Tetra(sodium/potassium)-7-[(E)-{2-acetamido-4-[(E)-(4-{[4-chlor-6-({2-[(4-fluor-6-{[4-(vinylsulfonyl)phenyl]amino}-1,3,5-triazin-2-yl)amino]propyl}amino)-1,3,5-triazin-2-yl]amino}-5-sulfonato-1-naphthyl)diazenyl]-5-methoxyphenyl}diazenyl]-1,3,6-naphthalintrisulfonate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test item was found positive in Chromosome aberration test, but was found negative in Ames and HPRT In vitro study.

Link to relevant study records

Reference

Reference 1

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 - 11 May 2006; Experiment start date - 08 June 2006; Experiment completion date - 24 July 2006; Study completion date - 19 September 2006.

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:

other: Commission Directive 2000/32/EC, L1362000, Annex 4A: "Mutagenicity - In vitro Mammalian Chromosome Aberration Test", dated May 19, 2000

Deviations:

no

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

"Kanpoan No. 287 - Environmental Agency" "Eisei No. 127 - Ministry of Health & Welfare" "Heisei 09/10/31 Kikyoku No. 2 - Ministry of International Trade & Industry".

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 40827/A
Batch: T2 5572 BOP 01/06
Purity: determined in this study
Appearance: black sticky powder
Expiration date: 28.02.2011
Storage: at room temperature

Target gene:

Structural chromosome aberrations

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 - 1 2 weeks old male Wistar Hanlbm rats, weight approx. 220 - 320 g induced by applications of 80 mg/kg b.w. Phenobarbital i.p. (Desitin; 22335 Hamburg, Germany) and ß-Naphthoflavone p.o. (Aldrich, 89555 Steinheim, Germany), 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 KCl solution (1:3 parts) 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 30.6 mg/mL (Lot no. 091205) in the pre-experiment and 29.3 mg/mL (Lot no. 190506) in the main experiment.

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:
8mM MgCI2
33 mM KCl
5 mM glucose-6-phosphate
4mM NADP
in 100 mM sodium-ortho-phosphate-buffer, pH 7.4.
During the experiment the S9 mix was stored in an ice bath. The S9 mix preparation was performed according to Ames et al.

Test concentrations with justification for top dose:

The applied concentrations in the pre-test on toxicity were 39.1, 78.1, 156.3, 312.5, 625.0, 1250.0, 2500.0 and 5000 µg/mL.
The applied concentrations in the cytogenetic experiments were 19.5, 39.1, 78.1, 156.3, 312.5 and 625.0 µg/mL.

Vehicle / solvent:

deionised water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

deionised water

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Without metabolic activation - 400 µg/mL (3.2 mM)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

deionised water

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

With metabolic activation - 1.4 µg/mL(5.0 µM)

Details on test system and experimental conditions:

Large stocks of the V79 cell line were stored in liquid nitrogen in the cell bank of RCC. Before freezing each batch was screened for mycoplasm contamination and checked for karyotype stability. Consequently, the parameters of the experiments remain similar because of standardized characteristics of the cells. Thawed stock cultures were propagated at 37 °C in 80 cm² plastic flasks. About 5x 105 cells per flask were seeded into 15 mL of MEM supplemented with 10 % fetal calf serum. The cells were sub-cultured twice weekly. The cell cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % carbon dioxide.

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

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 41530 cells/slide
- Test substance added in medium: preincubation.

