Sodium 1-amino-9,10-dihydro-9,10-dioxo-4-[[3-[(1-oxopropyl)amino]phenyl]amino]anthracene-2-sulphonate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

FAT 20242 was found to have mutagenic action in bacterial cells in presence of metabolic activation. FAT 20242 also found to have clastogenic potential when tested in vitro. However, source chemical did not induce mutations in the mammalian cells in the HPRT assay.

Link to relevant study records

Reference

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1988

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

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

Name: FAT 20'242/B
Batch No.: FC-83/5T
Aggregate State at RT: Solid
Molecular Weight: 487.45
Purity: cf. Analytical Certificate in the sponsor file
Analysis: cf. Analytical Certificate in the sponsor file
Stability: Pure: stable for 24 months. In solvent: > 8 hours in H20, ethanol, acetone, DMSO and DMF
Storage: light protected
Expiration Date: March 1993

Target gene:

Not applicable

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

Large stocks of the V79 cell line 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. 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 80 cm2 plastic flasks (GREINER, D-7440 Nürtingen, F.R.G.). Seeding is done with about 5 x 10s cells per flask in 15 ml of MEM medium supplemented with 10 % fetal calf serum (FCS; SEROMED, D-1000 Berlin). The cells are subcultured twice weekly. The cell cultures are incubated at 37°C and 4.5 % carbon dioxide atmosphere.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 liver microsomal fraction from the liver of old male Wistar rats.

Test concentrations with justification for top dose:

1)Pre-experiment for toxicity:
without S9 mix:
7 h: 25; 50; 100; 150 µg/ml
18 h: 2.5; 10; 25; 50; 100; 150 µg/ml
28 h: 25; 50; 100; 150 µg/ml

with S9 mix:
7 h: 100; 200; 300; 400 µg/ml
18 h: 10; 50; 100; 200; 300; 400 µg/ml
28 h: 100; 200; 300; 400 µg/ml

2)Experiment:
Without S9 mix:
7 h: 50 µg/ml
18 h: 2.5; 25; 50 µg/ml
28 h: 150 µg/ml

With S9 mix:
7 h: 100 µg/ml
18 h: 10; 100; 200 µg/ml
28 h: 400 µg/ml

Vehicle / solvent:

On the day of the experiment, the test article was dissolved in culture medium without fetal calf serum.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Concurrent solvent controls were performed.

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Remarks:

Ethylmethanesulfonate: without metabolic activation; Cyclophosphamide: With metabolic activation.

Details on test system and experimental conditions:

METHOD OF APPLICATION: Plastic flask 25 cm3

NUMBER OF REPLICATIONS: 4 per dose

NUMBER OF CELLS EVALUATED: Determination of the mitotic index by scoring 1000 cells of each slide

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

Analysis of metaphase cells:
Evaluation of the slides were 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 not included in the calculation of the aberration rates. At least 100 well spread metaphases per slide were scored for cytogenetic damage on coded slides. Only metaphases with characteristic chromosome number of 22 ± 1 were included in the analysis. To describe a cytotoxic effect the mitotic index (% cells in mitosis) was determined.

Evaluation criteria:

A test article is classified as mutagenic if it induces either a significant dose-related increase in the number of structural chromosomal aberrations or a significant and reproducible positive response for at least one of the test points.

A test article producing neither a significant dose-related increase in the number of structural chromosomal aberrations nor a significant and reproducible positive response at any one of the test points is considered non-mutagenic in this system.
This can be confirmed by means of the nonparametric Mann-Whitney test. However, both biological and statistical significance should be considered together.

Statistics:

Statistical significance at the five per cent level (p < 0.05) was evaluated by means of the non-parametric Mann-Whitney test.

