Disodium [2-hydroxy-3-[(2-hydroxy-1-naphthyl)azo]-5-nitrobenzene-1-sulphonato(3-)][1-[(2-hydroxy-4-nitrophenyl)azo]-2-naphtholato(2-)]chromate(2-)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

An Ames test was performed to investigate the potential of FAT 20013/B to induce gene mutations according to the plate incorporation test using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100. The assay was performed in two independent experiments both with and without liver microsomal activation. Under the experimental conditions defined in the protocol, and employing a doubling of the spontaneous reversion rate and dose-effect relationship as criteria of mutagenicity, the product FAT 20013/B was found mutagenic for S. typhimurium strain TA 98. No mutagenic effect was observed with the strains TA 1535, TA 1537 and TA 100. With TA 98, the evidence of mutagenicity existed at 2000 µg in the presence of S-9 mix, and at 200 µg and 2000 ug of product per Petri dish without S-9 mix. In the absence of S-9 mix, at 2000 µg of product per plate, a toxic effect was noted with a dispersed background lawn. A complete disappearance of the background lawn was observed with the strains TA 1535 and TA 1537 at 2000 µg in the absence of S-9 mix. It is concluded that the metabolites of FAT 20013/B exerted a mutagenic action on strain S. typhimurium TA 98.

A read-across study was performed wherein FAT 45089 was evaluated for its ability to induce forward mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus (hprt) of Chinese hamster ovary (CHO) cells, in the presence and absence of an exogenous metabolic activation system, as assayed by colony growth in the presence of 6-thioguanine (TG resistance, TG). Water was used as the vehicle. The assay was performed in two independent experiments, using identical procedures, both with and without liver microsomal activation. The test article was tested with the following concentrations:

without S9 mix: 0.01; 0.03; 0.10 and 0.15µg/ml

with. S9 mix: 0.01; 0.05; 0.15 and 0.25µg/ml.

In conclusion it can be stated that during the described mutagenicity test and under the experimental conditions reported the test article did not induce mutations at the mouse lymphoma thymidine kinase locus assay using the cell line L517 8Y.

Another read-across study was performed, wherein FAT 45089/B was assessed for its mutagenic potential in the chromosome aberration test in human lmphocytes in vitro in the absence and presence of S9 mix.

The treatment interval was 4 hours. The test article was tested at the following concentrations:

without S9 mix: fixation time 24 h: 40; 250; 500µg/ml fixation time 48 h: 500 µg/ml

with S9 mix: fixation time 24 h : 40; 250; 400µg/ml fixation time 48 h: 500 µg/ml

In conclusion, it can be stated that in the described study and under the experimental conditions reported, the test article FAT 45089/B did not induce structural chromosome aberrations in human lymphocytes in vitro.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Study period:

Experiment start date: 09 February 1987; Experiment end date: 21 July 1987; Study completion date: 27 August 1987

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Read across with Acid Blue 349 for the registration of Acid Black 207 is claimed because of the following similarities of the chemical structures: Both substances are azo dyes with chromium complex and belong to the group of acid dyes. For Acid Black 207, the database for genetic toxicity consists of a positive in vitro bacterial reverse mutation assay and a negative in vivo micronucleus assay. Hence, in order to complete the assessment of genetic toxicity potential of Acid Black 207, further information is required. The basis for the read-across approach for Acid Black 207 is the chemical analogy with Acid Blue 349. This read-across strategy is aiming to help in assessment of genetic toxicity potential of Acid Black 207. For details on justification of the read-across please refer the document attached to chapter 13 of this dossier.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

Name: FAT 45089/B
Batch No: Op 2 UO-V 1018/84
Supplier: CIBA-GEIGY AG, CH-4002 Basel, Switzerland
Molecular Weight: 962.5
Purity: 89.0 % (chromium determination, water free)
Analysis No.: FC-84/5 T
Stability: Pure: 12 months
In solvent: at least 8 h in aqueous solution
Storage: room temperature
Expiration Date: July 1989

