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in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17 January to 28 February 2005
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study conducted in accordance with international guideline

Data source

Reference Type:
study report
Report Date:

Materials and methods

Test guideline
according to
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay

Test material

Details on test material:
- Description: white powder
- Analytical purity: 99.2%
- Lot/batch No.: G140421
- Expiration date of the lot/batch: May 2005

Test animals

Details on test animals and environmental conditions:
- Source: a well established supplier
- Weight at study initiation: (P) Males: 29-40 g
- Diet: free access to standard rodent diet
- Water: free access to potable water taken from the public supply
- Acclimation period: minimum of 5 days

- Temperature (°C): 19 to 25°C
- Humidity (%): 40 to 70%
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 17 January 2005 to 28 February 2005

Administration / exposure

Route of administration:
oral: gavage
- Vehicle used: carboxymethyl cellulose (CMC)
- Justification for use and choice of vehicle: not reported
- Concentration of test material in vehicle: 25, 50, 100 mg/ml
- Amount of vehicle (if gavage): 20mL/kg/day
- Lot/batch no. (if required): 012K0060
- Purity: 0.5% w/v carboxymethylcellulose
Details on exposure:

- Rate of preparation of diet (frequency): on each day of dosing
Duration of treatment / exposure:
Frequency of treatment:
All animals were dosed once on Day 1 and on Day 3
Doses / concentrations
Doses / Concentrations:
0, 500, 1000, 2000 mg/kg/day
actual ingested
No. of animals per sex per dose:
7 male mice except for positive control (5 male mice)
Control animals:
yes, concurrent vehicle
Positive control(s):
Mitomycin C
- Justification for choice of positive control(s): none reported
- Route of administration: oral gavage
- Doses / concentrations: 0.6mg/ml, volume of 20ml/kg/day, dosage of 12mg/kg/day


Tissues and cell types examined:
Bone marrow/erythtroctes
Details of tissue and slide preparation:
BPS-MAE was administered once by oral gavage to one group of animals (2 males, 2 females) at 2000 mg/kg/day (the standard limit dose for the micronucleus test). Clinical signs of toxicity included piloerection, underactive behaviour, flattened and hunched posture, fast respiration and abnorma gait.
On the basis of these results 2000 mglkg/day was considered to be the maximum tolerated dose (MTD) in both male and female animals. In line with current guidelines, no substantial difference in toxicity was observed between the sexes, therefore male animals only were used in the main micronucleus test. Dose levels of 500, 1000 and 2000 mg/kg/day were selected.

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields): males from the vehicle control and test substance groups were sacrificed 24 hours after administration of the second dose. In addition males in the positive control group were sacrificed 24 hours after a single dose.

The bone marrow of both femurs from each animal was flushed out and pooled in a total volume of 2 ml of pre-filtered foetal calf serum by aspiration. The cells were sedimented by centrifugation, the supernatant was discarded and the cells were resuspended in a small volume of fresh serum. A small drop of the cell suspension was transferred to a glass microscope slide and a smear was prepared in the conventional manner (Schmid 1976). Four smears were made from each animal.

Fixation and staining of slides
1. Fixed for a minimum of 10 minutes in methanol and allowed to air-dry.
2. Rinsed in purified water.
3. Stained in acridine orange solution (0.01 mg/ml using purified water) for 3 minutes.
4. Washed in purified water for 5 minutes.
5. Rinsed in cold tap water for 2 minutes.
6. Stored in the dark at ca 4°C for a minimum of 1 hour until required.
7. Immediately prior to scoring, slides are wet mounted with 50 mm glass coverslips using purified water.
Evaluation criteria:
One smear from each animal was examined for the presence of micronuclei in 2000 immature erythrocytes. The proportion of immature erythrocytes was assessed by examination of at least 1000 erythrocytes from each animal. A record of the incidence of micronucleated mature erythrocytes was also kept.The following criteria was applied for assessment of assay acceptability:

1. Each treated and control group should include at least 5 analysable animals.
2. Vehicle control values for micronucleated polychromatic erythrocytes must be consistent with the laboratory historical negative control data.
3. Positive controls must show clear unequivocal positive responses.
4. The proportion of immature erythrocytes among total erythrocytes in treated groups is not less than 20% of the control value.
The results for each treatment group were compared with the results for the vehicle control group using non-parametric statistics.
For incidences of rnicronucleated immature erythrocytes, exact one-sided P-values are calculated by permutation (StatXact, CYTEL Software Corporation, NC, USA). Comparison of several dose levels are made with the vehicle control using the Linear by Linear Association test for trend in a step-down fashion if significance is detected (StatXact, CYTEL Software Corporation, NC, USA); for individual inter-group comparisons (ie the positive control group) this procedure simplifies to a straightforward permutation test (Agresti et al. 1990, Gibbons 1985). For assessment of effects on the proportion of immature erythrocytes, equivalent permutation tests based on rank scores are used, ie exact versions of Wilcoxon's sum of ranks test (Wilcoxon 1945) and lonckheere's test for trend (Jonckheere 1954).

Results and discussion

Test results
Vehicle controls validity:
Negative controls validity:
not applicable
Positive controls validity:
Additional information on results:
The test substance did not cause any statistically significant increases in the number of micronucleated immature erythrocytes. The test substance did not cause any substantial increases in the incidence of micronucleated mature erythrocytes. The test substance failed to cause any significant decreases in the proportion of immature erythrocytes.

Animals were treated with the test substance at dose levels of 500, 1000 and 2000 mg/kg/day. No clinical signs were noted for the vehicle and positive control group animals over the duration of the test. Animals dosed with BPS-MAE at 500, 1000 and 2000 mg/kg/day showed clinical signs of underactive behaviour, flattened posture, abnormal gait and fast respiration. In addition, one animal in the low treatment group, 500 mg/kg/day, showed overactive behaviour at the terminal observation. All animals survived until scheduled termination. Some incidences of weight loss were recorded but these were small and not considered to be treatment related.

Applicant's summary and conclusion

Interpretation of results (migrated information): negative
BPS-MAE did not show evidence of causing an increase in the induction of micronucleated immature erythrocytes or bone marrow cell toxicity in CD-l mice, when administered orally by gavage in this in vivo test procedure at a dose level of 2000 mg/kg/day.