Reaction mass of 6-Chloro-2-{4[ethyl(2-hydroxyethyl)amino]phenyl}-1-methylbenzo[cd]indolium hydroxide and 6-chloro-2-[4-[ethyl(2-hydroxyethyl)amino]phenyl]-1-methylbenz[cd]indolium acetate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The substance induced mutations in bacterial reverse mutation assays.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Study completion date: 18 November, 1983

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: As per the methodology proposed by Ames et al

Deviations:

not specified

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Name: FAT 31064/E
Purity: >95 %

Target gene:

Histidine requiring genes of Salmonella strains

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

microsomal enzymes from rat liver

Test concentrations with justification for top dose:

0.2, 2, 20, 200 and 2000 µg/petri dish;

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: distilled water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

Without S9

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: N-methyl-N'-nitrosoguanidine

Remarks:

Without S9

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: daunomycine

Remarks:

Without S9

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-anthramine

Remarks:

With S9

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Triplicate
- Number of independent experiments : One

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Background growth inhibition

METHODS FOR MEASUREMENTS OF GENOTOXICIY : Background growth inhibition

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: Salmonella typhimurium TA 98 and TA 1537

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In the mutagenicity tests without and with metabolic activation, due to toxicity of the test material, a slight decline in the number of revertant colonies was occasionally observed with all strains at the highest concentrations.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

other: Salmonella typhimurium TA 98 and TA 1537

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In the mutagenicity tests without and with metabolic activation, due to toxicity of the test material, a slight decline in the number of revertant colonies was occasionally observed with all strains at the highest concentrations.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In the mutagenicity tests without and with metabolic activation, due to toxicity of the test material, a slight decline in the number of revertant colonies was occasionally observed with all strains at the highest concentrations.

Vehicle controls validity:

valid

Positive controls validity:

valid

No mutagenic effect was observed with the strains TA 1535 and TA 100.

The evidence for mutagenicity existed only in the presence of a liver microsomal enzyme preparation (S-9 mix) from male rats pretreated with Aroclor 1254 at the concentrations of 20 and 200 µg of product per Petri dish with TA 1537, and at 200 µg with TA 98. For these two strains a toxic effect was noted at the highest concentration of the product with a complete disappearance of the background lawn for TA 1537 and a dispersed lawn of auxotrophic bacteria for TA 98.

A toxic effect was also noted with strain TA 100 in the absence of metabolic activation at 2000 µg of product per Petri dish.

Conclusions:

FAT 31064/F was found to exert a mutagenic action on strains S. typhimurium TA 98 and TA 1537 only in presence of metabolic activation.

Executive summary:

The mutagenic potential of FAT 31064/E was investigated in a bacterial reverse mutation assay according to procedure prescribed by Ames et al.

The substance FAT 31064/E was tested with the Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 at concentrations from 0.2 to 2000 µg (or nl) per petri dish - both in the presence and absence of metabolic activation.

A mutagenic effect was observed with strains TA 1537 and TA 98 only in presence of metabolic activation. No mutagenic effect was observed with strains TA 100 and TA 1535. A toxic effect was noted with strains TA 1537 and TA 100 at the highest tested concentrations.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Test start date: 12 Jan, 1990; Test end date: 09 Feb, 1990; Study completion date: 10 May 1990

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)

Version / remarks:

EEC Directive 84/449, L 251, B 14, p. 143-145

Deviations:

no

Principles of method if other than guideline:

None

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Identified as: FAT 31064/F
Batch No.: EN 158496.82 / HEW 133/6
Aggregate State at RT: solid
Colour: dark blue
Storage: room temperature
Expiration Date: November, 1994

Target gene:

Histidine requiring genes of Salmonella strains

Species / strain / cell type:

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

Species / strain / cell type:

S. typhimurium TA 1538

Metabolic activation:

with and without

Metabolic activation system:

S9 liver microsomal fraction was obtained from the liver of 8 - 1 2 weeks old male Wistar rats

Test concentrations with justification for top dose:

Exp. I and II; without S9 mix; TA 1535: 3.3; 10.0; 33.3; 66.6; 100.0; and 333.3 µg/plate
Exp. I and II; with S9 mix; TA 1535: 10.0; 33.3; 100.0; 333.3; 666.6; and 1000.0 µg/plate
Exp. I and II; without S9 mix; TA 1537 + TA 1538: 1.0; 3.3; 10.0; 33.3; 66.6; and 100.0 µg/plate
Exp. I and II; with S9 mix; TA 1537 + TA 1538: 10.0; 33.3; 100.0; 333.3; 1000.0; and 5000.0 µg/plate
Exp. I and II; with and without S9 mix; TA 98 + TA 100: 10.0; 33.3; 100.0; 333.3; 1000.0; and 5000.0 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: distilled water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

Without metabolic activation:- TA 100 and TA 1535: sodium azide; TA 98, TA1538, TA 1537: 4-nitro-o-phenylene-diamine; With metabolic activation: all strains: 2-aminoanthracene

Positive control substance:

sodium azide

other: 4-nitro-ophenylene-diamine; 2-aminoanthracene

Evaluation criteria:

The generally accepted conditions for the evaluation of the results are:
- corresponding background growth on both negative control and test plates
- normal range of spontaneous reversion rates.

