Reaction products of disodium 3,5-diamino-2-[(2-sulfonatophenyl)diazenyl]benzoate and  diazotised sodium 2-[(4-aminophenyl)sulfonyl]ethyl sulfate

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experiment start date - 02 July 2003; Experiment end date - 23 July 2003; Study completion date - 10 September 2003.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

yes

Remarks:

Ten animals (5 males, 5 females) per test group were evaluated In the present study the relative humidity under which the experiment was conducted ranged between 30 - 85 %

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

Ten animals (5 males, 5 females) per test group were evaluated In the present study the relative humidity under which the experiment was conducted ranged between 30 - 85 %

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

Identity: FAT 40'810/A
Batch no.: WP 6/02
Aggregate state at room temperature: solid
Colour: brown
Purity: approx. 75%
Stability in solvent: 7 days in water, saline, PEG and CMC, 1 day in vaseline and FCA
Storage: At room temperature
Expiry date: December 12, 2009

Specific details on test material used for the study:

Identity: FAT 40810/A
Batch: WP 6/02
Purity: approx. 75 %
Appearance: Solid, dark brownish powder
Expiration date: 12 December 2010
Storage: At room temperature at about 20 °C

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

Strain: NMRI
Source: RCC Ltd., CH-4414 Füllinsdorf
Number of animals: 72 (36 males/36 females)
Initial age at start of acclimatization: Males: 5 - 7 weeks, Females: 7 - 9 weeks
Acclimatization: minimum 5 days
Initial body weight at Start of Treatment: males mean value 33.2 g (SD ±1.7 g), females mean value 29.5 g (SD ±3.0 g)
According to the suppliers assurance the animals were in healthy condition. The animals were under quarantine in the animal house of RCC - CCR for a minimum of five days after their arrival. During this period the animals did not show any signs of illness or altered behaviour. The animals were distributed into the test groups at random and identified by cage number.
Husbandry: The animals were kept conventionally. The experiment was conducted under standard laboratory conditions.
Housing: Single
Cage Type: Makrolon Type l, with wire mesh top (EHRET GmbH, D-79302 Emmendingen)
Bedding: granulated soft wood bedding (ALTROMIN, D-32791 Lage/Lippe)
Feed: pelleted standard diet, ad libitum (ALTROMIN 1324, D-32791 Lage/Lippe)
Water: tap water, ad libitum, (Gemeindewerke, D-64380 Rolldorf)
Environment: temperature 22 ± 3 °C
relative humidity: 30 - 70 %
artificial light: 6.00 a.m. - 6.00 p.m.

Administration / exposure

Route of administration:

oral: unspecified

Vehicle:

Deionised water

Details on exposure:

single oral exposure of 10 ml/kg bw equivalent to 2000 mg test substance/kg bw

Duration of treatment / exposure:

single exposure

Frequency of treatment:

single application

Post exposure period:

48 hours in pre-experiment for assessment of toxicity, 24 hours in main experiment .

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

Negative control group

Doses / concentrations 2

Dose / conc.:

500 mg/kg bw/day (actual dose received)

Remarks:

Low dose group

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

Medium dose group

Doses / concentrations 4

Dose / conc.:

2 000 mg/kg bw/day (actual dose received)

Remarks:

High dose group

No. of animals per sex per dose:

Male: 500 mg/kg; No. of animals: 5; Sacrifice time: 24 hours
Male: 1000 mg/kg; No. of animals: 5; Sacrifice time: 24 hours
Male: 2000 mg/kg; No. of animals: 5; Sacrifice time: 24 hours
Male: 2000 mg/kg; No. of animals: 5; Sacrifice time: 48 hours
Female: 500 mg/kg; No. of animals: 5; Sacrifice times: 24 hours
Female: 1000 mg/kg; No. of animals: 5; Sacrifice times: 24 hours
Female: 2000 mg/kg; No. of animals: 5; Sacrifice times: 24 hours
Female: 2000 mg/kg; No. of animals: 5; Sacrifice times: 48 hours

Control animals:

yes, concurrent vehicle

Positive control(s):

40 mg/kg cyclophosphamide application orally to 5 males and 5 females

Examinations

Tissues and cell types examined:

bone marrow samples

Details of tissue and slide preparation:

The animals will be sacrificed using CO2. The femora were removed, the epiphyses were cut off and the marrow was ushed out with fetal calf serum, using a syringe. The cell suspension was centrifuged at 1500 rpm (390 x g) for 10 minutes and the supernatant was discarded. A small drop of the resuspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Grunwald (MERCK, D-64293 Darmstadt) / Giemsa (Gurr, BDH Limited Poole, Great Britain). Cover slips were mounted with EUKITT (KINDLER, D-79110 Freiburg). At least one slide was made from each bone marrow sample.

Evaluation criteria:

A test item is classified as mutagenic if it induces either a dose-related increase or a clear increase in the number of micronucleated polychromatic erythrocytes in a single dose group. Statistical methods (nonparametric Mann-Whitney test will be used as an aid in evaluating the results. However, the primary point of consideration is the biological relevance of the results. A test item that fails to produce a biological relevant increase in the number of micronucleated polychromatic erythrocytes is considered non-mutagenic in this system.

