Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010-03-26 to 2010-05-06
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

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:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
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): DHDPS
- CAS- No.: 80-09-1
- Physical state: Solid, white
- Analytical purity: 99.87 %
- Lot/batch No.: Batch identification: BB 1020

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Germany GmbH
- Age at study initiation: 5 – 8 weeks
- Weight at study initiation: 28.03 g (mean body weight)
- Housing: Makrolon cages, type M II; single housing
- Diet (e.g. ad libitum): Type Lignocel PS 14 fibres, dustfree bedding, supplied by SSNIFF, Soest, Germany
- Water (e.g. ad libitum): Drinking water from bottles was available ad libitum
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24°C
- Humidity (%): 30 - 70%
- Photoperiod (hrs dark / hrs light): 12 hours light from 6.00 - 18.00 hours; 12 hours darkness from 18.00 - 6.00 hours

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Due to the limited solubility of the test substance in water, DMSO was selected as the vehicle, which had been demonstrated to be suitable in the in vivo micronucleus test and for which historical data are available.
- Type and concentration of dispersant aid (if powder): DMSO is used up to 4 mL/kg body weight only
Duration of treatment / exposure:
The animals were treated once orally (gavage) with a volume of 10 mL/kg body weight of the test substance and the vehicle. The positive controls, both dissolved in purified water were administered to male animals once orally (CPP) or intraperitoneally (VCR) each in a volume of 10 mL/kg body weight. The animals were sacrificed 24 hours (all test substance concentrations, vehicle, both positive controls) and 48 hours (highest test substance concentration, vehicle) after the treatment, respectively.
Frequency of treatment:
24, 48 hours after dose administration
Doses / concentrationsopen allclose all
Dose / conc.:
500 other: mg/L bw (nominal)
Dose / conc.:
1 000 other: mg/L bw (nominal)
Dose / conc.:
2 000 other: mg/L bw (nominal)
No. of animals per sex per dose:
5 male animals per dose
Control animals:
yes, concurrent vehicle
Positive control(s):
- cyclophosphamide (CPP); vincristine sulfate (VCR)
- Doses / concentrations: 20 mg/kg bw (CPP); 0.15 mg/kg bw (VCR)

Examinations

Tissues and cell types examined:
The micronucleus study evaluated the potential of the test article to increase the incidence of micronucleated polychromatic erythrocytes in bone marrow of male mice.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
In a pretest for the determination of the acute oral toxicity, at 2 000 mg/kg body weight recommended as the highest dose according to the OECD Guideline all animals (male and female) survived. Weak signs of toxicity (piloerection) were observed over two days. However, there were no distinct differences in the clinical observations between males and females. Thus, only male animals were used for the cytogenetic investigations. Therefore, a dose of 2 000 mg/kg body weight was selected as the highest dose in the present cytogenetic study. 1 000 mg/kg and 500 mg/kg body weight were administered as further doses.

METHOD OF ANALYSIS:
For the determination of the test substance concentrations in the vehicle, 6 samples of each dose were taken from the test substance preparations. These were kept at room temperature until the treatment of the last animal (approximately 1 hour) and then were kept deep-frozen. The determination of the concentrations in the vehicle was carried out by means of HPLC The homogeneity of the samples and the stability of the test substance in the vehicle mixture DMSO/corn oil (ratio 2:3) were confirmed indirectly based on these data.
Evaluation criteria:
The mouse micronucleus test is considered valid if the following criteria are met:
- The quality of the slides must allow the evaluation of a sufficient number of analyzable cells; i. e. ≥ 2 000 PCEs per animal and a clear differentiation between PCEs and NCEs.
- The ratio of PCEs/NCEs in the concurrent vehicle control animals has to be within the normal range for the animal strain selected.
- The number of cells containing micronuclei in vehicle control animals has to be within the range of the historical vehicle control data both for PCEs and for NCEs.
- The two positive control substances have to induce a distinct increase in the number of PCEs containing small and/or large micronuclei within the range of the historical positive control data or above.
Statistics:
The statistical evaluation of the data was carried out using the program system MUKERN. The asymptotic U test according to MANN-WHITNEY (modified rank test according to WILCOXON) was carried out to clarify the question whether there are statistically significant differences between the untreated control group and the treated dose groups with regard to the micronucleus rate in polychromatic erythrocytes. The relative frequencies of cells containing micronuclei of each animal were used as a criterion for the rank determination for the U test. Statistical significances were identified as follows:
* p ≤ 0.05
** p ≤ 0.01
However, both biological relevance and statistical significance were considered together.

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
No dose-dependent inhibition of erythropoiesis induced by the treatment of mice with 4,4`-sulphonyldiphenol was detected. The ratio of polychromatic to normochromatic erythrocytes was in the range of the vehicle control values in all dose groups.
The administration of the test substance led to weak clinical signs of toxicity.

Any other information on results incl. tables

No other information

Applicant's summary and conclusion

Conclusions:
Interpretation of results: negative
The test substance 4,4`-sulphonyldiphenol has no chromosome-damaging (clastogenic) effect nor does it lead to any impairment of chromosome distribution in the course of mitosis (aneugenic activity) in bone marrow cells of NMRI mice in vivo.
Executive summary:

4,4`-sulphonyldiphenol was tested in a mouse micronucleus assay according to OECD guideline 474 and EU-method B.12. The test substance, dissolved in DMSO and emulsified in corn oil, was administered once orally to male animals at dose levels of 500 mg/kg, 1000 mg/kg and 2000 mg/kg body weight in a volume of 10 mL/kg body weight in each case. The animals were sacrificed and the bone marrow of the two femora was prepared 24 and 48 hours after administration in the highest dose group of 2000 mg/kg body weight and in the vehicle controls. In the test groups of 1000 mg/kg and 500 mg/kg body weight and in the positive control groups, the 24-hour sacrifice interval was investigated only. After staining of the preparations, 2000 polychromatic erythrocytes were evaluated per animal and investigated for micronuclei. The normocytes with and without micronuclei occurring per 2000 polychromatic erythrocytes were also recorded.

According to the results of the present study, the single oral administration of 4,4`-sulphonyldiphenol did not lead to any relevant increase in the number of polychromatic erythrocytes containing either small or large micronuclei. The rate of micronuclei was within the range of the concurrent vehicle control in all dose groups and at all sacrifice intervals and within the range of the historical vehicle control data. Thus, under the experimental conditions of this study, the test substance 4,4`-sulphonyldiphenol does not induce cytogenetic damage in bone marrow cells of NMRI mice in vivo. The administration of the test substance led to weak clinical signs of toxicity.