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 (Preparation by C C R)
The S9 liver microsomal fraction was obtained from the liver of 8 -12 weeks old male Wistar rats, strain CFHB (Süddeutsche Versuchstierfarm GmbH & Co. KG, D-7200 Tuttlingen, F.R.G.; weight ca. 150 - 200 g) which received a single i.p. injection of 500 mg/kg b.w. Aroclor 1254 (Antechnika, D-7500 Karlsruhe, F.R.G.) in olive oil 5 days previously. After cervical dislocation the livers of the animals were removed, washed in 150 mM KCl and homogenised. The homogenate, diluted 1:3 in KCl was centrifuged cold at 9,000 g for 10 minutes. A stock of the supernatant containing the microsomes was frozen in ampoules of 2 or 5 ml and stored at -70° C. Small numbers of the ampoules will be kept at -20 °C for only several weeks before use. The standardization of the protein content was made using the analysis kit of Bio-Rad Laboratories, D-8000 München: Bio-Rad protein assay, Catalog 500 000 6. The protein concentration in the S9 preparation was between 20 and 45 mg/mL.

Test concentrations with justification for top dose:

40; 250; 400; 500 and 800 µg/mL

Vehicle / solvent:

Vehicle: nutrient medium

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:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments : one

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor (cytogenetic assays): 3 hours before harvesting, Colcemide was added to the cultures (final concentration 0.2 µg/mL
- Methods of slide preparation and staining technique used including the stain used: Slides were prepared by dropping the cell suspension on to
a clean microscope slide. The cells were stained with acetoorcein.
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored):
- Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives. Breaks, fragments, deletions, exchanges and chromosomal disintegrations were recorded as structural chromosome aberrations. Gaps were recorded as well, but they were not included in the calculation of the aberration rates. At least 100 well spread metaphases per culture were scored for cytogenetic damage on coded slides. Only metaphases with characteristic chromosome number of 46 were included in the analysis. To describe a cytotoxic effect the mitotic index (% cells in mitosis) was determined.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Mitotic index (MI)

METHODS FOR MEASUREMENTS OF GENOTOXICIY : Increased aberration rate

Evaluation criteria:

The test article will be classified as mutagenic if it induces a significantly increased aberration rate with at least one of the concentrations tested as compared with the negative control. A test article which produce no significant positive response at any test point will be regarded as non-mutagenic in this assay.

Key result

Species / strain:

lymphocytes: Human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Conclusions:

The read across substance, FAT 45089/B, did not induce structural chromosome aberrations in human lymphocytes in vitro.

Executive summary:

The read across substance, FAT 45089/B was assessed for its mutagenic potential in the chromosome aberration test in human lmphocytes in vitro in the absence and presence of S9 mix. This test was performed according to OECD test guideline 473 and EEC Directive 84/449, Method B.10. In each experimental group, two parallel cultures were used. Per culture 100 metaphases were scored for structural chromosome aberrations. The following structural aberration types were recorded: Breaks, deletions, exchanges, partial or total disintegrations of chromosomes. Gaps were recorded and documented as well but not included in the calculation of the aberration rates. Preparation of chromosomes was done 24 h (low, medium and high dose) and 48 h (high dose) after start of the treatment with the test article. The treatment interval was 4 hours.

The test article was tested at the following concentrations:

without S9 mix: fixation time 24 h: 40; 250; 500 µg/mL fixation time 48 h: 500 µg/mL

with S9 mix: fixation time 24 h : 40; 250; 400 µg/mL fixation time 48 h: 500.00 µg/mL

In the pre-experiment on toxicity (scoring the mitotic index), with and without S9 mix treatment with 500 µg/ml reduced the mitotic index to about 50 % as compared to the negative controls. With this concentration the mitotic index in the main experiment was reduced after treatment with the test article in the presence and absence of S9 mix, indicating that FAT 45089/B had cytotoxic properties. The test article FAT 45089/B did not relevantly increase the number of cells carrying structural chromosome aberrations neither in the absence nor in the presence of S9 mix. Clear-cut enhanced numbers of structural chromosomal aberrations were obtained with the positive control articles EMS at a concentration of 1.24 mg/ml in the absence of metabolic activation, and CPA at a concentration of 9.90 µg/ml in the presence of metabolic activation, at fixation interval 24 h. In conclusion, it can be stated that in the described study and under the experimental conditions reported, the test article (read across substance) FAT 45089/B did not induce structural chromosome aberrations in human lymphocytes in vitro.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Study period:

