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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:
06 November 2009 - 15 June 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Compliant to GLP and testing guideline; adequate coherence between data, comments and conclusions.

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
micronucleus assay

Test material

Constituent 1
Reference substance name:
Post crystallization of ammonium sulphate aqueous phase products resulting from ammoniac neutralisation of sulphuric acid waste waters formed during methylmetacrylate synthesis
IUPAC Name:
Post crystallization of ammonium sulphate aqueous phase products resulting from ammoniac neutralisation of sulphuric acid waste waters formed during methylmetacrylate synthesis
Details on test material:
- Physical state: brown black liquid
- Lot/batch No.: 01/07/09
- Expiration date of the lot/batch: 01/07/2011
- Storage conditions of test material: at room temperature
- Purity/Impurities: not applicable (complex composition)

Test animals

Species:
mouse
Strain:
Swiss
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, l'Arbresle, France
- Age at study initiation: 6 weeks old
- Weight at study initiation: 32.5 g for males (ranging from 30.1 to 35.1 g) and 26.1 g for females (ranging from 24.2 to 28.1 g)
- Housing: polycarbonate cages
- Diet (e.g. ad libitum): SSNIFF R/M-H (Soest, Germany)
- Water (e.g. ad libitum): drinking water filtered by a FG Millipore membrane
- Acclimation period: 5 days before the day of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2°C
- Humidity (%): 30 to 70%
- Air changes (per hr): 12 cycles/hour
- Photoperiod (hrs dark / hrs light): 12 h/12 h

IN-LIFE DATES: From: 16 November 2009 To: 16 December 2009

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle used: water for injections
- Concentration of test material in vehicle: 50, 100 and 200 mg/mL
- Amount of vehicle (if gavage or dermal): 10 mL/kg
- Lot/batch no. (if required): 9F1823
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Before use, each sample of the test item was put in a water-filled container on a heating magnetic
stirrer settled at the temperature allowing dissolution of crystallized particles (80°C) until the
obtention of a solution.
For the main test, the test item was ground dissolved in the vehicle in order to achieve the
concentrations of 50, 100 and 200 mg/mL and then homogenized using a magnetic stirrer.
Duration of treatment / exposure:
A period of 2 days (2 treatments)
Frequency of treatment:
One treatment per day.
Post exposure period:
24 hours.
Doses / concentrations
Remarks:
Doses / Concentrations:
500, 1000 and 2000 mg/kg/day
Basis:
nominal conc.
No. of animals per sex per dose:
5 animals per sex and per dose.
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide

- Route of administration: oral
- Doses / concentrations: 50 mg/kg/day

Examinations

Tissues and cell types examined:
Bone marrow: polychromatic (PE) and normochromatic (NE) erythrocytes
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: The top dose-level for the cytogenetic test was selected according to the criteria specified in the international
guidelines; since no toxic effects were observed, the top dose-level was 2000 mg/kg/day. The two other selected dose-levels were 500 and
1000 mg/kg/day.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): Two treatments and one sampling times.

DETAILS OF SLIDE PREPARATION: At the time of sacrifice, all the animals were deeply anesthetized by an intraperitoneal injection of sodium
pentobarbital, then killed by cervical dislocation. The femurs of the animals were removed and the bone marrow was flushed out using fetal calf
serum.
After centrifugation, the supernatant was removed and the cells in the sediment were resuspended by shaking. A drop of this cell suspension was
placed and spread on a slide. The slides were air-dried and stained with Giemsa. The slides were coded so that the scorer is unaware of the treatment group of the slide under evaluation ("blind" scoring).

METHOD OF ANALYSIS: 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).
Evaluation criteria:
For a result to be considered positive, a statistically significant increase in the frequency of MPE must be demonstrated when compared to the
concurrent vehicle control group. Reference to historical data or other considerations of biological relevance was also taken into account in the
evaluation of data obtained.
Statistics:
Normality and homogeneity of variances will be tested using a Kolmogorov Smirnov test and a Bartlett test.
If normality and homogeneity of variances were demonstrated, the statistical comparisons was performed using a Student t-test (two groups) or a one-way analysis of variance (> or = 3 groups) followed by a Dunnett test (if necessary).
If normality or homogeneity of variances was not demonstrated, a Mann/Whitney test (two groups) or a Kruskall Wallis test (> or = 3 groups) was
performed followed by a Dunn test (if necessary).

All these analyses were performed using the software SAS Enterprise Guide V2 (2.0.0.417, SAS Institute Inc), with a level of significance of 0.05 for
all tests.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 2000 mg/kg/day
- Clinical signs of toxicity in test animals: none
- Evidence of cytotoxicity in tissue analyzed: none.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
The test item did not induce damage to the chromosomes or the mitotic apparatus of mice bone marrow cells after two oral administrations,
at a 24-hour interval, at the dose-levels of 500, 1000 and 2000 mg/kg/day.
Executive summary:

The potential of the test item to induce structural or numerical damage was evaluated in bone marrow cells of mice. The study was performed according to the international guidelines (OECD 474 and Commission Directive No. B12) and in compliance with the Principles of Good Laboratory Practices.

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 femaleSwiss Ico: OF1 (IOPS Caw) mice were given oral administrations of the test item at dose-levels of 500, 1000 and 2000 mg/kg/day, over a 2-day period. One group of five males and five females received the vehicle (water for injections) under the same experimental conditions, and acted as control group. One group of five males and five females received the positive control (cyclophosphamide) once by oral route at the dose-level of 50 mg/kg/day. The animals of the test item treated and vehicle control groups were killed 24 hours after the last treatment and the animals of the positive control group were killed 24 hours after the single treatment. Bone marrow smears were then prepared. 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).

Neither mortality, nor clinical signs were observed in the animals of either sex given 500, 1000 or 2000 mg/kg/day, throughout the observation period.  

The mean values of MPE as well as the PE/NE ratio for the vehicle and positive controls were consistent with our historical data. Cyclophosphamide induced a statistically significant increase in the frequency of MPE (p<0.01), indicating the sensitivity of the test system under our experimental conditions. The study was therefore considered valid. The mean values of MPE as well as the PE/NE ratio in the groups treated with the test item, were not found different from those of the vehicle group.

The test item did not induce damage to the chromosomes or the mitotic apparatus of mice bone marrow cells after two oral administrations, at a 24-hour interval, at the dose-levels of 500, 1000 and 2000 mg/kg/day.