A test article is considered as positive if either a significant dose-related increase in the number of revertants or a significant and reproducible increase for at least one test concentration is induced.
A test article producing neither a significant dose-related increase in the number of revertants nor a significant and reproducible positive response at any one of the test points is considered non-mutagenic in this system.
A significant response is described as follows:
A test article is considered as mutagen if in strain TA 100 the number of reversions is at least twice as high and in strains TA 1535, TA 1537, TA 1538, and TA 98 it is at least three times higher as compared to the spontaneous reversion rate.
Also, a dose-dependent increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of the test article regardless whether the highest dose induced the above described enhancement factors or not.

Statistics:

No appropriate statistical method is available.

Key result

Species / strain:

other: Salmonella typhimurium TA 98, TA 1538 and TA 1537

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

The background growth was reduced at the highest investiagted doses in all strains used

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

other: TA 1535 and TA 100

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

The background growth was reduced at the highest investiagted doses in all strains used

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

other: TA 1535, TA 1537, TA 1538, TA 98 and TA 100

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

The background growth was reduced at the highest investiagted doses in all strains used

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Reduced revertant rates indicating bacteriotoxicity occured in nearly all strains with and without metabolic activation in experiment I and II. The background growth was reduced at the highest investiagted doses in all strains used. In strain TA 100 an increase of revertant colony numbers was observed in experiment I and II in the presence of metabolic activation. In exp. II this increase reached a significant value at 33.3 µg/plate. In the strains TA 1537, TA 1538, and TA 98 (all with metabolic activation) a significant increase of revertant colonies was observed from the lowest dose level up to 333.3 µg/plate. At higher concentrations the numbers of revertants decreased due to toxic effects of the test article. Up to the highest investigated dose, neither a significant and reproducible increase in the number of revertants was found in the strains TA 1535 (with and without S9 mix), TA 1537, TA 1538, TA 98, and TA 100 (all without metabolic activation) as compared to the solvent control nor a concentration-dependent enhancement of the revertant number exists. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

Conclusions:

FAT 31064/F is considered to be mutagenic Salmonella typhimurium reverse mutation assay.

Executive summary:

This study was performed to investigate the potential of FAT 31064/F to induce gene mutations according to the plate incorporation test using the Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100. The assay was performed in two independent experiments, using identical procedures, both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. Reduced revertant rates indicating bacteriotoxicity occured in nearly all strains with and without metabolic activation in experiment I and II. The background growth was reduced at the highest investiagted doses in all strains used. In strain TA 100 an increase of revertant colony numbers was observed in experiment I and II in the presence of metabolic activation. In exp. II this increase reached a significant value at 33.3 µg/plate. In the strains TA 1537, TA 1538, and TA 98 (all with metabolic activation) a significant increase of revertant colonies was observed from the lowest dose level up to 333.3 µg/plate. At higher concentrations the numbers of revertants decreased due to toxic effects of the test article. Up to the highest investigated dose, neither a significant and reproducible increase in the number of revertants was found in the strains TA 1535 (with and without S9 mix), TA 1537, TA 1538, TA 98, and TA 100 (all without metabolic activation) as compared to the solvent control nor a concentration-dependent enhancement of the revertant number exists. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies. In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test article induced point mutations by base pair changes and frameshifts in the genome of the strains TA 1537, TA 1538, and TA 98 only in the presence of metabolic activation. Therefore, FAT 31064/F is considered to be mutagenic Salmonella typhimurium reverse mutation assay.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

FAT 31064/F was considered to be devoid of clastogenic potential 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:

Test start date: 02 January, 1990; Test end date: 12 February, 1990; Study completion date: 19 March 1990

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

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:

mammalian erythrocyte micronucleus test

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Identification of the test material as used in the study report: FAT 31'064/F
- Source and batch No.of test material: EN 158496.82/ HEW 133/6
- Expiration date of the batch: November 1994
- Aggregate State at RT: Solid
- Colour: Dark blue

