Tetrasodium [2-({4-fluoro-6-[(2-{[4-fluoro-6-({5-(hydroxykappaO)-6-[(2-{[2-(hydroxy-kappaO)-5-sulfophenyl] diazenyl-kappaN1}-4,5-dimethoxyphenyl) diazenyl-kappaN2]-7-sulfo-2-naphthyl} amino)-1,3,5-triazin-2-yl] amino} propyl) amino]-1,3,5-triazin-2-yl} amino) benzene-1,4- disulfonato(6-)] cuprate(4-)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test item was found to be clastogenic in chromosome aberration test but was found to be non-mutagenic in Ames and HPRT test.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Study initiation date - 05 December 2012;
Experiment start date - 12 December 2012;
Experiment completion date - 28 March 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:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Specific details on test material used for the study:

Name: FAT 40825/B TE
Batch No.: BOP 01-12
Physical State: powder
Colour: Black
Storage Conditions: at room temperature
Purity: 84.8% all coloured organic constituents, main constituent: 63.7%
Expiry Date: 28 January 2017
Safety Precautions: The routine hygienic procedures were sufficient to assure personnel health and safety.

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 and Sprague Dawley Phenobarbital and ß-Naphthoflavone-induced rat liver S9 mix

Test concentrations with justification for top dose:

Pre-experiment for experiment I (with and without metabolic activation):
5, 10, 25, 50, 100, 250, 500, 1000, 2500, 5000 µg/mL

Experiment I
without metabolic activation:
10, 25, 50, 100, 1000, 2000, 3500 and 5000 µg/mL
and with metabolic activation:
50, 100, 250, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000 and 5000 µg/mL

Experiment II
without metabolic activation:
25, 50, 100, 250, 500, 1000, 2000, 3500 and 5000 µg/mL
and with metabolic activation:
200, 400, 800, 1400, 2000, 2600, 3200, 3800, 4400 and 5000 µg/mL

Vehicle / solvent:

Vehicle (Solvent) used: For the pre-experiment the test item was dissolved in cell culture medium (MEM + 0% FBS).

For the main experiments 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

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

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. / medium
DURATION: 4 h (short-term exposure), 20 h (long-term exposure)
Expression time (cells in growth medium): 5 days
Selection time (if incubation with selection agent): about one week

SELECTION AGENT (mutation assay) 11 µg/mL 6-thioguanine (TG)
NUMBER OF REPLICATIONS: two separate experiments (I+II) with single exposure; 5 individual flasks were seeded and evaluated
NUMBER OF CELLS EVALUATED: 400000 cells per flask
DETERMINATION OF CYTOTOXICITY: Method: relative growth

Evaluation criteria:

A test is considered to be negative if there is no biologically relevant increase in the number of mutants.
There are several criteria for determining a positive result:
-a reproducible three times higher mutation frequency than the solvent control for at least one of the concentrations;
-a concentration related increase of the mutation frequency; such an evaluation may be considered also in the case that a three-fold increase of
the mutant frequency is not observed;
-if there is by chance a low spontaneous mutation rate in the corresponding negative and solvent controls a concentration related increase of the mutations within their range has to be discussed.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Experiment I without S9: at 100 and 1000 μg/mL; experiment I with S9: at 50-250 μg/mL and ≥ 2000 μg/mL; Experiment II without S9: ≥ 500 μg/mL; Experiment II with S9: at 200, 400, 2600 and ≥ 3800 μg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Precipitation: No precipitation of the test item was noted in any of the experiments.

Toxicity:
A biologically relevant growth inhibition (reduction of relative growth below 70%) was observed after the treatment with the test item in experiment I and II with and without metabolic activation.

In experiment I without metabolic activation the relative growth was 73.2% for the be highest concentration (5000 µg/mL) evaluated, a slightly reduced growth was observed at concentrations of 100 and 1000 µg/ml, respectively. The highest biologically relevant concentration evaluated with metabolic activation was 5000 µg/mL with a relative growth of 17.9%.

