Zeolite, cuboidal, crystalline, synthetic, non-fibrous

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

There are six reliable studies in genetic toxicity in vitro. Half of them with a Ag-exchanged type X zeolite, the other half with sodium aluminosilicate. None of these showed mutagenic effects. Only one of those (Zeostop X, OECD 473, Marshall 2000, CHO, RL 1) showed positive cytotoxic effects. But this is disproved by an in vivo study in cytotoxicity with the same substance (Zeostop X, OECD 474, Haddouk 2000, Mouse Bone Marrow, RL 2) which did not show any mutagenic effects. Therefore, zeolites should not be considered as genotoxic.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods with acceptable restrictions

Justification for type of information:

A structural analogue substance was used for assessment since the performed study is well documented, according to current guidelines and GLP compliant. The "Structural Analogues Substances Approach" is provided in chapter 13.2.

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

no guideline followed

Principles of method if other than guideline:

Cytogenetic study to determe chromosomal damage: This is accomplished
by observing cells in anaphase. As the chromatids separate and move along the spindle, aberrations may occur.

GLP compliance:

not specified

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

Sodium aluminosilicate (Compound FDA 71-45, Synthetic Silica, Lot Number SR-1621, as supplied by the Food and Drug Administration)

Species / strain / cell type:

mammalian cell line, other: human embryonic lung cultures (WI-38)

Metabolic activation:

without

Test concentrations with justification for top dose:

1, 10, 100 µg/ml

Vehicle / solvent:

saline

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

triethylenemelamine

Key result

Species / strain:

mammalian cell line, other: human embryonic lung cultures (WI-38)

Metabolic activation:

without

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The negative controls, medium level and low level tested exhibited 2, 0, and 1 percent acentric fragments, respectively. The high level had one cell with an acentric fragment and one cell with a bridge. This was not considered significant. The positive controls contained four cells with pulverization together with other aberrations.

Conclusions:

The compound produced no significant aberration in the anaphase chromosomes of human tissue culture cells when tested at the dosage levels employed in this study.

Executive summary:

This study investigated into the potential of Sodium aluminosilicate to produce cytotoxicity in human embryonic lung cultures (WI-38). No significant changes to the control were observed.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May 4 - Jul. 23, 1999; experimental phase: May 5 - Jun. 10, 1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

Jul. 1997

Deviations:

not specified

GLP compliance:

yes

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Specific details on test material used for the study:

ZEOSTOP X (a type X zeolite), batch number MR 453 136, was a white powder. It was Reived on 28 April 1999 and stored at room temperature in the dark. Test article purity was stated as 100% and an expiry date was given as May 2000.

Target gene:

tk locus (5-trifluorothymidine resistance)

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

from male Sprague Dawley rats

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver post-mitochondrial fraction (S-9)

Test concentrations with justification for top dose:

first experiment: 0, 2.5, 5, 10, 20, 25 µg/ml (without activation); 0, 10, 20, 40, 80 µg/ml (with activation)
second experiment: 0, 5, 10, 15, 17.5, 20, 22.5 µg/ml (without activation); 0, 10, 20, 40, 50, 60, 70 µg/ml (with activation)

Vehicle / solvent:

tissue culture medium RPMI 1640

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

benzo(a)pyrene

Rationale for test conditions:

1) The mutant frequencies in the negative (vehicle) control cultures fell within the normal range (above 60 mutants per 10^6 viable cells but not more than three times the historical mean value).
2) At least one concentration of each of the positive control chemicals induced a clear increase in mutant frequency (the difference between the positive and negative control mutant frequencies was greater than half the historical mean value).
3) The plating efficiencies of the negative controls from the mutation experiments were between the range of 60% to 140% on Day 0 and 70% to 130% on Day 2.

Evaluation criteria:

- The mutant frequency at one or more doses was significantly greater than that of the negative control (p<0.05).
- There was a significant dose-relationship as indicated by the linear trend analysis (p<0.05).