Experiment start date: 10 April 1989; Experiment end date: 09 May 1989; Study report date: 17 August 1989.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Read across with Acid Blue 349 for the registration of Acid Black 207 is claimed because of the following similarities of the chemical structures. Both substances are azo dyes with chromium complex and belong to the group of acid dyes. For Acid Black 207, the database for genetic toxicity consists of a positive in vitro bacterial reverse mutation assay and a negative in vivo micronucleus assay. Hence, in order to complete the assessment of genetic toxicity potential of Acid Black 207, further information is required. The basis for the read-across approach for Acid Black 207 is the chemical analogy with Acid Blue 349. This read-across strategy is aiming to help in assessment of genetic toxicity potential of Acid Black 207. For details on justification of the read-across please refer the document attached to chapter 13 of this dossier.

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Specific details on test material used for the study:

Name: FAT 45089/B
Batch No: Op 2 UO-V 1018/84
Supplier: CIBA-GEIGY AG, CH-4002 Basel, Switzerland
Molecular Weight: 962.5
Purity: 89.0 % (chromium determination, water free)
Analysis No.: FC-84/5 T
Stability: Pure: 12 months
In solvent: at least 8 h in aqueous solution
Storage: room temperature
Expiration Date: July 1989

Target gene:

Forward gene mutations at the autosomal thymidine kinase (TK) locus.

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

Large stocks of the cleansed L5178Y cell line (supplied by RCC/Notox, s'Hertogenbosch, NL) are stored in liquid nitrogen in the cell bank of C C R allowing the repeated use of the same cell culture batch in experiments. Before freezing, each batch is screened for mycoplasma contamination and checked for karyotype stability. Consequently, the parameters of the experiments remain similar because of the reproducible characteristics of the cells.

Thawed stock cultures are propagated at 37 °C in 175 sq.cm plastic
flasks (GREINER, D-7440 Nürtingen, F.R.G.). Seeding is done with about 4 x 10E6 cells per flask in 30 ml of RPMI 1640 medium supplemented with 10 % fetal calf serum (FCS; SEROMED, D-1000 Berlin). The cells are subcultured according to their growth density. The cell cultures are incubated at 37 °C and 4.5 % carbon dioxide atmosphere.

Metabolic activation:

with and without

Metabolic activation system:

S9 (Preparation by C C R)
The S9 liver microsomal fraction was obtained from the liver of 8 - 1 2 weeks old male Wistar rats, strain WU (SAVO-Ivanovas, med. Versuchstierzuchten GmbH, D-7964 Kisslegg, F.R.G.; weight ca. 150 - 200 g) which received a single i.p. injection of 500 mg/kg b.w.
Aroclor 1254 (Antechnika, D-7500 Karlsruhe, F.R.G.) in olive oil 5 days previously.
After cervical dislocation, the livers of the animals were removed, washed in 150 mM KCl and homogenised. The homogenate, diluted 1:3 in KCl was centrifuged cold at 9,000 g for 10 minutes. A stock of the supernatant containing the microsomes was frozen in ampoules of 2 or 5 ml and stored at - 70 °C. Small numbers of the ampoules were kept at - 20 °C for only several weeks before use. The standardization of the protein content was made using the analysis kit of Bio-Rad Laboratories, D-8000 München: Bio-Rad protein assay, Catalogue 500 000 6.
The protein concentration in the S9 preparation is usually between 20 and 45 mg/mL.

Test concentrations with justification for top dose:

Based on the results of this pre-experiment at least four adequate concentrations extending at least over one decadic logarithm to be applied in the mutation assay were chosen. The highest dose level should be 10 mM unless limited by the solubility of the test article or that producing some indication of cytotoxicity (reduced cell culture growth).