In experiment II without metabolic activation the relative growth was 28.7% for the highest concentration (5000 µg/mL) evaluated. The highest concentration evaluated with metabolic activation was 5000 µg/mL with a relative growth of 34.3%. Furthermore, in the experiments with metabolic activation toxic effects were observed also in the lowest concentrations (experiment I; between 50 and 250 µg/ml; experiment II: 200 and 400 µg/ml).

Mutagenicity:
In experiment I without metabolic activation most mutant values of the negative controls, the solvent controls and test item concentrations found were within the historical control data of the test facility BSL BIOSERVICE (about 5-43 mutants per cells). No dose-response relationship could be observed. The mutation frequencies found in the groups treated with the test item did not show a biologically relevant increase as compared to the solvent controls.
Mutation frequencies with the negative control were found to be 21.34 and 21.00, of the solvent control 23.08 and 13.68 mutants/10E6 cells and in the range of 4.90 to 45.14 mutants/10E6 cells with the test item, respectively. The highest mutation rate (compared to the solvent control values) of 2.46 was found at a concentration of 5000 µg/mL with a relative growth of 73.2%.

With metabolic activation most mutant values of the negative controls, the solvent controls and test item concentrations found were within the historical control data of the test facility BSL BIOSERVICE (about 5-44 mutants per 10E6 cells). No dose-response relationship could be observed. The mutation frequencies found in the groups treated with the test item did not show a biologically relevant increase as compared to the
solvent controls.
Mutation frequencies with the negative control were found to be 25.97 and 31.22, of the solvent control 22.13 and 29.55 mutants/10E6 cells and in the range of 14.29 to 54.83 mutants/10E6 cells with the test item, respectively. The highest mutation rate (compared to the solvent control values) of 2.12 was found at a concentration of 3500 µg/mL with a relative growth of 54.6%.
In experiment II without metabolic activation most mutant values of the negative controls, the solvent controls and test item concentrations found were within the historical control data of the test facility BSL BIOSERVICE (about 5-43 mutants per 10E6 cells). No dose-response relationship could be observed. The mutation frequencies found in the groups treated with the test item did not show a biologically relevant increase as compared to the solvent controls.

Mutation frequencies with the negative control were found to be 17.21 and 33.59, of the solvent control 42.28 and 32.74 mutants/10E6 cells and in the range of 27.24 to 61.61 mutants/10E6 cells with the test item, respectively. The highest mutation rate (compared to the solvent control values) of 1.64 was found at a concentration of 2000 µg/mL with a relative growth of 54.2%.
In experiment Il with metabolic activation most mutant values of the negative controls, the solvent controls and test item concentrations found were within the historical control data of the test facility BSL BIOSERVICE (about 5-44 mutants per 10E6 cells). No dose-response relationship could be observed. The mutation frequencies found in the groups treated with the test item did not show a biologically relevant increase as compared to
the solvent controls.

Mutation frequencies with the negative control were found to be 16.22 and 26.72, of the solvent control 30.57 and 34.82 mutants/10E6 cells and in the range of 4.55 to 57.14 mutants/10E6 cells with the test item, respectively. The highest mutation rate (compared to the solvent control values) of 1.75 was found at a concentration of 5000 µg/mL with a relative growth of 34.3%.

DMBA (1.0 and 1.5 µg/mL) and EMS (300 µg/mL) were used as positive controls and showed distinct and biologically relevant effects in mutation frequency.

Conclusions:

In conclusion, in the described mutagenicity test under the experimental conditions reported, the test item FAT 40825/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, V79 cells cultured in vitro were exposed to FAT 40825/B TE at concentrations of

- 10, 25, 50, 100, 1000, 2000, 3500 and 5000 µg/mL (without metabolic activation, Experiment I)

- 50, 100, 250, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000 and 5000 µg/mL (with metabolic activation, Experiment I)

- 25, 50, 100, 250, 500, 1000, 2000, 3500 and 5000 µg/mL (without metabolic activation, Experiment II)

- 200, 400, 800, 1400, 2000, 2600, 3200, 3800, 4400 and 5000 µg/mL (with metabolic activation, Experiment II).

