Isobutyl 4-chloro-3,5-diaminobenzoate

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

October 1986 - March 1987

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1987

Report date:

1987

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

not applicable

GLP compliance:

yes

Remarks:

OECD-Grundsätze der Guten Laborpraxis (Bundesanzeiger Nr. 42a vom 4. Februar 1983)

Type of assay:

other: mammalian soma cell micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

Isobutyl 4-chloro-3,5-diaminobenzoate

EC Number:

251-311-5

EC Name:

Isobutyl 4-chloro-3,5-diaminobenzoate

Cas Number:

32961-44-7

Molecular formula:

C11H15ClN2O2

IUPAC Name:

2-methylpropyl 3,5-diamino-4-chlorobenzoate

Test material form:

solid: granular

Remarks:

brown crystalline granulate

Details on test material:

Test item was confirmed to be stable during study duration

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: refrigerated
- Stability under test conditions: confirmed to be stable

Test animals

Species:

mouse

Strain:

NMRI

Remarks:

Bor:NMRI (SPF Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Breeder Winkelmann, Borchen
- Age at study initiation: ca. 9-12 weeks
- Weight at study initiation: 31-44 g
- Assigned to test groups randomly: yes
- Fasting period before study: not stated
- Housing: in groups of max. 3 resp. 5 mice, divided according to sex and treatment groups, in macrolone cages type I resp. II, on wooden soft bedding (Fa. Bogner GmbH, 5650 Solingen-Ohligs)
- Diet (e.g. ad libitum): "fixed -formula" ― Standarddiat Altromin 1324; Hersteller Altromin GmbH, Lage) ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: at least 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-22°C (set as 22±2°C)
- Humidity (%): 45-54% (set as min. 50%)
- Air changes (per hr): at least 10/h
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: polyethylene glycol (Lutrol)
- Amount of vehicle (if gavage or dermal): 40 mL/kg initially, which were reduced to 24 mL/kg as the first treated animals died due to the inherent toxicity of Lutrol

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Test item was suspended in Lutrol and stirred with a magnetic stirrer until application.

Duration of treatment / exposure:

single gavage, animals were decapitated either 24, 48, or 72 h after gavage

Frequency of treatment:

single gavage

Post exposure period:

animals were decapitated either 24, 48, or 72 h after gavage

Doses / concentrations

Dose / conc.:

20 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5/sex/dose

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide
- Route of administration: oral: gavage
- Doses / concentrations: 20 mg/kg

Examinations

Tissues and cell types examined:

femur bone marrow erythroblasts

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: Preliminary toxicity test with doses of 5, 10, 20, and 25 g/kg. All doses led to symptoms as apathy, diminished motility, ruffled fur, face-down position, cramps, difficulties breathing, orange coloured urine, diarrhea and lacking defecation. In the 25 g/kg dose group, 3 of 5 animals died.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): Single treatment, tissue was collected 24, 48, or 72 h after gavage (Controls only after 24h).
Sampling and preparation was done according to Schmid (SCHMID, W. Der Mikrokerntest. Deutsche Forschungsgemeinschaft. Kommission fur Mutagenitätsfragen. Mitteilung II 53—61 1975; Schmid W. The micronucleus—test for cytogenetic analysis. In: Hollander, A. (ed.), Chemical Mutagens, Principles and Methods for their Detection, Vol. 4 31-53 Plenum Press New York (1976)

DETAILS OF SLIDE PREPARATION:
Of each treated animal (no pretreatment with a spindle poison), at least one femur was preparated, in brief, using a syringe with calf serum and purgeing the bone. The collected cells were centrifuged and the sediment was spread onto the slide. Slides were stained with a staining machine Ames Hema-Tek Slide stainer by the company Miles.

METHOD OF ANALYSIS: As a rule, 1000 polychromatic erythrocytes per animals were analysed for micronuclei in a meandering course. In adition it is reasonable to determine the ratio of polychromatic and normochromatic cells. In case there were way more than 3000 normochromatic cells with 1000 polychromatic ones, without similar effects in other animals of the same treatment group, a pathologic, non-substance-related event is detected and the animal is excluded from assessment.
In addition to the number of normochromatic cells the number of normochromatic cells with micronuclei was determined.

