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Toxicological information

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
Study period:
June- September 1990
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Although less erythrocytes were scored for micronuclei, the study was conducted according to valid methods, therefore it is considered adequate, reliable and relevant for classification.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
yes
Principles of method if other than guideline:
Two harvesting times were used after repeated treatment schedule; ratio of normochromatic to polychromatic erythrocytes was evaluated in 200 erythrocytes per animal.
GLP compliance:
no
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): Chloramin B; benzenesulphonamide, sodium
- Substance type: Biocide
- Stability under test conditions: Stable (Stability:140°C) stability in vehicle min. 30 min.
- Storage condition of test material: In the dry and dark place during the study

Test animals

Species:
mouse
Strain:
ICR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Breeding Farm VELAZ, Prague
- Age at study initiation: 8-12 weeks
- Weight at study initiation: 20-30 g
- Assigned to test groups randomly: Yes, under following basis: the weight variation of animals was minimal and did not exceed ±20% of the mean weight for each sex
- Fasting period before study: About 20 hours before ORAL administration the animals were not fed, water was given ad libitum. The feed was given back to animals 3 hours after administration procedure. Application was INTRAPERITONEALLY.
- Housing: Animal room with monitored conditions; 5-6 animals of one sex in one plastic cage Velaz T3; bedding of sterilized shavings of soft wood
- Diet (e.g. ad libitum): Stazndard pelleted diet DOS 2B ad libitum (VELAZ, Prague)
- Water (e.g. ad libitum): Drinking tap water ad libitum
- Acclimation period: At minimum 5 days

ENVIRONMENTAL CONDITIONS:
- Temperature (°C): 21-23°C
- Humidity (%): 40-70%
- Air changes (per hr): Not provided
- Photoperiod (hrs dark / hrs light): Natural periods of light and dark

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: distilled water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Test substance application form was the solution of test substance of appropriate concentration in distilled water, prepared just before the administration.

APPLICATION VOLUME:
The volume administered was held constant in all groups – 0.1 mL/10 g of body weight by adequate adjusting the concentration. Negative control animals were administered by water for injections in the same volume. Also the positive control substance (cyclophosphamide) was administered intraperitoneally in the same volume as solution in distilled water in the dose of 20 mg/kg.

APPLICATION SCHEDULE
For test substance: 3 doses with 24-hours time spacing with cell harvesting times 24 and 48 hours after last dosing
For controls: single dose administration
Test substance, negative and positive control were administered by intraperitoneal injection in the morning between 8th and 9th h morning.
Duration of treatment / exposure:
The test substance was administered to the mice of ICR strain intraperitoneally in multiple dose regime: 3 doses with 24-hour interval between dosing. Cytogenetic effect was evaluated at two cell harvesting times: 24, 48 hours after last administration.
Frequency of treatment:
3 doses with 24-hour interval between dosing
Post exposure period:
24 or 48 hours
Doses / concentrations
Remarks:
Doses / Concentrations:
20 - 30- 40 - 65 mg/kg
Basis:
other: intraperioneal
No. of animals per sex per dose:
TEST GROUPS MAIN STUDY
1. distilled water, cell harvesting after 24 h: 5 males and 5 females
2. test substance 3 x 20 mg/kg, cell harvesting after 24 h: 6 males
3. test substance 3 x 20 mg/kg, cell harvesting after 48 h: 6 males
4. test substance 3 x 30 mg/kg, cell harvesting after 24 h: 6 males
5. test substance 3 x 30 mg/kg, cell harvesting after 48 h: 6 males
6. test substance 3 x 40 mg/kg, cell harvesting after 24 h: 6 females
7. test substance 3 x 40 mg/kg, cell harvesting after 48 h: 6 females
8. test substance 3 x 65 mg/kg, cell harvesting after 24 h: 6 females
9. test substance 3 x 65 mg/kg, cell harvesting after 48 h: 6 females
10. cyclophosphamide, 20 mg/kg, cell harvesting after 24 h: 5 males
11. cyclophosphamide, 20 mg/kg, cell harvesting after 24 h: 5 females
Control animals:
yes
Positive control(s):
cyclophosphamide
- Justification for choice of positive control(s): known positive
- Route of administration: intraperitoneal injection
- Dose: 20 mg/kg

Examinations

Tissues and cell types examined:
Bone marrow cells were obtained from the both femora immediately after the sacrifice of an animal.
After excising and careful cleansing of femur the upper end (proximal epiphysis) of the bone was clipped off. Bone marrow was gently flushed from the bone by 1 mL of calf serum into the beaker. The bone marrow was several times mixed by syringe with thin needle and then iot was poured out into the tube. The same procedure was used with the second femur.

