[No public or meaningful name is available]

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Reactive Red 280 was not mutagenic in the bacterial reverse mutation assay, however, a clastogenic effect was seen in the in vitro chromosomal aberration assay. The source chemical, Reactive Red 238, was negative in the in vitro mammalian cell gene mutation assay.

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 - 13 December 2004; Experiment start date - 06 January 2005; Experiment end date - 11 May 2005; Study completion date - 30 May 2005.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Japanese Guideline: Kanpoan No. 287 - Environmental Agency

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Japanese Guideline: Eisei No. 127 - Ministry of Health & Welfare

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Japanese Guideline: Heisei 09/10/31 Kikyoku No. 2 - Ministry of International Trade & Industry

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 40819/A
Description: Red brown powder
Batch number: Red ROE 420 BOP 01/04
Purity: approx. 77 %
Stability of test item: Stable under storage condition
Expiry date: 02 November 2009
Stability of test item dilution: Stable in PEG 300 for at least 7 days at room temperature
Storage conditions: At room temperature.

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, D-64287 Darmstadt) 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 cm2 plastic flasks (GREINER, D-72632 Frickenhausen). About 5 x10E5 cells per flask were seeded into 15 mL of MEM (Minimal Essential Medium; SEROMED; D-12247 Berlin) supplemented with 10 % fetal calf serum (FCS; PAA Laboratories GmbH, D-35091 Cölbe). The cells were subcultured 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:

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 (supplied from RCC Ltd; Laboratory Animal Services, CH- 4414 Füllinsdorf) 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 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 35.3 mg/mL (Lot. no. 221004) in all experimental parts.

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:

See any other information on materials and methods

Vehicle / solvent:

Deionised water. The final concentration of deionised water in the culture medium was 10 % (v/v). The solvent was chosen to its solubility properties and its relative nontoxicity to the cell cultures.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

With metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

RANGE-FINDER
A pre-test on cell growth inhibition with 4 hrs and 24 hrs treatment was performed in order to determine the toxicity of the test item. Cytotoxicity was determined using concentrations separated by no more than a factor of 2 - √10. The general experimental conditions in this pre-test were the same as described below for the cytogenetic main experiment. The following method was used: In a quantitative assessment, exponentially growing cell cultures (seeding about 40,000 cells/ slide, with regard to the culture time 48 hrs) were treated with the test item for simulating the conditions of the main experiment. A qualitative evaluation of cell number and cell morphology was made 4 hrs and 24 hrs after start of treatment. The cells were stained 24 hrs after start of treatment. Using a 400 fold microscopic magnification the cells were counted in 10 coordinate defined fields of the slides (2 slides per treatment group).

EXPERIMENTAL PERFORMANCE
- Exponentially growing stock cultures more than 50 % confluent are treated with trypsin-EDTA-solution at 37 °C for approx. 5 minutes. Then the enzymatic treatment is stopped by adding complete culture medium and a single cell suspension is prepared. The trypsin concentration for all subculturing steps is 0.5 % (w/v) in Ca-Mg-free salt solution (Invitrogen GIBCO, D-76131 Karlsruhe). Prior to the trypsin treatment the cells are rinsed with Ca-Mg-free salt solution. The cells were seeded into Quadriperm dishes (Heraeus, D-63450 Hanau) which contained microscopic slides (at least 2 chambers per dish and test group). In each chamber 1E4 - 6E4 cells were seeded with regard to the preparation time. The medium was MEM with 10 % FCS (complete medium).
- Exposure duration: Exposure period 4 hours: The culture medium of exponentially growing cell cultures was replaced with serum-free medium (for treatment with S9 mix) or complete medium (for treatment without S9 mix) with 10 % FCS (v/v), containing the test item. For the treatment with metabolic activation 50 µL S9 mix per mL medium were used. Concurrent negative, solvent, and positive controls were performed. After 4 hrs the cultures were washed twice with "Saline G" and then the cells were cultured in complete medium for the remaining culture time. Exposure period 18 and 28 hours: The culture medium of exponentially growing cell cultures was replaced with complete medium (with 10 % FCS) containing different concentrations of the test item without S9 mix. The medium was not changed until preparation of the cells. All cultures were incubated at 37° C in a humidified atmosphere with 1.5 % CO2 (98.5 % air).
- Preparation of the cultures: 15.5 hrs and 25.5 hrs, respectively after the start of the treatment colcemid was added (0.2 µg/mL culture medium) to the cultures. 2.5 hrs later, the cells on the slides were treated 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, D-64293 Darmstadt).
- 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') using NIKON microscopes with 100x oil immersion objectives. Breaks, fragments, deletions, exchanges, and chromosome disintegrations were recorded as structural chromosome aberrations. Gaps were recorded as well but not included in the calculation of the aberration rates. 100 well spread metaphase plates per culture were scored for cytogenetic damage on coded slides, except for the highest evaluated concentration in Experiment II in the absence of metabolic activation, where 200 metaphase plates were scored. Only metaphases with characteristic chromosome numbers of 22 ± 1 were included in the analysis. To describe a cytotoxic effect the mitotic index (% cells in mitosis) was determined. In addition, the number of polyploid cells in 500 metaphase cells per culture was determined (% polyploid metaphases; in the case of this aneuploid cell line polyploid means a near tetraploid karyotype).

