2-Naphthalenesulfonic acid, 7-amino-4-hydroxy-8-[2-[2-sulfo-4-[[2-(sulfooxy)ethyl]sulfonyl]phenyl]diazenyl]-, potassium sodium salt (1:?:?), coupled with diazotized 2-[(4-amino-5-methoxy-2-methylphenyl)sulfonyl]ethyl hydrogen sulfate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Reactive Red 278 did not lead to mutations in the in vitro mammalian cell gene mutation assay nor caused DNA damage in the unscheduled DNA synthesis assay. But positive outcomes were reported with E. coli WP2 uvrA strain in the bacterial reverse mutation assay and the in vitro chromosomal aberration assay with Chinese Hamster V79 cells.

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 May 2003; Experiment start date - 21 May 2003; Experiment end date - 02 July 2003; Study completion date - 10 September 2003.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 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

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 40812/A
Batch: WP 8/03
Purity: approx. 75 %
Appearance: Solid, dark red-brownish powder
Expiration date: 23 April 2010
Storage: At room temperature at about 20 °C

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, 0-72632 Frickenhausen). About 5 x 10E5 cells per flask were seeded into 15 ml of MEM (Minimal Essential Medium; SEROMED; D-12247 Berlin) supplemented with 10 % fetal calf serum (FCS; PM Laboratories GmbH, D-35091 Colbe). 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:

S9 preparation:
Phenobarbital/ß-Naphthoflavone induced rat liver S9 was used as the metabolic activation system. The S9 was prepared from 8 - 12 weeks old male Wistar Hanlbm rats, weight approx. 220 - 320 g (supplied from RCC Ltd; Biotechnology & Animal Breeding Division, CH-4414 Fullinsdorf) 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 KCI solution (1:3 parts respectively) followed by centrifugation at 9000 g. Aliquots of the supernatant were frozen and stored in ampoules at -20 °C. Small numbers of the ampoules were kept at -20 °C for up to one week. The protein concentration was 26.2 mg/ml (Lot. no. 070303) in the pre-test and in the main experiment.

S9 Mix
An appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution to result in a final protein concentration of 0.75 mg/ml in the cultures. Cofactors were added to the S9 mix to reach the following concentrations:
8 mM MgCl2
33 mM KCI
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 incl. tables.

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-FINDING
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 37,630 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. 24 hrs after start of treatment the cells were stained. Using a 400 fold microscopic magnification the cells were counted in 10 coordinate defined fields of the slides (2 slides per treatment group). The cell number of the treatment groups is given as % cells in relation to the control.

DOSE SELECTION
The highest concentration used in the cytogenetic experiments was chosen with regard to the current OECD Guideline for in vitro mammalian cytogenetic tests requesting for the top concentration clear toxicity with reduced cell numbers or mitotic indices below 50 % of control, whichever is the lowest concentration, and/or the occurrence of precipitation. In case of nontoxicity the maximum concentration should be 5 mg/mL, 5 µL/mL or 10 mM, whichever is the lowest, if formulability in an appropriate solvent is possible. 5000 µg/mL of FAT 40812/A were applied as top concentration for treatment of the cultures in the pre-test. Test item concentrations between 39.1 and 5000 µg/mL (with and without S9 mix) were chosen for the evaluation of cytotoxicity.
Using reduced cell numbers as an indicator for toxicity in the pre-test, clear toxic effects were observed after 4 hrs treatment with 2500 µg/mL and above in the absence of S9 mix and with 1250 µg/mL and above in the presence of S9 mix. In addition, 24 hours treatment with 625 µg/mL and above in the absence of S9 mix induced strong toxic effects. Considering the toxicity data of the pre-test, 2500 µg/mL (without S9 mix) and 1500 µg/mL (with S9 mix) were chosen as top concentration in the main experiment. Since the test item was considered to be clastogenic after 4 hours treatment a second experiment was not performed.

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 1 x 10E4 - 6 x 10E4 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.
- Preparation of the Cultures: 15.5 hrs 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 additional two cultures per test item and solvent control group, not treated with colcemid, were set up in parallel. These cultures were stained after 18 hrs 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 test item concentration 500 µg/mL with 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 %.
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 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.

Although the inclusion of the structural chromosome aberrations is the purpose of this study, it is important to include the polyploids and endoreduplications. A test item can be classified as mutagenic 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. 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.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

- In a range finding pre-test on toxicity, clear toxic effects were observed after 4 hrs treatment with 2500 µg/mL and above in the absence of S9 mix and with 1250 µg/mL and above in the presence of S9 mix. In addition, 24 hours treatment with 625 µg/mL and above in the absence of S9 mix induced strong toxic effects.

- In the pre-experiment, neither precipitation in cell culture medium nor relevant influence of the test item on the pH value or osmolarity was observed (solvent control 297 mOsm, pH 7.4 versus 315 mOsm and pH 7.4 at 5000 µg/mL).