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Range finding experiment: The top doses where cells survived treatment were 20 µg/mL in the absence of S-9 (28.53% relative survival) and 156 µg/mL in the presence of S-9 (17.91% relative survival).
First main experiment: The top doses selected were 25 mg/mL in the absence of S-9 (3.08% relative survival [2% RTG]) and 80 µg/mL the presence of S-9 (10.03% relative survival [7% RTG]).
Second main experiment: The top doses selected to determine TFT resistance in this experiment were 22.5 µg/mL in the absence of S-9 (5.33% relative survival [5% RTG]) and 70 µg/mL in the presence of S-9 (9.25% relative survival [3% RTG]).

In the absence of S-9, no statistically significant increases in mutant frequency were observed following treatment with ZEOSTOP X at any dose level tested, in Experiment 1 or 2.

In the presence of S-9, in Experiment 1, a small but statistically significant increase (1.4-fold), in mutant frequency (136 mutants per 10^6 viable cells [dose], compared to 94 mutants per 10^6 viable cells [control]) was observed at the top dose analysed (80 µg/mL).

However, 80 µg/mL was a highly toxic dose (10.03 % relative survival and 7 % relative total growth) and the increase in mutant frequency was related to a depression in Day 2 viability counts rather than a marked increase in mutant numbers. Furthermore, in Experiment 2, when ZEOSTOP X was tested up to doses of similar toxicity, no statistically significant increases in mutant frequency were observed. The small increase in mutant frequency seen at 80 µg/mL in Experiment 1 was considered to be due to a chance event, probably related to the high toxicity observed at this dose, but of no biological significance.

When tested up to toxic doses, ZEOSTOP X did not induce mutation at the tk locus of L5178Y mouse lymphoma cells in two independent experiments, in the absence or presence of S-9.

Conclusions:

It was concluded that, under the conditions employed in this study, Zeostop X was not mutagenic in this test system.

Executive summary:

Zeostop X was assayed for its ability to induce mutation at the tk locus (5-trifluorothymidine resistance) in mouse lymphoma cells using a fluctuation protocol. The study consisted of a cytotoxicity range-finding experiment followed by two independent experiments, each conducted in the absence and presence of metabolic activation by an Aroclor 1254 induced rat liver post-mitochondrial fraction (S-9).

When tested up to toxic doses, Zeostop X did not induce mutation at the tk locus of L5178Y mouse lymphoma cells in two independent experiments, in the absence or presence of S-9.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May 4 - Jul. 23, 1999; experimental phase: May 11-25, 1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

name: ZEOSTOP X (X Zeolite)
batch number: MR 453 136
appearance: white powder
reived on 28 April 1999
storage: stored at room temperature in the dark
purity: approximately 100%
expiry date: May 2000.

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver post-mitochondrial fraction (S-9)

Test concentrations with justification for top dose:

range finding: 8, 40, 200, 1000, 5000 µg/plate
experiment 1: 0.032, 0.16, 0.8, 4, 20, 100 µg/plate
experiment 2 with activation: 0.09766, 0.39063, 1.5625, 6.25, 25, 100 µg/plate
experiment 2 without activation (strains TA98 and TA100): 0.03906, 0.15625, 0.625, 2.5, 10 µg/plate
experiment 2 without activation (strains TA102, TA1535, TA1537): 0.07813, 0.3125, 1.25, 5, 20 µg/plate

Vehicle / solvent:

DMSO

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

other: glutaraldehyde, 2-aminoanthracene

Rationale for test conditions:

The assay was considered valid if the following criteria were met:
- the mean negative control counts fell within the normal ranges .
- the positive control chemicals induced clear increases in revertant numbers confirming discrimination between different strains, and an active S-9 preparation
- no more than 5% of the plates were lost through contamination or some other unforeseen event.

Evaluation criteria:

The test article was considered to be mutagenic if a dose related and reproducible increase in the number of revertants was observed, or a significant and reproducible increase in the number of revertants was induced at one or more test concentration. An increase in revertant numbers was considered to be significant if the number of revertant colonies was at least two times the mean negative control counts in strains TA98, TA100 and TA102, or three times in strains TA1535 and TA1537.

