Zeolite, cuboidal, crystalline, synthetic, non-fibrous

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Sep. 9, 1999 - Mr. 29, 2000; experimental phase: Oct. 4 - Nov. 12, 1999

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2000

Report date:

2000

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

1997-07-21

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian bone marrow chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

Zeolite, cuboidal, crystalline, synthetic, non-fibrous

EC Number:

930-915-9

Cas Number:

1318-02-1

Molecular formula:

M2/nO • Al2O3 • ySiO2 • wH2O (n is the valency of the cation M, predominantly Na, y can range from 0.64 to 8.8, and w is the number of water molecules (general formula) Na: 1.34 - 24.02%, Al: 2.20 - 39.51%, Si: 15.52 - 68.64% (general composition); additionally, depending on the water quality: Ca, Mg and K might be present below 6%

IUPAC Name:

Zeolite, cuboidal, crystalline, synthetic, non-fibrous

Test material form:

solid: particulate/powder

Remarks:

no surface treatment

Specific details on test material used for the study:

name: Zeostop X (X Zeolite)
batch number: MR 453 136
description: white powder
date of receipt: 3 September 1999
storage conditions: at room temperature and protected from light
purity: 100%
expiry date: May 2000

Test animals

Species:

mouse

Strain:

Swiss

Details on species / strain selection:

Number: three male and three female mice for the preliminary toxicity test; 56 mice: 28 males andd 28 females for the cytogenetic study (first test); 35 female mice for the cytogenetic study (second test)
Strain: Swiss Ico: OF1 (IOPS Caw)

Sex:

male/female

Details on test animals or test system and environmental conditions:

Breeder: Iffa Crédo, L'Arbresle, France
Age: on the day of treatment, the animals were at least 5 weeks old
temperature: 21 ±2°C,
relative humidity: 30 to 70%
light/dark cycle: 12 h/12 h (07:00 - 19:00)
ventilation: about 12 cycles/hour of filtered non-recycled fresh air
All animals had free access to A04C pelleted maintenance diet and tap water.

Administration / exposure

Route of administration:

oral: unspecified

Vehicle:

0.5% aqueous methylcellulose solution: methylcellulose, batch No. 118H0286

Duration of treatment / exposure:

four times at a 24-hour interval

Frequency of treatment:

four times at a 24-hour interval

Post exposure period:

24 hours after last treatment

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

1 250 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

2 500 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

5 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5 (first test) and 5 (females only, second test)

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide (CPA, batch No 114552) dissolved in distilled water at a concentration of 5 mg/ml

Examinations

Tissues and cell types examined:

For each animal, the number of the micronucleated polychromatic erythrocytes (MPE) was counted in 2000 polychromatic erythrocytes; the polychromatic (PE) and normochromatic (NE) erythrocyte ratio was established by scoring a total of 1000 erythrocytes(PE + NE).

Details of tissue and slide preparation:

The femurs of the animals were removed and the bone marrow was eluted out using fetal calf serum. After centrifugation, the supernatant was removed and the cells in the sediment were suspended by shaking. A drop of this cell suspension was placed and spread on a slide. The slides were air-dried and stained with Giemsa. All the slides were coded for scoring.

Evaluation criteria:

For a result to be considered positive, a statistically significant increase in the frequency of MPEhad to be demonstrated when compared to the concurrent vehicle control group. Reference to historical data, or other considerations of biological relevance were also taken into account in the evaluation of data obtained.

Statistics:

When there was no significant within-group heterogeneity, using the heterogeneity chi-square test value, the frequencies of MPE in each treated group were compared with those in the concurrent vehicle control groups by using a 2 x 2 contingency table to determine the value).
When there was significant within group heterogeneity, then that group was compared with the control group using a non-parametric analysis, the Mann-Whitney test.
The student *'!" test was usedfor the PE/NE ratio comparison.
Probability values of p <= 0.05 were considered as significant.

Results and discussion

Test results

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

Although toxic effects were observed, these were also documented for the control group (cf. below).

Vehicle controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

PRELIMINARY TOXICITY TEST

In order to select the top dose-level for the cytogenetic study, 5000 mg/kg/day were administered four times, to three males and three females. The interval between each administration was 24 hours. No clinical sign and no mortality were noted in both males and females.

 

CYTOGENETIC EXPERIMENT

For males, no mortality was noted. The clinical signs were as follows:

-   At 1250 mg/kg/day, piloerection in all animals from 24 hours following the second or third treatment and until sacrifice of the animals.

-   At 2500 and 5000 mg/kg/day, piloerection in 4/5 animals from 24 hours following the second treatment and until sacrifice.

 

For females, mortality and clinical signs were as follows:

-   At 1250 mg/kg/day, 2/5 animals were found dead 24 hours after the second treatment. No clinical signs were recorded prior to death. Piloerection was noted in 2/3 surviving animals from 2 or 24 hours following the third treatment and until sacrifice.

-   At 2500 mg/kg/day, 1/5 animals died 2 hours after the third treatment. Piloerection, dyspnea and hypoactivity were noted from 24 hours following the second treatment up to death. Piloerection was also recorded in 1/5 animals from 24 hours following the second treatment up to sacrifice.         

-   At 5000 mg/kg/day, 2/5 animals were found dead 24 hours after the first treatment. No clinical signs were observed prior to death. Piloerection was also noted in 1/3 surviving animals, from 24 hours following the second treatment up to sacrifice.

-   In the control group, 1/5 animals was found dead 24 hours after the second treatment. For this female, the cause of death could not be established. However, since mortality in the vehicle group is unusual, regurgitation followed by accidental aspiration of the vehicle in the lungs could not be excluded.

Due to the low number of available animals in the vehicle control group of females (1/5 females dead and 1/5 females showed a very low PE/NE ratio (0.2), this experiment was rejected and was repeated for females only, at the same dose-levels.

In this second test, no clinical signs and no mortality were noted except at 2500 mg/kg/day, at which 1/5 females showing piloerection 24 hours after the first treatment died within the 2 hours following the second treatment.

 

For both males and females, the mean values of MPE in the groups treated with the test substance, were equivalent to those of the vehicle control group, and no significant difference was noted. In males given 5000 mg/kg/day, the PE/NE ratio was significantly lower (p < 0.05) compared to that of the vehicle group, providing evidence of exposure of bone marrow to the test substance. The mean values of MPE as well as the PE/NE ratio for the vehicle and positive controls were consistent with the historical data.

 

Cyclophosphamide induced a highly significant increase (p < 0.001) in the frequency of MPE, indicating the sensitivity of the test system under the experimental conditions. The study was therefore considered valid.      

Applicant's summary and conclusion

Conclusions:

Under the experimental conditions, the test substance ZEOSTOP X did not induce damage to the chromosomes or the mitotic apparatus of mice bone marrow cells after four oral administrations, with a 24-hour interval, at the dose levels of 1250, 2500 and 5000 mg/kg/day.

Executive summary:

The objective of this study was to evaluate the potential of the test substance to induce damage to the chromosomes or the mitotic apparatus in bone marrow cells of mice.

Therefore, a preliminary toxicity test was performed to define the dose-levels to be used for the cytogenetic study. In the main study, three groups of five male and five female Swiss Ico: OF1 (IOPS Caw) mice received four oral treatments of Zeostop X at dose-levels of 1250, 2500 and 5000 mg/kg/day, at a 24-hour interval. Due the low number of available animals in the female vehicle control group, the experiment was repeated for females only, at the same dose-levels.

Zeostop X did not induce damage to the chromosomes or the mitotic apparatus of mice bone marrow cells.