamino-2-[(2-sulfonatophenyl)diazenyl]benzoate and  diazotised sodium 2-[(4-aminophenyl)sulfonyl]ethyl sulfate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test item was found to be "non-mutagenic".

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Study initiation date - 04 February 2003; Experiment start date - 05 February 2003; Experiment end date - 21 February 2003; Study completion date - 17 March 2003;

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

2000/32/EC, Annex 4D

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Identity: FAT 40810/A
Batch: WP 6/02
Purity: approx. 75 %
Appearance: Solid, dark brownish powder
Expiration date: 12 December 2010
Storage: At room temperature at about 20 °C

Target gene:

Histidine

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Details on mammalian cell type (if applicable):

The histidine-auxotrophic strains of Salmonella typhimurium TA 98 and TA 1535 were obtained from Prof. B. Ames, Berkeley, USA (1983). Strain TA 100 was obtained from Dr. M. Schupbach, Hoffmann-La Roche Limited, Basel, Switzerland (1986). Strain TA 1537 was obtained from Dr. S. Albertini, Hoffmann-La Roche Limited, Basel, Switzerland (1994). The tryptophan-auxotrophic strain of Escherichia coli (WP2 uvrA) was obtained from the National Collection of Industrial Bacteria, Aberdeen, Scotland (1977). The strain cultures were stored as stock cultures in ampoules with nutrient broth + DMSO (10 ml + 1ml) in a deep freezer at about -80 °C.

Metabolic activation:

with and without

Metabolic activation system:

S9 (Preparation in-house)
Rat-liver post mitochondrial supernatant (S9 fraction) was prepared in advance from male rats (HanBrl:WIST SPF), delivered by RCC Ltd, Animal Breeding and Biotechnology, Füllinsdorf, Switzerland. The animals were treated with Aroclor 1254 (Analabs Inc., delivered from Antechnika, Karslruhe, Germany), 500 mg/kg, i.p. 5 days prior to sacrifice. The livers were homogenized with 3 volumes of 150 mM KCI. The homogenate was centrifuged for 15 minutes at 9000x g and the resulting supernatant (S9 fraction) was stored at approximately -80 °C for no longer than one year. The protein content of the S9 fraction was 31.31 mg/ml.

S9 Mix
On day of experiment an appropriate quantity of S9 supernatant was thawed and mixed with S9 co-factor solution. The amount of S9 supernatant was 10 % v/v in the mixture. Cofactors were added to the S9 mix to reach the following concentrations in the S9 mix:
8 mM MgCl2
33 mM KCI
5 mM Glucose-6-phosphate
4 mM NADP
in 100 mM sodium phosphate-buffer, pH 7.4.
Before starting the experiment the S9 mix was sterile filtered and stored in a refrigerator. The S9 mix preparation was performed according to Ames et al.

Test concentrations with justification for top dose:

Concentration range in the main test (with and without metabolic activation): 312.5, 625, 1250, 2500 and 5000 µg/plate

Vehicle / solvent:

Solvent: dimethylsulfoxide (DMSO)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA100 and TA1535, without S9

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

TA98 without S9

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

TA1537 without S9

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

E. coli WP2 uvrA without S9

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

TA1535 with S9

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

TA98, TA100, TA1537 and E. coli WP2 uvrA with S9

Details on test system and experimental conditions:

Storage: The strain cultures were stored as stock cultures in ampoules with nutrient broth + DMSO (10 ml + 1ml) in a deep freezer at about -80 °C.
Precultures: 60 µl aliquots from a frozen stock were grown in 60 ml nutrient broth medium in a 300 ml Erlenmeyer flask (Nutrient broth No. 2, Oxoid Ltd., Basingstoke, U.K) for 8 hours by orbital shaking at 37 °C, 130 - 140 rounds per minute and then used for the experiment. The bacterial cultures were incubated in a temperature / time controlled incubator.
Selective Agar: The plates with the selective agar (minimal agar plates) were made in-house. Each Petri dish contained about 20.0 ml of minimal agar (1.5 % agar supplemented with 2 % salts of the Vogel-Bonner Medium E and 2 % glucose). The agar used was Select AGAR, GIBCO BRL, Switzerland. Glucose was delivered from Merck, Germany [D(+)glucose, anhydrous]. The Vogel-Bonner Medium E was prepared in-house.
Overlay Agar: The overlay agar contained per litre: 6.0 g GIBCO BRL Select Agar and 6.0 g NaCl (Merck, Germany). Sterilisation was performed at 121 °C in an autoclave, cooled down to 50 °C and dispensed into glass bottles. On day of test performance the agar was molten in a water bath and 10% aliquots (v/v) of 0.5 mM L-histidine / 0.5 mM d-biotin for Salmonella strains or 0.5 mM tryptophan, dissolved in bidistilled water, for E. coli strains were added sterile filtered.
Mammalian Microsomal Fraction S9 Mix: The bacteria used in this assay do not possess the enzyme systems which, in mammals, are known to convert promutagens into active DNA damaging metabolites. In order to overcome this major drawback an exogenous metabolic system is added in form of mammalian microsome enzyme activation mixture.
S9 (Preparation in-house): Rat-liver post mitochondrial supernatant (S9 fraction) was prepared in advance from male rats (HanBrl:WlST SPF), delivered by RCC Ltd, Animal Breeding and Biotechnology, Füllinsdorf, Switzerland. The animals were treated with Aroclor 1254 (Analabs lnc., delivered from Antechnika, Karslruhe, Germany), 500 mg/kg, i.p. 5 days prior to sacrifice. The livers were homogenized with 3 volumes of 150 mM KCI. The homogenate was centrifuged for 15 minutes at 9000x g and the resulting supernatant (S9 fraction) was stored at approximately -80 °C for no longer than one year. The protein content of the S9 fraction was 31.31 mg/ml.
S9 Mix: On day of experiment an appropriate quantity of S9 supernatant was thawed and mixed with S9 co-factor solution. The amount of S9 supernatant was 10 % v/v in the mixture. Cofactors were added to the S9 mix to reach the following concentrations in the S9 mix: 8 mM MgCI2 ; 33 mM KCI ; 5 mM Glucose-6-phosphate; 4 mM NADP in 100 mM sodium phosphate-buffer, pH 7.4. Before starting the experiment the S9 mix was sterile filtered and stored in a refrigerator. The S9 mix preparation was performed according to Ames et aI.