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Negative (solvent) and positive control treatments were included for all strains in both experiments. The mean numbers of revertant colonies on negative control plates all fell within acceptable ranges, and were significantly elevated by positive control treatments.
No ZEOSTOP X treatments of any of the test strains resulted in an increase revertant numbers sufficient to be considered indicative of mutagenic activity.

Conclusions:

It was concluded that ZEOSTOP X did not induce mutation in five histidine-requiring strains of Salmonella typhimurium (TA98, TA100, TA1535, TA1537, TA102) when tested under the conditions employed in this study. These conditions included treatments at concentrations that were approaching, or extended into the toxic range, in the absence and in the presence of a rat liver metabolic activation system (S-9).

Executive summary:

ZEOSTOP X was assayed for mutation to five histidine-requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium, both in the absence and in the presence of metabolic activation by an Aroclor 1254-induced rat liver post-mitochondrial fraction (S-9), in two separate experiments. No ZEOSTOP X treatments of any of the test strains resulted in an increase revertant numbers sufficient to be considered indicative of mutagenic activity.

Reference 4

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

May 4, 1999 - May 22, 2000; experimental phase: May 5 - Jun. 21, 1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)

Version / remarks:

1998

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

name: Zeostop X (X Zeolite)
batch number: MR 453136
appearance: white powder
received: 28 April 1999
storage: stored at room temperature in the dark
purity: approximately 100%
expiry date: May 2000

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CHO cells, supplied by Dr S Galloway, West Point, PA, USA, were maintained at Covance Laboratories Limited in tissue culture flasks containing McCoy’s 5A medium including 10% (v/v) foetal calf serum (FCS), and 100 µg/ml gentamycin.

Metabolic activation:

with and without

Metabolic activation system:

S-9 prepared from a rat liver post-mitochondrial fraction from Aroclor 1254-induced animals

Test concentrations with justification for top dose:

The following doses were selected for analysis:
Experiment 1
3+17 hours, -S-9: 27.49, 42.95, 67.11 µg/ml (reduction in cell number at highest analysed dose 48%)
3+17 hours, +S-9: 163.8, 320, 400 µg/ml (reduction in cell number at highest analysed dose 63%)

Experiment 2
20+0 hours, -S-9: 37.87, 61.66, 72.54 µg/ml (reduction in cell number at highest analysed dose 54%)
3+17 hours, +S-9: 226.3, 266.2, 313.2 µg/ml (reduction in cell number at highest analysed dose 49%)

Vehicle / solvent:

Due to the solubility limitations of Zeostop X, treatments were performed using a suspension of the test article. Test article formulations were prepared by suspending Zeostop X in supplemented McCoy’s 5A medium, with the aid of vortex-mixing, immediately prior to assay. Formulations were not filter-sterilized and further dilutions were made using culture medium. The test article suspensions wereprotected from light and used within 2 hours of the initial formulation.

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

cyclophosphamide

Rationale for test conditions:

The highest dose for chromosone analysis from cultures sampled at 20 hours should be one at which at least 50% reduction in cell number has occurred or should be the highest dose tested. Analysis of slides from highly cytotoxic concentrations was avoided if possible. Slides from cultures treated with heavily precipitating doses were checked to confirm that the presence of precipitate did not preclude analysis. Slides from the highest selected dose and two lower doses, such that a range of toxicity from maximum to little or none is covered, were taken for micfescope analysis.

Evaluation criteria:

One hundred metaphases from each code were analysed for chromosome aberrations. Only cells with 19-23 chromosomes were considered acceptable for analysis of structural aberrations. Any cell with more than 23 chromosomes 'that is polyploid, endoreduplicated and hyperdiploid cells, observed during this search was noted and recorded separately. Classification of structural aberrations was based on the scheme described by ISCN. Under this scheme, a gap is defined as a discontinuity less than the width of the chromatid with no evidence of displacement of the fragment and a deletion is deftned as a discontinuity greater than the width of the chromatid and/or evidence ofj displacement of the fragment.