Solvent control versus fixation interval S9 mix p-value

Test group 50.0 µg 7 h - n.t.
" 100 µg 7 h + n.t.
" 2.5 µg 18 h - n.t.
" 25 µg 18 h - n.t.
" 50 µg 18 h - n.t.
" 10 µg 18 h + n.t.
" 100 µg 18 h + n.t.
" 200 µg 18 h + 0.0147*
" 150 µg 28 h - 0.0000*
" 400 µg 28 h + 0.0000*

n.t. = Not tested
* Aberration rate is statistically significant higher than the control rate.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

None

Dose Selection:

In the pre-experiment for 'toxicity the colony forming ability of the V79 cells was reduced to about 20% after treatment with 150.0 and 400.0 µg/ml*, respectively, in the absence and presence of S9 mix.

In the main experiment, in the absence of S9 mix at fixation intervals 7 and 18 h with concentrations higher than 50 µg/ml no metaphases could be found. At fixation interval 28 h cells after treatment with 150 µg/ml as maximum concentration were evaluated. In the presence of S9 mix after treatment with concentrations higher than 100 µg/ml (fixation interval 7 h) and 200 µg/ml (fixation interval 18 h) not enough scorable cells could be found. But at fixation interval 28 h. 400 µg/ml as maximum dose level could be evaluated for cytogenetic damage.

With the highest dose level applied in the absence and presence of S9 mix the mitotic index was clearly suppressed.

Conclusions:

FAT 20242/B induced structural chromosome aberrations in the V79 Chinese Hamster cell line.

Executive summary:

The test article FAT 20'242/B was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese Hamster in vitro in accordance with OECD Guideline 473.

Preparation of chromosomes was done 7 h (high dose), 18 h (low, medium and high dose), and 28 h (high dose) after start of treatment with the test article. The treatment interval was 4 h. In each experimental group, except the positive controls, four parallel cultures were used. Per culture 100 metaphases were scored for structural chromosomal aberrations. The following dose levels were evaluated:

without S9 mix: with S9 mix:

7 h

18 h

28 h

50.0 ug/ml 7 h: 100.0 ug/ml

2.5; 25.0; 50.0 ug/ml 18 h: 10.0; 100.0; 200.0 ug/ml

150.0 ug/ml 28 h: 400.0 ug/ml

The concentration range of the test article applied had been determined in a pre-experiment using the plating efficiency assay as indicator for toxicity response.

Treatment of the cells with 150.0 and 400.0 ug/ml, respectively, reduced clearly the plating efficiency of the V79 cells. Also the mitotic index was reduced after treatment with the highest concentrations at all fixation intervals in the presence and absence of S9 mix.

There were relevant enhancements of cells with structural aberrations after treatment with the highest dose levels at fixation intervals 18 and 28 h with and without metabolic activation by S9 mix.

Appropriate reference mutagens were used as positive controls and showed distinct increases of cells with structural chromosome aberrations.

CONCLUSION:

In conclusion, it can be stated that in the study described and under the experimental conditions reported, the test article induced structural chromosome aberrations as determined by the chromosomal aberration test in the V7 9 Chinese Hamster cell line. Therefore, FAT 20'242/B is considered to be clastogenic in this chromosomal aberration test.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

FAT 20242 was not clastogenic in a micronecleus assay.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: other: Chromosome mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1988

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EEC Directive 84/449, L 251, B12, p.137-139

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Environmental Protection agency, code of federal regulations, title 40, subpart F-genetic Toxicity, Revision July 1, 1986 "In vivo mammalian bone marrow cytogenetics tests: Micronucleus assay":

Deviations:

no

GLP compliance:

yes

Type of assay:

micronucleus assay

Specific details on test material used for the study:

Name: FAT 20'242/B
Batch No.: FC-83/5T
Aggregate State at RT: Solid
Colour : not indicated by the sponsor.
Purity: cf. Analytical Certificate in the sponsor file
Analysis: cf. Analytical Certificate in the sponsor file
Stability: Pure: stable for 24 months. In solvent: > 8 hours in H20, ethanol, acetone, DMSO and DMF
Storage: light protected
Expiration Date: March 1993