- In the cytogenetic experiment, toxic effects indicated by reduced cell numbers of below 50 % of control were observed in after 4 hours treatment with 1500 µg/mL (42 % of control) in the absence of S9 mix and with 500 µg/mL (49 % of control) in the presence of S9 mix. In contrary, no clearly reduced mitotic indices of below 50 % of control could be observed up to the highest evaluated concentrations of the test item. Additionally, at cytotoxic test item concentrations indicated by reduced cell numbers extremely high mitotic rates were found. In detail, after 4 hours treatment with 2000 µg/mL in the absence of S9 mix and with 750, 1000, and 1500 µg/mL in the presence of S9 mix the mitotic indices were strongly increased (155 %, 308 %, 313 %, and 255 % of control, respectively). This means that surviving cells have an extremely high mitotic activity. This is no usual finding and gives an evidence for test item induced cell transformation.

- In the absence of S9 mix, the aberration rates were statistically significant (p<0.05) increased after treatment with 500 and 1500 µg/mL as compared to the corresponding solvent control (0.0 %). Besides the responses after treatment with 1500 µg/mL (9.0 % aberrant cells, exclusive gaps) was biologically relevant clearly exceeding our historical control data ratio: 0.0 – 4.0 % aberrant cells, exclusive gaps. Also, at this concentration the number of cells carrying exchanges was distinctly increased (2.5 %) as compared to the solvent control (0.0 %) and give additional evidence for a clastogenic potential of the test item. Therefore, these observations have to be regarded as being biologically relevant.
In the presence of S9 mix, a dose related increase in the number of cells carrying structural chromosome aberrations (1.0 %, 1.5 %, and 4.3 % of control) was observed in the evaluated concentration range (125, 250, and 500 µg/mL, respectively). The response of the highest concentration, confirmed in an increased sample for evaluation of 400 metaphase plates in total, slightly exceeded our historical control data range: 0.0 - 4.0 % aberrant cells, exclusive gaps. Additionally, at pre-evaluation of the slides distinct increased numbers of micronucleated cells and of cells containing fragmented nuclei were observed at the three highest test item concentrations not evaluated for cytogenetic damage (750, 1000, and 1500 µg/mL). Therefore, the sum of observations has to be regarded as biologically relevant.
No biologically relevant increase in the rate of polyploid metaphases was found after treatment with the test item (2.0 - 3.2 %) as compared to the rates of the solvent control (2.8 - 3.0 %). EMS (200 µg/mL) and CPA (0.7 µg/mL) was used as positive controls and showed distinct increases in cells with structural chromosome aberrations.

Conclusions:

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

Executive summary:

In a GLP-compliant chromosome aberration test, tested according to OECD guideline 473, Chinese hamster V79 cells (in vitro), were exposed to the test substance, with and without metabolic activation by S9 mix. Two independent experiments were performed. The exposure period was 4 hrs with and without metabolic activation. In each experimental group two parallel cultures were set up. Per culture 100 metaphase plates were scored for structural chromosome aberrations, except for the test concentration 500 µg/mL with metabolic activation where 200 metaphase plates were scored. 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 of the cytogenetic experiments was performed considering the toxicity data. In both experiments, clear toxic effects indicated by reduced mitotic indices or reduced cell numbers were observed after treatment with the test item. Clear toxic effects indicated by reduced cell numbers of below 50 % of control were observed in both experimental parts at the highest evaluated concentrations. In contrary no clearly reduced mitotic indices were observed at the test item concentrations evaluated. But after treatment with 2000 µg/mL in the absence of S9 mix and with 750, 1000, and 1500 µg/mL in the presence of S9 mix extremely high mitotic indices were observed. In the absence and presence of S9 mix, biologically relevant increases in the number of cells carrying structural chromosomal aberrations were observed after treatment with the test item at concentrations showing clear reduced cell numbers. No increase in the frequencies of polypoid metaphases were found after treatment with the test item as compared to the frequencies of the controls. In conclusion, the test substance is considered to be clastogenic in this chromosome aberration test in the absence and 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 - 21 May 2003; Experiment start date - 13 June 2003; Experiment end date - 24 July 2003; Study completion date - 05 August 2003.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

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 40812/A
Batch: WP 8/03
Purity: approx. 75 %
Appearance: Solid, dark red-brownish powder
Expiration date: 23 April 2010
Storage: At room temperature at about 20 °C

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):

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 Ames et al.

The bacterial strains TA 1535, TA 1537, and TA 100 were obtained from Ames
(University of California, 94720 Berkeley, U.S.A.). The bacterial strain TA 98 was
obtained from E. Merck (D-64293 Darmstadt). The Escherichia coli strain WP2 uvrA was obtained from RCC Ltd. (CH-4332 Stein).

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/ß-Naphthoflavone induced rat liver S9 mix.