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 18h
- Exposure duration/duration of treatment: 4h


FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor: Colchicine (0.2 µg/mL) - 2.5 hrs.
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays):
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored):
- Criteria for scoring micronucleated cells (selection of analysable cells and micronucleus identification):
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable):
- Criteria for scoring chromosome aberrations (selection of analysable cells and aberration identification):
- Determination of polyploidy:
- Determination of endoreplication:

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection):
- Selection time (if incubation with a selective agent):
- Fixation time (start of exposure up to fixation or harvest of cells):
- Method used: agar or microwell plates for the mouse lymphoma assay.
- If a selective agent is used (e.g., 6-thioguanine or trifluorothymidine), indicate its identity, its concentration and, duration and period of cell exposure.
- Number of cells seeded and method to enumerate numbers of viable and mutants cells:
- Criteria for small (slow growing) and large (fast growing) colonies:

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: background growth inhibition; mitotic index (MI); relative population doubling (RPD); relative increase in cell count (RICC); replication index; cytokinesis-block proliferation index; cloning efficiency; relative total growth (RTG); relative survival (RS); other:
- Any supplementary information relevant to cytotoxicity:

METHODS FOR MEASUREMENTS OF GENOTOXICIY

- OTHER:

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 (9) (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 at the five per cent level (p < 0.05) was evaluated by means of the
Fisher's exact test.

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 specified

Positive controls validity:

valid

Conclusions:

Under the experimental conditions reported, the test item induced structural chromosome aberrations as determined by the chromosome aberration test in V79 cells (Chinese hamster cell line) in vitro.

Executive summary:

In a GLP compliant study conducted according to OECD test guideline 473, 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 one experiment.

 

The following study design was performed:

Exposure period: 4hrs; Recovery: 14hrs; Preparation interval: 18hrs; With and without S9 mix.

In each experimental group two parallel cultures were set up. Per culture at least 100 metaphase plates were scored for structural chromosome aberrations.

 

The applied concentrations in the pre-test on toxicity were 39.1, 78.1, 156.3, 312.5, 625.0, 1250.0, 2500.0 and 5000 µg/mL.

The applied concentrations in the cytogenetic experiments were 19.5, 39.1, 78.1, 56.3, 156.3, 312.5 and 625.0 µg/mL.

 

In the main experiment, in the absence and presence of S9 mix, concentrations showing clear cytotoxicity were not evaluable for cytogenetic damage. In the absence and the presence of S9 mix, statistically significant and biologically relevant increases in the number of cells carrying structural chromosomal aberrations were observed after treatment with the test item. No relevant increase in the frequencies of polyploid metaphases was found after treatment with the test item as compared to the frequencies of the controls. Appropriate mutagens were used as positive controls. They induced statistically significant increases (p <0.05) in cells with structural chromosome aberrations.

 

Therefore, test item was considered to be clastogenic in this chromosome aberration test in the absence and presence of S9 mix.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Study initiation date - 10 May 2006; Experiment start date - 20 May 2006; Experiment completion date -13 June 2006; Study completion date - 19 July 2006.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Commission Directive 2000/32/EC, L1362000, Annex 4D",

Deviations:

no

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

"Kanpoan No. 287 - Environment Protection Agency" "Eisei No. 127 -- Ministry of Health & Welfare" "Heisei 09/10/31 Kikyoku No. 2 -- Ministry of International Trade & Industry

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 40827/A
Batch: T2 5572 BOP 01/06
Purity: determined in this study
Appearance: black sticky powder
Expiration date: 28.02.2011
Storage: at room temperature

Target gene:

The Salmonella typhimurium histidine (his) and the E. coli tryptophan (trp) reversion system

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):

Regular checking of the properties of the strains regarding the membrane permeability and ampicillin resistance as well as spontaneous mutation rates is performed in RCC Cytotest Cell Research according to B. Ames et al. (1) and D. Maron and B. Ames.
In this way it was ensured that the experimental conditions set down by Ames were fulfilled.
The bacterial strains TA 1535, TA 1537, TA 98, TA 100, and WP2 uvrA were obtained from Trinova Biochem GmbH (35394 Gießen, Germany).

