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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:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The reliability is rated Klimish 1

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report date:
2010

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
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): CIMENT FONDU ®
- Substance type: inorganic
- Physical state:dark grey fine powder
- Analytical purity: 100 %
- Composition of test material, percentage of components: Ciment Fondu® is a mixture of more than 99.5% milled clinker of Ciment Fondu® which is the same substance as the registered substance here, and a grinding agent
- Lot/batch No.:91478
- Expiration date of the lot/batch: 20/10/2010
- Storage condition of test material: sealed at ambient temperature

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: OFA Sprague-Dawley rats (Charles RIVER origin, Saint-Germain-sur-l'Arbresle - FRANCE)
- Age at study initiation: 5 to 10 weeks old
- Weight at study initiation:approximately 200 g
- Assigned to test groups randomly: yes
- Fasting period before study: no
- Housing: polypropylene cages measuring 42.5 x 26.6 x 15 cm, covered by a stainless steel netted lid, in which they were placed in groups of 3 or 2 by random-distribution.
- Diet :the feedstuff used were No. 801175 RM1(P) DU IRR 9KGy (toxicity assay) and No. 811020 RM1 (E) SQC IRR 25kGy (main assay) irradiated rat/mouse feed from Special Diets Services (ENGLAND).
- Water :Drinking water softened, treated by osmosis and filtered on 0.2 μm membrane was provided ad libitum.
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C):22 ± 3
- Humidity (%): 55 ± 15
- Air changes (per hr): 20
- Photoperiod (hrs dark / hrs light): 12h/12h

IN-LIFE DATES: until the 01/07/2010

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle used: CMC (carboxymethyl cellulose) at 0.5% in distilled water
- Concentration of test material in vehicle: For the preliminary and confirmatory toxicity assays, suspensions at a maximum concentration of 200
mg/mL were prepared and administered to the animals at a dose volume of 10 mL/kg, giving a final dose of 2000 mg/kg. In the main genotoxicity
assay, three suspensions of the test item at the concentrations of 200 - 100 and 50 mg/mL were prepared, giving final doses of 2000 - 1000 and 500 mg/kg, respectively when administered at 10 mL/kg.
- Amount of vehicle (if gavage or dermal): 10 mL/kg
- Lot/batch no.:(Sigma, batch 039K0040)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item CIMENT FONDU® was suspended in CMC at 0.5% in distilled water, previously heated at 37°C. The suspension was also maintained at 37°C.
For the preliminary and confirmatory toxicity assays, suspensions at a maximum concentration of 200 mg/mL were prepared and administered to
the animals at a dose volume of 10 mL/kg, giving a final dose of 2000 mg/kg. In the main genotoxicity assay, three suspensions of the test item at
the concentrations of 200 - 100 and 50 mg/mL were prepared, giving final doses of 2000 - 1000 and 500 mg/kg, respectively when administered at 10 mL/kg.
Taking into account the nature of the test item, it rapidly hardened. The suspensions were thus maintained in constant agitation. However, even maintained under constant agitation, solidification under a gelatinous piece was noted, but the test item could be homogeneously resuspended after a
vigorous agitation.
Duration of treatment / exposure:
2 daily treatments at 24-hour intervals. Samples were taken at 24 hours after the last treatment.
Doses / concentrations
Remarks:
Doses / Concentrations:

Basis:
actual ingested
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
other: Positice controls
Positive control(s):
cyclophosphamide
- Route of administration:intraperitoneal
- Doses / concentrations:25 mg/kg/day , single administration

Examinations

Details of tissue and slide preparation:


TREATMENT AND SAMPLING TIMES :Samples were taken at 24 hours after the last treatment.

DETAILS OF SLIDE PREPARATION:
At the sampling time, the animals were sacrificed by CO2 asphyxia; the femurs were removed, and the bone marrow was extracted with foetal calf
serum (1 mL per animal). The cell suspensions were centrifuged for 5 minutes at 1000 rpm. The supernatant was eliminated.
The centrifugate was spread on slides. The smears were stained using a technique, derived from the May Grunwald Giemsa technique (SCHMID, 1975), which makes it possible to distinguish between polychromatic (PCE) and normochromatic erythrocytes (NCE): PCE are purple whereas NCE are red.
After coding the slides by a person not involved in the study, two slides per animal were read by two independent operators; for each animal, the
number of polychromatic erythrocytes having one or more Howell-Jolly bodies (micronuclei) was determined for at least 2000 polychromatic erythrocytes. (In case of divergence, 2000 new polychromatic erythrocytes are examined; the retained value was the mean of all readings).
Evaluation criteria:
For a test item to be considered negative in the micronucleus test, there must be no statistically significant increase in the number of micronuclei observed compared with negative control animals.
For a test item to be considered positive in the micronucleus test, there must be seen a statistically significant increase in the number of micronuclei, with at least a doubling of the number of micronuclei compared with the negative control animals at one of three doses tested.
Statistics:
The statistical comparison for the polychromatic/normochromatic erythrocyte ratio and for the weight homogeneity within the sex of each group
was performed using the Student's t test.
Statistical analysis was performed for micronucleus number using a non-parametric test, the Mann Whitney U rank test. An analysis of a large
number of control results has shown that the distribution of the numbers of micronuclei does not correspond to a Gaussian distribution, but to a Poisson-type distribution. This makes it necessary to use a non-parametric statistical test, and the Mann Whitney U rank test is recommended by
UKEMS (LOVELL et al., 1989). Statistical analysis for micronucleus number was conducted, males and females separately and two sexes combined (if using the same doses for the two sexes) in case of homogeneity in the results within the sex of each group.
Furthermore, when the high and/or mid and/or low tested doses are different in male and in female rats, the mean PCE/NCE ratios, student’s t test for the comparison of the ratio PCE/NCE to control, the calculation of the mean number of micronuclei and the statistical comparison to control are not
performed for both sexes combined.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range:2000 mg/kg/bw
- Solubility: suspension
- Clinical signs of toxicity in test animals: no