Test concentrations with justification for top dose:

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:

With metabolic activation: Strain TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Methyl methane sulfonate, MMS

Remarks:

Without metabolic activation: Strain WP2 uvrA,

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Without metabolic activation - Strain TA 1535 and TA 100

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

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

Remarks:

Without metabolic activation - Strain TA 1537 and TA 98

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. The concentration range included two logarithmic decades. The following concentrations were tested: 33; 100; 333; 1000; 2500; and 5000 µg/plate

EXPERIMENTAL PERFORMANCE
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. The colonies were counted using the AUTOCOUNT (Artek Systems Corporation, BIOSYS GmbH, D-61184 Karben).

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.

Statistics:

No statistical evaluation of the data is required.

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

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:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

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 S9 mix in both experiments. A minor toxic effect, evident as a reduction in the number of revertants was only observed in strain TA 1535 at 2500 µg/plate in the presence of metabolic activation in experiment II.
- No substantial increase in revertant colony numbers of any of the Salmonella typhimurium strains was observed following treatment with FAT 40812/A at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). The Escherichia coli strain showed an increase in revertant colony numbers in the preincubation test. In the first pre-incubation test the threshold of two was exceeded at 5000 µg/plate with and without metabolic activation. In the confirmatory experiment (pre-incubation test) the threshold was exceeded at 5000 µg/plate only without metabolic activation but there was also 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:

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

Executive summary:

In a GLP-compliant Ames test, 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. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration and the controls were tested in triplicate. The test item was tested at 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 S9 mix in both experiments. A minor toxic effect, evident as a reduction in the number of revertants was only observed in strain TA 1535 at 2500 µg/plate in the presence of metabolic activation in experiment II. No substantial increase in revertant colony numbers of any of the Salmonella typhimurium strains was observed at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). The Escherichia coli strain showed an increase in revertant colony numbers in the preincubation test. In the first pre-incubation test the threshold of two was exceeded at 5000 µg/plate with and without metabolic activation. In the confirmatory experiment (pre-incubation test) the threshold was exceeded at 5000 µg/plate only without metabolic activation but there was also tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls. They showed a distinct increase of induced revertant colonies. In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did induce gene mutations by base pair changes and others in the genome of the Escherichia coli strain WP2 uvrA used.

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 - 15 August 2003; Experiment start date - 19 August 2003; Experiment end date - 17 October 2003; Study completion date - 09 December 2003.

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:

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

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:

Identity: FAT 40812/A
Batch: WP 8/03
Purity: approx. 75 %
Appearance: Solid, dark red-brownish powder
Expiration date: 23 April 2010
Storage: At room temperature at about 20 °C

Target gene:

hypoxanthine-guanine phosphoribosyl transferase (HPRT)

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

Large stocks of the V79 cell line (supplied by Laboratory for Mutagenicity Testing; Technical University; D-64287 Darmstadt) are stored in liquid nitrogen in the cell bank of RCC-CCR allowing the repeated use of the same cell culture batch in experiments. Before freezing, the level of spontaneous mutants was depressed by treatment with HAT-medium. Each batch is screened for mycoplasma contamination and checked for karyotype stability and spontaneous mutant frequency. Consequently, the parameters of the experi­ ments remain similar because of the reproducible characteristics of the cells.

Thawed stock cultures are propagated at 37 °C in 80 sq.cm plastic flasks (GREINER, D-72632 Frickenhausen). About 5 x 10E5 cells are seeded into each flask with 15 ml of MEM (minimal essential medium; SEROMED, D-12247 Berlin) supplemented with 10 % fetal calf serum (FCS; PAA Laboratories GmbH, D-35091 Colbe). The cells are subcultured twice weekly. The cell cultures are incubated at 37 °C in a 4.5 % carbon dioxide atmosphere (95.5% air).

For the selection of mutant cells the medium is supplemented with 11 µg/mL thioguanine (6TG, SIGMA GmbH, D-82041 Deisenhofen).

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/β-Naphthoflavone induced rat liver S9 was used as the metabolic activation system. The S9 was prepared from 8 - 12 weeks old male Wistar Han rats, weight approx. 220 - 320 g induced by applications of 80 mg/kg b.w. Phenobarbital i.p. (Desitin; D- 22335 Hamburg) and (β-Naphthoflavone p.a. (Aldrich, D-89555 Steinheim) each on three consecutive days. The livers were prepared 24 hours after the last treatment. The S9 fractions were produced by dilution of the liver homogenate with a KCI solution (1+3) 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 in the S9 preparation was 26.2 mg/ml (Lot No.: 070303) in the pre-experiment and in experiment I. 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 following concentrations:

8mM - MgCl2
33mM - KCl
5mM - 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:

Experiment I
without S9 mix: 37.5, 75, 150, 300, 450, 600 µg/mL
with S9 mix: 75, 150, 300, 600, 1200, 2400 µg/mL

Experiment II
without S9 mix: 25, 50, 100, 200, 400, 600 µg/mL

Vehicle / solvent:

Water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

with metabolic activation

Details on test system and experimental conditions:

DETERMINATION OF CYTOTOXICITY
A pre-test was performed in order to determine the concentration range for the mutagenicity experiments. The general culture conditions and experimental conditions in this pre-test were the same as described for the mutagenicity experiment below. In this pre-test the colony forming ability of approximately 500 single cells (duplicate cultures per concentration level) after treatment with the test item was observed and compared to the controls. Toxicity of the test item is indicated by a reduction of the cloning efficiency (CE).

