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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:
16 January to 13 February 1989
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study (available at the time; 1983), and to GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1989
Report Date:
1989

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Remarks:
Carried out according to the 1983 version which recommended the counting of 1000 cells (not the 2000 recommended in the current version)
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Chlorowax 500C
- Substance type: technical product
- Physical state: colourless liquid
- Analytical purity: "100%"
- Impurities (identity and concentrations): none
- Composition of test material, percentage of components: C10-13 chlorinated parrafin (58% chlorination)
- Isomers composition: no data
- Purity test date: no data
- Lot/batch No.: 036895; ICI Y00987/003/001
- Expiration date of the lot/batch: no data
- Stability under test conditions: no data
- Storage condition of test material: in dark at 4oC

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Hoechst AG, Kastengrund, Germany
- Age at study initiation: 7 weeks
- Weight at study initiation: males 27-33 g; females 21-27 g
- Assigned to test groups randomly: no data
- Fasting period before study: no data
- Housing: 5 of same sex/cage in Macrolon cages with softwood granulate bedding
- Diet (e.g. ad libitum): conventional, ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22+/- 2
- Humidity (%): 55 +/- 10
- Air changes (per hr): no data; "fully air-conditioned"
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 16 January 1989 To: 19 January 1989

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: sesame oil
- Justification for choice of solvent/vehicle: no data
- Concentration of test material in vehicle: 500 mg/mL
- Amount of vehicle (if gavage or dermal): 10 ml/kg bw
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: 12500 mg weighed into a beaker, mixed with sesame oil, washed out into a 25 mL flask and made up to the calibration mark with sesame oil

Duration of treatment / exposure:
single exposure
Frequency of treatment:
single treatment
Post exposure period:
24, 48 or 72 h
Doses / concentrations
Remarks:
Doses / Concentrations:
5000 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
5/sex/dose for each of the three time points
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Justification for choice of positive control(s): on list of recommended substances
- Route of administration: oral
- Doses / concentrations: 50 mg/kg bw

Examinations

Tissues and cell types examined:
bone marrow erythrocytes
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: on basis of a range-finding study and considered to be the maximum applicable dose

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): treated and vehicle controls animals sampled at 24, 48 or 72 h after dosing. Positive control sampled at 24 h.

DETAILS OF SLIDE PREPARATION: bone marrow was flushed out of femurs into foetal calf serum and centrifuged. The cell pellets were smeared onto slides and air-dried for 24 h before staining with May-Grunwalds/Giema solutions

METHOD OF ANALYSIS: 1000 polychromatic erythrocytes were counted and the number with micronuclei were recorded. 1000 mature erythrocytes were also counted and examined for micronuclei.

Evaluation criteria:
A clear increase in the incidence of micronuclei compared with the vehicle controls corroberated by statistical analysis
Statistics:
The number of of polychromatic cells with micronuclei and the number of mature cells with micronuclei were compared with the control groups according to the method of Wilcoxon (paired, one-sided, increase). The ratio of polychromatic to mature erythrocytes were compared using the method of Wilcoxon (paired, two-sided analysis).

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
All animals survived the treatment and showed no clinical signs of toxicity or evidence of gross macroscopic changes at necropsy.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
In a GLP study, conducted according to OECD Guideline 474 (1983), Chlorowax 500C (a C10-13 chlorinated paraffin; 58% chlorination) at 5 g/kg bw showed no evidence of genotoxic potential in an in vivo bone marrow micronucleus assay in male and female mice when sampled at 24, 48 or 72 h after dosing.
Executive summary:

In a GLP study, conducted according to OECD Guideline 474 (1983), Chlorowax 500C (a C10-13 chlorinated paraffin; 58% chlorination) was assessed for its ability to cause chromosome damage in the bone marrow erythrocytes of NMRI mice after oral exposure. Groups of five mice of each sex were dosed by oral gavage with 5 g/kg bw of the test compound in sesame oil and bone marrow erythrocytes were sampled from the femurs after 24, 48 or 72 h. Further groups of mice (vehicle controls) received sesame oil alone and samples were taken at the same time points. The positive control, cyclophosphamide was given orally at 50 mg/kg bw and the bone marrow cells were sampled at 24 h. After slide preparation and differential staining of the cells with May-Grunwalds/Giema, 1000 cells each of polychromatic and mature erythrocytes were counted and the number with micronuclei were recorded. The ratio of polychromatic to mature erythrocytes was used to assess toxicity.

No increase in the incidence of micronucleated polychromatic erythrocytes or mataure erythrocytes was evident in any of the treatment groups compared to the controls. The ratio of polychromatic to mature erythrocytes was similar in treated and untreated animals indicating that the test substance was not toxic to the bone marrow cells. The positive control induced the expected increase in micronuclei.

Under the conditions of the study, Chlorowax 500C showed no evidence of genotoxic activity in an in vivo bone marrow micronucleus assay in male and female mice.