Evaluation criteria:

- Acceptability of the test: The chromosome aberration test is considered acceptable if it meets the following criteria:
a) The number of structural aberrations found in the negative and/or solvent controls falls within the range of our historical laboratory control data: 0.0 - 4.0 % aberrant cells, exclusive gaps.
b) The positive control substances should produce significant increases in the number of cells with structural, chromosome aberrations, which are within the range of the laboratory's historical control data.

- Evaluation of results: 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 the 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.
A test item can be classified as aneugenic if: the number of induced numerical aberrations is not in the range of our historical control data (0.0 - 8.5 % polyploid cells).

Statistics:

Statistical significance was confirmed by means of the Fisher's exact test (p < 0.05). However, both biological and statistical significance should be considered together.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with

Genotoxicity:

positive

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:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE FINDING STUDY: In a range finding pre-test on toxicity cell numbers 24 hrs after start of treatment were scored as an indicator for cytotoxicity. Concentrations between 39.1 and 5000 µg/mL were applied. Clear toxic effects were observed after treatment with 1250 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 312.5 µg/mL and above in the absence of S9 mix induced strong toxic effects.

TEST-SPECIFIC CONFOUNDING FACTORS: In the pre-experiment, neither precipitation nor relevant influence of the test item on the pH value or osmolarity was observed (solvent control 291 mOsm, pH 7.4 versus 317 mOsm and pH 7.4 at 5000 µg/mL).

MAIN TEST: In Experiment I, in the absence and the presence of S9 mix and in Experiment II, in the absence of S9 mix, no biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed. The aberration rates of the cells after treatment with the test item (0.0 - 3.8 % aberrant cells, exclusive gaps) were close-to the range of the solvent control values (1.0- 3.0 % aberrant cells, exclusive gaps) and within the range of our historical control data: 0.0 - 4.0 % aberrant cells, exclusive gaps.
In Experiment II, in the absence of S9 mix, at preparation interval 28 hrs a single significant (p < 0.05) increase after evaluation of 200 metaphase plates per culture was observed after treatment with 500 µg/mL. Although this value of 3.8 % aberrant cells, exclusive gaps, was statistically significant compared to the low response (1.0 % aberrant cells) in the solvent control data, the response is within the historical control data range (0.0- 4.0 % aberrant cells). Therefore, the statistical significance has to be regarded as being biologically irrelevant. However, in Experiment II in the presence of S9 mix at preparation interval 28 hrs, a dose related increase in the number of aberrant metaphase cells was observed in the concentration range evaluated: 125, 250, and 500 µg/mL (3.0%, 8.0%, and 15.0%, respectively). The values of the two highest scored concentrations were, statistically sigificant as compared with the respective solvent control group (1.5 % aberrant cells, exclusive gaps) and clearly exceeded our historical control data range (0.0 - 4.0 aberrant cells, exclusive gaps). The dose-dependency of metaphases carrying exchanges in these three test groups (0.5 %, 2.0 %, and 9.0 %, respectively) give additional evidence for a clastogenic potential of the test item. Finally; the dose-dependencies and statistical significances observed in Experiment II in the presence of S9 mix at preparation interval 28 hrs have to be regarded as biologically relevant. In both experiments, no biologically relevant increase in the rate of polyploid metaphases was found after treatment with the test item (0.9 - 3.7 %) as compared to the rates of the solvent controls (0.8 - 3.2 %). In both experiments, EMS (300 and 400 µg/mL, respectively) and CPA (1.0 and 1.4 µg/mL, respectively) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations, except in Experiment II in the presence of S9 mix after exposure with CPA. At this experimental part after 4 hrs treatment with 1.4 µg/mL CPA the aberration rate (2.5 %) was lower than expected and was not within our historical positive control data range: 8.5 % - 22.0 % aberrant cells, exclusive gaps. It has to be proposed that any technical error occurred in this test group. However, the observation of clastogenicity in Experiment II in the presence of S9 mix at preparation interval 28 hrs after treatment with two test item concentrations (250 and 500 µg/mL) give proof for the validity of this experimental part. Therefore, this deviation has no detrimental impact on the validity of the study.