MAIN EXPERIMENT
Seeding: Three days old exponentially growing stock cultures (more than 50 % confluent) were trypsinized at 37 °C for 5 minutes. Then the enzymatic digestion was stopped by adding complete culture medium and a single cell suspension was prepared. The trypsin concentration for all subculturing steps was 0.2 % in Ca-Mg-free salt solution (Trypsin: Difco Laboratories, Detroit, USA). Prior to the trypsin treatment the cells were rinsed with Ca-Mg-free salt solution containing 200 mg/L EDTA (ethylene diamine tetraacetic acid). The cell suspension was seeded into plastic culture flasks (Greiner, D-72632 Frickenhausen). Approximately 1.5 x 10E6 (single culture) and 5 x 10E2 cells (in duplicate) were seeded in MEM with 10 % FCS (complete medium) for the determination of mutation rate and toxicity, respectively.
Treatment: After 24 hours the medium was replaced with serum-free medium containing the test item, either without S9 mix or with 50 µL/mL S9 mix. Concurrent negative and positive controls were treated in parallel. After 4 hours this medium was replaced with complete medium following two washing steps with "saline G". In the second experiment the cells were exposed to the test item for 24 hours in complete medium in the absence of metabolic activation. The pH was adjusted to 7.2. The colonies used to determine the cloning efficiency (survival) were fixed and stained approx. 7 days after treatment. Three days after treatment 1.5 x 10E6 cells per experimental point were subcultivated in 175 cm² flasks containing 30 mL medium. Following the expression time of 7 days, five 80 cm² cell culture flasks were seeded with about 3 - 5 x 10E5 cells each in medium containing 6-TG. Two additional 25 cm² flasks were seeded with approx. 500 cells each in non-selective medium to determine the viability. The cultures are incubated at 37 °C in a humidified atmosphere with 4.5 % CO2 for about 8 days. The colonies were stained with 10 % methylene blue in 0.01 % KOH solution (E. MERCK, D-64293 Darmstadt). The stained colonies with more than 50 cells were counted. In doubt the colony size was checked with a preparation microscope (Nikon, D-40407 Düsseldorf). Subculturing of a log-phase culture showed an initial spontaneous mutation rate at the beginning of the experiment of 8.0 mutant colonies (Experiment I) and 7.0 (Experiment II) mutant colonies per 1 x 10E6 cells.

Evaluation criteria:

- Acceptability of the assay: The gene mutation assay is considered acceptable if it meets the following criteria: the numbers of mutant colonies per 1 x 10E6 cells found in the negative and/or solvent controls fall within the laboratory historical control data range; the positive control substances must produce a significant increase in mutant colony frequencies; the cloning efficiency II (absolute value) of the negative and/or solvent controls must exceed 50 %.

- Evaluation of results: A test item is classified as positive if it induces either a concentration-related increase of the mutant frequency or a reproducible and positive response at one of the test points. A test item producing neither a concentration- related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered non-mutagenic in this system.

A positive response is described as follows: A test item is classified as mutagenic if it reproducibly induces a mutation frequency that is three times above the spontaneous mutation frequency at least at one of the concentrations in the experiment. The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed. However, in a case by case evaluation this decision depends on the level of the corresponding negative control data. If there is by chance a low spontaneous mutation rate in the range normally found (0.5 - 31.8 mutants per 1 x 10E6 cells) a concentration-related increase of the mutations within this range has to be discussed. The variability of the mutation rates of negative and solvent controls within all experiments of this study was also taken into consideration.

Statistics:

Since the distribution of mutant cells does not follow known statistical models, an adequate statistical method is not available.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS GENOTOXICITY:
- No relevant and reproducible increase in mutant colony numbers, 10E6 cells was observed in the main experiments up to the maximal concentration. All mutant frequencies remained well within the historical range of negative and solvent controls. At the concentration of 1200 µg/mL with metabolic activation a single increase of 37.0 mutant colonies/ 1 x 10E6 cells occurred, exceeding the range of the historical data of the solvent control (0.7-24.9 mutant colonies/ 1 x 10E6). Since the induction factor of 3 times the corresponding solvent control was not reached and the effect was not observed in the parallel culture, this effect was judged to be biologically irrelevant.