Metabolic activation:

with and without

Metabolic activation system:

S9 (Preparation by RCC - CCR)
Phenobarbital/ß-Naphthoflavone induced rat liver S9 is used as the metabolic activation system. The S9 is prepared from 8 - 1 2 weeks old male Wistar Hanlbm rats, weight approx. 220 - 320 g induced by applications of 80 mg/kg b.w. Phenobarbital i.p. (Desitin; D-22335 Hamburg) and ß-Naphthoflavone p.o. (Aldrich, D-89555 Steinheim) each on three consecutive days. The livers are prepared 24 hours after the last treatment. The S9 fractions are produced by dilution of the liver homogenate with a KCl solution (1+3) followed by centrifugation at 9000 g. Aliquots of the supernatant are frozen and stored in ampoules at -80° C. Small numbers of the ampoules can be kept at -20 °C for up to one week. Each batch of S9 mix is routinely tested with 2-aminoanthracene as well as benzo(a)pyrene.
The protein concentration in the S9 preparation was 31.0 mg/mL (lot no. R 030306) in both experiments.

S9 Mix
Before the experiment an appropriate quantity of S9 supernatant was thawed and mixed with S9 co-factor solution. The amount of S9 supernatant was 15 % v/v in the S9 mix.
Cofactors are added to the S9 mix to reach the following concentrations in the S9 mix:
8 mM MgCI2
33 mM KCl
5 m M Glucose-6-phosphate
5mM NADP
in 100 mM sodium-ortho-phosphate-buffer, pH 7.4.

During the experiment the S9 mix was stored in an ice bath. The S9 mix preparation was performed according to Ames et al.

Test concentrations with justification for top dose:

Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate;
Experiment II: 33; 100; 333; 1000; 2500; and 5000 µg/plate

Vehicle / solvent:

deionised water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Deionised water

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Without metabolic activation - TA 1535, TA 100 (10 µg/plate)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylene-diamine, 4-NOPD

Remarks:

Without metabolic activation - TA 1537 (10 µg/plate), TA 98 (50 µg/plate)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Deionised water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Methyl methane sulfonate, MMS

Remarks:

Without metabolic activation - WP2 uvrA (4 µL/plate)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene, 2-AA

Remarks:

With metabolic activation - 2.5 µg/plate (TA 1535, TA 1537, TA 98, TA 100), 10 µg/plate (WP2uvrA)

Details on test system and experimental conditions:

The bacterial strains (S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and Escherichia coli WP2 uvrA) were stored as stock cultures in ampoules with nutrient broth + 5 % DMSO in liquid nitrogen. The broth was incubated in a shaking water bath at 37 ℃ for 4h.

The S9 was prepared from 8 - 1 2 weeks old male Wistar Hanlbm rats, weight approx. 220 - 320 g induced by applications of 80 mg/kg b.w. Phenobarbital i.p. and ß-Naphthoflavone p.o. each on three consecutive days. The livers are prepared 24 hours after the last treatment. The S9 fractions are produced by dilution of the liver homogenate with a KCl solution followed by centrifugation at 9000 g. Aliquots of the supernatant are frozen and stored in ampoules at -80° C. Small numbers of the ampoules can be kept at -20°C for up to one week. Each batch of S9 mix is routinely tested with 2-aminoanthracene as well as benzo(a)pyrene.

For each strain and dose level including the controls, three plates were used.
The following materials were mixed in a test tube and poured onto the selective agar plates:
100 µL Test solution at each dose level, solvent (negative control) or reference mutagen solution (positive control),
500 µL S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation),
100 µL Bacteria suspension (cf. test system, pre-culture of the strains),
2000 µL Overlay agar
In the pre-incubation assay 100 µL test solution, 500 µL S9 mix / S9 mix substitution buffer and 100 µL bacterial suspension were mixed in a test tube and shaken at 37 °C for 60 minutes. After pre-incubation 2.0 mL overlay agar (45 °C) was added to each tube. The mixture was poured on selective agar plates.
After solidification the plates were incubated upside down for at least 48 hours at 37 °C in the dark.

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 TA 1535 and TA 1537) 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.

Species / strain:

other: Salmonella typhimurium TA 1535, TA 1537, TA 98, and TA 100, E. coli WP2 uvrA.