ADDITIONAL INFORMATION ON CYTOTOXICITY: In both cytogenetic experiments, cytotoxicity indicated by reduced cell numbers or mitotic indices below 50 % of control was observed. In detail, in the absence of S9 mix, clearly reduced cell numbers were observed after 4 hrs treatment with 1500 µg/mL (22.0% of control) in Experiment I, and after 28 hrs treatment with 500 µg/mL (37.4 % of control) in Experiment II. In addition, in Experiment II in the presence of S9 mix, at preparation interval 28 hrs the cell numbers were distinctly reduced after treatment with 500 µg/mL (47.8 % of control). Besides, in Experiment II in the absence of S9 mix, the mitotic indices were clearly reduced after 28 hrs continuous treatment with 500 µg/mL (38.5 % of control). However, in Experiment I in the presence of S9 mix and in Experiment II after 18 hrs treatment in the absence of S9 mix, concentrations showing clear toxic effects were not scorable for cytogenetic damage.

Conclusions:

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

Executive summary:

In a GLP-compliant chromosome aberration test, performed according to OECD guideline 473, Chinese hamster V79 cells, were exposed to the test substance, with and without metabolic activation by S9 mix in two 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. Dose selection for the cytogenetic experiments was performed considering the toxicity data available from the pre-test. Toxic effects indicated by reduced cell numbers or mitotic indices of below 50 % of control were observed in both cytogenetic experiments. However, in Experiment I in the presence of S9 mix and in Experiment II after 18 hrs treatment in the absence of S9 mix, concentrations showing clear cytotoxicity were not scorable for cytogenetic damage. No biologically relevant increase of the aberration rates was observed in Experiment I and II in the absence of S9 mix and in Experiment I in the presence of S9 mix. In contrast, in Experiment II, in the presence of S9 mix, a dose-related increase in the number of cells carrying structural chromosomal aberrations with statistically significant and biologically relevant values 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. In conclusion, it can be stated that under the experimental conditions reported, the test item did induce structural chromosome aberrations. Therefore, the test substance is considered to be clastogenic in the 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 - 14 December 2004; Experiment start date - 25 January 2005; Experiment end date - 04 February 2005; Study completion date - 14 March 2005.

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:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Japanese Guideline: Kanpoan No. 287 - Environment Protection Agency

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Japanese Guideline: Eisei No. 127 - Ministry of Health & Welfare

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Japanese Guideline: 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 40819/A
Description: Red brown powder
Batch number: Red ROE 420 BOP 01/04
Purity: approx. 77 %
Stability of test item: Stable under storage condition
Expiry date: 02 November 2009
Stability of test item dilution: Stable in PEG 300 for at least 7 days at room temperature
Storage conditions: At room temperature.

Target gene:

histidine and tryptophan

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 according to B. Ames et al. and D. Maron and B. Ames. In this way it was ensured that the experimental conditions set down by Ames were fulfilled.
Storage:
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:

Phenobarbital/ß-Naphthoflavone induced rat liver S9 is used as the metabolic activation system. The S9 is prepared from 8 - 12 weeks old male Wistar Hanlbm rats, weight approx. 220 - 320 g induced by applications of 80 mg/kg b.w. Phénobarbital 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. 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. The protein concentration in the S9 preparation was 27.1 mg/mL (lot no. R 041104) in the pre-experiment and in experiment I and 36.7 mg/mL (lot no. R 100904) in experiment II.

S9Mix
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 mM 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:

- Vehicle(s)/solvent(s) used: deionised water
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene, 2-AA

Remarks:

Strain TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA, with metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Strain WP2 uvrA, without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Strain TA 1535 and TA 100, without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

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

Remarks:

Strain TA 1537 and TA 98, without metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar

NUMBER OF REPLICATIONS: 3

STORAGE AND PRECULTURE
The strain cultures were stored as stock cultures in ampoules with nutrient broth +5 % DMSO (MERCK, D-64293 Darmstadt) in liquid nitrogen. From the thawed ampoules of the strains 0.5 mL suspension was transferred into 250 mL Erlenmeyer flasks containing 20 mL nutrient medium. A solution of 20 µL ampicillin (25 µg/mL) was added to the strains TA 98 and TA 100. The bacterial cultures were incubated in a shaking water bath for 4 hours at 37 °C.