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

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium; in agar (plate incorporation) and preincubation

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period:
48 hours


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


METHODS FOR MEASUREMENTS OF GENOTOXICIY
: A biologically relevant increase in the number of revertants.

Evaluation criteria:

- A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA1535 and TA1537) the colony count of the corresponding solvent control is observed.
- A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
- An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
- A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

A statistical analysis was not required. At present the use of statistical methods concerning this particular test system is not generally recommended.

Species / strain:

other: Salmonella typhimurium strains TA 100, TA 1535, TA 98, TA 1537, and the Escherichia coli strain WP2 uvrA.

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

(< 5000 µg/plate)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Observations: A precipitation of the test item was not observed on the surface of any agar plate.

Conclusions:

FAT 40810/A did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Executive summary:

The test item FAT 40810/A was assessed for its potential to induce gene mutations according to the plate incorporation test (first experiment with and without metabolic activation, second experiment without activation) and the pre-incubation test (second experiment with metabolic activation) using Salmonella typhimurium strains TA 100, TA 1535, TA 98, TA 1537, and the Escherichia coli strain WP2 uvrA. This study was conducted in accordance with OECD test guideline 471 and EU method B.13/14. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, the negative (solvent control) and the positive controls were tested in triplicate. The test item was tested at the following concentrations: 312.5, 625, 1250, 2500 and 5000 µg/plate. In both mutagenicity tests normal background growth was observed with all strains at all concentrations tested. No toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation. No precipitation of the test item was observed on the surface of the agar plates. No substantial increase in revertant colony numbers of any of the five tester strains occurred following treatment with FAT 40’810/A at any concentration level, neither in the presence nor in the absence of an metabolic activation system. There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls. They showed a distinct increase of induced revertant colonies. In conclusion, it can be stated that during the described mutagenicity test and under the given experimental conditions reported, FAT 40810/A did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Study initiation date - 24 January 2003; Experiment start date - 05 February 2003; Experiment end date - 23 May 2003; Study completion date - 18 June 2003.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

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:

Identity: FAT 40810/A
Batch: WP 6/02
Purity: approx. 75 %
Appearance: Solid, dark brownish powder
Expiration date: 12 December 2010
Storage: At room temperature at about 20 °C

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Metabolic activation system:

S9 (Preparation by RCC Cytotest Cell Research)
Phenobarbital/ß-Naphthoflavone induced rat liver S9 was used as the metabolic activation system. The S9 was prepared from 8 - 12 weeks old male Wistar Hanlbm rats, weight approx 220 - 320 g (supplied from RCC Ltd; Biotechnology & Animal Breeding Division, CH-4414 Fulillinsdorf) induced by applications of 80 mg/kg b.w. Phenobarbital i.p. (Desitin; D-22335 Hamburg) and β-Naphthoflavone p.o. (Aldrich, D-89555 Steinheim) each on three consecutive days. The livers were prepared 24 hours after the last treatment. The S9 fractions were produced by dilution of the liver homogenate with a KCI solution (1:3 parts respectively) followed by centrifugation at 9000 g. Aliquots of the supernatant were frozen and stored in ampoules at -80 °C. Small numbers of the ampoules were kept at -20 °C for up to one week. The protein concentration was 34.6 mg/ml (Lot. no. 311002) in the pre-test and in experiment I and II.


S9 Mix
An appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution to result in a final protein concentration of 0.75 mg/ml in the cultures. Cofactors were added to the S9 mix to reach the following concentrations:
8 mM MgCl2
33 mM KCI
5 mM glucose-6-phosphate
4 mM 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. (1).