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

During the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Executive summary:

In a GLP study conducted according to OECD test guideline 473, the potential of the test article to induce gene mutations was evaluated in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without metabolic activation in both independent experiments. No toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test article at any dose level, neither in the presence nor 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 biological relevance. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies. So the test article was considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Study initiation date - 14 January 2013; Experiment start date - 29 January 2013; Experiment completion date- 05 April 2013; Study completion date - 06 May 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:

Name: FAT 40827/B
Batch No: BOP 01-12 (Lot BS-1118392)
Physical state at RT: Solid powder.
Colour: Black
Expiry date - 17 April 2017
Storage conditions: At room temperature.

Target gene:

hypoxanthine-guanine-phosphoribosyl-transferase (HPRT)

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

V79 cells in vitro have been used to examine the ability of chemicals to induce cytogenetic changes and thus identify potential carcinogens or mutagens. These cells are characterized by their high proliferation rate (12 - 14h doubling time of the BSL Bioservice stock cultures) and their high cloning efficiency of untreated cells, usually more than 50 %. These facts are necessary for the appropriate performance of this study.
The V79 cells were stored over liquid nitrogen (vapour phase) in the cell bank of BSL Bioservice. Each cell batch was routinely check for mycoplasma infections (PCR). Thawed stock cultures were maintained in plastic culture flasks in minimal essential medium (MEM).

-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 Sprague Dawley Phenobarbital and ß-Naphthoflavone-induced rat liver S9 mix:
The S9 liver microsomal fraction was obtained from Trinova Biochem GmbH, Giessen, Germany. Male SD rats were induced with phenobarbital/ ß-Naphthoflavone. A stock of the supernatant containing the microsomes is frozen in aliquots of 5 mL and stored at <-75 °C.

S9 Mix:
An appropriate quantity of the 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 concentration below;
8 mM Mg Cl2
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):
10, 25, 50, 100, 250, 500, 1000, 1750, 2500 and 5000 µg/mL

Experiment I
without metabolic activation:
1, 2.5, 5, 10, 20, 30, 40, 50, 60 and 80 µg/mL
and with metabolic activation:
0.5, 1, 2.5, 10, 25, 50, 100, 125 and 150 µg/mL

Experiment II
without metabolic activation:
2.5, 5, 10, 25, 50, 100, 200, 300, 400 and 500 µg/mL
and with metabolic activation:
6, 13, 20, 40, 60, 80, 120, 140, 160 and 180 µg/mL

Vehicle / solvent:

Vehicle (Solvent) used:
A stock solution of the test item in Aqua ad injectabilia was 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

Remarks:

Medium MEM

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:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

with metabolic activation; (1.0 and 1.5 µg/mL)

Details on test system and experimental conditions:

METHOD OF APPLICATION: dissolved in Aqua ad inj.
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
METHODS FOR MEASUREMENTS OF GENOTOXICIY: Biological relevant increase in the number of mutants.

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: ≥ 30 μg/mL; experiment I with S9: ≥ 50 μg/mL; Experiment II without S9: ≥ 200 μg/mL; Experiment II with S9:≥ 40 μg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

In conclusion, in the described mutagenicity test under the experimental conditions reported, the test item FAT 40827/B 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, V79 cells cultured in vitro were exposed to FAT 40827/B at concentrations;

- 1.0, 2.5, 5, 10, 20, 30, 40, 50, 60 and 80 µg/mL (without metabolic activation, Experiment I)

- 0.5, 1.0, 2.5, 10, 25, 50, 100, 125 and 150 µg/mL (with metabolic activation, Experiment I)

- 2.5, 5.0, 10, 25, 50, 100, 200, 300, 400 and 500 µg/mL (without metabolic activation, Experiment II)

- 6, 13, 20, 40, 60, 80, 120, 140, 160 and 180 µg/mL (with metabolic activation, Experiment II). 