Any other information on results incl. tables

Genotoxic assay

SAMPLING

TIME

(24 h after

last treatment)  

 TEST ITEM

Doses in mg/kg/day

(x2)  

 SEX   

 PCE/NCE RATIO   

  MICRONUCLEI

FOR 1000 PCE   

 Mean

 Student's t

Test (p)

 Mean

 Mann Whitney (p)

Negative

control group      

 VEHICLE

10 mL/kg

 M

1.78

 

 1.20

 

 F

1.22

 

 0.60

 

 M+F

1.50

 

 0.90

 

Positive

control group       

 Cyclophosphamide

25 mg/kg/day (x1)      

 M

0.63

<0.01

 9.6

p<0.01

 F

0.58

<0.01

 8.5

p<0.01

 M+F

0.60

<0.001

 9.05

p<0.001

 CIMENT FONDU®

TREATED GROUPS

 2000      

 M

1.70

N.S.

 1.20

N.S.

 F

1.30

N.S. 

 0.50

N.S.

 M+F

1.50

N.S. 

 0.85

N.S.

 1000      

 M

1.52

N.S. 

1.60 

N.S.

 F

1.11 

N.S.

1.10

N.S.

 M+F

1.32 

N.S.

1.35

N.S.

 500      

 M

2.13

N.S.

1.40

N.S.

 F

1.07

N.S.

1.10 

N.S.

 M+F

1.60 

N.S.

1.25 

N.S.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
CIMENT FONDU® induced no genotoxic activity under the experimental conditions described.
Executive summary:

The potential clastogenic activity of CIMENT FONDU ® (batch 91478) provided by KERNEOS was tested using the in vivo micronucleus test in the rat, in compliance with the Commission Regulation (EC) No. 440/2008 and the OECD Guideline 474 (1997), by oral route, using 2 successive daily

treatments at the maximum dose recommended by OECD guidelines.

The test item CIMENT FONDU ® was suspended in CMC at 0.5% in distilled water, previously heated at 37°C. The suspension was also maintained at 37°C. For the preliminary and confirmatory toxicity assays, suspensions at a maximum concentration of 200 mg/mL were prepared and administered to the animals at a dose volume of 10 mL/kg, giving a final dose of 2000 mg/kg. In the main genotoxicity assay, three suspensions of the test item at the concentrations of 200 - 100 and 50 mg/mL were prepared, giving final doses of 2000 - 1000 and 500 mg/kg, respectively when administered at 10 mL/kg.

Taking into account the nature of the test item (i.e.cement), it rapidly hardened. The suspensions were thus maintained in constant agitation. However, even maintained under constant agitation, solidification under a gelatinous piece was noted, but the test item could be homogeneously resuspended after a vigorous agitation.

Animals were treated twice with 2000 mg/kg/day: preliminary test: 2 males and 2 females and confirmatory test: 5 males and 5 females.

The results of the toxicity assays (observed up to 24 hours after the preliminary and second treatment) indicate that this dose induced neither mortality nor clinical signs.

The highest dose retained for the micronucleus assay was set at 2000 mg/kg/day (x2).Two inferior doses of 1000 and 500 mg/kg/day (x2) were also tested.

The weight homogeneity of the animals used in this test after random-distribution, was verified for males and females separately, by comparing the weight mean of the treatment groups with that of the control group. There was no statistically significant difference between the weights of males treated with the test item and those of control rats, except in the case of the positive control in male rats, and the low dose treated group in female rats. However, it was considered that this slight deviation affected neither the quality nor the integrity of the current study.

The ratio of polychromatic (PCE) to normochromatic erythrocytes (NCE) was established at each dose level. No statistically significant decrease in the ratio PCE to NCE was noted in the 3 CIMENT FONDU ® treatment groups when compared to the negative control group, either in treated male and female separately or with both sexes pooled. Thus, no proof of systemic exposure was evidenced.

The results obtained on negative control animals and those treated with the positive reference substance were similar to those generally obtained in the laboratory. A statistically significant increase in the frequency of micronuclei was noted in the group treated with Cyclophosphamide, demonstrating the sensitivity of the animal strain used to a clastogenic agent. The validity criteria for the test were fulfilled and the test was validated.

Regarding the frequency of micronuclei, no statistically significant increase in the frequencies of micronucleated polychromatic erythrocytes was found in the animals treated withCIMENT FONDU® at any dose, both sexes combined or males and females separately, when compared with the control group.

The validity criteria for the study were fulfilled. The current study was thus considered as valid. The test item CIMENT FONDU® (batch 91478) provided by KERNEOS was investigated by means of thein vivomicronucleus test, in male and female OFA Sprague Dawley rats treated orally twice with 2000 – 1000 and 500 mg/kg/day, followed by one sampling time 24 hours after the last treatment. As a conclusion, CIMENT FONDU® induced no genotoxic activity under these experimental conditions.