Test concentrations with justification for top dose:

Concentration range in the main test (with metabolic activation) Experiment I & II: 25, 50, 100, 150, 200, 300 µg/ml
Concentration range in the main test (without metabolic activation) Experiment I (4 hrs): 125, 250, 500, 750, 1000, 1500 µg/ml
Concentration range in the main test (without metabolic activation) Experiment II (18 hrs): 250, 500, 750, 1000, 1500, 2000 µg/ml
Concentration range in the main test (without metabolic activation) Experiment II (82 hrs): 250, 500, 750, 1000 µg/ml

Vehicle / solvent:

deionised water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation

Untreated negative controls:

yes

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:

Exposure period (with metabolic activation): 4 hours
Exposure period (without metabolic activation): 28 hours

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Single
- Number of independent experiments: Duplicate

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 1 - 6 x 10E4


FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
Evaluation of the cultures was performed (according to standard protocol of the "Arbeitsgruppe der lndustrie, Cytogenetik" [9]) using NIKON microscopes with 100x oil immersion objectives. Breaks, fragments, deletions, exchanges, and chromosome disintegrations were recorded as structural chromosome aberrations. Gaps were recorded as well but not included in the calculation of the aberration rates. 100 well spread metaphase plates per culture were scored for cytogenetic damage on coded slides. Only metaphases with characteristic chromosome numbers of 22 ± 1 were included in the analysis. To describe a cytotoxic effect the mitotic index (% cells in mitosis) was determined. In addition, the number of polyploid cells in 500 metaphase cells per culture was determined (% polyploid metaphases; in the case of this aneuploid cell line polyploid means a near tetraploid karyotype).


METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: relative increase in cell count (RICC)

METHODS FOR MEASUREMENTS OF GENOTOXICIY
- number of cells with structural chromosome aberrations.

Evaluation criteria:

A test item is classified as non-clastogenic if:
- the number of induced structural chromosome aberrations in all evaluated dose groups is in the range of our historical control data (0.0 - 4.0 % aberrant cells, exclusive gaps). and/or
— no significant increase of the number of structural chromosome aberrations is observed.
A test item is classified as clastogenic if:
— the number of induced structural chromosome aberrations is not in the range of our historical control data (0.0 - 4.0 % aberrant cells, exclusive gaps) and
— either a concentration-related or a significant increase of the number of structural chromosome aberrations is observed.
Statistical significance was confirmed by means of the Fisher's exact test (10) (p <0.05). However, both biological and statistical significance should be considered together. If the criteria mentioned above for the test item are not clearly met, the classification with regard to the historical data and the biological relevance is discussed and/or a confirmatory experiment is performed. Although the inclusion of the structural chromosome aberrations is the purpose of this study, it is important to include the polyploids and endoreduplications. The following criteria is valid:
A test item can be classified as aneugenic if:
— the number of induced numerical aberrations is not in the range of our historical control data (0.0 - 8.5 % polyploid cells).

Statistics:

Statistical significance was confirmed by means of the Fisher's exact test (10) (p <0.05).

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

200 µg/ml

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

200 µg/ml

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Observations:
Precipitation was observed at concentrations of ≥1000
µg/ml (without S9-mix) and ≥200 µg/ml (with S9-mix).

Conclusions:

FAT 40810/A is considered to be clastogenic in this chromosome aberration test in the absence and the presence of S9 mix

Executive summary:

FAT 40810/A was evaluated for its potential to induce chromosome aberration according to OECD test guideline 473 and EU method B.10 in a GLP certified laboratory. The test item, dissolved in deionised water, was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro in the absence and presence of metabolic activation by S9 mix. Two independent experiments were performed. In experiment I, the exposure period was 4 hrs with and without metabolic activation. In experiment II the exposure period was 4 hrs with S9 mix and 18 hrs and 28 hrs without S9 mix. The chromosomes were prepared 18 hrs (exp. I and II) and 28 hrs (exp. II) after start of treatment with the test item. In each experimental group two parallel cultures were set up. Per culture 100 metaphase plates were scored for structural chromosome aberrations. In a range finding pre-test on toxicity cell numbers 24 hrs after start of treatment were scored as an indicator for cytotoxicity. Concentrations between 39.1 and 5000 µg/mL were applied. Clear toxic effects were observed after 4 and 24 hrs treatment with 1250 µg/mL and above in the absence of S9 mix. In addition, 4 hrs treatment with 312.5 µg/mL and above in the presence of S9 mix induced strong toxic effects. In the pre-experiment, precipitation of the test item in culture medium was observed after treatment with 312.5 µg/mL and above in the absence and the presence of S9 mix. In experiment I, precipitation of the test item in culture medium was observed after 4 hrs treatment with 1000 µg/mL and above in the absence of S9 mix and with 200 µg/mL and above in the presence of S9 mix. In experiment II, in the absence of S9 mix precipitation occurred at preparation interval 18 hrs with 1000 µg/mL and above and at preparation interval 28 hrs with 750 µg/mL and above. In the cytogenetic experiments, toxic effects indicated by reduced cell numbers of about and below 50 % of control were observed in experiment I in the absence of S9 mix after 4 hrs treatment with 1500 µg/mL (51 % of control) and in experiment ll in the presence of S9 mix after 4 hrs treatment with 200 µg/mL (40 % of control) at the 28 hrs preparation interval. In contrary, no cytotoxicity indicated by clearly reduced mitotic indices were observed at the test groups evaluated. However, concentrations showing clear toxic effects were not evaluable for cytogenetic damage. In experiment I, in the absence and presence of S9 mix, no statistically significant and biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed. The aberration rates of the cells after treatment with the test item (0.0 - 3.0 % aberrant cells, exclusive gaps) were near the range of the solvent control values (1.0 - 3.5 % aberrant cells, exclusive gaps) and within the range of our historical control data: 0.0 - 4.0 % aberrant cells, exclusive gaps. In experiment ll, after 28 hrs continuous treatment in the absence of S9 mix, no statistically significant and biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed. The aberration rate of the cells after treatment with the test item (1.5 % aberrant cells, exclusive gaps) was identical to the respective solvent control value (1.5 % aberrant cells, exclusive gaps) and within the range of our historical control data: 0.0 - 4.0 % aberrant cells, exclusive gaps. In experiment II, after 18 hrs continuous treatment in the absence of S9 mix the aberration rates were dose-related increased (0.5, 2.5, and 7.0 %) at the concentration range evaluated (250 - 750 µg/mL). The value (7.0 %) after treatment with 750 µg/mL clearly exceeded our historical control data range (0.0 - 4.0 %) and it was statistically significant as compared to the corresponding solvent control (1.0 %). Therefore, these observations have to be regarded as biologically relevant. Additionally, in experiment ll at 28 hrs preparation interval in the presence of S9 mix, a single statistically significant and biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed after treatment with 200 µg/mL (12.5 %) clearly exceeding our historical control data range. The cells carrying exchanges observed (6.0 %) give additional evidence for a clastogenic potential of the test item. In addition, at pre-evaluation of the slides distinct increased numbers of micronucleated cells and of cells containing fragmented nuclei were observed. Thus, these findings have to be regarded as biologically relevant. In both experiments, no biologically relevant increase in the rate of polyploid metaphases was found after treatment with the test item (0.9 - 4.2 %) as compared to the rates of the solvent controls (0.8 - 3.2 %). In both experiments, EMS (200 µg/mL) and CPA (0.7 and 1.0 µg/mL, respectively) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations. In conclusion, it can be stated that under the experimental conditions reported, the test item FAT 40810/A induced structural chromosome aberrations in V79 cells (Chinese hamster cell line) in the absence and the presence of metabolic activation.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experiment start date - 30 April 2013; Experiment completion date - 07 October 2013; Study completion date - 04 November 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)