A test article was considered as positive in this assay if:
1) the proportion of cells with structural aberrations at one or more concentrations exceeded the normal range in both replicate cultures, and
2) a statistically significant increase in the proportion of cells with structural aberrations (excluding gaps) occured at such doses.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Appropriate negative (vehicle) control cultures were included in the test system in both experiments under each treatment condition. The proportion of cells with structural aberrations in these cultures fell within historical solvent control ranges. 4-Nitroquinoline 1-oxide and cyclophosphamide were employed as positive control chemicals in the absence and presence of liver S-9, respectively. Cells receiving these were sampled in each experiment, 20 hours after the start of treatment; both compounds induced statistically significant increases in the proportion of cells with structural aberrations.

In experiment 1, the highest concentrations chosen for analysis, 67.11 and 400 µg/mL, induced approximately 48% and 63% reduction in cell number in the absence and presence of S-9, respectively.

In experiment 2, the highest concentrations chosen for analysis were, 72.54 and 313.2 µg/mL, which induced approximately 54% and 49% reduction in cell number in the absence and presence of S-9 respectively.

Treatment of cells with Zeostop X in the absence and presence of S-9 resulted in increased frequents of cells with aberrations. Frequencies which exceeded historical negative control ranges in both replicates and where the total number of aberrant cells was significantly higher than the concurrent negative control were observed at either the high or intermediate concentration analysed from all treatment regimes. The total number of aberrations seen was clearly elevated in most treated cultures and exchanges, which are observed only infrequently in negative controls, were induced.

Increased frequencies of cells with numerical aberrations which exceeded historical negative control ranges were observed under all treatment conditions. The effect was attributable to endoreduplication. It should be noted that the biological significance of endoreduplication and its likely occurrence in vivo is not clear.

Conclusions:

It was concluded that Zeostop X induced structural chromosome aberrations in cultured CHO cells in both the absence and presence of S-9 when tested to cytotoxic doses at which undissolved material was present.

Executive summary:

Zeostop X was tested in an in vitro cytogenetics assay using duplicate cultures of CHO cells in two independent experiments. Treatment of cells with Zeostop X in the absence and presence of S-9 resulted in increased frequents of cells with aberrations. Frequencies which exceeded historical negative control ranges in both replicates and where the total number of aberrant cells was significantly higher than the concurrent negative control were observed at either the high or intermediate concentration analysed from all treatment regimes.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Three studies assessed genetic toxicity in vivo. Two with sodium aluminosilicate and one with a type X zeolite. In all three studies the substances were tested up to 5000 mg/kg bw/d. None of them showed genotoxic potential. 

The study with type X zeolite in cytotoxicity (Zeostop X, OECD 474, Haddouk 2000, Mouse Bone Marrow, RL 2) takes precedence over the in vitro one with the same substance (Zeostop X, OECD 473, Marshall 2000, CHO, RL 1). The former concludes that under the experimental conditions, the test substance 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.

 

Link to relevant study records

Reference

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

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

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

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.

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

Dose / conc.:

1 250 mg/kg bw/day (nominal)

Dose / conc.:

2 500 mg/kg bw/day (nominal)

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

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.

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

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.      

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

None of the in vivo studies showed genotoxic potential, and only one of the six reliable in vitro studies did. But this positive response to cytotoxicity is overruled by an in vivo study in cytotoxicity with the same test substance, that as an in vivo one takes precedence over an in vitro study, and which did not induce damage to the chromosomes or the mitotic apparatus.

In conclusion, there is no need for classification.

This is also supported by the MAK report on workplace safety (cf. chapter 7.12) that states: "In the available genotoxicity studies, neither gene mutations nor chromosomal aberrations or dominant lethal mutations were induced. The structure [of zeolites] also does not give rise to a corresponding suspicion, so that there is no classification in a category for germ cell mutagens."