In case of toxic effects, the highest concentration should reduce if possible the relative cell growth to approximately 10 - 50 %.
In the pre-experiment for toxicity the cell culture growth of the mouse lymphoma cells was clearly reduced after treatment with 0.10 mg/ml. With higher concentrations (see table I page 18) cell growth was decreased in such a way that the complete performance of the main experiment could be inhibited.
Because of the steep increase in toxicity in the range of 0.10 0.30 mg/ml 8 (without S9 mix) and 6 (with S9 mix) dose levels instead of 4 were chosen for the main experiment I.

without S9 mix: 0.01; 0.03; 0.06; 0.10; 0.15; 0.20; 0.25 and 0.30 mg/ml
with S9 mix: 0.01; 0.05; 0.10; 0.15; 0.20; and 0.25 mg/ml

After determination of cell culture growth 48 h after treatment, 0.15 mg/ml (without S9 mix) and 0.25 mg/ml (with S9 mix) were chosen as highest dose levels for the further performance of the main experiments. With higher concentrations the cell growth was not adequate for performing the experiment.

Vehicle / solvent:

Vehicle: DMSO, dimethylsulfoxide

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:

3-methylcholanthrene

Remarks:

with metabolic activation

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate)
- Number of independent experiments : Two


FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 2 day
- Selection time (if incubation with a selective agent): BrDU
- If a selective agent is used: RPMI 1640 complete culture medium supplemented with 50 ug/ml BrdU transformed into a gel by addition of 0.35 % agar noble.

METHODS FOR MEASUREMENT OF CYTOTOXICITY: Method.: Reduced cell culture growth

METHODS FOR MEASUREMENTS OF GENOTOXICIY : Significant dose-related increase in the mutant frequency.

Evaluation criteria:

The gene mutation assay is considered acceptable if it meets the following criteria:
a) the number of mutant colonies per 10s cells found in the negative and/or solvent controls fall within the laboratory historical control data range: 1-32 mutants/10E6 cells.
b) the positive control substances should produce a significant increase in mutant colony frequencies.
c) the plating efficiency (absolute value) of the negative and/or solvent controls should exceed 50 %.

Statistics:

A statistical evaluation of the results was not necessary to perform as the mutant colony numbers per 10E6 cells of the groups treated with the test article were in the range of the negative controls.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Conclusions:

Under the experimental conditions, the test article did not induce mutations at the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y. Therefore, FAT 45089/B is considered to be non-mutagenic in this mouse lymphoma thymidine kinase locus assay.

Executive summary:

The experiments were performed to assess the potential of the test article to induce gene mutations by means of two independent mouse lymphoma thymidine kinase locus assays using the cell line L5178Y. The assay was performed in two independent experiments, using identical procedures, both with and without liver microsomal activation. The test article was tested with the following concentrations:

without S9 mix: 0.01; 0.03; 0.10 and 0.15 µg/mL

with. S9 mix: 0.01; 0.05; 0.15 and 0.25 µg/mlL.

In both experiments, treatment with the highest concentrations of the test article reduced clearly the growth of the mouse lymphoma cells. The mutation rates found in the groups treated with the test article were considered not to be enhanced significantly as compared with the negative controls. The test article did not induce a reproducible concentration-related increase in mutant colony numbers. The mutant values of the groups treated with the test article were in the range of the negative controls. In this study the range of the negative controls was from 16.2 up to 27.2 mutants per 10E6 cells; the range of the groups treated with the test article was from 17.1 up to 31.1 mutants per 10E6 cells. EMS (0.8 mg/ml) and 3-MC (3 (µg/ml) were used as positive controls and showed a distinct increase in induced mutant colonies. In conclusion, the test article did not induce mutations at the mouse lymphoma thymidine kinase locus assay using the cell line L517 8Y.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Study completion date: 05 May 1983

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

only four tester strains tested; no tester strain to detect cross-linking mutagens was included

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Name: FAT 20013/B
Purity: >90 %

Target gene:

Histidine for Salmonella

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Rat-liver post mitochondrial supernatant (S9 fraction): Three male Sprague-Dawley rats weighing approximately 200 grams were given an intraperitoneal injection of Aroclor 1254 (Monsanto) in peanut oil at a dose of 500 mg/kg. Five days after the injection, the rats were anaesthetized with ether, decapitated and the livers removed in a sterile manner. The livers were homogenized in twice their volume of sterile 0.15 M KCl, pooled together and centrifuged for 15 min at 9000 xg (Beekman refrigerated ultracentrifuge). The supernatant (termed the S-9 fraction) was aliquoted into sterile tubes and frozen at -70 °C.