Statistics:

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

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

Doses producing toxicity (reduced spontaneous activity: pre-test = 2000 mg/kg main test = 2000 mg/kg and 1000 mg/kg

Vehicle controls validity:

valid

Negative controls validity:

not valid

Positive controls validity:

valid

Additional information on results:

See "Any other information on results incl. tables"

Any other information on results incl. tables

Pre-Experiment for Toxicity

In a pre-experiment 4 animals (2 males, 2 females) received orally a single dose of 2000 mg/kg b.w. FAT 40810/A formulated in deionised water. The volume administered was 10 ml/kg b.w.

The animals treated with 2000 mg/kg b.w. expressed toxic reactions as shown in the table:

 

Toxic reactions	Hours post-treatment male/female
	1 h	2-4 h	6h	24 h	30 h	48 h
reduction of spontaneous activity	0/2	2/2	2/2	2/2	2/2	1/1
eyelid closure	0/0	2/2	2/2	2/2	1/1	1/1
urine colour	-	orange	orange	orange	orange	-

On the basis of these data 2000 mg/kg b.w. were estimated to be suitable.

Toxic symptoms in the Main Experiment

In the main experiment for the highest dose group 24 animals (12 males, 12 females) received orally a single dose of 2000 mg /kg b.w. FAT 40810/A formulated in deionised water. The volume administered was 10 ml/kg b.w. The animals treated with 2000 mg /kg b.w. expressed toxic reactions as shown in the table

Toxic reactions	Hours post-treatment male/female
	1 h	2-4 h	6h	24 h
reduction of spontaneous activity	5/8	an	12/12	5/6
ruffled fur	0/4	8/6	9/11	4/4
urine colour	-	orange	orange	orange/ -

 

For the mid dose group 12 animals (6 males, 6 females) received orally a single dose of 1000 mg /kg b.w. FAT 40810/A formulated in deionised water. The volume administered was 10 ml/kg b.w.

The animals treated with 1000 mg /kg b.w. expressed toxic reactions as shown in the table:

Toxic reactions	Hours post-treatment male/female
	1 h	2-4 h	6h	24 h
reduction of spontaneous activity	4/0	4/4	6/6	2/1
ruffled fur	0/0	010	1/0	010

 

For the low dose group 12 animals (6 males, 6 females) received orally a single dose of 500 mg /kg b.w. FAT 40810/A formulated in deionised water. The volume administered was 10 ml/kg b.w.

The animals treated with 500 mg /kg b.w. expressed toxic reactions as shown in the table:

Toxic reactions	Hours post-treatment male/female
	1 h	2-4 h	6h	24 h
reduction of spontaneous activity	010	1/0	010	010

 

 

Summary of Micronucleus Test Results

Test group	Dose mg/kg b.w.	Sampling time (h)	PCEs with micronuclei (%)	Range	PCE per 2000 erythrocytes
vehicle	0	24	0.055	0-2	1157
test item	500	24	0.070	0-5	1091
test item	1000	24	0.060	0 -3	1129
test item	2000	24	0.050	0-3	1080
positive control	40	24	1.320	7-40	1079
test item	2000	48	0.060	0-3	1089

Applicant's summary and conclusion

Conclusions:

FAT 40810/A is considered to be non-mutagenic in this in vivo micronucleus assay.

Executive summary:

The test item FAT 40810lA was assessed in the micronucleus assay for its potential to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse. This study is conducted in accordance with OECD test guideline 474 and EU method B.12 in a GLP-certified laboratory. The test item was formulated in deionised water, which was also used as vehicle control. The volume administered orally was 10 ml/kg bw. 24 h and 48 h after a single administration of the test item the bone marrow cells were collected for micronuclei analysis. Ten animals (5 males, 5 females) per test group were evaluated for the occurrence of micronuclei. At least 2000 polychromatic erythrocytes (PCEs) per animal were scored for micronuclei. To describe a cytotoxic effect due to the treatment with the test item the ratio between polychromatic and total erythrocytes was determined in the same sample and reported as the number of PCEs per 2000 erythrocytes. The following dose levels of the test item were investigated:

24 h preparation interval: 500, 1000, and 2000 mg/kg bw.

48 h preparation interval: 2000 mg/kg bw.

 

As estimated by a pre-experiment 2000 mg/kg bw (the maximum guideline-recommended dose) was suitable. The mean number of polychromatic erythrocytes was not increased after treatment with the test item as compared to the mean value of PCEs of the vehicle control indicating that FAT 40810/A had no cytotoxic properties in the bone marrow. However, the urine of the treated animals turned orange indicating the systemic distribution of the test item and thus, its bioavailability. In comparison to the corresponding vehicle controls there was no statistically significant or biologically relevant enhancement in the frequency of the detected micronuclei at any preparation interval and dose level after administration of the test item. The mean values of micronuclei observed after treatment with FAT 40810/A were below or near to the value of the vehicle control group. 40 mg/kg bw cyclophosphamide administered orally 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 item did not induce micronuclei as determined by the micronucleus test in the bone marrow cells of the mouse.