FAT 40827/B was tested up to cytotoxic concentrations. Biologically relevant growth inhibition was observed in experiment I and II without and with metabolic activation. In experiment I without metabolic activation the relative growth was 16.0 % for the highest concentration (80 µg/mL) evaluated. The highest biologically relevant concentration evaluated with metabolic activation was 150 µg/mL with a relative growth of 13.5 %. In experiment II without metabolic activation the relative growth was 12.4 % for the highest concentration (500 µg/mL) evaluated. The highest concentration evaluated with metabolic activation was 180 µg/mL with a relative growth of 12.9 %. In experiment I without metabolic activation the highest mutation rate (compared to the solvent control values) of 2.16 was found at a concentration of 60 µg/mL with a relative growth of 20.7 %. In experiment I with metabolic activation the highest mutation rate (compared to the solvent control values) of 1.48 was found at a concentration of 50 µg/mL with a relative growth of 62.3 %. In experiment II without metabolic activation the highest mutation rate (compared to the solvent control values) of 1.26 was found at a concentration of 25 µg/mL with a relative growth of 101.6 %. In experiment II with metabolic activation the highest mutation rate (compared to the solvent control values) of 2.04 was found at a concentration of 60 µg/mL with a relative growth of 61.2 %. The positive controls did induce the appropriate response. There was no evidence of a concentration related positive response of induced mutant colonies over background. In conclusion, in the described mutagenicity test under the experimental conditions reported, the test item FAT 40827/B is considered to be non-mutagenic in the HPRT locus using V79 cells of the Chinese Hamster.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

The test item was considered to be non-mutagenic in this micronucleus assay

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Study initiation date - 23 October 2006; Experiment start date - 01 November 2006; Experiment completion date - 19 December 2006; Study completion date - 20 March 2007.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Commission Directive 2000/32/EC, Annex 4C, dated May 19, 2000.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Specific details on test material used for the study:

Identity: FAT 40827/A
Batch: T2 5572 BOP 01/06
Purity: determined in this study
Appearance: black sticky powder
Expiration date: 28.02.2011
Storage: at room temperature

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

Strain: NMRI
Source Harlan Winkelmann GmbH D-33178 Borchen
Number of Animals: 72 (36 males/36 females)
Initial Age at Start of Acclimatisation: 6 - 9 weeks
Acclimatization: minimum 5 days
Initial Body Weight at Start of Treatment: males mean value 31.1 g (SD ± 1.5 g) females mean value 31.2 g (SD ± 1.6 g);
The animals were kept conventionally. The experiment was conducted under standard laboratory conditions.
Housing: single
Cage Type: Makrolon Type I, with wire mesh top
Bedding: granulated soft wood bedding
Feed: pelleted standard diet, ad libitum
Water: tap water, ad libitum
Environment: temperature 22 ± 3 °C
relative humidity 30 - 73 %
artificial light 6.00 a.m. - 6.00 p.m.

Route of administration:

oral: unspecified

Vehicle:

deionised water

Details on exposure:

On the day of the experiment, the test item was formulated in deionised water and the animals (including the controls) were weighed and the individual volume to be administered was adjusted to the animal’s body weight (All animals received a single standard volume of 10 mL/kg body weight orally). The animals received the test item, the vehicle or the positive control substance once.

Duration of treatment / exposure:

24 and 48 hours

Frequency of treatment:

once

Post exposure period:

Sampling of the bone marrow was done 24 and 48 hours after treatment, respectively.

Dose / conc.:

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

Remarks:

24h interval

Dose / conc.:

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

Remarks:

24h interval

Dose / conc.:

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

Remarks:

24 and 48h interval

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

CPA, Cyclophosphamide, dissolved in deionised water, dosing: 40 mg/kg b.w., route and frequency of administration: orally, once, volume administered: 10 mL/kg bw

Tissues and cell types examined:

The marrow of the femora;
Two types of erythrocytes were observed in the bone marrow smears: normochromatic (mature red blood cells about to pass into the blood stream) and polychromatic (immature red blood cells).