TEST-SPECIFIC CONFOUNDING FACTORS:
- No precipitation of the test item was observed up to the maximal concentration in all experiments.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- In experiment I relevant toxic effects indicated by a strongly reduced relative cloning efficiency occurred at 150 µg/mL and above in the absence and at 1200 µg/mL and above in the presence of metabolic activation (both cultures).
- In experiment II a strong toxic effect was observed at the highest concentration of 600 µg/mL at both cultures.

Conclusions:

The test substance is considered to be non-mutagenic in this HPRT assay.

Executive summary:

In a GLP-compliant mammalian cell gene mutation test, performed according to OECD guideline 476, V79 cells of the Chinese hamster were exposed to the test substance with and without metabolic activation to investigate the potential of the test substance to induce gene mutations at the HPRT locus.

The assay was performed in two independent experiments. The cells were exposed to the test item for 4 hours in the first experiment with and without metabolic activation. The second experiment was solely performed in the absence of metabolic activation with a treatment period of 4 hours.

The cell cultures were evaluated at the following concentrations based on the results of the range-finding pre-test:

Experiment I:

without S9 mix: 75; 150; 300; 450; and 600 µg/ml

with S9 mix: 150; 300; 600; 1200.0; and 2400 µg/ml

Experiment II:

without S9 mix:50; 100; 200; 400; and 600 µg/ml

No precipitation of the test item was observed up to the maximal concentration in all experiments.

In experiment I relevant toxic effects indicated by a strongly reduced relative cloning efficiency I occurred at 150.0 µg/ml and above in the absence and at 1200 µg/ml and above in the presence of metabolic activation (both cultures).

In experiment II a strong toxic effect was observed at the highest  concentration of 600 µg/ml at both cultures.

No relevant and reproducible increase in mutant colony numbers cells was observed in the main experiments up to the maximal concentration. All mutant frequencies remained well within the historical range of negative and solvent controls.

In both experiments of this study (with and without S9 mix) the range of the negative and solvent controls was from 4.5 up to 19.4 mutants per 1 x 10E6 cells; the range of the groups treated with the test item was from 0.0 up to 37.0 mutants per 1 x 10E6 cells.

EMS (0.15 mg/ml) and DMBA (2.7 µg/ml) were used as positive controls and showed a distinct increase in induced mutant colonies.

In conclusion, the test substance did not induce gene mutations at the HPRT locus in V79 cells. Therefore, FAT 40812/A is considered to be non-mutagenic in this HPRT assay.

Reference 4

Endpoint:

in vitro DNA damage and/or repair study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Study initiation date - 18 March 2004; Experiment start date - 25 March 2004; Experiment end date - 30 April 2004; Study completion date - 03 September 2004.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 482 (Genetic Toxicology: DNA Damage and Repair, Unscheduled DNA Synthesis in Mammalian Cells In Vitro)

Version / remarks:

See " Any other information on materials and methods incl. tables"

Deviations:

yes

Remarks:

Deviations did not affect the validity of the study

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5550 - Unscheduled DNA Synthesis in Mammalian Cells in Culture

Version / remarks:

See " Any other information on materials and methods incl. tables"

Deviations:

yes

Remarks:

Deviations did not affect the validity of the study

GLP compliance:

yes (incl. QA statement)

Type of assay:

sister chromatid exchange assay in mammalian cells

Specific details on test material used for the study:

Identity: FAT 40812/A
Batch: WP 8/03
Purity: approx. 75 %
Appearance: Solid, dark red-brownish powder
Expiration date: 23 April 2010
Storage: At room temperature at about 20 °C

Species / strain / cell type:

hepatocytes: Wistar rats

Metabolic activation:

not applicable

Test concentrations with justification for top dose:

500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98 and 0.49 µg/mL

Vehicle / solvent:

Williams Medium E / 1 % FCS. The solvent was chosen according to their solubility properties and their relative non-toxicity for the cells.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-acetylaminofluorene

Details on test system and experimental conditions:

- Isolation of the Primary Hepatocytes: After narcotizing the rat with Na-thiopental the liver was perfused through the vena portae with Hanks' balanced salt solution (HBSS, Gibco/BRL, D-76344 Eggenstein), supplemented with collagenase (0.05 % (w/v), Roche Diagnostics, D-68305 Mannheim) adjusted to pH 7.4 and maintained at 37 °C. The hepatocytes were isolated from the liver and washed twice with the perfusion solution without collagenase (HBSS). The crude cell suspension was filtered through a stainless steel mesh (94 µm) to yield a single cell suspension. The viability of the actual performed perfusion was determined by the trypan blue dye exclusion method. In addition, the number of the isolated cells was determined.

- Pre-Experiment for Toxicity: To evaluate the toxicity of the test item a pre-experiment was performed with 11 concentrations. The experimental conditions in the pre-experiment were the same as described below for the repair assay except the omission of ³HTdR. Toxicity could be evidenced by altered cell morphology and/or reduced number of adherent cells. In addition, the capability of the cells to incorporate a vital dye was determined by the neutral red absorption assay.