Evaluation criteria:

The treatment group with the highest mean value, provided that this value was above the value of the negative control group, as well as the positive control group, were evaluated with the distribution-free Wilcoxon rank test, with regard to the amount of polychromatic cells with micronuclei and of normochromatic cells. A difference is statistically significantly different if the error probability is <5% that the value of the treatment group is higher than the one of control. Evaluation of the rate of normochromatic cells with micronuclei was performed if already an elevated rate of polychromatic cells with micronuclei was seen. In this case, the group with the highest mean value was compared to control with the one-sided Chi²-Test.
In addition, the 1s ranges for all mean values were calculated.

Statistics:

See evaluation criteria

Results and discussion

Test results

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

Treated mice showed signs of toxicity incl. apathy, diminished motility, ruffled fur, diarrhea and orange stained urine, 2/30 animals died.

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
In the preliminary toxicity test with doses of 5, 10, 20, and 25 g/kg, all doses led to symptoms as apathy, diminished motility, ruffled fur, face-down position, cramps, difficult breathing, orange coloured urine, diarrhea and lacking defecation. In the 25 g/kg dose group, 3 of 5 animals died.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): Not significantly over control
- Ratio of PCE/NCE (for Micronucleus assay): Treatment with the test item caused an inhibition of erythropoesis, Ratios of PCE/NCE are:
1000 : 1783 (1s = 771) (negative control)
1000 : 3189 (1s = 1741) (treatment group, 24h)
1000 : 2224 (1s = 1077) (treatment group, 48h)
1000 : 1755 (1s = 688) (treatment group, 72h)
- Appropriateness of dose levels and route: Treated mice showed signs of toxicity incl. apathy, diminished motility, ruffled fur, diarrhea and orange stained urine, 2/30 animals died.

Applicant's summary and conclusion

Conclusions:

The study was conducted similar to OECD guideline 474 on the registered substance itself. The method is to be considered scientifically reasonable with no deficiencies in documentation or any deviations, the validity criteria are fulfilled, positive and negative controls gave the appropriate response. Hence, the results can be considered as reliable to assess the potential of Isobutyl 4-chloro-3,5-diaminobenzoate to induce micronuclei in vivo in NMRI mice.
There were no biologically or statistically significant differences between the treatment groups (20 g/kg test item) and the negative control with regard to polychromatic cells with micronuclei, relevant biological differences were not observed.
Hence, Isobutyl 4-chloro-3,5-diaminobenzoate should be regarded as non-clastogenic in vivo.

Executive summary:

Using the micronucleus test similar to OECD TG 474, Isobutyl 4-chloro-3,5-diaminobenzoate was investigated in male and female mice for a possible clastogenic effect on the chromosomes of bone marrow erythroblasts. The known clastogen and cytostatic agent cyclophosphamide served as a positive control.

The animals received a single administration. 24, 48 and 72 hours after the administration the femoral marrow of the Isobutyl 4-chloro-3,5-diaminobenzoate groups was prepared. Negative and positive control were sacrificed after 24 hours only. The dose of Isobutyl 4-chloro-3,5-diaminobenzoate was 20000 mg/kg body weight, and of the positive control cyclophosphamide 20 mg/kg. The test substance and the positive control cyclophosphamide were administered orally.

The animals treated with Isobutyl 4-chloro-3,5-diaminobenzoate showed lasting symptoms of toxicity after the administration. 2 of 30 treated animals died until the end of the test.

Erythrocyte formation, as measured by the ratio polychromatic to normochromatic erythrocytes, was time-dependent affected.

No indications of a clastogenic effect of Isobutyl 4-chloro-3,5-diaminobenzoate were found after a single treatment with 20000 mg/kg per os.

Cyclophosphamide, the positive control, had a clear clastogenic effect, as can be seen from the biologically relevant increase in polychromatic erythrocytes with micronuclei. An inhibition of erythropoiesis was not found here.