FAT 40825/B TE was tested up to cytotoxic concentrations.

Biologically relevant growth inhibition was observed in experiment I and II with and without metabolic activation. In experiment I without metabolic activation the relative growth was 73.2% for the highest concentration (5000 µg/mL) evaluated. The highest biologically relevant concentration evaluated with metabolic activation was 5000 µg/mL with a relative growth of 17.9%. In experiment II without metabolic activation the relative growth was 28.7% for the highest concentration (5000 µg/mL) evaluated. The highest concentration evaluated with metabolic activation was 5000 µg/mL with a relative growth of 34.3%.

 

In experiment I without metabolic activation the highest mutation rate (compared to the solvent control values) of 2.46 was found at a concentration of 5000 µg/mL with a relative growth of 73.2%.

In experiment I with metabolic activation the highest mutation rate (compared to the solvent control values) of 2.12 was found at a concentration of 3500 µg/mL with a relative growth of 54.6%.
In experiment II without metabolic activation the highest mutation rate (compared to the solvent control values) of 1.64 was found at a concentration of 2000 µg/mL with a relative growth of 54.2%.
In experiment II with metabolic activation the highest mutation rate (compared to the solvent control values) of 1.75 was found at a concentration of 5000 µg/mL with a relative growth of 34.3%.

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.

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 - 14 March 2006;
Experiment start date - 29 March 2006;
Experiment completion date - 04 January 2007;
Study completion date - 19 Jaunary 2007.

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 40825/A
Batch number: CHU 297 / BOP 04/05
Purity: Organic part (Na-salt): approx. 83.7 %; All coloured components: approx. 80.54 %; Main component: approx. 59.1 %.
Appearance: Solid, black powder
Storage conditions: At room temperature at about 20 °C
Expiration date: December 31, 2010

Target gene:

chromosome

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

Large stocks of the V79 cell line (supplied by Laboratory for Mutagenicity Testing, LMP, Technical University Darmstadt, 64287 Darmstadt, Germany) were stored in liquid nitrogen in the cell bank of RCC Cytotest Cell Research GmbH allowing the repeated use of the same cell culture batch in experiments. 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 sq.cm plastic flasks (GREINER, 72632 Frickenhausen, Germany). About 5 x 10E5 cells per flask were seeded into 15 mL of MEM (Minimal Essential Medium; SEROMED; 12247 Berlin, Germany) supplemented with 10 % fetal calf serum (FCS; PAA Laboratories GmbH, 35091 Cölbe, Germany). The cells were sub-cultured twice weekly. The cell cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % carbon dioxide (98.5 % air).

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 on three consecutive days from 8-12 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. 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 32.6 mg/mL (Lot no. 021205) in the pre-test, 30.6 mg/mL (Lot no. 091205) in Experiment IA, 29.3 mg/mL (Lot no.190506) in Experiment IB, and 25.3 mg/mL (Lot no. 061006) in Experiment 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 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:

IA: Exposure period 4 hrs without S9 mix 156.3, 312.5, 625.0, 1250.0, 2500.0, 5000.0 µg/mL
II: Exposure period 18 hrs without S9 mix 78.1, 156.3, 312.5, 625.0, 1250.0, 2500.0 µg/mL
II: Exposure period 28 hrs without S9 mix: 312.5, 625.0, 1250.0, 2500.0 µg/mL
IA: Exposure period 4 hrs and Preparation interval 18 hrs with S9 mix: 4.9, 9.8, 19.5, 39.1, 78.1, 156.3 µg/mL
IB: Exposure period 4 hrs and Preparation interval 18 hrs with S9 mix 25.0, 50.0, 75.0, 100.0, 125.0, 150.0, 175.0, 200.0 µg/mL
II: Exposure period 4 hrs and Preparation interval 28 hrs with S9 mix: 9.8, 19.5, 39.1, 78.1, 156.3, 312.5 µg/mL
II: Exposure period 4 hrs and Preparation interval 28 hrs with S9 mix: 31.3, 62.5, 125.0, 250.0 , 500.0, 1000.0 µg/mL

Vehicle / solvent:

deionised water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Without metabolic activation - 300 - 500 µg/mL (1.6 - 4.0 mM)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

With metabolic activation - 1.4 - 2.0 µg/mL (5.0 - 7.0 mM)

Details on test system and experimental conditions:

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

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): The cells were seeded into Quadriperm dishes (Heraeus, 63450 Hanau, Germany) that contained microscopic slides (at least 2 chambers per dish and test group). In each chamber 1 x10E4 to 6x10E4 cells were seeded with regard to the preparation time. The medium was MEM with 10 % FCS (complete medium).