- Dose Selection: According to the results from the pre-experiments the concentrations to be applied in the repair assay were chosen. The highest concentration normally used should be 10 mM or 5 mg/mL (whatever is less) unless limited by the solubility of the test item or that producing some indication of cytotoxicity. The cytotoxicity should not suppress the incorporation of neutral red completely. According to the criteria mentioned above at least five adequately spaced concentrations were tested. The treatment interval was 18 hours. Two independent experiments were performed.

- Treatment of the Cells: Aliquots of 2.5 mL with freshly isolated hepatocytes in complete culture medium (2 x 10E5 living cells/mL) were added to 35 mm six-well cluster dishes (Greiner, D-72603 Nürtingen) containing one gelatinized 25 mm round plastic coverslip (Thermanox, Nunc, D-65203 Wiesbaden) per well. After an attachment period of approximately 1.5 h in a 95 % air/ 5 % CO2 humidified incubator at 37 °C the culture medium was discarded. Then the cell layer was rinsed once with PBS to remove non-adherent cells. Subsequently, treatment was initiated by adding the dissolved test item together with ³HTdR (5 µCi/mL, specific activity 20 Ci/mmol; New England Nuclear, D-63033 Dreieich) in 2.0 mL culture medium (WME, 1 % FCS). Each concentration including the positive and negative/solvent control was tested in 8 replicates (6 cultures for the UDS analysis and 2 cultures for the concurrent cytotoxicity analysis). The exposure period (18 h) was terminated by rinsing the cultures twice with PBS to remove all traces of free radiolabel. A hypotonic solution of 1 % sodium citrate was added to each culture for 10 minutes to swell the nuclei for better grain quantification. The cells on the cover slips were then fixed by three changes of methanohacetic acid (3+1 v/v) for 20 minutes each, rinsed with 96 % ethanol, and air dried. For each concentration two cultures were treated as described above except the omission of 3HTdR. These cultures served for determination of toxicity with the neutral red absorption assay.

- Autoradiographic Processing: The cover slips were mounted cell surface up on glass slides and coated with KODAK NTB2 photographic emulsion (Tecnomara, D-35463 Fernwald) in the dark. The coated slides were stored in light-proof boxes in the presence of a drying agent for 7 days at 4°C. The photographic emulsion was then developed with llford Phenisol (Ilford Imaging GmbH, 63265 Dreieich) at room temperature, fixed in Rapid Fixer (llford Imaging GmbH, 63265 Dreieich) and stained with hematoxylin/eosin.

- Quantification of UDS: Evaluation was performed microscopically on coded slides using NIKON microscopes with oil immersion objectives. The number of silver grains above the nucleus were counted automatically using the Scorecerer UDS device version 2.0 DT3152 (Perceptive Instruments). In addition, the number of grains of one nuclear-sized cytoplasm area adjacent to the nucleus was counted. At least two slides per concentration and 50 cells per slide were evaluated. Heavily labeled S-phase cells were excluded from counting.

Evaluation criteria:

- Nuclear and net grain counts are taken into consideration together. Increased net grains which are based on enhanced nuclear grain counts are considered relevant. Increased net grain values which are mainly due to decreased cytoplasmic grains are considered non-relevant.
- The result of a particular test point is classified positive if the mean nuclear grain count is statistically significant increased over the corresponding control value and if the mean net grain value is higher than five per nucleus.
- A test item is classified positive if it induces either a significant concentration-related increase in radiolabel incorporation expressed as nuclear and net grains or, in the absence of a concentration response, a reproducible and statistically significant positive response for at least one of the test points.
- A test item producing neither a significant concentration related increase in radiolabel incorporation expressed as nuclear and net grains nor a statistically significant and reproducible positive response at anyone of the test points is considered non-effective in this system.
- However, both biological and statistical significance should be considered together in the evaluation.

Statistics:

Statistical significance can be evaluated by means of the non-parametric Mann-Whitney test. A statistical evaluation of the results was not necessary to perform as the number of net grain counts of the groups treated with the test item were in the range of the corresponding controls.

Species / strain:

hepatocytes: Wistar rat

Metabolic activation:

not applicable

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

- The treatment of the hepatocytes with FAT 40812/A at higher concentrations (125, 250, 500 µg/mL) induced substantial cytotoxic effects. However, the interference of the test item colour with the neutral red assay disguised the quantity of the toxic effects observed at the higher concentrations. In the second experiment this was not as much of a problem as in the first experiment. The microscopical analysis of the cells after the treatment period may in this study be a more useful tool for indicating the toxicity pattern of the test item.