DETERMINATION OF CYTOTOXICITY
To evaluate the toxicity of the test item a pre-experiment was performed with strains TA 1535, TA 1537, TA 98, TA 100, and WP2 uvrA. Eight concentrations were tested for toxicity and mutation induction with three plates each. The experimental conditions in this pre-experiment were the same as for the experiment I (plate incorporation test). Toxicity of the test item results in a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn. The pre-experiment is reported as main experiment I, if the following criteria are met: Evaluable plates (>0 colonies) at five concentrations or more in all strains used.

DOSE SELECTION
In the pre-experiment the concentration range of the test item was 3 - 5000 µg/plate. The pre-experiment is reported as experiment I since no relevant toxic effects were observed and 5000 µg/plate were chosen as maximal concentration.

EXPERIMENTAL PERFORMANCE
In the pre-incubation assay 100 µL test solution, 500 µL S9 mix / S9 mix substitution buffer, 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. The colonies were counted using the Petri Viewer Mk2 (Perceptive Instruments Ltd, Suffolk CB 7BN, UK) with the software program Ames Study Manager.

Evaluation criteria:

ACCEPTABILITY OF THE ASSAY:
The Salmonella typhimurium and Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria: regular background growth in the negative and solvent control, the spontaneous reversion rates in the negative and solvent control are in the range of our historical data, the positive control substances should produce a significant increase in mutant colony frequencies.

EVALUATION OF RESULTS
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:

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

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

The test substance is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Executive summary:

In a GLP-compliant reverse mutation assay, performed according to OECD guideline 471, 4 Salmonella typhimurium strains (TA 1535, TA 1537, TA 98, and TA 100) and 1 Escherichia coli strain WP2 uvrA, were used to the test the mutagenic potential of the test substance (33, 100, 333, 1000, 2500, 5000 µg per plate), both with and without metabolic activation. 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 occurred in the test groups with and without metabolic activation, and no substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test substance at any dose level, neither in the presence nor absence of metabolic activation. There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. 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. Therefore, the test substance is 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 - 05 Devember 2012; Experiment start date - 12 December 2012; Experiment completion date - 14 March 2013; Study completion date - 16 April 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 40348/F TE
Batch No: BOP 01-12 (Lot: BS-1109708)
Physical State: solid powder
Colour: red-brown
Storage Conditions: at room temperature
Molecular weight: 982.96
Active components: sum of all coloured substance: 82.37 %
Main constituents: 46.60 %
Date of Analysis: 21 August 2012
Expiry Date: 06 February 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

Source:
V79 cells in vitro have been widely 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 - 14 h 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 the study. The V79 cells (ATCC, CCL-93) were stored over liquid nitrogen (vapour phase) in the cell bank of BSL BIOSERVICE. This allows the repeated use of the same cell culture batch in experiments. Each cell batch was routinely checked for mycoplasma infections (PCR). Thawed stock cultures were maintained in plastic culture flasks in minimal essential medium (MEM). For purifying the cell population of pre-existing HPRT" mutants cells were exposed to HAT medium containing 100 µM hypoxanthine, 0.4 µM aminopterin, 16 µM thymidine and 10.0 µM glycine for several cell doublings (2-3 days).

Metabolic activation:

with and without

Metabolic activation system:

Liver S9 of Wistar Phenobarbital and ß-Naphthoflavone-induced rat liver S9 mix and Liver S9 of Sprague Dawley Phenobarbital and ß-Naphthoflavone-induced rat liver S9 mix.