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Wiga GmbH, D-8741 Sulzfeld, F.R.G.
- Age at study initiation: minimum 10 weeks
- Weight at study initiation: Approx. 30 g.
- Assigned to test groups randomly: Yes
- Acclimation period: minimum 5 days.
- Housing: single
- Cage type: Makrolon Type I, with wire mesh top (EBECO, D-4620 Castrop-Rauxel, F.R.G.)
- Bedding: granulated soft wood bedding (ALTROMIN, D-4937 Lage/Lippe, F.R.G.)
- Feed: pelleted standard diet (ALTROMIN, D-4937 Lage/Lippe, F.R.G.)
- Water: tap water, ad libitum (Südhessische Gas- und Wasser AG, D-6100 Darmstadt)

According to the suppliers assurance the animals were in healthy condition. The animals were under quarantine in the animal house of L M P for two weeks after their arrival. During this period the animals did not show any signs of illness or altered behaviour.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 9°C
- Humidity (%): relative humidity not regulated
- Photoperiod (hrs dark / hrs light): Artificial light 6:00 am. - 6 pm

Route of administration:

oral: unspecified

Vehicle:

Name: Carboxymethylcellulose (CMC)
Supplier: SERVA D-6900 Heidelberg, F.R.G.
Catalogue no.: 16110
Route and Frequency of Administration: orally, singly
Volume Administered: 20 ml/kg b.w.

CMC was used as negative control.

On the day of the experiment, the test article was suspended in 4 % Carboxymethylcellulose. The vehicle was chosen to its nontoxicity for the animals. All animals received a single standard dose volume of 20 ml/kg body weight orally.

Details on exposure:

The test article was suspended in 4 % Carboxymethylcellulose. This suspending agent was used as negative control. The volume administered orally was 20 ml/kg b.w.. 24 h, 48 h and 72 h after a single application 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.

Duration of treatment / exposure:

3500 mg/kg b.w. at 24, 48 and 72 hours preparation interval.

Frequency of treatment:

single application of the test article.

Post exposure period:

72 hours

Remarks:

Doses / Concentrations:
3500 mg/kg b.w.
Basis:
nominal conc.

No. of animals per sex per dose:

- Pre-experiment 1: 3 males and 3 females at 5000 mg/kg b.w.
- Pre-experiment 2: 3 males and 3 females at 2000, 3000 and 4000 mg/kg b.w.
- Pre-experiment 3: 3 males and 3 females at 3500 mg/kg b.w.

Micronucleus assay: 5 males and 5 females at 3500 mg/kg

Control animals:

yes, concurrent vehicle

Positive control(s):

Name: CPA; Cyclophosphamide
Supplier: SERVA, D-6900 Heidelberg, F.R.G
Catalogue no.: 17681
Dissolved in: physiological saline
Dosing: 30 mg/kg b.w.
Route and Frequency of Administration: orally, singly
Volume Administered: 10 ml/kg b.w.

Solution prepared on day of administration.
The stability of CPA at room temperature is good. At 20°C only 1 % of CPA is hydrolysed per day in aqueous solution.

Tissues and cell types examined:

Polychromatic erythrocytes (PCE) in the bone marrow of the mouse.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
It is recommended to use the maximum tolerated dose or the highest dose that can be formulated and administered reproducibly.
The volume to be administered should be compatible with physiological space available. The maximum tolerated dose level was determined to be the dose that caused toxic reactions without having major effects on survival within 72 hours.

