Calcium 4-chloro-2-(5-hydroxy-3-methyl-1-(3-sulfonatophenyl)pyrazol-4-ylazo)-5-methylbenzenesulfonate

Genetic toxicity: in vivo

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Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Well performed GLP and OECD guideine study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1989

Report date:

1989

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Test material form:

solid: bulk

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain specifics: Hoe: NMRKf (SPF71)
- Source: Hoechst AG breeding colony
- Age at study initiation: 7 weeks
- Weight at study initiation: males: 29-36 g, mean 32.1 g, females: 21-29 g, mean 24.7 g
- Housing: in groups of five in Macrolon type 3 cages in fully air-conditioned room, softwood granulate
- Diet (e.g. ad libitum): rat/mouse standard diet Altromin 1324, ad libitum
- Water (e.g. ad libitum): tap water in plastic bottles, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2 °C
- Humidity (%): 55 ± 10 %
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: sesame oil (Oleum Sesami Ph. Eur. III, Pharm. Fabrik GmbH, Frankfurt/Main, Germany)
- Concentration of test material in vehicle: 10 % (w/v)
- Amount of vehicle (if gavage or dermal): 20 mL/kg body weight

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

the test substance suspensions were prepared fresh each day. 5000 mg were weight in a beaker, mixed with sesame oil, washed out in a 50 mL flask and topped up to the calibration mark.

Duration of treatment / exposure:

oral administration of two equal parts within 2 hours for 24 h and 48 h dosed group; one single oral administration for the 72 h dosed group.

Frequency of treatment:

See above

Post exposure period:

24 hours (dosed group, negative control and positive control), 48 hours (dosed group and negative control) and 72 hours (dosed group and negative control)

Doses / concentrations

Remarks:

Doses / Concentrations:
2000
Basis:
nominal conc.
mg/kg body weight

No. of animals per sex per dose:

5 males and 5 females (10 animals) per dose group

Based on the results of a preliminary range finding study, a dose of 2000 mg/kg bw was the maximum applicable dose level.

Control animals:

yes, concurrent vehicle

Positive control(s):

For the cyclophosphamide stock solution 5 mL distilled water were added to 100 mg cyclophosphamide in an injection phial and shaken to form a clear solution. For administration 2 mL of the 2 % stock solution were mixed with 6 mL distilled water.
50 mg/kg body weight cyclophosphamide (Endoxan (R), 5 males and 5 females)

Examinations

Tissues and cell types examined:

1000 polychromatic erythrocytes from the femoral bone marrow were counted for each animal.

Details of tissue and slide preparation:

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
Animals were killed 24, 48 or 72 h after application

DETAILS OF SLIDE PREPARATION:
Removal of femoral bones and bone marrow from the proximal ends flushed into centrifuge tube containing about 3 ml foetal bovine serum, centrifugation (5 min at 1200 rpm), one drop of thoroughly mixed sediment smeared on a cleaned slide, air-dried for approx. 24 h, staining (methanol, May-Grünwalds solution, Giemsa) and coating with Entellan

METHOD OF ANALYSIS:
Number of cells with micronuclei and ratio of polychromatic to normochromatic erythrocytes
Statistical evaluation (see below)

Evaluation criteria:

The results of the treatment groups at each killing time were compared with corresponding control values.
95 % level of significance for comparisons.
Actual data were also compared with historical controls.

Statistics:

Wilcoxon (paired, two-sided) for the ratio of polychromatic to normochromatic erythrocytes.
Wilcoxon (paired, on-sided, increase) for the proportion of polychromatic and normochromatic erythrocytes with micronuclei

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Remarks:

all animals free of clinical signs of toxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The dissection of the animals revealed yellow coloured content in stomach and intestinum.

RESULTS OF RANGE-FINDING STUDY

- Dose range: 2000 mg/kg body weight
- Other: lethality: 0 out of 3 males and 0 out of 3 females

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): no statistically significant increase of micronucleated polychromatic erythrocytes in dosed animals
- Ratio of PCE/NCE (for Micronucleus assay): the ratio remained essentially unaffected by the test substance
- Statistical evaluation: see above

Any other information on results incl. tables

Mean mutation indices in polychromatic erythrocytes:

sex	sampling after dosing	dose [mg/kg bw]	mean mutation index
male	24 h	control	1.0
	24 h	2000	1.5
	24 h	positive control	29.0
female	24 h	control	1.0
	24 h	2000	1.8
	24 h	positive control	22.7
male	48 h	control	1.0
	48 h	2000	1.7
female	48 h	control	1.0
	48 h	2000	1.3
male	72 h	control	1.0
	72 h	2000	0.7
female	72 h	control	1.0
	72 h	2000	1.1

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
Oral administration of the test item did not lead to a substantial increase of micronucleated polychromatic erythocytes. Therefore, the test item was not mutagenic in the in vivo mouse micronucleus test

Executive summary:

The test item was tested in the in vivo micronucleus test according to OECD 474. The test substance was suspended in sesame oil and dosed orally at 2000 mg per kg bodyweight to male and female mice, based on the results of the previously conducted dose range finding assay. According to the test procedure the animals were killed 24, 48 or 72 hours after administration.

Endoxan® was used as positive control substance and was administered orally at a dose of 50 mg per kg bodyweight.

 

The number of polychromatic and normochromatic erythrocytes containing micronuclei was not increased. The ratio of polychromatic/normochromatic erythrocytes in both male and female animals remained unaffected by the treatment with the test item and was statistically not different from the control values.

Endoxan® induced in both males and females a marked statistically significant increase in the number of polychromatic cells with micronuclei, indicating the sensitivity of the system.

The results indicate that, under the conditions of the present study, the test item is not mutagenic in the micronucleus test.