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability of the test substance in the solvent/vehicle: Pure: stable for five years; In vehicle: stable in water, ethanol, acetone, DMSO, DMF for 48 hours

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: BRL Tierfarm Flillinsdorf, CH- 4414 Villinsdorf/Basel, Switzerland
- Age at study initiation: 10 weeks
- Weight at study initiation: approximately 30 g

- Housing: single in Makrolon Type I, with wire mesh top (EBECO, D-4620 Castrop-Rauxel, F.R.G.)
- Diet: pelleted standard diet, ad libitum (ALTROMIN, D-4937 Lage/Lippe, F.R.G.)
- Water: tap water, ad libitum, (SUdhessische Gas- and Wasser AG, D-6100 Darmstadt)
- Acclimation period: one week

ENVIRONMENTAL CONDITIONS
- Temperature: 21 ± 3°C
- Photoperiod: artificial light 6.00 a.m. - 6.00 p.m.

IN-LIFE DATES: From: January 02, 1990 to February 12, 1990

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: Carboxymethycellulose (1 %)

Details on exposure:

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.

Duration of treatment / exposure:

Once orally (gavage)

Frequency of treatment:

Single dose

Post exposure period:

Sampling of the bone marrow was done 24, 48 and 72 hours after treatment.

Dose / conc.:

5 000 mg/kg bw (total dose)

No. of animals per sex per dose:

Six males and six females were assigned to each test group. Ten animals (5 males, 5 females) per test group were evaluated for the occurrence of micronuclei.

Control animals:

yes, concurrent vehicle

Positive control(s):

- Positive control used: Cyclophosphamide
- Route of administration: Orally once
- Dissolved in: Physiological saline
- Doses: 40 mg/kg bw
- Volume administered: 10 ml/kg b.w.

Tissues and cell types examined:

Bone marrow and polychromatic erythrocytes (PCE)

Details of tissue and slide preparation:

Preparation of the Animals:
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 5 ml syringe. The cell suspension was centrifuged at 1,500 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-Grunwald/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.

Analysis of Cells:
Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives.. 1,000 polychromatic erythro¬cytes (PCE) were analysed per animal for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and normochro-matic erythrocytes was determined in same sample and expressed in hormochromatic erythrocytes per 1000 PCEs. The analysis was per¬formed with coded slides. Five animals per sex and group were evaluated as described. The remaining animal of each test group was evaluated in case an animal had died in its test group spontaneously or due to gavage error.

Evaluation criteria:

EVALUATION OF RESULTS
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 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.

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:

PRE-EXPERIMENT FOR TOXICITY
In a pre-experiment 4 animals (2 males, 2 females) received orally a single dose of 5000 mg/kg b.w., FAT 31064/F suspended' in carboxymethylceilulose (1 %).
The volume administered was 20 ml/kg b.w.
All treated animals expressed slight toxic reactions: reduction of spontaneous activity.

Higher dosing was not attainable:
a) Appropriate suspensions (homogeneity, viscosity) could be obtained only up to 250 mg/ml.
b) Application volumes higher than 20 ml/kg b.w. were not justifiable for the rodents used.

LETHALITIES IN THE MICRONUCLEUS ASSAY
At preparation interval 24 hours one male and one female died. At preparation interval 48 hours one male and one female died. At preparation interval 72 hours one male died.

Summary of Micronucleus Test Results:

Test Group	Dose mg/kg b.w	sampling time (h)	PCEs with micronuclei (%)	Range	PCE/NCE
Vehicle	0	24	0.092	0 -3	1000/827
Test Article	5000	24	0.092	0-3	1000/925
cyclo-phosphamide	40	24	6.692	3-13	1000/964
Vehicle	0	48	0.072	0 -2	1000/714
Test article	5000	48	0.062	0 -2	1000/1358
Vehicle	0	72	0.09	0 -3	1000/780
Test article	5000	72	0.092	0 -3	1000/821

In comparison to the corresponding negative controls there was no enhancement in the frequency of the detected micronuclei at any preparation interval after application of the test article. The mean values of micronuclei observed after treatment with FAT 31'064/F were in the same range as compared to the negative control groups.

40 mg/kg b.w. cyclophosphamide (positive control) which showed a distinct increase of induced micronuleus frequency.

Conclusions:

FAT 31064/F was considered to be devoid of clastogenic potential in this micronucleus assay.