Test concentrations with justification for top dose:

0.2, 2, 20, 200 and 2000 µg

Vehicle / solvent:

Bidistilled water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: N-methyl-N'-nitro-N-nitrosoguanidine

Remarks:

TA 1535 and TA 100

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

TA 1537

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Daunomycin

Remarks:

TA 98

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-anthramine

Remarks:

TA 1535, TA 1537, TA 98, TA 100

Details on test system and experimental conditions:

TEST SUBSTANCE AND CONCENTRATIONS
A sample of the product FAT 20013/B (a brown dyestuff) was tested in S. typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 at five dose levels: 0.2, 2, 20, 200 and 2000 (µg) per Petri dish. The test substance was dissolved in distilled water and every concentration was tested in triplicate.

Evaluation criteria:

The criteria of mutagenicity used in this test are a doubling of the spontaneous reversion rate and a dose-effect relationship.

Key result

Species / strain:

other: For strains TA 1535, TA 1537 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:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Conclusions:

It is concluded that the metabolites of FAT 20013/B exerted a mutagenic action on strain S. typhimurium TA 98.

Executive summary:

A study was performed to investigate the potential of FAT 20013/B to induce gene mutations according to the plate incorporation test using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100. The assay was performed in two independent experiments both with and without liver microsomal activation. Each dose was tested in triplicate. Under the experimental conditions defined in the protocol, and employing a doubling of the spontaneous reversion rate and dose-effect relationship as criteria of mutagenicity, the product FAT 20013/B was found mutagenic for S. typhimurium strain TA 98. No mutagenic effect was observed with the strains TA 1535, TA 1537 and TA 100. With TA 98, the evidence of mutagenicity existed at 2000 µg in the presence of S-9 mix, and at 200 µg and 2000 µg of product per Petri dish without S-9 mix. In the absence of S-9 mix, at 2000 µg of product per plate, a toxic effect was noted with a dispersed background lawn. A complete disappearance of the background lawn was observed with the strains TA 1535 and TA 1537 at 2000 µg in the absence of S-9 mix. It is concluded that the metabolites of FAT 20013/B exerted a mutagenic action on strain S. typhimurium TA 98.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

An in vivo study was performed to investigate the potential of FAT 20013/C to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse. This study was conducted in accordance with OECD test guideline 474 and EEC Directive 92/69, L 383, Annex V, B 12. The test article was formulated in deionised water. Deionised water was used as vehicle control. The volume administered orally was 20 ml/kg b.w.. 24 h and 48 h after a single administration of the test article 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. 1000 polychromatic erythrocytes (PCE) per animal were scored for micronuclei. To describe a cytotoxic effect due to the treatment with the test article the ratio between polychromatic and normochromatic erythrocytes (NCE) was determined in the same sample and reported as the number of NCE per 1000 PCE. The following dose levels of the test article were investigated: 24 h preparation interval: 200, 670 and 2000 mg/kg b.w.. 48 h preparation interval: 2000 mg/kg b.w.. The highest guideline-recommended dose (2000 mg/kg) was estimated by a pre-experiment to be suitable. None of the animals expressed toxic reactions. After treatment with the highest dose of test article the number of NCEs was increased as compared to the corresponding vehicle controls thus indicating that FAT 20013/C had cytotoxic effectiveness in the bone marrow. In comparison to the corresponding vehicle controls there was no significant enhancement in the frequency of the detected micronuclei at any preparation interval after administration of the test article and with any dose level used. 40 mg/kg b.w. cyclophosphamide was used as positive control which showed a statistically significant increase of induced micronucleus frequency. In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test article did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse. Therefore, FAT 20013/C is considered to be non-mutagenic in this micronucleus assay.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Start of Experiment: 05 December 1997; End of Experiment: 16 December 1997; Study completion date: 13 January 1997.

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

Type of assay:

mammalian erythrocyte micronucleus test

Specific details on test material used for the study:

Identification: FAT 20'013/C
Description: Grey solid
Batch Number: 220
Purity / Formulation: 53.15 %
Stability of Test Article: Stable under specified storage conditions; expiration date: DEC-1999
Storage Conditions: In the original container at room temperature (approx. 20 °C), away from direct sunlight.
Safety Precautions: Gloves, goggles and face mask were obligatory to ensure the health and safety of the personnel.