For the pre-experiment the S9 liver microsomal fraction was prepared at BSL BIOSERVICE GmbH. Male Wistar rats were induced with Phenobarbital (80 mg/kg bw) and J3-Naphthoflavone (100 mg/kg bw) for three consecutive days by oral route.
The following quality control determinations were performed:
a) Biological activity in:
- the Salmonella typhimurium assay using 2-aminoanthracene and benzo[a]pyrene
- the mouse lymphoma assay using benzo[a]pyrene
- the chromosome aberration assay using cyclophosphamide.
b) Sterility Test
A stock of the supernatant containing the microsomes was frozen in ampoules of 2 and 4 mL and stored at ≤- 75 °C.
The protein concentration in the S9 preparation (Lot: 190712) was 37 mg/mL. The S9 mix preparation was performed according to Ames et al. For the main experiments the S9 liver microsomal fraction was obtained from Trinova Biochem GmbH, Giessen, Germany. Male Sprague Dawley rats were induced with phenobarbital ß-Naphthoflavone. The following quality control determinations were performed by Trinova Biochem GmbH:
a) Alkoxyresorufin-0-dealkylase activities
b) Test for the presence of adventitious agents
c) Promutagen activation (including biological activity in the Salmonella typhimurium assay using 2-aminoanthracene and benzo[a]pyrene)
The following additional quality control determination was performed by BSL BIOSERVICE GmbH:
Biological activity in:
- the mouse lymphoma assay using benzo[a]pyrene
- the HPRT assay using 7,12-Dimethylbenz[a]anthracene
A stock of the supernatant containing the microsomes is frozen in aliquots of 5 mL and stored at ≤-75 °C. The protein concentration in the S9 preparation (Lot: 2996) was 47. 7 mg/mL.

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
Pre-experiment for experiment II (only without metabolic activation, 20 h long-term exposure assay): 2.5, 5, 10, 25, 50, 75, 100, 250, 500, 750 µg/mL

Experiment I
without metabolic activation: 2.5, 5, 10, 25, 50, 100, 200, 300 and 400 µg/mL
and with metabolic activation: 0.25, 0.5, 1, 10, 20, 40, 60 and 80 µg/mL
Experiment II
without metabolic activation: 25, 50, 100, 250, 500, 750, 1000, 1400, 1800 and 2200 µg/mL
and with metabolic activation: 0.4, 0.8, 1.5, 3, 6, 12, 25, 50, 75 and 100 µg/mL

Vehicle / solvent:

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

Medium MEM

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

With metabolic activation; 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: ≥25 μg/mL; experiment I with S9: ≥10 μg/mL; Experiment II without S9: ≥500 μg/mL; Experiment II with S9:≥12 μg/mL

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

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 10.9 % for the highest concentration (400 µg/mL) evaluated. The highest biologically relevant concentration evaluated with metabolic activation was 80 µg/mL with a relative growth of 12.8 %.

In experiment II without metabolic activation the relative growth was 15.0 % for the highest concentration (2200 µg/mL) evaluated. The highest concentration evaluated with metabolic activation was 100 µg/mL with a relative growth of 16.7 %.

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 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 26.57 and 10.68, of the solvent control 11.11 and 25.00 mutants/10E6 cells and in the range of 2.40 to 33.18 mutants/10E6 cells with the test item, respectively. The highest mutation rate (compared to the solvent control values) of 1.84 was found at a concentration of 400 µg/mL with a relative growth of 10.9 %.
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 30.30 and 22.66, of the solvent control 21.67 and 18.60 mutants/10E6 cells and in the range of 1.91 to 39.80 mutants/ 10E6 cells with the test item, respectively. The highest mutation rate (compared to the solvent control values) of 1.98 was found at a concentration of 20 µg/mL with a relative growth of 29.4 %.
In experiment II without metabolic activation all 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 15.38, and 25.20 of the solvent control 21.09 and 10.77 mutants/10E6 cells and in the range of 8.73 to 31.80 mutants/10E6 cells with the test item, respectively. The highest mutation rate (compared to the solvent control values) of 2.00 was found at a concentration of 1000 µg/mL with a relative growth of 35.8 %.
In experiment II with metabolic activation all 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 29.37 and 28.85, of the solvent control 30.71 and 17.67 mutants/10E6 cells and in the range of 11.07 to 43.03 mutants/10E6 cells with the test item, respectively. The highest mutation rate (compared to the solvent control values) of 1.78 was found at a concentration of 75 µg/mL with a relative growth of 26.1 %. DMBA (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:

FAT 40348/F TE is considered to be non-mutagenic in the HPRT locus using V79 cells of the Chinese Hamster.

Executive summary:

The test item FAT 40348/F TE was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster. This study was conducted according to OECD test guideline 476 in a GLP-certified laboratory. V79 cells cultured in vitro were exposed to FAT 40348/F TE at concentrations of

- 2.5, 5, 10, 25, 50, 100, 200, 300 and 400 µg/mL (without metabolic activation, Experiment I)

- 0.25, 0.5, 1, 10, 20, 40, 60 and 80 µg/mL (with metabolic activation, Experiment I)

- 25, 50, 100, 250, 500, 750, 1000, 1400, 1800 and 2200 µg/mL (without metabolic activation, Experiment II)

- 0.4, 0.8, 1.5, 3, 6, 12, 25, 50, 75 and 100 µg/mL (with metabolic activation, Experiment II).