Preparation of the Cultures:
Colcemid was added (0.2 µg/mL culture medium) to the cultures 15.5 hrs and 25.5 hrs, respectively after the start of the treatment. The cells on the slides were treated 2.5 hrs later, in the chambers with hypotonic solution (0.4 % KCl) for 20 min at 37° C. After incubation in the hypotonic solution the cells were fixed with a mixture of methanol and glacial acetic acid (3:1 parts, respectively). Per experiment two slides per group were prepared. After preparation the cells were stained with Giemsa (E. Merck, 64293 Darmstadt, Germany).

Evaluation of Cell Numbers:
For evaluation of cytotoxicity indicated by reduced cell numbers two additional cultures per test item and solvent control group, not treated with colcemid, were set up in parallel. These cultures were stained after 18 hrs and 28 hrs, respectively, in order to determine microscopically the cell number within 10 defined fields per coded slide. The cell number of the treatment groups is given in percentage compared to the respective solvent control.

Analysis of Metaphase Cells:
Evaluation of the cultures was performed (according to standard protocol of the "Arbeitsgruppe der Industrie, Cytogenetic' [5]) 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. At least 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 plates per culture was determined (% polyploid metaphases; in the case of this aneuploid cell line polyploid means a near tetraploid karyotype).

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 criterion 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 - 5.6 % polyploid cells).

Statistics:

Statistical significance at the five per cent level (p <0.05) was evaluated by means of the Fisher's exact test. Evaluation was performed only for cells carrying aberrations exclusive gaps.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

in the absence of S9 mix

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with

Genotoxicity:

negative

Remarks:

in the presence of S9 mix

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Precipitation of the test item in culture medium was observed in the presence of S9 mix, in Experiment IA, at preparation interval 18 hrs with 78.1 µg/mL and above, in Experiment IB, at preparation interval 18 hrs with 50 µg/mL and above, and in Experiment II at preparation interval 28 hrs with 62.5 µg/mL and above.