200 erythrocytes per animal were evaluated for the proportion of immature and mature erythrocytes in bone marrow: “index of cytotoxicity”= PCE: PCE+NCE.
PCE= polychromatic (=immature) erythrocytes
NCE= normochromatic (=mature) erythrocytes
1000 polychromatic erythrocytes per animal were scored for the incidence of micronucleated immature erythrocytes (PCE)

Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
On the basis of results of pilot acute toxicity experiments.
The maximum tolerated dose was determined as follows. From the data the LD50 values were estimated by graphic extrapolation. The estimated values were ca. 3 x 40 mg/kg for males and 3 x 80 for females.
For the administration in the main experiment the doses equivalent to ca. 50% and 80% of estimated LD50 were chosen; i.e. 3 x 20 and 3 x 30 mg/kg for males and 3 x 40 and 3 x 65 mg/kg for females.

DETAILS OF SLIDE PREPARATION:
The bone marrow with serum in tubes was centrifugated (5 min, 1000 rpm). The supernatant was gently removed, two drops of bovine serum were added to the sediment and this cell suspension was mixed on test tube mixer.
Clean and degreased slides were marked by study number and by animal code. One drop of cell suspension was placed onto the slide and using a pusher slide a smear was prepared. Three slides per animal were prepared.
After drying (24 h at laboratory temperature) the smears were fixed by ethanol 5 minutes. Then they were twice rinsed by distilled water and stained by 5% solution of Giemsa for 15 minutes.

METHOD OF ANALYSIS:
Stained smears were examined by light microscope.
200 erythrocytes per animal were evaluated for the proportion of immature and mature erythrocytes in bone marrow: “index of cytotoxicity”= PCE: PCE+NCE.
PCE= polychromatic (=immature) erythrocytes
NCE= normochromatic (=mature) erythrocytes
1000 polychromatic erythrocytes per animal were scored for the incidence of micronucleated immature erythrocytes (PCE)


OTHER: CRITERIA FOR SCORING MICRONUCLEATED IMMATURE ERYTHROCYTES
Micronuclei were identified according to the criteria established by Schmid and Heddle and Salamone:
Colour of micronuclei: purple
Form: generally round or almond shaped (occasional lightly stained and ring shaped)
Borders: sharp
Size: 5-20% size of polychromatic erythrocyte (PCE)
Unit of scoring: number of immature erythrocytes with micronuclei (number of micronuclei per PCE is not a result, occasional more than one micronucleus may appear per PCE).
Statistics:
The results were evaluated for statistical significance using the tables of mutation frequencies by Kastenbaum and Bowman. (Tables for determining the statistical significance of mutation)

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls valid:
yes
Positive controls valid:
yes

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
Under the experimental design described above, the result of mouse micronucleus test with test substance Chloramin B trihydrate, was negative. The test substance does not induce the micronuclei in the immature erythrocytes in bone marrow of mice therefore it is considered non-mutagenic in this test.
Executive summary:

The test substance Chloramin B trihydrate was tested in in vivo micronucleus test for the detection of chromosome damage induction.

The test was performed according to OECD Test Guideline No. 474 – Genetic Toxicology: Micronucleus Test. The test substance was administered to the mice of ICR strain intraperitoneally in multiple dose regime: 3 doses with 24-hour interval between dosing. The doses administered were as follows: 3 x 20 and 3 x 30 mg/kg in males, resp. 3 x 40 and 3 x 65 mg/kg in females.

Cytogenetic effect was evaluated at two cell harvesting times: 24, 48 hours after last administration. Under the experimental design described above, the result of mouse micronucleus test with test substance Chloramin B, was negative. The test substance did not induce the micronuclei in the immature erythrocytes in bone marrow of mice therefore it is considered non-mutagenic in this test.