FAT 40348/F 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 10.9 % for 400 µg/mL evaluated. The highest biologically relevant concentration evaluated with metabolic activation was 80 µg/mL with a relative growth of 12.8 %. In experiment II without metabolic activation the relative growth was 15.0% for the highest concentration (2200 µg/mL) evaluated. The highest concentration evaluated with metabolic activation was 100 µg/mL with a relative growth of 16.7 %. In experiment I without metabolic activation the highest mutation rate (compared to the solvent control values) of 1.84 was found at a concentration of 400 µg/mL with a relative growth of 10.9 %. In experiment I with metabolic activation the highest mutation rate (compared to the solvent control values) of 1.98 was found at a concentration of 20 µg/mL with a relative growth of 29.4 %. In experiment II without metabolic activation the highest mutation rate (compared to the solvent control values) of 2.00 was found at a concentration of 1000 µg/mL with a relative growth of 35.8 %. In experiment II with metabolic activation the highest mutation rate (compared to the solvent control values) of 1.78 was found at a concentration of 75 µg/mL with a relative growth of 26.1 %. The positive controls induced the appropriate response. There was no evidence of a concentration related positive responseof induced mutant colonies over background. This study is classified as acceptable. This study satisfies the requirement for Test Guideline OPPTS 870.5300, OECD 476 for in vitro mutagenicity (mammalian forward gene mutation) data.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

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

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 June 2005; Experiment start date - 14 June 2005; Experiment end date - 01 August 2005; Study completion date - 06 October 2005.

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:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Specific details on test material used for the study:

Identity: FAT 40819/A
Description: Red brown powder
Batch number: Red ROE 420 BOP 01/04
Purity: approx. 77 %
Stability of test item: Stable under storage condition
Expiry date: 02 November 2009
Stability of test item dilution: Stable in PEG 300 for at least 7 days at room temperature
Storage conditions: At room temperature.

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Winkelmann GmbH, D-33178, Borchen
- Age at start acclimatization: 8-10 weeks
- Weight at start of treatment: Males 40.0 g (SD ± 3.4 g), Females: 31.5 g (SD ± 1.6 g)
- Assigned to test groups randomly: yes
- Housing: Individually, in Makrolon Type I cages, with wire mesh top (EHRET GmbH, D-79302 Emmendingen) with granulated soft wood bedding (Harlan Winkelmann GmbH, D-33178 Borchen).
- Diet: Pelleted standard diet, ad libitum (Harlan Winkelmann GmbH, D-33178 Borchen).
- Water: tap water, ad libitum, (Gemeindewerke, D-64380 Roßdorf)
- Acclimation period: minimum 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

Deionised water. The vehicle was chosen to its relative non-toxicity for the animals.
Volume: 20 mL/kg bw

Details on exposure:

On the day of the experiment, the test item was formulated.

Frequency of treatment:

Single treatment

Post exposure period:

24 hours for all doses, 48 hours for the 2000 mg/kg bw dose group.

Dose / conc.:

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

Remarks:

Negative control

Dose / conc.:

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

Remarks:

Low dose

Dose / conc.:

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

Remarks:

Mid dose

Dose / conc.:

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

Remarks:

High dose

No. of animals per sex per dose:

6

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide;
- Route of administration: orally
- Doses: 40 mg/kg bw
- Volume administrated: 10 mL/kg bw

Tissues and cell types examined:

Normochromatic and polychromatic erythrocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
- A preliminary study on acute toxicity was performed with two animals per sex under identical conditions as in the mutagenicity study concerning: animal strain; vehicle; route, frequency, and volume of administration. The animals were treated orally with the test item and examined for acute toxic symptoms at intervals of around 1 h, 2-4 h, 6 h, 24 h, 30 h, and 48 h after administration of the test item.
- The maximum tolerated dose level is determined to be the dose that causes toxic reactions without having major effects on survival within 48 hours.
- Three adequate spaced dose levels spaced by a factor of 2 were applied at the central sampling interval 24 h after treatment. For the highest dose level an additional sample was taken at 48 h after treatment.