TREATMENT AND SAMPLING TIMES:
Approximately 18 hours before treatment with the test article the animals received no food but water ad libitum. At the beginning of the treatment
the animals were weighed and the individual volume to be administered was adjusted to the animal's body weight. The animals received the test article once. Twelve animals, six males and six females, were treated per dose group. Sampling of the bone marrow was done 24, 48 and 72 hours 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 2.0 ml fetal calf serum, using a 5 ml syringe, into 1 ml fetal calf serum. The cell suspension was centrifuged at 1,000 rpm for 5 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/Giemsa. Cover slips were mounted with EUKITT (KINDLER, D-7800 Freiburg F.R.G.). 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. 1,000 polychromatic erythrocytes (PCE) were analysed per animal for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined in same sample and expressed in normochromatic erythrocytes per 1000 PCEs. The analysis was performed with coded slides.

Evaluation criteria:

A test article is classified as mutagenic if it induces either a statistically significant dose-related increase in the number of micronucleated polychromatic erythrocytes or a reproducible statistically significant positive response for at least one of the test points.

A test article producing neither a statistically significant dose-related increase in the number of micronucleated polychromatic erythrocytes nor a statistically significant and reproducible positive response at anyone of the test points is considered nonmutagenic in this system.

This can be confirmed by means of the nonparametric Mann-Whitney test (6 ) .
However, both biological and statistical significance should be considered together.

Statistics:

Statistical significance at the five per cent level (p < 0.05) was evaluated by means of the non-parametric Mann-Whitney test.

*Negative control versus test group: 3500 mg/kg; 24 h
Significance: n.t.

*Negative control versus test group: 3500 mg/kg; 48 h
Significance: n.t.

*Negative control versus test group: 3500 mg/kg; 72 h
Significance: -

-: Not significant
+: Significant
n.t.: Not tested

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

One female died within 72 hours after administration of the test article

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

None

PRE-EXPERIMENT FOR TOXICITY:

1. In a first pre-experiment 6 animals ( 3 males, 3 females) received orally a single dose of 5000 mg/kg b.w. FAT 20'242/B suspended in 4 % CMC. The volume administered was 20 ml/kg b.w.. All treated animals expressed toxic reactions: reduction of spontaneous activity, eyelid closure and apathy. Two males and one female died.

2. In a second pre-experiment 6 animals (3 males, 3 females) per dose level received orally a single dose of 2000, 3000 or 4000 mg/kg b.w., respectively, FAT 20'242/B suspended in 4 % CMC. The volume administered was 20 ml/kg b.w..

4000 mg/kg b.w. induced lethalities: One male and one female died. After administration of 2000 and 3000 mg/kg b.w. the animals expressed reduction of spontaneous activity, eyelid closure and apathy.

3. In a third pre-experiment 6 animals ( 3 males, 3 females) received orally a single dose of 3500 mg/kg b.w. FAT 20'242/B suspended in 4 % CMC. The volume administered was 20 ml/kg b.w.. All treated animals expressed toxic reactions: reduction of spontaneous activity, eyelid closure and apathy. To avoid the loss of animals for evaluation in the mutagenicity assay the maximum tolerated dose was estimated to be 3500 mg/kg body weight.

TOXIC EFFECTS IN THE MICRONUCLEUS ASSAY:

After application of 3500 mg/kg b.w. FAT 20'242/B the animals expressed the same toxic symptoms as observed in the pre-experiment. One female died at preparation interval 72 hours.

SUMMARY OF RESULTS:

Test group	Dose mg/kg b.w.	Sampling time (h)	PCE with micronuclei	Range	PCE/NCE
Suspending agent	0	24	0.04%	0 -1	1000/626
Test article	3500	24	0.03%	0 -2	1000/673
Cyclophosphamide	30	24	0.58%	2 -10	1000/808
Suspending agent	0	48	0.09%	0 -2	1000/684
Test article	3500	48	0.05%	0 -2	1000/809
Suspending agent	0	72	0.04%	0 -2	1000/830
Test article	3500	72	0.10%	0 -2	1000/717

Conclusions:

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 in the bone marrow cells of the mouse.

Executive summary:

This study was performed to investigate the potential of FAT 20'242/B to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse. The test article was suspended in 4 % Carboxymethylcellulose. This suspending agent was used as negative control. The volume administered orally was 20 ml/kg b.w.. 24 h, 48 h and 72 h after a single application 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.