Details of tissue and slide preparation:

The animals were sacrificed using CO2 followed by bleeding. The femora were removed, the epiphyses were cut off and the marrow was flushed out with foetal 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 re-suspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Grünwald /Giemsa. Cover slips were mounted with EUKITT.

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:

no effects

Remarks:

no cytotoxic properties in the bone marrow

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The urine of the treated animals was stained red to orange indicating the systemic distribution of the test article and hence confirming its bioavailability.

Micronuclei in polychromatic erythrocytes (PCE) and relationship PCE/ total erythrocytes scoring 24 hours after treatment

Test group	Dose mg/kg b.w.	Micronucleated cells per 2000 PCEs per animal	Percent cells with micronuclei	PCE per 2000 erythrocytes
Vehicle (a. deion.)	0	2.5	0.125	1110
Test item	500	3.5	0.175	1108
	1000	2.0	0.100	1100
	2000	2.7	0.135	1114
Positive control(CPA)	40	43.2	2.160	971

 

Micronuclei in polychromatic erythrocytes (PCE) and relationship PCE/ total erythrocytes scoring 48 hours after treatment

Test group	Dose mg/kg b.w.	Micronucleated cells per 2000 PCEs per animal	Percent cells with micronuclei	PCE per 2000 erythrocytes
Test item	2000	1.6	0.080	1211

Conclusions:

During the study described and under the experimental conditions reported, the test article did not induce micronuclei as determined by the
micronucleus test in the bone marrow cells of the mouse.

Executive summary:

A GLP-compliant study was performed according to OECD test guideline 474 to investigate the potential of the test item 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 normochromatic 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.

The highest dose (2000 mg/kg; maximum guideline-recommended dose) was estimated by pre-experiments to be suitable. After treatment with the test item the number of PCEs was not substantially decreased as compared to the mean value of PCEs of the vehicle control thus indicating that the test item did not exert any cytotoxic effects in the bone marrow. However, the urine of the treated animals was stained red to orange indicating the systemic distribution of the test item and hence confirming its bioavailability. In comparison to the corresponding vehicle controls there was no biologically relevant or statistically significant enhancement in the frequency of the detected micronuclei at any preparation interval after administration of the test item and with any dose level used. 40 mg/kg bw cyclophosphamide administered orally was used as positive control which showed a substantial increase of induced micronucleus frequency. In conclusion, the test item was considered to be non-mutagenic in this micronucleus assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Three in vitro tests and one in vivo test were performed with the test substance, including gene mutation assay, chromosome aberration test and micronucleus assay.

 

Ames Study:

In a GLP study conducted according to OECD test guideline 473, the potential of the test article to induce gene mutations was evaluated in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without metabolic activation in both independent experiments. No toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test article at any dose level, neither in the presence nor 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 biological relevance. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies. So the test article was considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

 

HPRT:

In a mammalian cell gene mutation assay (HPRT locus) conducted according to OECD test guideline 476, V79 cells cultured in vitro were exposed to FAT 40827/B at concentrations;

- 1.0, 2.5, 5, 10, 20, 30, 40, 50, 60 and 80 µg/mL (without metabolic activation, Experiment I)

- 0.5, 1.0, 2.5, 10, 25, 50, 100, 125 and 150 µg/mL (with metabolic activation, Experiment I)

- 2.5, 5.0, 10, 25, 50, 100, 200, 300, 400 and 500 µg/mL (without metabolic activation, Experiment II)

- 6, 13, 20, 40, 60, 80, 120, 140, 160 and 180 µg/mL (with metabolic activation, Experiment II).

FAT 40827/B was tested up to cytotoxic concentrations.