DETAILS OF SLIDE PREPARATION:
- The animals were sacrificed using CO2 following by bleeding. The femora were removed, the epiphyses were cut off and the marrow was flushed out with fetal calf serum, using a syringe. The cell suspension was centrifuged at 1500 rpm (390 x g) for 10 minutes and the supernatant was discarded. A small drop of the resuspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Grünwald (MERCK, D-64293 Darmstadt)/Giemsa (Gurr, BDH Limited Poole, Great Britain). Cover slips were mounted with EUKITT (KINDLER, D-79110 Freiburg). At least one slide was made from each bone marrow sample.

METHOD OF ANALYSIS:
- Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives. At least 2000 polychromatic erythrocytes (PCE) were analysed per animal for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and expressed in polychromatic erythrocytes per 2000 erythrocytes. The analysis was performed with coded slides. Ten animals (5 males, 5 females) per test group were evaluated as described.

Evaluation criteria:

The study was considered valid as the following criteria are met: the negative controls are in the range of our historical control data; the positive controls are in the range of our historical control data; at least 4 animals per group and sex can be evaluated; PCE to erythrocyte ratio should not be less than 20 % of the negative control.

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. 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 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.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Conclusions:

The test substance did not induce micronuclei in bone marrow cells of the mouse.

Executive summary:

In a GLP-compliant erythrocyte micronucleus test, tested according to OECD guideline 474, 6 NMRI mice per sex were treated once by oral gavage with the test substance (500, 1000, 2000 mg/kg bw) dissolved in water followed by a 24 or 48 hours post exposure period. Ten animals (5 males, 5 females) per test group were evaluated for the occurrence of micronuclei and at least 2000 polychromatic erythrocytes (PCEs) per animal were scored. The mean number of polychromatic erythrocytes was not decreased after treatment with the test item as compared to the mean value of PCEs of the vehicle control indicating that the test substance had no cytotoxic properties in the bone marrow. The analysis of the plasma samples of the animals treated with 2000 mg/kg b.w., showed, that significant levels (38.8 - 67.7 µg/mL) of the test item could be detected in the plasma 1 hour after treatment. The level dropped to 2.1 - 4.1 µg/mL at 4 hours sampling interval. Thus, the bioaviability of the test item could be confirmed. In comparison to the corresponding vehicle controls there was no statistically significant or biologically relevant enhancement in the frequency of the detected micronuclei at any preparation interval and dose level after administration of the test item. The mean values of micronuclei observed after treatment with the test substance were below or near to the value of the vehicle control group. In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test item did not induce micronuclei as determined by the micronucleus test in the bone marrow cells of the mouse.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Genetic toxicity in vitro, Reverse mutation assay:

In a GLP-compliant reverse mutation assay, performed according to OECD guideline 471, 4 Salmonella typhimurium strains (TA 1535, TA 1537, TA 98, and TA 100) and 1 Escherichia coli strain WP2 uvrA, were used to the test the mutagenic potential of the test substance (33, 100, 333, 1000, 2500, 5000 µg per plate), both with and without metabolic activation (RCC 2005). 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 occurred in the test groups with and without metabolic activation, and no substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test substance at any dose level, neither in the presence nor absence of metabolic activation. There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. 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 pairchanges or frame shifts in the genome of the strains used. Therefore, the test substance is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Genetic toxicity in vitro, Chromosomal aberration test:

In a GLP-compliant chromosome aberration test, performed according to OECD guideline 473, Chinese Hamster V79 cells, were exposed to the test substance, with and without metabolic activation by S9 mix in two independent experiments (RCC 2005). In each experimental group two parallel cultures were set up. Per culture at least 100 metaphase plates were scored for structural chromosome aberrations. Dose selection for the cytogenetic experiments was performed considering the toxicity data available from the pre-test. Toxic effects indicated by reduced cell numbers or mitotic indices of below 50 % of control were observed in both cytogenetic experiments. However, in Experiment I in the presence of S9 mix and in Experiment II after 18 hrs treatment in the absence of S9 mix, concentrations showing clear cytotoxicity were not scorable for cytogenetic damage. No biologically relevant increase of the aberration rates was observed in Experiment I and II in the absence of S9 mix and in Experiment I in the presence of S9 mix. In contrast, in Experiment II, in the presence of S9 mix, a dose-related increase in the number of cells carrying structural chromosomal aberrations with statistically significant and biologically relevant values 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. In conclusion, it can be stated that under the experimental conditions reported, the test item did induce structural chromosome aberrations. Therefore, the test substance is considered to be clastogenic in the presence of S9 mix.

