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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:
2008-01-24 to 2008-03-14
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study according to OECD TG No. 474 and in compliance with GLP Regulations.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report Date:
2008

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
21 Jul 1997
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Name of test material: 2-Propylheptylacrylat rein
-Test substance number: 07/0846-1
- Analytical purity: 98.5 area-%
- Isomers composition: about 87 % 2 Propylheptylacrylate and 10 % 4-methyl-2-propylhexylacrylate
- Analytical report no.: 07L00384
- Lot/batch No.: B4112/13 - 03122007
- Expiration date of the lot/batch: 2008-12-03
- pH-value: Ca. 5.5 (undiluted test substance)

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Mice / Crl:NMRI from Charles River Laboratories Germany GmbH.
- Age at study initiation: 5 – 8 weeks
- Weight at study initiation: 29.3 g (mean bw)
- Assigned to test groups randomly: The animals were assigned to the test groups according to a randomization plan prepared with an appropriate computer program.
- Housing: Makrolon cages, type M I; single housing
- Diet: Standardized pelleted feed (Maus/Ratte Haltung "GLP", Provimi Kliba SA, Kaiseraugst, Switzerland)
- Water: Drinking water from bottles was available ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 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: corn oil
- Justification for choice of solvent/vehicle: Due to the limited solubility of the test substance in water, corn oil 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.
- Concentration of test material in vehicle: 500 mg/kg body weight or 10 mL/kg body weight of a test substance solution with a concentration of 50 mg/mL.
1000 mg/kg body weight or 10 mL/kg body weight of a test substance solution with a concentration of 100 mg/mL.
2000 mg/kg body weight or 10 mL/kg body weight of a test substance solution with a concentration of 200 mg/mL.
- Amount of vehicle (if gavage or dermal): 10 mL.
-Stability: The stability of the test substance at room temperature in the vehicle corn oil was determined analytically.
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: To achieve a solution of the test substance in the vehicle, the test substance preparation was
shaken thoroughly.

The animals were treated once orally (gavage) with a volume of 10 mL/kg body weight of the test substance, the vehicle and the positive control substances
Duration of treatment / exposure:
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:
The animals were treated once.
Post exposure period:
None.
Doses / concentrations
Remarks:
Doses / Concentrations:
500, 1000, and 2000 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
5 males per dose
Control animals:
yes, concurrent vehicle
Positive control(s):
-Cyclophosphamide, Vincristine sulfate
- Justification for choice of positive control(s): well-established reference clastogens and aneugens
- Route of administration: oral, gavage (Cyclophosphamide), peritoneal (Vincristine sulfate)
- Doses / concentrations: Cyclophosphamide: 20 mg/kg body weight, Vincristine sulfate: 0.15 mg/kg body weight

Examinations

Tissues and cell types examined:
Bone marrow of femora (normochromatic and polychromatic erythrocytes)
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. The only clinical sign observed was hunched posture. However, there were no distinct differences in the symptoms between males and females. Thus, only male animals were used for the cytogenetic investigations. Therefore, a dose of 2000 mg/kg body weight was selected as the highest dose in the present cytogenetic study. 1000 mg/kg and 500 mg/kg body weight were administered as further doses.

DETAILS OF 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 preheated up to 37°C (about 2 mL/femur).
- The suspension was mixed thoroughly with a pipette and centrifuged at 300 x g for 5 minutes. The supernatant was removed and the precipitate was resuspended in about 50 μL fresh FCS.
- One 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 with eosin and methylene blue (modified May-Grünwald solution or Wrights solution) for about 5 minutes.
- After briefly rinsing in purified water, the preparations were soaked in purified water for about 2 - 3 minutes.
- Subsequently, the slides were stained with Giemsa solution (15 mL Giemsa plus 185 mL purified water) for about 15 minutes.
- After rinsing twice in purified water and clarifying in xylene, the preparations were mounted in Corbit-Balsam.
Evaluation criteria:
In general, 2 000 polychromatic erythrocytes (PCE) were evaluated for the occurrence of micronuclei from each animal of every test group, so in total 10 000 PCEs were scored per test group. The normochromatic erythrocytes (= normocytes / NCE) were also scored.

Acceptance criteria:
-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.

Assessment criteria
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 vehicle control value and the range of the historical vehicle 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 increased above the concurrent vehicle control value and is within the range of the historical vehicle control data.
Statistics:
The statistical evaluation of the data was carried out using the program system MUKERN (BASF SE). 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:
yes
Remarks:
weak clinical signs of toxicity (hunched posture)
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
The single oral administration of the vehicle corn oil in a volume of 10 mL/kg bw led to 1.2 ‰ polychromatic erythrocytes containing micronuclei after the 24-hour sacrifice interval or to 1.3‰ after the 48-hour sacrifice interval, respectively.
After the single administration of the highest dose of 2000 mg/kg body weight, 1.3‰ polychromatic erythrocytes containing micronuclei were found after 24 hours and 3.0‰ after 48 hours.
In the two lower dose groups, rates of micronuclei of about 1.9‰ (1000 mg/kg bw group) and 2.4‰ (500 mg/kg bw group) were detected after a sacrifice interval of 24 hours in each case.
The positive control substance for clastogenicity, cyclophosphamide, led to a statistically significant increase (14.4‰) in the number of polychromatic erythrocytes containing exclusively small micronuclei, as expected.
Vincristine sulfate, a spindle poison, produced a 66.4‰ increase in micronuclei in polychromatic erythrocytes. A significant portion increase, 14.6‰ was attributable to large micronuclei.
The number of normochromatic erythrocytes containing micronuclei did not differ to any appreciable extent in the vehicle control group or in the various dose groups at any of the sacrifice intervals.
No inhibition of erythropoiesis was observed.

Clinical signs:
All animals in all dose groups showed a hunched posture up to 2 and 4 h after treatment.

Any other information on results incl. tables

Number of total PCE and total NCE and number of PCE/NCE with micronuclei:

 

Interval 24 h

Interval 48 h

 

Total No of

MN (o/oo) in

Total No of

MN (o/oo) in

 

PCE

NCE

PCE

NCE

PCE

NCE

PCE

NCE

Vehicle corn oil

10000

4338

1.2

0.7

10000

4494

1.3

0.2

500 mg/kg

10000

4188

2.4

0.5

 

 

 

 

1000 mg/kg

10000

5239

1.9

1.3

 

 

 

 

2000 mg/kg

10000

4103

1.3

0.2

10000

5109

3.0*

1.2

CPP 20 mg/kg

10000

2835

14.4**

1.8

 

 

 

 

VCR 0.15 mg/kg

10000

5687

66.4**

1.1

 

 

 

 

Proportion of small and large micronuclei per total of 10000 PCEs:

 

Interval 24 h

Interval 48 h

 

Total No of

cells (o/oo) with

Total No of

cells (o/oo) with

 

PCE

 

MN d<D/4

MN

d>=D/4

PCE

 

MN d<D/4

MN

d>=D/4

Vehicle corn oil

10000

 

1.1

0.1

10000

 

1.3

0

500 mg/kg

10000

 

2.4

0

 

 

 

 

1000 mg/kg

10000

 

1.9

0

 

 

 

 

2000 mg/kg

10000

 

1.3

0

10000

 

2.9*

0.1

CPP 20 mg/kg

10000

 

14.4**

0

 

 

 

 

VCR 0.15 mg/kg

10000

 

51.8**

14.6**

 

 

 

 

*: p<=0.05

**: p<=0.01

Applicant's summary and conclusion