In a range finding pre-test on toxicity, cell numbers were scored 24 hrs after start of treatment as an indicator for cytotoxicity. Concentrations between 39.1 and 5000 µg/mL were applied. Clear toxic effects were observed after treatment with 2500 µg/mL and above in the absence of S9 mix and with 78.1 µg/mL and above in the presence of S9 mix. In addition, 24 hrs continuous treatment with 1250 ug/mL and above in the absence of S9 mix induced strong toxic effects. In the pre-experiment, no precipitation of the test item in culture medium was observed after treatment up to 1250 µg/mL in the absence of S9 mix. At the concentrations 2500 and 5000 µg/mL, precipitation could not be assessed due to strong colouration. No relevant influence of the test item on the pH value or osmolarity was observed (solvent control 283 mOsm, pH 7.4 versus 299 mOsm and pH 7.3 at 5000 µg/mL). Precipitation of the test item in culture medium was observed in the presence of S9 mix, in Experiment IA, at preparation interval 18 hrs with 78.1 µg/mL and above, in Experiment 1B, at preparation interval 18 hrs with 50 µg/mL and above, and in Experiment II at preparation interval 28 hrs with 62.5 µg/mL and above. In Experiment 1A, in the absence of S9 mix and in Experiments IB and II, in the presence of S9 mix, no toxic effects indicated by reduced cell numbers and/or mitotic indices of below 50 % of control were observed up to the highest applied concentrations. In contrast, in Experiment II, in the absence of S9 mix, the mitotic index was reduced after 28 hrs continuous treatment with 625 µg/mL. In addition, in Experiment IA, in the presence of S9 mix, reduced cell numbers were observed after 4 hrs treatment with 156.3 µg/mL. In Experiment II, in the absence of S9 mix, at preparation interval 18 hrs, concentrations showing clear cytotoxicity were not scorable for cytogenetic damage. In Experiment IA, in the absence of S9 mix, no biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed at the scored concentrations. However, in the presence of S9 mix, a statistically significant and biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed at the highest evaluated concentration (6.0 % aberrant cells, excluding gaps). The number of aberrant cells showed a dose-related increase (2.5, 5.0, and 6.0 %), with the two highest concentrations exceeding our historical control value (0.0 - 4.0 % aberrant cells, excluding gaps). This observation provided additional evidence for the clastogenic potential of the test item. To verify the results obtained in Experiment IA, a confirmatory Experiment IB, in the presence of S9 mix was performed. In this experiment, at 50, 100, and 200 µg/mL, the increases in the number of aberrant cells (6.0, 5.0, and 5.3 % aberrant cells, excluding gaps, respectively), exceeded our historical control range of 0.0 - 4.0 % aberrant cells, excluding gaps. However, this increase was not dose related increase and the values were not significantly higher compared to the corresponding control (3.5 %). Therefore, this observation is considered to be biologically irrelevant and the result of Experiment I was not confirmed. In Experiment II, in the presence of S9 mix, no statistically significant and biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed at the scored concentrations. In contrast, in the absence of S9 mix, two statistically significant increases in the number of aberrant cells, excluding gaps (both 5.5 %) exceeding our historical control data (0.0 - 4.0 % aberrant cells, excluding gaps) were observed after treatment 18 hrs continuous treatment with 312.5 and 625 µg/mL, respectively. In addition, at preparation interval 28 hrs, two statistically significant and biologically relevant increases in the number of aberrant cells, excluding gaps (9.0 and 10.5 %) were observed at the scored concentrations (312.5 and 625 µg/mL, respectively). In both experiments, no biologically relevant increase in the rate of polyploid metaphases was found after treatment with the test item (0.9 - 3.4 %) as compared to the rates of the solvent controls (1.1 -3.3%).
In both experiments, either EMS (300 or 500 µg/mL) or CPA (1.4 or 2.0 µg/mL) were used as positive controls and showed distinct increases in the number of cells with structural chromosome aberrations.

Conclusions:

Under the experimental conditions reported, the test item induced structural chromosome aberrations in V79 cells (Chinese hamster cell line) in the absence of S9 mix.

Executive summary:

A GLP-compliant study was conducted according to OECD test guideline 473 to assess the potential of test substance to induce structural chromosome aberration. The test item, suspended (pre-experiment and) or dissolved (experiments IB and II) in deionised water, was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro in three independent experiments. In each experimental group two parallel cultures were set up. Per culture at least 100 metaphase plates were scored for structural chromosome aberrations. The highest applied concentration in the pre-test on toxicity (5000 µg/mL) was chosen with respect to the current OECD Guideline 473. Dose selection for the cytogenetic experiments was performed considering the toxicity data and the occurrence of precipitation. In the absence of S9 mix and in Experiment IB and II, in the presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration. In Experiment II, in the absence of S9 mix, at preparation interval 18 hrs, concentrations showing clear cytotoxicity were not scorable for cytogenetic damage. In contrast, in the presence of S9 mix and in Experiment II, in the absence of S9 mix, at preparation interval 28 hrs, cytotoxicity was observed at the highest scorable concentrations. In the absence of S9 mix, and in Experiment IB and II in the presence of S9 mix, neither a statistically significant nor a biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item. In contrast, in the presence of S9 mix, a statistically significant and biologically relevant increase (6.0 % aberrant cells, excluding gaps) was observed at the highest scored concentration. The number of aberrant cells showed a dose-related increase, with the two highest concentrations exceeding our historical control data range (0.0 - 4.0 % aberrant cells, excluding gaps). This observation was not verified in the confirmatory Experiment IB. In Experiment II, in the absence of S9 mix, at preparation interval 18 hrs, two statistically significant increases in the number of aberrant cells (both 5.5 %) exceeding our historical control data range (0.0 - 4.0 % aberrant cells, excluding gaps) were observed. In addition, at preparation interval 28 hrs, two statistically significant and biologically relevant increases in the number of aberrant cells, excluding gaps (9.0 and 10.5 %, respectively) were observed at the scored concentrations. 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. In conclusion, it can be stated that 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. Therefore, the test item is considered to be clastogenic in this chromosome aberration test in the absence of S9 mix.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Study initiation date - 09 March 2006;
Experiment start date - 19 April 2006;
Experiment completion date - 08 May 2006;
Study completion date - 09 June 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", dated May 19, 2000