 

Genetic toxicity in vitro HPRT (read across with Reactive Red 238)

Currently no in vitro mammalian cell gene mutation assay is available for 40819, however, a similar substance, Reactive Red (238) FAT 40348/F was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese Hamster. This study was conducted according to OECD test guideline 476 in a GLP-certified laboratory. V79 cells cultured in vitro were exposed to FAT 40348/F at concentrations of 

- 2.5, 5, 10, 25, 50, 100, 200, 300 and 400 µg/mL (without metabolic activation, Experiment I)

- 0.25, 0.5, 1, 10, 20, 40, 60 and 80 µg/mL (with metabolic activation, Experiment I)

- 25, 50, 100, 250, 500, 750, 1000, 1400, 1800 and 2200 µg/mL (without metabolic activation, Experiment II)

- 0.4, 0.8, 1.5, 3, 6, 12, 25, 50, 75 and 100 µg/mL (with metabolic activation, Experiment II).

FAT 40348/F 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 10.9 % for 400 µg/mL evaluated. The highest biologically relevant concentration evaluated with metabolic activation was 80 µg/mL with a relative growth of 12.8 %. In experiment II without metabolic activation the relative growth was 15.0 % for the highest concentration (2200 µg/mL) evaluated. The highest concentration evaluated with metabolic activation was 100 µg/mL with a relative growth of 16.7 %. In experiment I without metabolic activation the highest mutation rate (compared to the solvent control values) of 1.84 was found at a concentration of 400 µg/mL with a relative growth of 10.9 %. In experiment I with metabolic activation the highest mutation rate (compared to the solvent control values) of 1.98 was found at a concentration of 20 µg/mL with a relative growth of 29.4 %. In experiment II without metabolic activation the highest mutation rate (compared to the solvent control values) of 2.00 was found at a concentration of 1000 µg/mL with a relative growth of 35.8 %. In experiment II with metabolic activation the highest mutation rate (compared to the solvent control values) of 1.78 was found at a concentration of 75 µg/mL with a relative growth of 26.1 %. The positive controls induced 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.

 

Genetic toxicity in vivo, Micronucleus test:

In a GLP-compliant erythrocyte micronucleus test, tested according to OECD guideline 474, 6 NMRI mice per sex were treated once by oral gavage with the test substance (500, 1000, 2000 mg/kg bw) dissolved in water followed by a 24 or 48 hours post exposure period (RCC 2005). Ten animals (5 males, 5 females) per test group were evaluated for the occurrence of micronuclei and at least 2000 polychromatic erythrocytes (PCEs) per animal were scored. The mean number of polychromatic erythrocytes was not decreased after treatment with the test item as compared to the mean value of PCEs of the vehicle control indicating that the test substance had no cytotoxic properties in the bone marrow. The analysis of the plasma samples of the animals treated with 2000 mg/kg b.w. showed, that significant levels (38.8 - 67.7 µg/mL) of the test item could be detected in the plasma 1 hour after treatment. The level dropped to 2.1 - 4.1 µg/mL at 4 hours sampling interval. Thus, the bioavailibility of the test item could be confirmed. In comparison to the corresponding vehicle controls there was no statistically significant or biologically relevant enhancement in the frequency of the detected micronuclei at any preparation interval and dose level after administration of the test item. The mean values of micronuclei observed after treatment with the test substance were below or near to the value of the vehicle control group. In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test item did not induce micronuclei as determined by the micronucleus test in the bone marrow cells of the mouse.

Conclusion:

Reactive Red 280 was not mutagenic in the bacterial reverse mutation assay. Though a clastogenic effect is seen in the in vitro chromosomal aberration assay, it could not be reproduced in the in vivo micronucleus assay. Hence, Reactive Red 280 was concluded to be not genotoxic and to fulfill the requirements of Annex VIII of REACH regulation. Similarly, the source chemical, Reactive Red 238, was found to be non-mutagenic in the bacterial reverse mutation assay as well as in vitro mammalian cell gene mutation assay. Similar to the target substance, it was clastogenic in the in vitro chromosomal aberration assay, however this effect could not be reproduced in an in vivo micronucleus assay. As the source chemical was found to be not mutagenic in the in vitro mammalian cell gene mutation assay, this study was used for read-across, to add strength to already available data.

Justification for classification or non-classification

Based on the available genotoxicity studies, the test substance does not need to be classified for genotoxicity according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.