The following dose level of the test article was investigated: 24 h, 48 h, and 72 h preparation interval: 3500 mg/kg b.w.

In a pre-experiment this dose level was estimated to be the maximum tolerated dose. The animals expressed toxic reactions. One female died within 72 h after administration of the test article.

After treatment with the test article, the ratio between PCEs and NCEs was not affected as compared to the corresponding negative controls thus indicating no cytotoxic effects. In comparison with the corresponding negative controls there was no substantial enhancement in the frequency of the detected micronuclei at any preparation interval after application of the test article.

An appropriate reference mutagen was used as positive control which showed a distinct 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 20'242/B is considered to be non-clastogenic in this micronucleus assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

A study was carried out for FAT 20242 to test the mutagenic effects on histidine-auxotrophic mutants of Salmonella typhimurium. The investigations were performed with the following concentrations of the trial substance without and with microsomal activation: 25, 75, 225, 675 and 2025 µg/0.1 ml. In order to confirm the results, the experiments on strains TA 98 and TA 1537 were repeated. These tests permit the detection of point mutations in bacteria induced by chemical substances. Any mutagenic effects of the substances are demonstrable on comparison of the number of bacteria in the treated and control cultures that have undergone back-mutation to histidine-prototrophism. To ensure that mutagenic effects of metabolites of the test substances formed in mammals would also be detected, experiments were performed in which the cultures were additionally treated with an activation mixture (rat liver microsomes and co-factors).

In the experiments performed without microsomal activation none of the tested concentrations of FAT 20242/A caused an increase in the number of back-mutant colonies.In the experiments in which activation mixture was added to the cultures, treatment with FAT 20242/A led to an increase in the number of back-mutant colonies of strains TA 98 and TA 1537 at the concentrations of 25 µg/0.1 ml and above. A slight increase was also observed with strain TA 100 at the concentrations of 675 and 2025 µg/0.1 ml. The metabolites of FAT 20242/A formed as a result of microsomal activation thus exerted a clear-cut mutagenic action in this test system.

FAT 20242/B was tested for mutagenic effects on histidine-auxotrophic strains of Salmonella typhimurium and on a tryptophan auxotrophic strain of E. coli. The investigations were performed with the following concentrations of the trial substance without and with microsomal activation: 5, 10, 50, 100, 500, 1000 and 5000 µg/0.1 ml. These tests permit the detection of point mutations in bacteria induced by chemical substances. Any mutagenic effects of the substances are demonstrable on comparison of the number of bacteria in the treated and control cultures that have undergone back-mutation to histidine- or tryptophan-prototrophism. To ensure that mutagenic effects of metabolites of the test substance formed in mammals would also be detected, experiments were performed in which the cultures were additionally treated with an activation mixture (rat liver microsomes and co-factors).

In the experiments performed without microsomal activation, comparison of the number of back-mutant colonies in the controls and the cultures treated with the various concentrations of FAT 20 242/B revealed no marked deviations. In the experiments carried out with microsomal activation treatment with FAT 20242/B led to an increase in the number of back mutant colonies of strains TA 98, TA 1537 and TA 1538. The metabolites of FAT 20242/B as a result of microsomal activation thus exerted a mutagenic action in this test system.

Similarly, FAT 20242/C and FAT 20242/D was tested for mutagenic effects on histidine-auxotrophic mutants of Salmonella typhimurium. The metabolites of FAT 20242/C formed as a result of microsomal activation thus displayed a mutagenic effect in this test system.

