C,C'-azodi(formamide)

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

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:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

C,C'-azodi(formamide)

EC Number:

204-650-8

EC Name:

C,C'-azodi(formamide)

Cas Number:

123-77-3

Molecular formula:

C2H4N4O2

IUPAC Name:

diazene-1,2-dicarboxamide

Details on test material:

- Name of test material (as cited in study report): Unifoam AZ SO-NL
- Substance type: yellow powder
- Physical state: solid
- Analytical purity: 100%
- Lot/batch No.: 23.3.88/4
- Storage condition of test material: room temperature in the dark
- Other: suspensions of unifoam az so-nl were prepared in 1% methylcellulose using a Silverson high-speed mixer, and prepared on the morning of the test at the concentrations shown overleaf.

Test animals

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source:Charles River U.K. Limited, Margate, Kent, England
- Age at study initiation:35 days old
- Weight at study initiation:22 and 24 grams
- Assigned to test groups randomly: yes
- Fasting period before study: The animals were deprvied of diet overnight prior to and for two hours after oral dosing.
- Housing:Each group of 2 or 5 mice was kept in a plastic disposable cage
- Diet (e.g. ad libitum):free access to pelleted Labsure LAD 1 rodent breeding diet.
- Water (e.g. ad libitum):free access to tap water.
- Acclimation period:4 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C):22
- Humidity (%):no data
- Air changes (per hr):30
- Photoperiod (hrs dark / hrs light):12/12

IN-LIFE DATES: From: To: no data

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

1% methylcellulose

Details on exposure:

Preliminary toxicity test:
Increasing dosages of Unifoam AZ SO-NL were administered up to a maximum of 5000 mg/kg bodyweight (0, 312.5, 625, 1250, 2500, 5000mg/kg). Twelve male and twelve female mice were used in this experiment (2mice/sex/group).
Following dosing, the animals were observed regularly during the working day for a period of 72 hours, and any mortalities or signs of malreaction during the experiment were recorded.

Duration of treatment / exposure:

single treatment

Frequency of treatment:

single treatment

Post exposure period:

24, 48, and 72 hours for Unifoam AZ SO-NL treated groups
24 hours for positive control group (Mitomycin C)

Doses / concentrations

Remarks:

Doses / Concentrations:
5000mg/kg
Basis:
actual ingested

No. of animals per sex per dose:

Vehicle comtrol group: 15 males and 15 females
Unifoam AZ SO-NL group: 19 males and 17 females
Mitomycin C positive control group: 5 males and 5 females

Control animals:

yes

Positive control(s):

Mitomycin C, obtained from Sigma London Chemical Company Limited (batch number 96F-0547-1) was tested at 12mg/kg.
It was prepared as a solution in sterile 0.9% saline at a concentration of 0.6 mg/ml.

Examinations

Tissues and cell types examined:

Micronucleated cells

Details of tissue and slide preparation:

Following dosing, the animals were examined daily and weighted prior to dosing, and any mortalities or clinical signs of reaction to the test compounds were recorded. Five males and five females from the negative control and test compound groups were sacrificed 24, 48, and 72 hours after dosing. The positive control group was sacfrificed 24 hours after dosing. The animals were killed by cervical dislocation and both femurs dissected out from each animal. The femurs were cleared of tissue and one epiphysis removed from each bone.

A direct bone marrow smear was made onto a slide containing a drop of calf serum. One smear was made from each femur. The prepared smears were air-dried and stained for 10 minutes in 10% Giemsa. Following rinsing and differentiation in buffered distilled water for 10 minutes, the smears were air-dried and mounted with coverslips using DPX.

Stained smears were examined by light microscopy to determine the incidence of micronucleated cells per 1000 polychromatic erythrocytes per animal. The ratio of polychromatic to normochromatic erythrocytes for each animal was assessed by examination of at least 1000 erythrocytes. A record of the number of micronucleated normochromatic erythrocytes was also kept.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Remarks:

but limit comcentration tested

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 0-5000mg/kg
- Solubility: no data
- Clinical signs of toxicity in test animals:hunched posture and piloerection in the high dose groups until 4 hours after dosing

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay):Unifoam AZ SO-NL did not cause any statistically significant increases in the number of micronucleated polychromatic erythrocytes at any of the three kill times - (P>0.05 using Wilcoxon's sum of ranks test. Unifoam AZ SO-NL did not cause any substantial increases in the incidence of micronucleated normochromatic erythrocytes at any of the three kill times.
Mitomycin C caused large, highly significant increases (P<0.001) in the frequency of micronucleated polychromatic erythrocytes.
- Ratio of PCE/NCE (for Micronucleus assay):Unifoam AZ SO-NL failed to cause any significant decreases in the ratio of polychromatic to normochromatic erythrocytes (P>0.05 using Wilcoxon's sum of ranks test). Mitomycin C caused statistically significant decreases in the ratio (P<0.05).

Applicant's summary and conclusion

Conclusions:

Negative

Executive summary:

In this assessment of the effect of Unifoam AZ SO-NL on the incidence of micronucleated polychromatic erythrocytes in mice, a dosage of 5000 mg/kg bodyweight was administered orally, by intragastric gavage (HLS 1988, OCI79/88802). (A preliminary toxicity test had been carried out to determine the toxicity of Unifoam AZ SO-NL). As no toxicity was observed at a dose level of 5000 mg/kg, the highest dose recommended for acute toxicity tests, this dosage was chosen for the main test.

Negative and positive control groups were dosed in an identical manner, orally by intragastric gavage. The negative control group received the vehicle, 1% methylcellulose. The positive control group was treated with mitomycin C, at 12 mg/kg.

Bone marrow smears were obtained from the negative control and test compound groups at 3 sampling times; these being 24, 48 or 72 hours after dosing. Bone marrow smears were obtained from the positive control group 24 hours after dosing. One smear from each animal was examined for the presence of micronuclei in 1000 polychromatic erythrocytes. The ratio of polychromatic to normochromatic erythrocytes was assessed by examination of at least 1000 erythrocytes from each animal. A record of the incidence of micronucleated normochromatic erythrocytes was also kept.

 

At all sampling times, mice treated with Unifoam AZ SO-NL showed no significant increase in the frequency of micronucleated polychromatic erythrocytes. There was no significant decrease in the ratio of polychromatic to normochromatic erythrocytes after treatment of the animals with Unifoam AZ SO-NL. The positive control compound, mitomycin C, produced large, highly significant increases in the frequency of micronucleated polychromatic erythrocytes together with decreases in the ratio of polychromatic to normochromatic erythrocytes.

It is concluded from the results obtained that Unifoam AZ SO-NL shows no evidence of mutagenic potential or bone marrow cell toxicity when administered orally in this in vivo test procedure.