Biologically relevant growth inhibition was observed in experiment I and II without and with metabolic activation. In experiment I without metabolic activation the relative growth was 16.0 % for the highest concentration (80 µg/mL) evaluated. The highest biologically relevant concentration evaluated with metabolic activation was 150 µg/mL with a relative growth of 13.5 %. In experiment II without metabolic activation the relative growth was 12.4 % for the highest concentration (500 µg/mL) evaluated. The highest concentration evaluated with metabolic activation was 180 µg/mL with a relative growth of 12.9 %. In experiment I without metabolic activation the highest mutation rate (compared to the solvent control values) of 2.16 was found at a concentration of 60 µg/mL with a relative growth of 20.7 %. In experiment I with metabolic activation the highest mutation rate (compared to the solvent control values) of 1.48 was found at a concentration of 50 µg/mL with a relative growth of 62.3 %. In experiment II without metabolic activation the highest mutation rate (compared to the solvent control values) of 1.26 was found at a concentration of 25 µg/mL with a relative growth of 101.6 %. In experiment II with metabolic activation the highest mutation rate (compared to the solvent control values) of 2.04 was found at a concentration of 60 µg/mL with a relative growth of 61.2 %. The positive controls did induce the appropriate response. There was no evidence of a concentration related positive response of induced mutant colonies over background. In conclusion, in the described mutagenicity test under the experimental conditions reported, the test item FAT 40827/B is considered to be non-mutagenic in the HPRT locus using V79 cells of the Chinese Hamster.

 

Chromosome abberation:

In a GLP-compliant study conducted according to OECD test guideline 473, 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 one experiment.

The following study design was performed:

Exposure period: 4hrs; Recovery: 14hrs; Preparation interval: 18hrs; With and without S9 mix.

In each experimental group two parallel cultures were set up. Per culture at least 100 metaphase plates were scored for structural chromosome aberrations. The applied concentrations in the pre-test on toxicity were 39.1, 78.1, 156.3, 312.5, 625.0, 1250.0, 2500.0 and 5000 µg/mL. The applied concentrations in the cytogenetic experiments were 19.5, 39.1, 78.1, 56.3, 156.3, 312.5 and 625.0 µg/mL. In the main experiment, in the absence and presence of S9 mix, concentrations showing clear cytotoxicity were not evaluable for cytogenetic damage. In the absence and the presence of S9 mix, statistically significant and biologically relevant increases in the number of cells carrying structural chromosomal aberrations were observed after treatment with the test item. No relevant increase in the frequencies of polyploid metaphases was found after treatment with the test item as compared to the frequencies of the controls. Appropriate mutagens were used as positive controls. They induced statistically significant increases (p <0.05) in cells with structural chromosome aberrations.

Therefore, test item was considered to be clastogenic in this chromosome aberration test in the absence and presence of S9 mix.

 

Micronucleus study

A GLP-compliant study was performed according to OECD test guideline 474 to investigate the potential of the test item 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 normochromatic 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.

The highest dose (2000 mg/kg; maximum guideline-recommended dose) was estimated by pre-experiments to be suitable. After treatment with the test item the number of PCEs was not substantially decreased as compared to the mean value of PCEs of the vehicle control thus indicating that the test item did not exert any cytotoxic effects in the bone marrow. However, the urine of the treated animals was stained red to orange indicating the systemic distribution of the test item and hence confirming its bioavailability. In comparison to the corresponding vehicle controls there was no biologically relevant or statistically significant enhancement in the frequency of the detected micronuclei at any preparation interval after administration of the test item and with any dose level used. 40 mg/kg bw cyclophosphamide administered orally was used as positive control which showed a substantial increase of induced micronucleus frequency. In conclusion, the test item was considered to be non-mutagenic in this micronucleus assay.

 

Conclusion on genotoxicity:

Reactive Brown 051 was not mutagenic in the bacterial cell reverse mutationa assay as well as the mammalian cell gene mutation assay in vitro. It was found to lead to clastogenic effects in the mammalian cell in vitro, but this effect could not be reproduced in the in vivo micronucleus assay, hence, Reactive Brown 051 was considered to be not clastogenic and thereby not geneotoxic.

Justification for classification or non-classification

Based on the assessment of three in vitro mutagenicity studies and one in vivo study, the test substance is not subject to classification for mutagenicity according to CLP (Regulation EC No 1272/2008).