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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:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: No details on test substance identity. Guideline study without complete GLP status. Scientifically acceptable.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report Date:
2007

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
no
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Vitamin A Rohöl, (Batch: BK 2/8, date: 11.12.2006)
Sample represents a step in the vitamin A synthesis process.
Retinyl acetate is assumed to be the main component.
Impurities: Heptan, Kitol , ring closure compounds , unknown components
no further data

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 (information from the breeder)
- Weight at study initiation: About 30 g
- Housing: Makrolon cages, type MI
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: At least 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24°C
- Humidity (%): 30 - 70%
Fully air-conditioned rooms
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: unspecified
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The substance to be administered per kg body weight was dissolved in corn oil.
To achieve a solution of the test substance in the vehicle, the test substance preparation was
shaken thoroughly. All test substance formulations were prepared immediately before administration.
Duration of treatment / exposure:
48 hours
Frequency of treatment:
twice with 24 hour interval between administration
Post exposure period:
24 hours
Doses / concentrations
Remarks:
Doses / Concentrations:
1000, 2000 mg/kg bw
Basis:
nominal conc.
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
10 mg/kg bw cyclophosphamide (CPP)

Examinations

Tissues and cell types examined:
Polychromatic and normochromatic erythrocytes in bone marrow
Details of tissue and slide preparation:
• The two femora of the animals sacrificed by cervical dislocation were prepared by
dissection and removing all soft tissues.
• After cutting off the epiphyses, the bone marrow was flushed out of the diaphysis into a
centrifuge tube using a cannula filled with fetal calf serum (FCS) which was at 37°C (about
2 mL/femur).
• The suspension was mixed thoroughly with a pipette, centrifuged at 300 x g for 5 minutes,
the supernatant was removed and the precipitate was resuspended in about 50 μL fresh
FCS.
• 1 drop of this suspension was dropped onto clean microscopic slides, using a Pasteur
pipette. Smears were prepared using slides with ground edges, the preparations were
dried in the air and subsequently stained.
• The slides were stained in eosin and methylene blue (modified May-Grünwald solution or
Wrights solution) for about 5 minutes.
• After having briefly been rinsed in purified water, the preparations were soaked in purified
water for about 2 - 3 minutes.
• Subsequently, the slides were stained in Giemsa solution (15 mL Giemsa, 185 mL purified
water) for about 15 minutes.
• After having been rinsed twice in purified water and clarified in xylene, the preparations
were mounted in Corbit-Balsam.
Evaluation criteria:
The mouse micronucleus test is considered valid if the following criteria are met:
• The quality of the slides must allow the identification and evaluation of a sufficient number
of analyzable cells, i.e. ≥ 2 000 PCEs and a clear differentiation between PCEs and NCEs.
• The ratio of PCEs/NCEs in the untreated animals (vehicle control) has to be within the
range of the historical negative control data for the animal strain selected.
• The number of cells containing micronuclei in negative control animals has to be within the
range of the historical negative control data both for PCEs and for NCEs.
• The positive control substance has to induce a significant increase in the number of PCEs
containing micronuclei within the range of the historical positive control data or above.

A finding is considered positive if the following criteria are met:
• Statistically significant and dose-related increase in the number of PCEs containing
micronuclei.
• The number of PCEs containing micronuclei has to exceed both the concurrent negative
control value and the range of the historical negative control data.

A test substance is considered negative if the following criteria are met:
• The number of cells containing micronuclei in the dose groups is not statistically significant
above the concurrent negative control value and is within the historical negative control
data range.
Statistics:
The asymptotic U test according to MANN-WHITNEY (modified rank test according to
WILCOXON) was carried out to clarify the question whether there were significant differences
between the control group and 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.

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
slight inhibition of erythropoiesis at both dose levels
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid

Any other information on results incl. tables

The two oral administrations of the vehicle corn oil in a volume of 10 mL/kg body weight led to 1.2‰ polychromatic erythrocytes containing micronuclei after the 24-hour sacrifice interval.

Besides, after twice administrations of 2 000 mg/kg and 1 000 mg/kg body weight, 1.7‰ and 1.9‰ polychromatic erythrocytes containing micronuclei were found after 24 hours, respectively.

With 14.8‰ the positive control substance cyclophosphamide led to a expected statistically significant and biologically relevant increase in the number of polychromatic erythrocytes containing exclusively small micronuclei.

The number of normochromatic erythrocytes containing micronuclei did not differ to any appreciable extent in the vehicle control or in both dose groups.

A slight inhibition of erythropoiesis, induced by the treatment of mice with Vitamin A Rohöl was detected at both dose levels.

The two oral administrations of the vehicle in a volume of 10 mL/kg body weight were tolerated by all animals without any signs or symptoms. The administration of the test substance led to distinct clinical signs of toxicity, i.e. reduced general condition, piloerection, eyelid closure, hunched posture, urine/faeces discoloured by test substance. In addition, the single administration of the positive control substance cyclophosphamide in a dose of 20 mg/kg body weight caused no evident signs of toxicity.

Applicant's summary and conclusion