Executive summary:

The test article FAT 31064/F was assessed in the micronucleus assay for its potential to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse. The test article was suspended in Carboxymethylcellulose-solution (1 %). 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. To describe a cytotoxic effect due to the treatment with the test article the ratio between polychromatic and normochromatic erythrocytes (NCE)wasdetermined 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 h, 48 h, and 72 h preparation interval: 5000 mg/kg b.w. In a pre-experiment this dose level was estimated to be the maximum attainable dose. The animals expressed slight toxic reactions. In the micronucleus assay three males and two females died after administration of the test article. The mean number of normochromatic erythrocytes was increased after treatment with the test article at preparation interval 48 hours as compared to the mean value of NCEs of the corresponding negative control, indicating that FAT 31064/F had cytotoxic properties. In comparison to the corresponding negative controls there was no enhancement in the frequency of the detected micronuclei at any preparation interval after application of the test article. The mean values of micronuclei observed after treatment with FAT 31064/F were in the same rangeascompared to thenegativecontrol groups. 40 mg/kg b.w. cyclophosphamide administered per os was used as positive control which showed a distinct increase of induced micronuleus 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 in the bone marrow cells of the mouse.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Currently two bacterial reverse mutation assays and a micronucleus assay are available with the substance.

Bacterial reverse mutation assay with FAT 31064/E

The mutagenic potential of FAT 31064/E was investigated in a bacterial reverse mutation assay according to procedure prescribed by Ames et al. The substance FAT 31064/E was tested with the Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 at concentrations from 0.2 to 2000 µg (or nl) per petri dish - both in the presence and absence of metabolic activation. A mutagenic effect was observed with strains TA 1537 and TA 98 only in presence of metabolic activation. No mutagenic effect was observed with strains TA 100 and TA 1535. A toxic effect was noted with strains TA 1537 and TA 100 at the highest tested concentrations.

Bacterial reverse mutation assay with FAT 31064/F

This study was performed to investigate the potential of FAT 31'064/F to induce gene mutations according to the plate incorporation test using the Salmonella typhimurium strains

TA 1535, TA 1537, TA 1538, TA 98 and TA 100. The assay was performed in two independent experiments, using identical procedures, both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. Reduced revertant rates indicating bacteriotoxicity occured in nearly all strains with and without metabolic activation in experiment I and II. The background growth was reduced at the highest investiagted doses in all strains used. In strain TA 100 an increase of revertant colony numbers was observed in experiment I and II in the presence of metabolic activation. In exp. II this increase reached a significant value at 33.3 µg/plate. In the strains TA 1537, TA 1538, and TA 98 (all with metabolic activation) a significant increase of revertant colonies was observed from the lowest dose level up to 333.3 µg/plate. At higher concentrations the numbers of revertants decreased due to toxic effects of the test article. Up to the highest investigated dose, neither a significant and reproducible increase in the number of revertants was found in the strains TA 1535 (with and without S9 mix), TA 1537, TA 1538, TA 98, and TA 100 (all without metabolic activation) as compared to the solvent control nor a concentration-dependent enhancement of the revertant number exists. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies. In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test article induced point mutations by base pair changes and frameshifts in the genome of the strains TA 1537, TA 1538, and TA 98 only in the presence of metabolic activation. Therefore, FAT 31064/F is considered to be mutagenic Salmonella typhimurium reverse mutation assay.

Micronucleus assay with FA T 31064/E

The test article FAT 31064/F was assessed in the micronucleus assay for its potential to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse.The test article was suspended in carboxymethylcellulose-solution (1 %). 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. To describe a cytotoxic effect due to the treatment with the test article the ratio between polychromatic and normochromatic erythrocytes (NCE)wasdetermined in thesame sampleand reported as the number of NCE per 1000 PCE. The following dose level of the test article was investigated: 24 h, 48 h, and 72 h preparation interval: 5000 mg/kg b.w.. In a pre-experiment this dose level was estimated to be the maximum attainable dose. The animals expressed slight toxic reactions. In the micronucleus assay three males and two females died after administration of the test article. The mean number of normochromatic erythrocytes was increased after treatment with the test article at preparation interval 48 hours as compared to the mean value of NCEs of the corresponding negative control, indicating that FAT 31'064/F had cytotoxic properties. In comparison to the corresponding negative controls there was no enhancement in the frequency of the detected micronuclei at any preparation interval after application of the test article. The mean values of micronuclei observed after treatment with FAT 31'064/F were in the same rangeascompared to thenegativecontrol groups. 40 mg/kg b.w. cyclophosphamide administered per os was used as positive control which showed a distinct increase of induced micronuleus 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 in the bone marrow cells of the mouse.

Conclusion:

The bacterial reverse mutation assays were positive while the micronucleus assay returned negative. Hence, clastogenicity can be ruled out. However, further information is required for the assessment of mutagenic potential of the substance in mammalian cells. Hence, a testing proposal for conduct of UDS assay is included.

Justification for classification or non-classification

Insufficient information.