A similar substance (read across) FAT 20033/I was subjected for investigating gene mutations using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and TA 102. The assays were performed in two independent experiments both with and without liver microsomal activation. Both the studies were performed in accordance to OECD guidance and in compliance with GLP. The substance was tested up to 5000 µg/plate. In both the experiments the test substance induced point mutations in strains TA 1537, TA 98 and TA 100. However, the read across substance FAT 20033/I, when tested for follow-up studies of in vitro mammalian cell gene mutation test (HPRT-locus) and in vitro cytogenicity chromosome aberration assay showed negative results. The test substance was positive in the Ames test but negative in the in vitro mammalian gene mutation test and negative in the in vitro mammalian chromosome aberration test. Considering similar structural properties the current substance FAT 20242/B is suspected to have showed false-positive effects in bacteria Ames assay. 

A study was conducted with FAT 20242/B to assess structural chromosome aberrations by means of the chromosome aberration assay in the Chinese Hamster cell line V79. Preparation of chromosomes was done 7 h (high dose), 18 h (low, medium and high dose), and 28 h (high dose) after start of treatment with the test article. The treatment interval was 4 h. In each experimental group, except the positive controls, four parallel cultures were used. Per culture 100 metaphases were scored for structural chromosomal aberrations. In the main experiment, at all fixation intervals, the mitotic index was reduced after treatment with the highest dose levels in the absence and presence of S9 mix, indicating that FAT 20242/B had cytotoxic properties. In the absence of S9 mix, at fixation interval 18 h, there was a dose related significant increase of the aberration rate after treatment with 25.0 and 50.0 µg/ml. Additionally in both test groups 2.50 % and 3.75 %, respectively, of the cells were carrying exchanges as compared with 0.75 % in the corresponding solvent control. Also at fixation interval 28 h after treatment with 150.0 µg/ml the aberration rate was clearly increased to 18.25 % (15.25 % of the cells were carrying exchanges) as compared to the solvent control: 1.25 % (exchanges: 0.25 %). In the presence of S9 mix the aberration rates were slightly but statistically significant increased after treatment with the highest dose levels at the fixation intervals 18 and 28 h. Additionally, at both intervals the frequency of cells carrying exchanges were markedly increased, too. Both, in the absence and presence of S9 mix the test article showed increased the frequency of cells with aberrations at fixation intervals 18 and 28 h. In conclusion, it can be stated that in the study described and under the experimental conditions reported, the test article induced structural chromosome aberrations as determined by the chromosomal aberration test in the V79 Chinese Hamster cell line. Therefore, FAT 20242/B is considered to be clastogenic in this chromosomal aberration test.

Since the in vitro chromosomal aberration study showed positive response, an in vivo experiment was performed, to investigate the potential of FAT 20242/B to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse.

In an in vivo the test article was suspended in 4 % Carboxymethylcellulose. This suspending agent was used as negative control. The volume administered orally was 20 ml/kg b w. After 24,48 and 72 h single application 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 level of the test article was investigated: 24,48 and 72 h preparation interval: 3500 mg/kg bw. In a pre-experiment this dose level was estimated to be the maximum tolerated dose. The animals expressed toxic reactions. One female died within 72 h after administration of the test article. After treatment with the test article the ratio between PCEs and NCEs was not affected as compared to the corresponding negative controls thus indicating no cytotoxic effects. In comparison with the corresponding negative controls there was no substantial enhancement in the frequency of the detected micronuclei at any preparation interval after application of the test article. An appropriate reference mutagen was used as positive control which showed a distinct increase of induced micronucleus frequency. In conclusion, it can be stated that during the study described and under the in vivo 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 20242/B is considered to be non-mutagenic in this micronucleus assay.

Conclusion:

The target chemical as well as source chemical were found to have mutagenic action in bacterial cells, however the source chemical was found to not have mutagenic action in mammalian cells when tested in an HPRT assay. The target chemical was positive for chromosomal aberrations in vitro, however it could not be reproduced i the micronucleus assay in the mouse. Based on these findings, both target chemical as well as source chemical are considered to be not genotoxic.

Justification for classification or non-classification

FAT 20242 is considered to be not genotoxic, hence it does not require classification for mutagenicity as per the Regulation (EC) No. 1272/2008.