Type of assay:

mammalian cell gene mutation assay

Specific details on test material used for the study:

Identity: FAT 40810/A
Batch: WP 6/02
Purity: approx. 75 %
Appearance: Solid, dark brownish powder
Expiration date: 12 December 2010
Storage: At room temperature at about 20 °C

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:
The S9 liver microsomal fraction was prepared at BSL BIOSERVICE GmbH. Male Wistar rats were induced with Phenobarbital (80 mg/kg bw) and Beta-Napthoflavone (100 mg/kg bw) for three consecutive days by oral route.
The following quality control determinations were performed;
a) Biological activity in
- the Salmonella typhimurium assay using 2-aminoanthracene and benzo(aplha)pyrene.
- the mouse lymphoma assay using benzo(aplha)pyrene.
- the chromosome aberration assay using cyclophosphamide.
b) Sterility test
A stock of supernant containing the microsomes were frozen in ampoules of 2 and 4 mL and stored at ≤-75 °C. The protein concentration in the S9 preparation was 33 mg/mL. The S9 mix preparation was performed according to Ames et al.

S9 mix:
An appropriate quantity of the S9 supernant was thawed and mixed with S9 cofactor solution to result in a final protein concentration of 0.75 mg/ml in the cultures. Cofactors were added to the S9 mix to reach the concentration below;
8 mM MgCl2
33 mM KCl
5 mM Glucose-6-phosphate
5 mM NADP
in 100 mM sodium-phosphate-buffer pH 7.4. During the experiment the S9 mix was stored on ice.

Test concentrations with justification for top dose:

Pre-experiment for experiment I (with and without metabolic activation): 50, 100, 250, 500, 1000, 1750, 2500, 3750, 5000 µg/mL
Pre-experiment for experiment II (only without metabolic activation, 20 h long-term exposure assay): 5, 10, 25, 50, 100, 250, 500, 1000, 1500, 2000 µg/mL

Experiment I
without metabolic activation: 5, 10, 25, 50, 100, 200, 300, 400 and 500 µg/mL
and with metabolic activation: 50, 100, 200, 400, 600, 700, 800, 1000, 1100 and 1200 µg/mL

Experiment II
without metabolic activation: 50, 100, 200, 300, 400, 500, 600, 700, 725 and 750 µg/mL
and with metabolic activation: 60, 200, 480, 760, 900, 1040, 1180, 1320, 1460 and 1600 µg/mL

Experiment III
with metabolic activation: 1100, 1200, 1460, 1600 and 1670 µg/mL

Vehicle / solvent:

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

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; 0.8 and 1.0 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: three separate experiments (I, II and III) 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: ≥ 50 μg/mL; experiment I with S9: 200 and ≥ 600 μg/mL; Experiment II without S9: ≥ 400 μg/mL; Experiment II with S9:≥ 1040 μg/mL; Experiment III with S9:≥ 1100 μg/mL

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

FAT 40810/B 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) conducted according to OECD test guideline 476 and EC B.17, V79 cells cultured in-vitro were exposed to FAT 40810/B TE dissolved in cell culture medium at concentrations of

- 5, 10, 25, 50, 100, 200, 300, 400 and 500 µg/mL (without metabolic activation, Experiment I)

- 50, 100, 200, 400, 600, 700, 800, 1000, 1100 and 1200 µg/mL (with metabolic activation, Experiment I)

- 50, 100, 200, 300, 400, 500, 600, 700, 725 and 750 µg/mL (without metabolic activation, Experiment II)

- 60, 200, 480, 760, 900, 1040, 1180, 1320, 1460 and 1600 µg/mL (with metabolic activation, Experiment II)

- 1100, 1200, 1460, 1600 and 1670 µg/mL (with metabolic activation, Experiment III).

