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

Genetic toxicity: in vivo

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1988

Report date:

1988

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

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

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

289-550-2

EC Name:

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

Cas Number:

89923-62-6

Molecular formula:

C23H19N3O6S.Na

IUPAC Name:

sodium 1-amino-9,10-dioxo-4-[(3-propanamidophenyl)amino]-9,10-dihydroanthracene-2-sulfonate

Test material form:

other: solid

Details on test material:

None

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

Test animals

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

Administration / exposure

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

Doses / concentrations

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.

Examinations

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

Results and discussion

Test results

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

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.