Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: other: Chromosomal damage (clastogenicity)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2003-01-17 until 2003-03-06
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP - Guideline study
Cross-reference
Reason / purpose for cross-reference:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report date:
2003

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
Chemical structure
Reference substance name:
-
EC Number:
443-860-6
EC Name:
-
Cas Number:
302776-68-7
Molecular formula:
C24 H31 N O4
IUPAC Name:
hexyl 2-[4-(diethylamino)-2-hydroxybenzoyl]benzoate

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland GmbH.
- Age at study initiation: An age range of about 5 - 8 weeks
- Weight at study initiation: Mean weight of about 28 g
- Assigned to test groups randomly: yes
- Housing: For the duration of at least 5 days the animals were housed in Makrolon cages. Before the start of the treatment the animals were transferred to Makrolon cages, type Ml, and housed individually under the same conditions until the end of the test.
- Diet: Standardized pelleted feed (Ratte - Maus Diät, Provimi Kliba SA, Kaiseraugst, Switzerland), ad libitum.
- Water: Drinking water ad libitum.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24°C
- Humidity (%): Humidity range of 30 - 70%.
- Photoperiod (hrs dark / hrs light): The day/night rhythm was 12 hours (12 hours light from 6.00 am- 6.00 pm and 12 hours darkness from 6.00 pm - 6.00 am).

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Due to the insolubility of the test substance in water, DMSO was selected as the vehicle. DMSO was already demonstrated to be suitable in the in vivo micronucleus test. Historical data are also available.
- Concentration of test material in vehicle: The substance was dissolved in DMSO and was administered twice:
- 500 mg test substance/kg body weight or 4 mL/kg body weight of a solution with a concentration of 12.5 g/100 mL.
- 1000 mg test substance/kg body weight or 4 mL/kg body weight of a solution with a concentration of 25 g/100 mL.
- 2000 mg test substance/kg body weight or 4 mL/kg body weight of a solution with a concentration of 50 g/100 mL.
Duration of treatment / exposure:
Two intraperitoneal administration at a 24-hour interval.
Frequency of treatment:
Two intraperitoneal administration at a 24-hour interval.
Animals of the positive control groups were treated only once.
Post exposure period:
24 hours after last application
Doses / concentrationsopen allclose all
Dose / conc.:
500 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Dose / conc.:
2 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
5 male mice per group.
One vehicle control group.
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide (CPP): clastogenic effects
Vincristine (VCR): aneugenic effects
- Justification for choice of positive control(s): Both positive control articles (CPP and VCR) are well-defined clastogens and aneugens respectively.
- Route of administration: Intraperitoneal
- Doses / concentrations:
CPP 20 mg/kg bw , 10 mL test solution
VCR 0.15 mg/ kg bw, 10 mL test solution

Examinations

Tissues and cell types examined:
polychromatic erythrocytes from the bone marrow
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: Range-finding assay (see below, in "additional information on results")

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields): Samples of bone marrow from the animals of the vehicle and the dose groups were taken 24 hours after the last treatment.

DETAILS OF SLIDE PREPARATION: After the animals were sacrificed and the bone marrow was prepared according to the method described by Schmid (1976, 1977) and Salamone et al.,(1980), slides were prepared and stained:
The slides were stained in eosin and methylene blue solution for 5 minutes (May Grünwald solution modified = Wrights solution), rinsed in purified water and then placed in fresh purified water for 2 or 3 minutes. They were finally stained in 7.5% Giemsa solution for 15 minutes.
After being rinsed twice in purified water and clarified in xylene, the preparations were mounted using Corbit-Balsam.

METHOD OF ANALYSIS: Microscopic evaluation. Thereby the following parameters were recorded:
1. Number of polychromatic erythrocytes
2. Number of polychromatic erythrocytes containing micronuclei
3. Number of normochromatic erythrocytes
4. Number of normochromatic erythrocytes containing micronuclei
5. Ratio of polychromatic to normochromatic erythrocytes
6. Number of small micronuclei (d < D14) and of large micronuclei (d > D14) (d = diameter of micronucleus, D = cell diameter)
Evaluation criteria:
The test chemical is considered positive in this assay if the following criteria are met:
1. A dose-related and significant increase in the number of micronucleated polychromatic erythrocytes.
2. The proportion of cells containing micronuclei exceeded both the values of the concurrent negative control range and the negative historical control range.
A test substance is generally considered negative in this test system if:
1. There was no significant increase in the number of micronucleated polychromatic erythrocytes at any dose above concurrent control frequencies.
2. The frequencies of cells containing micronuclei were within the historical control range.
Statistics:
The statistical evaluation of the data was carried out using the program system MUKERN.
A comparison of the number of micronuclei in polychromatic erythrocytes in the dose groups with the vehicle control group was carried out using the Wilcoxon test (one-sided) for the hypothesis of equal medians. A significance of P < / = 0.05 or P < / =0.01 was used.

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
In a pretest for the determination of the acute intraperitoneal toxicity, all animals (male and female) survived the two treatments with 2,000 mg/kg body weight which was recommended as the highest dose according to the OECD Guideline. The clinical signs observed were piloerection, squatting posture and animals in a poor general state. Though there were no distinct symptomatic differences between the male and female animals, only male animals were used in the cytogenetic test.
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.

RESULTS OF DEFINITIVE STUDY

1. Clinical examinations:
1.1 The two intraperitoneal administrations of the vehicle in a volume of 4 mL/kg body weight were tolerated by all animals without any signs or symptoms.
1.2 Neither the single administration of the positive control substance, cyclophosphamide, in a dose of 20 mg/kg body weight nor that of vincristinein a dose of 0.15 mg/kg body weight caused any evident signs of toxicity.
1.3 The administration of the test substance led to evident signs of systemic toxicity.

2. Microscopic findings:
2.1 The two intraperitoneal administrations of DMSO in a volume of 4 mL/kg body weight led to 1.4 ‰ polychromatic erythrocytes containing micronuclei after the 24-hour sacrifice interval.
2.2 After two administrations of the highest dose of 2,000 mg/kg body weight, 1.1 ‰ polychromatic erythrocytes containing micronuclei were found after 24 hours.
2.3 In the two lower dose groups, rates of micronuclei of about 1.4 ‰ (500 mg/kg group) and 1.6 ‰ (1,000 mg/kg group) were detected.
2.4 With 18.5 ‰ the positive control substance- cyclophosphamide led to the expected clear increase in the number of polychromatic erythrocytes containing mainly small micronuclei.
2.5 With 76.6 ‰ the positive control vincristine responsible for induction of spindle poison effects also led to a clearly enhanced number of polychromatic erythrocytes containing micronuclei with the expected amount of large micronuclei, i.e. 18.7 ‰.
2.6 The number of normochromatic erythrocytes containing micronuclei did not differ to any appreciable extent in the negative control or in the various dose groups.
2.7 A slight and dose-dependent inhibition of erythropoiesis, induced by the treatment of mice with Uvinul A Plus was detected from about 500 mg/kg body weight onward.

Thus, the test substance Hexyl 2-(1-(diethylaminohydroxyphenyl)methanoyl)benzoate did not cause any increase in the rate of micronuclei, even at systemic toxic dosis.
The number of normochromatic or polychromatic erythrocytes containing small micronuclei (d < D/4) or large micronuclei (d D/4) did not deviate from the control value and was within the historical control range.

Applicant's summary and conclusion