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

Source: BRL, CH-4414 Füllinsdorf
Number of Animals: 72 (36 males/36 females)
Initial Age at Start of Acclimatization: 8-12 weeks
Acclimatization: minimum 5 days
Initial Body Weight at Start of Treatment: males mean value 31.5 g (SD ± 3.2 g); females mean value 25.1 g (SD ± 1.8 g)

Route of administration:

oral: gavage

Vehicle:

Vehicle: Deionised water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: On the day of the experiment, the test article was formulated in deionised water. The vehicle was chosen to its non-toxicity for the animals. All animals received a single standard volume of 20 mL/kg body weight orally.

Duration of treatment / exposure:

48 hours

Frequency of treatment:

single treatement

Dose / conc.:

200 mg/kg bw/day (nominal)

Remarks:

24 h interval

Dose / conc.:

670 mg/kg bw/day (nominal)

Remarks:

24 h interval

Dose / conc.:

2 000 mg/kg bw/day (nominal)

Remarks:

24h and 48h interval each

No. of animals per sex per dose:

6 animals per sex per dose

Control animals:

yes

Positive control(s):

Cyclophosphamide;
- Justification for choice of positive control(s): recommended by the guidelines
- Route of administration: oral
- Doses: 40 mg/kg b.w.
- Frequency: once

Tissues and cell types examined:

Bone marrow - polychromatic erythrocytes (PCE)

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: 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 extending over a single log range 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.

TREATMENT AND SAMPLING TIMES: Single dose treatment; 24h for 200 mg/kg bw and 670 mg/kg bw 24 and 48 hours for 2000 mg/kg bw.

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-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 1000 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 1000 the PCEs. The analysis was performed with coded slides.
Five animals per sex and group were evaluated as described. The remaining 6th animal of each test group was evaluated in case an animal had died in its test group.

Evaluation criteria:

The study is considered valid if the following criteria are met:
- the vehicle controls are in the range of our historical control data (0.03 - 0.26 % PCEs with micronuclei).
- the positive controls show substantially increased values
- more than 80 % of animals are évaluable

Statistics:

Nonparametric Mann-Whitney test

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

The mean number of normochromatic erythrocytes was increased after treatment with the highest dose of the test article as compared to the mean value of NCEs of the corresponding vehicle control, indicating that FAT 20013/C had cytotoxic properties in the bone marrow.

Conclusions:

FAT 20013/C is considered to be non-mutagenic in this micronucleus assay.

Executive summary:

An in vivo study was performed to investigate the potential of FAT 20013/C to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse. This study was conducted in accordance with OECD test guideline 474 and EEC Directive 92/69, L 383, Annex V, B 12. The test article was formulated in deionised water. Deionised water was used as vehicle control. The volume administered orally was 20 ml/kg b.w.. 24 h and 48 h after a single administration of the test article 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. 1000 polychromatic erythrocytes (PCE) per animal were scored for micronuclei. To describe a cytotoxic effect due to the treatment with the test article the ratio between polychromatic and normochromatic erythrocytes (NCE) was determined in the same sample and reported as the number of NCE per 1000 PCE. The following dose levels of the test article were investigated: 24 h preparation interval: 200, 670 and 2000 mg/kg b.w.. 48 h preparation interval: 2000 mg/kg b.w.. The highest guideline-recommended dose (2000 mg/kg) was estimated by a pre-experiment to be suitable. None of the animals expressed toxic reactions. After treatment with the highest dose of test article the number of NCEs was increased as compared to the corresponding vehicle controls thus indicating that FAT 20013/C had cytotoxic effectiveness in the bone marrow. In comparison to the corresponding vehicle controls there was no significant enhancement in the frequency of the detected micronuclei at any preparation interval after administration of the test article and with any dose level used. 40 mg/kg b.w. cyclophosphamide was used as positive control which showed a statistically significant increase of induced micronucleus frequency. In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test article did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse. Therefore, FAT 20013/C is considered to be non-mutagenic in this micronucleus assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

Based on negative results in in vitro chromosomal aberration assay and mammalian cell mutation assay (HPRT) with the read across substance as well as the in vivo micronucleus assay with FAT 20013, FAT 20013 can be considered to be non-genotoxic. Hence, the substance is not classified.