 

FAT 40810/B 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 14.9 % for the highest concentration (500 µg/mL) evaluated. The highest biologically relevant concentration evaluated with metabolic activation was 1200 µg/mL with a relative growth of 14.6 %. In experiment II without metabolic activation the relative growth was 13.5% for the highest concentration (750 µg/mL) evaluated. The highest concentration evaluated with metabolic activation was 1600 µg/mL with a relative growth of 13.9 %. In experiment III with metabolic activation the relative growth was 12.3 % for the highest concentration (1670 µg/mL) evaluated. In experiment I without metabolic activation the highest mutation rate (compared to the negative control values) of 2.29 was found at a concentration of 50 µg/mL with a relative growth of 64.8 %. In experiment I with metabolic activation the highest mutation rate (compared to the negative control values) of 2.05 was found at a concentration of 800 µg/mL with a relative growth of 43.7 %. In experiment II without metabolic activation the highest mutation rate (compared to the negative control values) of 2.73 was found at a concentration of 600 µg/mL with a relative growth of 35.4 %. In experiment II with metabolic activation the highest mutation rate (compared to the negative control values) of 5.79 was found at a concentration of 1600 µg/mL with a relative growth of 13.9 %.  In experiment III with metabolic activation the highest mutation rate (compared to the negative control values) of 1.88 was found at concentrations of 1670 µg/mL with a relative growth of 12.3 %. The observed elevated number of mutant colonies and the elevated mutation factors in experiment II (with metabolic activation) could not be reproduced in experiment III (with metabolic activation). At the same concentration ranges neither elevated numbers of mutant colonies nor mutation factors above the critical threshold value were observed. Due to these findings, the absence of a dose-response relationship as well as due to the overall integrity of the data the observed increased mutant value as well as the elevated mutation factor in experiment II (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 for in vitro mutagenicity (mammalian forward gene mutation) data.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

FAT40810/A was - negative in Ames test (with / without S9) - negative in in vitro gene mutation assay positive in in vitro micronucleus test, but negative in vivo.

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:

Experiment start date - 02 July 2003; Experiment end date - 23 July 2003; Study completion date - 10 September 2003.

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:

yes

Remarks:

Ten animals (5 males, 5 females) per test group were evaluated In the present study the relative humidity under which the experiment was conducted ranged between 30 - 85 %

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

Ten animals (5 males, 5 females) per test group were evaluated In the present study the relative humidity under which the experiment was conducted ranged between 30 - 85 %

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Specific details on test material used for the study:

Identity: FAT 40810/A
Batch: WP 6/02
Purity: approx. 75 %
Appearance: Solid, dark brownish powder
Expiration date: 12 December 2010
Storage: At room temperature at about 20 °C

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

Strain: NMRI
Source: RCC Ltd., CH-4414 Füllinsdorf
Number of animals: 72 (36 males/36 females)
Initial age at start of acclimatization: Males: 5 - 7 weeks, Females: 7 - 9 weeks
Acclimatization: minimum 5 days
Initial body weight at Start of Treatment: males mean value 33.2 g (SD ±1.7 g), females mean value 29.5 g (SD ±3.0 g)
According to the suppliers assurance the animals were in healthy condition. The animals were under quarantine in the animal house of RCC - CCR for a minimum of five days after their arrival. During this period the animals did not show any signs of illness or altered behaviour. The animals were distributed into the test groups at random and identified by cage number.
Husbandry: The animals were kept conventionally. The experiment was conducted under standard laboratory conditions.
Housing: Single
Cage Type: Makrolon Type l, 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 Rolldorf)
Environment: temperature 22 ± 3 °C
relative humidity: 30 - 70 %
artificial light: 6.00 a.m. - 6.00 p.m.

Route of administration:

oral: unspecified

Vehicle:

Deionised water

Details on exposure:

single oral exposure of 10 ml/kg bw equivalent to 2000 mg test substance/kg bw

Duration of treatment / exposure:

single exposure

Frequency of treatment:

single application

Post exposure period:

48 hours in pre-experiment for assessment of toxicity, 24 hours in main experiment .

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

Negative control group

Dose / conc.:

500 mg/kg bw/day (actual dose received)

Remarks:

Low dose group

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

Medium dose group

Dose / conc.:

2 000 mg/kg bw/day (actual dose received)

Remarks:

High dose group

No. of animals per sex per dose:

Male: 500 mg/kg; No. of animals: 5; Sacrifice time: 24 hours
Male: 1000 mg/kg; No. of animals: 5; Sacrifice time: 24 hours
Male: 2000 mg/kg; No. of animals: 5; Sacrifice time: 24 hours
Male: 2000 mg/kg; No. of animals: 5; Sacrifice time: 48 hours
Female: 500 mg/kg; No. of animals: 5; Sacrifice times: 24 hours
Female: 1000 mg/kg; No. of animals: 5; Sacrifice times: 24 hours
Female: 2000 mg/kg; No. of animals: 5; Sacrifice times: 24 hours
Female: 2000 mg/kg; No. of animals: 5; Sacrifice times: 48 hours

Control animals:

yes, concurrent vehicle

Positive control(s):

40 mg/kg cyclophosphamide application orally to 5 males and 5 females

Tissues and cell types examined:

bone marrow samples

Details of tissue and slide preparation:

The animals will be sacrificed using CO2. The femora were removed, the epiphyses were cut off and the marrow was ushed 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-Grunwald (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.