Deviations:

no

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

Kanpoan No. 287; Eisei No. 127; "Heisei 09/10/31 Kikyoku No. 2

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 40825/A
Batch number: CHU 297 / BOP 04/05
Purity: Organic part (Na-salt): approx. 83.7 %; All coloured components: approx. 80.54 %; Main component: approx. 59.1 %.
Appearance: Solid, black powder
Storage conditions: At room temperature at about 20 °C
Expiration date: December 31, 2010

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):

The bacterial strains TA 1535, TA 1537, TA 98, TA 100, and WP2 uvrA were obtained from Trinova Biochem GmbH (35394 Gießen, Germany).
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.
The strain cultures were stored as stock cultures in ampoules with nutrient broth + 5 % DMSO (MERCK, D-64293 Darmstadt) in liquid nitrogen.

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

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

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

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

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

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

Remarks:

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

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

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

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

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 4 h. The S9 was prepared from 8 - 12 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 (1+3) followed by centrifugation at 9000 g. Aliquotes 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. Then colonies were counted.

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.

Statistics:

According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

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. In experiment I, the data in the negative control with and without metabolic activation of strain WP2 uvrA and in experiment II, the data in the solvent control with metabolic activation of strain TA 98 were slightly above our historical control range. Since this deviation is rather small, this effect is considered to be based upon biologically irrelevant fluctuations in the number of colonies. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with FAT 40825/A 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. They showed a distinct increase of induced revertant colonies.

Conclusions:

Test item is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Executive summary:

In a GLP study performed according to OECD test guideline 471, to investigate the potential of the test item to induce gene mutations 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 test item was tested at the following concentrations:

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

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 item 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. In conclusion, it can be stated that 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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

The test substance was found to be non-mutagenic in invivo micronucleus study.

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 - 10 January 2007;
Experiment start date - 15 January 2007;
Experiment completion date - 16 February 2007;
Study completion date - 11 April 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 AC, dated May 19, 2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian erythrocyte micronucleus test

Specific details on test material used for the study:

Identity: FAT 40825/A
Batch number: CHU 297 / BOP 04/05
Purity: Organic part (Na-salt): approx. 83.7 %; All coloured components: approx. 80.54 %; Main component: approx. 59.1 %.
Appearance: Solid, black powder
Storage conditions: At room temperature at about 20 °C
Expiration date: December 31, 2010

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: 7 - 8 weeks
Acclimatisation: minimum 5 days
Initial Body Weight at Start of Treatment: males mean value 35.6 g (SD ±1.5 g) females mean value 27.6 g (SD ±1.1 g)

Husbandry:
The animals were kept conventionally. The experiment was conducted under standard laboratory conditions.
Housing: single
Cage Type: Makrolon Type I, with wire mesh top
Bedding: granulated soft wood bedding
Feed: pelleted standard diet, ad libitum
Water: tap water, ad libitum,
Environment: temperature 22 ±3 °C relative humidity 30 - 72 %
artificial light 6.00 a.m. - 6.00 p.m.

Route of administration:

oral: gavage

Vehicle:

deionised water

Details on exposure:

On the day of the experiment, the test item was formulated in deionised water. All animals received a single standard volume of 10 mL/kg body weight orally.