- In both experiments no increase in the number of nuclear and net grain counts was observed up to the highest concentration evaluated. Therefore, the net grain values obtained after treatment with the test item were consistently negative. There was also no relevant increase in the percentage of cells in repair.

- An appropriate reference mutagen (2-AAF 2.23 µg/ml) was used as positive control. 2-AAF showed reproducibly a distinct increase in nuclear and net grain counts.

Conclusions:

FAT 40'812/A did not induce DNA-damage leading to increased repair synthesis in the hepatocytes used.

Executive summary:

In a GLP-compliant unscheduled DNA synthesis assay, performed according to OECD guideline 482, the potential of the test substance to induce DNA repair synthesis in primary hepatocytes of rats in vitro was investigated. The test was performed in two independent experiments, using identical procedures. The freshly isolated hepatocytes were exposed to the test item for 18 h in the presence of 3HTdR (methyl-³H-thymidine). The uptake of radioactivity was determined by autoradiography. For each concentration, including the controls, 100 cells were evaluated. The following concentrations were evaluated in both main experiments: 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98 and 0.49 µg/mL. The concentration ranges for the main experiments were estimated by pre-experiments for toxicity. In the two independent experiments, after treatment with the test substance, no reproducible concentration dependent increase in the number of nuclear and net grain counts was observed up to the highest concentration evaluated. In conclusion, it can be stated that during the described study and under the experimental conditions reported, the test substance did not induce DNA-damage leading to increased repair synthesis in the hepatocytes used.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

FAT 40812/A was not clastogenic in the micronucleus assay.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Study initiation date - 11 August 2003; Experiment start date - 04 August 2003; Experiment end date - 04 September 2003; Study completion date - 16 October 2003.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

yes

Remarks:

In the present study, the relative humidity under which the experiment was conducted ranged between 30-78 % and not between 30-70 % as described in study plan.

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

In the present study, the relative humidity under which the experiment was conducted ranged between 30-78 % and not between 30-70 % as described in study plan.

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Specific details on test material used for the study:

Identity: FAT 40812/A
Batch: WP 8/03
Purity: approx. 75 %
Appearance: Solid, dark red-brownish powder
Expiration date: 23 April 2010
Storage: At room temperature at about 20 °C

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: RCC Ltd., CH-4414 Füllinsdorf
- Age at study acclimatizatuion: Males: 5-7 weeks, Females: 7-9 weeks
- Weight at start of treatment: Males 33.2 g (SD ± 3.6 g), Females: 29.5 g (SD ± 2.3 g)
- Assigned to test groups randomly: yes
- Fasting period before study: 18 hours
- Housing: Individually, in Makrolon Type I, with wire mesh top (EHRET GmbH, D-79302 Emmendingen) with granulated soft wood bedding (ALTROMIN, D-32791 Lage/Lippe).
- Diet: pelleted standard diet, ad libitum (ALTROMIN 1324, D-32791 Lage/Lippe)
- Water: tap water, ad libitum, (Gemeindewerke, D-64380 Roßdorf)
- Acclimation period: minimum 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 76
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

- water
- The vehicle was chosen to its relative non-toxicity for the animals.
- Volume administrated: 10 mL/kg bw

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 (nominal)

Remarks:

Negative control

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

Low dose

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

Mid dose

Dose / conc.:

2 000 mg/kg bw/day (nominal)

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: starvation period, 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 by cervical dislocation. 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-Grunwald (MERCK, D-64293 Darmstadt)/Giemsa (Gurr, BDH Limited Poole, Great Britain). Cover slips were mounted with EUKITT (KINDLER, D-79110 Freiburg). At least one slide was made from each bone marrow sample.

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 normochromatic erythrocytes per 2000 PCEs. The analysis was performed with coded slides. Ten animals (5 males, 5 females) per test group were evaluated as described.

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. 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 consideraton is the biological relevance of the results.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

animals treated with 2000 mg/kg b.w.

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:

A GLP-compliant erythrocyte micronucleus test was performed to investigate the potential of FAT 40812/A 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 total erythrocytes was determined in the same sample and reported as the number of PCEs per 2000 erythrocytes. The following dose levels of the test item were investigated:

24 h preparation interval: 500, 1000, and 2000 mg/kg 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 FAT 40812/A 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. Based on the findings of the study, FAT 40812/A is considered to be non-clastogenic in this micronucleus assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In vitro, Ames test :

In a GLP-compliant Ames test, performed according to OECD guideline 471, 4 Salmonella typhimurium strains (TA 1535, TA 1537, TA 98, and TA 100) and Escherichia coli strain WP2 uvrA, were used to the test the mutagenic potential of the test substance (RCC 2003). The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration and the controls were tested in triplicate. The test item was tested at 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 S9 mix in both experiments. A minor toxic effect, evident as a reduction in the number of revertants was only observed in strain TA 1535 at 2500 µg/plate in the presence of metabolic activation in experiment II. No substantial increase in revertant colony numbers of any of the Salmonella typhimurium strains was observed at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). The Escherichia coli strain showed an increase in revertant colony numbers in the preincubation test. In the first pre-incubation test the threshold of two was exceeded at 5000 µg/plate with and without metabolic activation. In the confirmatory experiment (pre-incubation test) the threshold was exceeded at 5000 µg/plate only without metabolic activation but there was also tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls. They showed a distinct increase of induced revertant colonies. In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did induce gene mutations by base pair changes and others in the genome of the Escherichia coli strain WP2 uvrA used.