Evaluation criteria:

A test item 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. Statistical methods (nonparametric Mann-Whitney test will be used as an aid in evaluating the results. However, the primary point of consideration is the biological relevance of the results. 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.

Statistics:

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

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

Doses producing toxicity (reduced spontaneous activity: pre-test = 2000 mg/kg main test = 2000 mg/kg and 1000 mg/kg

Vehicle controls validity:

valid

Negative controls validity:

not valid

Positive controls validity:

valid

Additional information on results:

See "Any other information on results incl. tables"

Pre-Experiment for Toxicity

In a pre-experiment 4 animals (2 males, 2 females) received orally a single dose of 2000 mg/kg b.w. FAT 40810/A formulated in deionised water. The volume administered was 10 ml/kg b.w.

The animals treated with 2000 mg/kg b.w. expressed toxic reactions as shown in the table:

 

Toxic reactions	Hours post-treatment male/female
	1 h	2-4 h	6h	24 h	30 h	48 h
reduction of spontaneous activity	0/2	2/2	2/2	2/2	2/2	1/1
eyelid closure	0/0	2/2	2/2	2/2	1/1	1/1
urine colour	-	orange	orange	orange	orange	-

On the basis of these data 2000 mg/kg b.w. were estimated to be suitable.

Toxic symptoms in the Main Experiment

In the main experiment for the highest dose group 24 animals (12 males, 12 females) received orally a single dose of 2000 mg /kg b.w. FAT 40810/A formulated in deionised water. The volume administered was 10 ml/kg b.w. The animals treated with 2000 mg /kg b.w. expressed toxic reactions as shown in the table

Toxic reactions	Hours post-treatment male/female
	1 h	2-4 h	6h	24 h
reduction of spontaneous activity	5/8	an	12/12	5/6
ruffled fur	0/4	8/6	9/11	4/4
urine colour	-	orange	orange	orange/ -

 

For the mid dose group 12 animals (6 males, 6 females) received orally a single dose of 1000 mg /kg b.w. FAT 40810/A formulated in deionised water. The volume administered was 10 ml/kg b.w.

The animals treated with 1000 mg /kg b.w. expressed toxic reactions as shown in the table:

Toxic reactions	Hours post-treatment male/female
	1 h	2-4 h	6h	24 h
reduction of spontaneous activity	4/0	4/4	6/6	2/1
ruffled fur	0/0	010	1/0	010

 

For the low dose group 12 animals (6 males, 6 females) received orally a single dose of 500 mg /kg b.w. FAT 40810/A formulated in deionised water. The volume administered was 10 ml/kg b.w.

The animals treated with 500 mg /kg b.w. expressed toxic reactions as shown in the table:

Toxic reactions	Hours post-treatment male/female
	1 h	2-4 h	6h	24 h
reduction of spontaneous activity	010	1/0	010	010

 

 

Summary of Micronucleus Test Results

Test group	Dose mg/kg b.w.	Sampling time (h)	PCEs with micronuclei (%)	Range	PCE per 2000 erythrocytes
vehicle	0	24	0.055	0-2	1157
test item	500	24	0.070	0-5	1091
test item	1000	24	0.060	0 -3	1129
test item	2000	24	0.050	0-3	1080
positive control	40	24	1.320	7-40	1079
test item	2000	48	0.060	0-3	1089

Conclusions:

FAT 40810/A is considered to be non-mutagenic in this in vivo micronucleus assay.

Executive summary:

The test item FAT 40810lA was assessed in the micronucleus assay for its potential to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse. This study is conducted in accordance with OECD test guideline 474 and EU method B.12 in a GLP-certified laboratory. The test item was formulated in deionised water, which was also used as vehicle control. The volume administered orally was 10 ml/kg bw. 24 h and 48 h after a single administration of the test item 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 (PCEs) per animal were scored for micronuclei. To describe a cytotoxic effect due to the treatment with the test item the ratio between polychromatic and total erythrocytes was determined in the same sample and reported as the number of PCEs per 2000 erythrocytes. The following dose levels of the test item were investigated:

24 h preparation interval: 500, 1000, and 2000 mg/kg bw.

48 h preparation interval: 2000 mg/kg bw.