Duration of treatment / exposure:

24 h and 48 h

Frequency of treatment:

1

Post exposure period:

24 h and 48 h

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Group 1 : Vehicle control (24 h)

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

Group 2: Test item (24 h)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

Group 3: Test item (24 h)

Dose / conc.:

2 000 mg/kg bw/day (nominal)

Remarks:

Group 4: Test item (24 h)

Dose / conc.:

2 000 mg/kg bw/day (nominal)

Remarks:

Group 5: Test item (48 h)

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

Name: CPA; Cyclophosphamide;
Dissolved in: deionised water
Dosing: 40 mg/kg bw
Route and frequency of administration: orally, once
Volume administered: 10 mL/kg bw

Tissues and cell types examined:

Tissue: The marrow of the femora;
Cells: Two types of erythrocytes-polychromatic and normochromatic erythrocytes.

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 resuspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Grünwald. Cover slips were mounted with EUKITT. 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 (8)) 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

Vehicle controls validity:

valid

Positive controls validity:

valid

Pre-Experiment for Toxicity
In the first pre-experiment 4 animals (2 males, 2 females) received orally a single dose of 100 mg/kg b.w. FAT 40825/A formulated in deionised water. The volume administered was 10 mL/kg b.w. The animals treated with 100 mg/kg b.w. did not express any toxic reactions. In the second pre-experiment 4 animals (2 males, 2 females) received orally a single dose of 1000 mg/kg b.w. FAT 40825/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 of ruffled fur after 2-4h interval. In the third pre-experiment 4 animals (2 males, 2 females) received orally a single dose of 2000 mg/kg b.w FAT 40825/A formulated in deionised water. The volume administered was 10 mL/kg b.w. The animals treated with 2000 mg/kg b.w. did not express any toxic reactions. On the basis of these data 2000 mg/kg b.w. were estimated to be suitable as the high dose.

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 40825/A formulated in deionised water. For the mid and low doses group 12 animals (6 males, 6 females) each received orally a single dose of 1000 or 500 mg/kg b.w. FAT 40825/A formulated in deionised water, respectively. The volume administered was 10 mL/kg b.w.. The treated with test item did not show, irrespective of the dosage any signs of toxicity. The treatment of the animals with the vehicle control also did not induce any clinical signs of toxicity.

Conclusions:

The test item did not induce micronuclei as determined by the micronucleus test in the bone marrow cells of the mouse.

Executive summary:

A 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 b.w. 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 b.w.

48 h preparation interval: 2000 mg/kg b.w.

The highest dose (2000 mg/kg; maximum guideline-recommended dose) was estimated by a pre-experiment 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. In comparison to the corresponding vehicle controls there was no biologically relevant or statistically significant enhancement in the frequency of the detected micronuclei at any preparation interval after administration of the test item and with any dose level used. 40 mg/kg b.w. cyclophosphamide administered orally was used as positive control which showed a substantial increase of induced micronucleus frequency. In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse. Therefore, the test item is 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 of the test substance, including Ames test, chromosome aberration test, HPRT test and micronucleus assay.

 

The Ames study was performed to investigate the potential of the test item to induce gene mutations 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. 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 item 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. In conclusion, it can be stated that 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.

 

In a second in vitro experiment ( mammalian cell gene mutation assay, HPRT locus), V79 cells cultured in vitro were exposed to FAT 40825/B TE at concentrations of

- 10, 25, 50, 100, 1000, 2000, 3500 and 5000 µg/mL (without metabolic activation, Experiment I)

- 50, 100, 250, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000 and 5000 µg/mL (with metabolic activation, Experiment I)

- 25, 50, 100, 250, 500, 1000, 2000, 3500 and 5000 µg/mL (without metabolic activation, Experiment II)

- 200, 400, 800, 1400, 2000, 2600, 3200, 3800, 4400 and 5000 µg/mL (with metabolic activation, Experiment II).

FAT 40825/B TE was tested up to cytotoxic concentrations.