In vitro, chromosome abberation test :

In a GLP-compliant chromosome aberration test, tested according to OECD guideline 473, Chinese hamster V79 cells (in vitro), were exposed to the test substance, with and without metabolic activation by S9 mix (RCC 2003). Two independent experiments were performed. The exposure period was 4 hrs with and without metabolic activation. In each experimental group two parallel cultures were set up. Per culture 100 metaphase plates were scored for structural chromosome aberrations, except for the test concentration 500 µg/mL with metabolic activation where 200 metaphase plates were scored. 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 of the cytogenetic experiments was performed considering the toxicity data. In both experiments, clear toxic effects indicated by reduced mitotic indices or reduced cell numbers were observed after treatment with the test item. Clear toxic effects indicated by reduced cell numbers of below 50 % of control were observed in both experimental parts at the highest evaluated concentrations. In contrary no clearly reduced mitotic indices were observed at the test item concentrations evaluated. But after treatment with 2000 µg/mL in the absence of S9 mix and with 750, 1000, and 1500 µg/mL in the presence of S9 mix extremely high mitotic indices were observed. In the absence and presence of S9 mix, biologically relevant increases in the number of cells carrying structural chromosomal aberrations were observed after treatment with the test item at concentrations showing clear reduced cell numbers. No increase in the frequencies of polypoid metaphases were found after treatment with the test item as compared to the frequencies of the controls. In conclusion, the test substance is considered to be clastogenic in this chromosome aberration test in the absence and in the presence of S9 mix.

 

In vitro, gene mutation assay :

In a GLP-compliant mammalian cell gene mutation test, performed according to OECD guideline 476, V79 cells of the Chinese hamster were exposed to the test substance with and without metabolic activation to investigate the potential of the test substance to induce gene mutations at the HPRT locus (RCC 2003). The assay was performed in two independent experiments. The cells were exposed to the test item for 4 hours in the first experiment with and without metabolic activation. The second experiment was solely performed in the absence of metabolic activation with a treatment period of 4 hours. No substantial and reproducible dose dependent increase in mutant colony numbers was observed in both main experiments. In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, the test substance is considered to be non-mutagenic in this HPRT assay.

 

In vitro, UDS:

In a GLP-compliant unscheduled DNA synthesis assay, performed according to OECD guideline 482, the potential of the test substance to induce DNA repair synthesis in primary hepatocytes of rats in vitro was investigated (RCC 2004). The test was performed in two independent experiments, using identical procedures. The freshly isolated hepatocytes were exposed to the test item for 18 h in the presence of 3HTdR (methyl-3H-thymidine). The uptake of radioactivity was determined by autoradiography. For each concentration, including the controls, 100 cells were evaluated. The following concentrations were evaluated in both main experiments: 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98 and 0.49 µg/mL. The concentration ranges for the main experiments were estimated by pre-experiments for toxicity. In the two independent experiments, after treatment with the test substance, no reproducible concentration dependent increase in the number of nuclear and net grain counts was observed up to the highest concentration evaluated. In conclusion, it can be stated that during the described study and under the experimental conditions reported, the test substance did not induce DNA-damage leading to increased repair synthesis in the hepatocytes used.

 

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 2003). 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. After treatment with the test substance, the number of PCEs was not substantially decreased as compared to the mean value of PCEs of the vehicle control indicating that the test substance has no cytotoxic properties in the bone marrow. 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 substance. Therefore, it can be stated that during the study described and under the experimental conditions reported, the test item did not induce micronuclei in the bone marrow cells of the mouse.

Short description of key information:
The test substance was reported to have produced significant increase in number of revertants at 5000 ug/plate with the Escherichia coli strain WP2 uvrA in the bacterial reverse mutation assay. However, it failed to produce mutagenic effect in the in vitro mammalian cell gene mutation assay. Also, the test substance did not lead to DNA damage when tested in the in vitro unscheduled DNA synthesis assay. Hence, Reactive Red 278 is considered to be not mutagenic. In the in vitro chromosomal aberration assay, Reactive Red 278 had clastogenic effect, but this effect could not be reproduced in the in vivo erythrocyte micronucleus test and hence, it is considered to be not clastogenic. Thus, Reactive Red 278 is neither muatgenic nor clastogenic and hence, considered to be not a genotoxicant.

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 the Directive 67/548/EEC and according to the EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008