 

As estimated by a pre-experiment 2000 mg/kg bw (the maximum guideline-recommended dose) was suitable. The mean number of polychromatic erythrocytes was not increased after treatment with the test item as compared to the mean value of PCEs of the vehicle control indicating that FAT 40810/A had no cytotoxic properties in the bone marrow. However, the urine of the treated animals turned orange indicating the systemic distribution of the test item and thus, its bioavailability. 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 40810/A were below or near to the value of the vehicle control group. 40 mg/kg bw cyclophosphamide administered orally was used as positive control which showed a statistically significant increase of induced micronucleus frequency. In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test item did not induce micronuclei as determined by the micronucleus test in the bone marrow cells of the mouse.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vivo:

Three in vitro tests and one in vivo study were performed. The results were as follows:

Ames test:

The test item FAT 40810/A was assessed for its potential to induce gene mutations according to the plate incorporation test (first experiment with and without metabolic activation, second experiment without activation) and the pre-incubation test (second experiment with metabolic activation) using Salmonella typhimurium strains TA 100, TA 1535, TA 98, TA 1537, and the Escherichia coli strain WP2 uvrA. This study was conducted in accordance with OECD test guideline 471 and EU method B.13/14. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, the negative (solvent control) and the positive controls were tested in triplicate. The test item was tested at the following concentrations: 312.5, 625, 1250, 2500 and 5000 µg/plate. In both mutagenicity tests normal background growth was observed with all strains at all concentrations tested. No toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation. No precipitation of the test item was observed on the surface of the agar plates. No substantial increase in revertant colony numbers of any of the five tester strains occurred following treatment with FAT 40810/A at any concentration level, neither in the presence nor in the absence of an metabolic activation system. There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls. They showed a distinct increase of induced revertant colonies.

In conclusion, it can be stated that during the described mutagenicity test and under the given experimental conditions reported, FAT 40810/A did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

 

Chromosome aberration test in vitro:

FAT 40810/A was evaluated for its potential to induce chromosome aberration according to OECD test guideline 473 and EU method B.10 in a GLP certified laboratory. The test item, dissolved in deionised water, was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro in the absence and presence of metabolic activation by S9 mix. Two independent experiments were performed. In experiment I, the exposure period was 4 hrs with and without metabolic activation. In experiment II the exposure period was 4 hrs with S9 mix and 18 hrs and 28 hrs without S9 mix. The chromosomes were prepared 18 hrs (exp. I and II) and 28 hrs (exp. II) after start of treatment with the test item. In each experimental group two parallel cultures were set up. Per culture 100 metaphase plates were scored for structural chromosome aberrations. In a range finding pre-test on toxicity cell numbers 24 hrs after start of treatment were scored as an indicator for cytotoxicity. Concentrations between 39.1 and 5000 µg/mL were applied. Clear toxic effects were observed after 4 and 24 hrs treatment with 1250 µg/mL and above in the absence of S9 mix. In addition, 4 hrs treatment with 312.5 µg/mL and above in the presence of S9 mix induced strong toxic effects. In the pre-experiment, precipitation of the test item in culture medium was observed after treatment with 312.5 µg/mL and above in the absence and the presence of S9 mix. In experiment I, precipitation of the test item in culture medium was observed after 4 hrs treatment with 1000 µg/mL and above in the absence of S9 mix and with 200 µg/mL and above in the presence of S9 mix. In experiment II, in the absence of S9 mix precipitation occurred at preparation interval 18 hrs with 1000 µg/mL and above and at preparation interval 28 hrs with 750 µg/mL and above. In the cytogenetic experiments, toxic effects indicated by reduced cell numbers of about and below 50 % of control were observed in experiment I in the absence of S9 mix after 4 hrs treatment with 1500 µg/mL (51 % of control) and in experiment ll in the presence of S9 mix after 4 hrs treatment with 200 µg/mL (40 % of control) at the 28 hrs preparation interval. In contrary, no cytotoxicity indicated by clearly reduced mitotic indices were observed at the test groups evaluated. However, concentrations showing clear toxic effects were not evaluable for cytogenetic damage. In experiment I, in the absence and presence of S9 mix, no statistically signicant and biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed. The aberration rates of the cells after treatment with the test item (0.0 — 3.0 % aberrant cells, exclusive gaps) were near the range of the solvent control values (1.0 - 3.5 % aberrant cells, exclusive gaps) and within the range of our historical control data: 0.0 - 4.0 % aberrant cells, exclusive gaps. In experiment ll, after 28 hrs continuous treatment in the absence of S9 mix, no statistically significant and biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed. The aberration rate of the cells after treatment with the test item (1.5 % aberrant cells, exclusive gaps) was identical to the respective solvent control value (1.5 % aberrant cells, exclusive gaps) and within the range of our historical control data: 0.0 - 4.0 % aberrant cells, exclusive gaps. In experiment II, after 18 hrs continuous treatment in the absence of S9 mix the aberration rates were dose-related increased (0.5, 2.5, and 7.0 %) at the concentration range evaluated (250 - 750 µg/mL). The value (7.0 %) after treatment with 750 µg/mL clearly exceeded our historical control data range (0.0 - 4.0 %) and it was statistically signicant as compared to the corresponding solvent control (1.0 %). Therefore, these observations have to be regarded as biologically relevant. Additionally, in experiment ll at 28 hrs preparation interval in the presence of S9 mix, a single statistically significant and biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed after treatment with 200 µg/mL (12.5 %) clearly exceeding our historical control data range. The cells carrying exchanges observed (6.0 %) give additional evidence for a clastogenic potential of the test item. In addition, at pre-evaluation of the slides distinct increased numbers of micronucleated cells and of cells containing fragmented nuclei were observed. Thus, these findings have to be regarded as biologically relevant.