Biologically relevant growth inhibition was observed in experiment I and II with and without metabolic activation. In experiment I without metabolic activation the relative growth was 73.2 % for the highest concentration (5000 µg/mL) evaluated. The highest biologically relevant concentration evaluated with metabolic activation was 5000 µg/mL with a relative growth of 17.9 %. In experiment II without metabolic activation the relative growth was 28.7 % for the highest concentration (5000 µg/mL) evaluated. The highest concentration evaluated with metabolic activation was 5000 µg/mL with a relative growth of 34.3 %.

In experiment I without metabolic activation the highest mutation rate (compared to the solvent control values) of 2.46 was found at a concentration of 5000 µg/mL with a relative growth of 73.2 %.

In experiment I with metabolic activation the highest mutation rate (compared to the solvent control values) of 2.12 was found at a concentration of 3500 µg/mL with a relative growth of 54.6 %.
In experiment II without metabolic activation the highest mutation rate (compared to the solvent control values) of 1.64 was found at a concentration of 2000 µg/mL with a relative growth of 54.2 %.
In experiment II with metabolic activation the highest mutation rate (compared to the solvent control values) of 1.75 was found at a concentration of 5000 µg/mL with a relative growth of 34.3 %. 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 a third in vitro experiment the test item, suspended (pre-experiment and) or dissolved (experiments IB and II) in deionised water, was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro in three independent experiments. In each experimental group two parallel cultures were set up. Per culture at least 100 metaphase plates were scored for structural chromosome aberrations. The highest applied concentration in the pre-test on toxicity (5000 µg/mL) was chosen with respect to the current OECD Guideline 473. Dose selection for the cytogenetic experiments was performed considering the toxicity data and the occurrence of precipitation. In the absence of S9 mix and in Experiment IB and II, in the presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration. In Experiment II, in the absence of S9 mix, at preparation interval 18 hrs, concentrations showing clear cytotoxicity were not scorable for cytogenetic damage. In contrast, in the presence of S9 mix and in Experiment II, in the absence of S9 mix, at preparation interval 28 hrs, cytotoxicity was observed at the highest scorable concentrations. In the absence of S9 mix, and in Experiment IB and II in the presence of S9 mix, neither a statistically significant nor a biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item.

In contrast, in the presence of S9 mix, a statistically significant and biologically relevant increase (6.0 % aberrant cells, excluding gaps) was observed at the highest scored concentration. The number of aberrant cells showed a dose-related increase, with the two highest concentrations exceeding our historical control data range (0.0 - 4.0 % aberrant cells, excluding gaps). This observation was not verified in the confirmatory Experiment IB. In Experiment II, in the absence of S9 mix, at preparation interval 18 hrs, two statistically significant increases in the number of aberrant cells (both 5.5 %) exceeding our historical control data range (0.0 - 4.0 % aberrant cells, excluding gaps) were observed. In addition, at preparation interval 28 hrs, two statistically significant and biologically relevant increases in the number of aberrant cells, excluding gaps (9.0 and 10.5 %, respectively) were observed at the scored concentrations. 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.

In conclusion, it can be stated that 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. Therefore, the test item is considered to be clastogenic in this chromosome aberration test in the absence of S9 mix.

 

The in vivo micronucleus study was performed 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 b.w..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.

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. In comparison to the corresponding vehicle controls there was no biologically relevant or statistically significant enhancement in the frequency of the detected micronuclei at any preparation interval after administration of the test item and with any dose level used. 40 mg/kg b.w. cyclophosphamide administered orally was used as positive control which showed a substantial increase of induced micronucleus frequency. In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse. Therefore, the test item is considered to be non-mutagenic in this micronucleus assay. The in vivo test indicated the negative result although the in vitro chromosome aberration test indicated a positive result. The in vivo test result is more reliable for its more similarity to the human metabolic environment. In addition, the Ames test and the HPRT test showed the negative results as well. Thus, it can be concluded that the test substance indicates no genetic toxicity under the experimental conditions used.

Justification for classification or non-classification

Based on the outcome of three in vitro and one in vivo mutagenicity experiment, it is concluded that the substance FAT 40825 is not mutagenic and thus does not require classification for mutagenicity according to CLP (Regulation EC No 1272/2008).