In both experiments, no biologically relevant increase in the rate of polyploid metaphases was found after treatment with the test item (0.9 - 4.2 %) as compared to the rates of the solvent controls (0.8 - 3.2 %). In both experiments, EMS (200 µg/mL) and CPA (0.7 and 1.0 µg/mL, respectively) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations.

In conclusion, it can be stated that under the experimental conditions reported, the test item FAT 40'810/A induced structural chromosome aberrations in V79 cells (Chinese hamster cell line) in the absence and the presence of metabolic activation.

 

Mammalian gene mutation study in vitro:

In a mammalian cell gene mutation assay (HPRT locus) conducted according to OECD test guideline 476 and EC B.17, V79 cells cultured in-vitro were exposed to FAT 40810/B TE dissolved in cell culture medium at concentrations of 

- 5, 10, 25, 50, 100, 200, 300, 400 and 500 µg/mL (without metabolic activation, Experiment I)

- 50, 100, 200, 400, 600, 700, 800, 1000, 1100 and 1200 µg/mL (with metabolic activation, Experiment I)

- 50, 100, 200, 300, 400, 500, 600, 700, 725 and 750 µg/mL (without metabolic activation, Experiment II)

- 60, 200, 480, 760, 900, 1040, 1180, 1320, 1460 and 1600 µg/mL (with metabolic activation, Experiment II)

- 1100, 1200, 1460, 1600 and 1670 µg/mL (with metabolic activation, Experiment III).

FAT 40810/B 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 14.9 % for the highest concentration (500 µg/mL) evaluated. The highest biologically relevant concentration evaluated with metabolic activation was 1200 µg/mL with a relative growth of 14.6 %.

In experiment II without metabolic activation the relative growth was 13.5 % for the highest concentration (750 µg/mL) evaluated. The highest concentration evaluated with metabolic activation was 1600 µg/mL with a relative growth of 13.9 %.

In experiment III with metabolic activation the relative growth was 12.3 % for the highest concentration (1670 µg/mL) evaluated.

In experiment I without metabolic activation the highest mutation rate (compared to the negative control values) of 2.29 was found at a concentration of 50 µg/mL with a relative growth of 64.8 %.

In experiment I with metabolic activation the highest mutation rate (compared to the negative control values) of 2.05 was found at a concentration of 800 µg/mL with a relative growth of 43.7 %.

In experiment II without metabolic activation the highest mutation rate (compared to the negative control values) of 2.73 was found at a concentration of 600 µg/mL with a relative growth of 35.4 %.

In experiment II with metabolic activation the highest mutation rate (compared to the negative control values) of 5.79 was found at a concentration of 1600 µg/mL with a relative growth of 13.9%  In experiment III with metabolic activation the highest mutation rate (compared to the negative control values) of 1.88 was found at concentrations of 1670 µg/mL with a relative growth of 12.3 %.

The observed elevated number of mutant colonies and the elevated mutation factors in experiment II (with metabolic activation) could not be reproduced in experiment III (with metabolic activation). At the same concentration ranges neither elevated numbers of mutant colonies nor mutation factors above the critical threshold value were observed. Due to these findings, the absence of a dose-response relationship as well as due to the overall integrity of the data the observed increased mutant value as well as the elevated mutation factor in experiment II (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 for in vitro mutagenicity (mammalian forward gene mutation) data.

 

Micronucleus assay in vivo:

The test item FAT 40810/A was assessed in the micronucleus assay for its potential to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse. The test item was formulated in deionised water, which was also used as vehicle control. The volume administered orally was 10 ml/kg bw. 24 h and 48 h after a single administration of the test item 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 (PCEs) per animal were scored for micronuclei. To describe a cytotoxic effect due to the treatment with the test item the ratio between polychromatic and total erythrocytes was determined in the same sample and reported as the number of PCEs per 2000 erythrocytes. The following dose levels of the test item were investigated: 24 h preparation interval: 500, 1000, and 2000 mg/kg bw. 48 h preparation interval: 2000 mg/kg bw. As estimated by a pre-experiment 2000 mg FAT 40810/A per kg bw (the maximum guideline-recommended dose) was suitable. The mean number of polychromatic erythrocytes was not increased after treatment with the test item as compared to the mean value of PCEs of the vehicle control indicating that FAT 40810/A had no cytotoxic properties in the bone marrow. However, the urine of the treated animals turned orange indicating the systemic distribution of the test item and thus, its bioavailability. 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 40810/A were below or near to the value of the vehicle control group. 40 mg/kg bw cyclophosphamide administered orally was used as positive control which showed a statistically significant increase of induced micronucleus frequency. In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test item did not induce micronuclei as determined by the micronucleus test in the bone marrow cells of the mouse. 

In the overall conclusion, FAT40810 is considered to be non-mutagenic, as the substance was found negative in an Ames test and an in vitro gene mutation study, whereas the positive findings in the in vitro micronucleus test could not be verified in an in vivo test which also turned out negative..

Justification for selection of genetic toxicity endpoint
in vivo study considered more relevant than available in vitro studies

Justification for classification or non-classification

Based on the assessment of three in vitro and one in vivo mutagenicity studies, the substance FAT 40810 is not considered mutagenic and thus not classified for mutagenicity according to CLP (Regulation EC No 1272/2008) or DSD (Directive 67/548/EEC).