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:
13 Aug 2001 - 05 Nov 2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2001
Report Date:
2001

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
other: micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
- Name of test material (as cited in study report): Tert.-Butylacrylat
- Physical state: liquid
- Lot/batch No.: Tank B 601
- Analytical purity: 99.69 % (Stability of the test substance in olive oil was determined analytically.)
- Stability under test conditions: Storage stability of the test substance covering the period of the study was determined by the sponsor by recharacterisation after completion of the experimental phase of the study.
- Storage condition of test material: Refrigerator, protected from light

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: RCC Ltd., Fuellinsdorf, CH
- Age at study initiation: 7-10 weeks
- Weight at study initiation: males 46.7±3.0 g; females 32.2 ± 4.9g
- Assigned to test groups randomly: yes
- Housing: individually
- Diet: standard diet, ad libitum (ALTROMIN 1324, D-32791 Lage/Lippe )
- Water: ad libitum, (Gemeindewerke, D-64380 Rossdorf)
- Acclimation period: minimum 5 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 4
- Humidity (%): 30 - 70
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: olive oil
- Justification for choice of solvent/vehicle: The vehicle was chosen to its relative non-toxicity for the animals
Details on exposure:
- i.p. injection of 10 mL/kg bw
Duration of treatment / exposure:
single treatment
Frequency of treatment:
single treatment
Post exposure period:
24 or 48 h
Doses / concentrationsopen allclose all
Dose / conc.:
100 other: mg/kg bw
Remarks:
males
Dose / conc.:
200 other: mg/kg bw
Remarks:
males
Dose / conc.:
400 other: mg/kg bw
Remarks:
males
Dose / conc.:
125 other: mg/kg bw
Remarks:
females
Dose / conc.:
250 other: mg/kg bw
Remarks:
females
Dose / conc.:
500 other: mg/kg bw
Remarks:
females
No. of animals per sex per dose:
- Pre-tests: 2 + 5 per sex and per dose
- Main-test: 12 per sex in the high dose and 6 per sex in the mid and low dose, each
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide, dissolved in water
- Route of administration: i.p.
- Dose: 40 mg/kg bw

Examinations

Tissues and cell types examined:
Bone marrow cells were used. At least 2000 PCEs per animal were scored for micronuclei.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
It is generally recommended to use the maximum tolerated dose or the highest dose that can be formulated and administered reproducibly or 2000 mg/kg as the upper limit for non-toxic test substances.
The maximum tolerated dose level is determined to be the dose that causes toxic reactions without having major effects on survival.
The maximum tolerated dose was therefore determined in extensive pre-tests in which doses of 400 to 1000 mg/kg were administered intraperitoneal to male and female mice.

Results of the pre-tests:
In a first pretest male mice (2 animals/dose) were administered i.p. 400, 500 and 1000 mg/kg. Both animals of the 1000 mg/kg dose died, also 1/2 animals of the 500 mg/kg dose. Clinical signs described were reduction of the spontaneous activity, abdominal position eyelid closure and apathy. To confirm the results 5 animals per dose were administered 400 and 500 mg/kg i.p.. Two of the animals in the 500 mg/kg dose died. All animals of both groups showed a reduction of the spontaneous activity and apathy, further clinical signs were abdominal position, and eyelid closure.
Based on this results a clear systemic availability of the test substance was proved in male mice.

Also in female mice the systemic availability and the maximum tolerated dose was determined in pre-tests.
Again in a first pretest 2 mice per dose received 500, 600, 750 and 1000 mg/kg i.p. The animals of the 750 and 1000 mg/kg dose died. Clinical signs in all groups were reduction of the spontaneous activity, apathy and eyelid closure. In confirming prestudies 5 female mice per group were administered i.p. 500 and 600 mg/kg. 4/5 mice in the 600 mg/kg dose died. All animals showed reduction of spontaneous activity, further clinical signs observed were abdominal position, eyelid closure and apathy.

The volume to be administered should be compatible with physiological space available. Three adequate spaced dose levels spaced by a factor of 2 were applied at the central sampling interval 24 h after treatment. For the highest dose level an additional sample was taken at 48 h after treatment.

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields):
Males:
Negative control and 400 mg/kg bw: sampling after 24 and 48 h
Positive control, 100 and 200 mg/kg bw: sampling after 24 h
Females:
Negative control and 500 mg/kg bw: sampling after 24 and 48 h
Positive control, 125 and 250 mg/kg bw: sampling after 24 h

DETAILS OF SLIDE PREPARATION:
The cell suspension was centrifuged at 1500 rpm (390 x g) for 10 minutes and the supernatant was discarded. A small drop of the resuspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Grünwald (MERCK, D-64293 Darmstadt)/Giemsa (Gurr, BDH Limited Poole, Great Britain). Cover slips were mounted with EUKITT (KINDLER, D-79110 Freiburg). At least one slide was made from each bone marrow sample.

OTHER:
To describe a cytotoxic effect due to the treatment with the test substance the ratio between polychromatic (PCE) and normochromatic erythrocytes (NCE) was determined in the sample and reported as number of NCEs per 2000 PCEs.
After treatment with the test substance the number of NCEs was not substantially changed as compared to the mean value of NCEs of the vehicle control thus indicating that tert-butyl acrylate had no cytotoxic effectiveness in the bone marrow.
Evaluation criteria:
A test substance is classified as mutagenic if it induces either a dose-related increase in the number of micronucleated polychromatic erythrocytes, which clearly exceeds the negative control range or a relevant positive response for at least one of the test points. Statistical methods (nonparametric Mann-Whitney test) can be used as an aid in evaluating the results. However, the primary point of consideration is the biological relevance of the results.
A test substance producing neither a dose-related increase in the number of micronucleated polychromatic erythrocytes nor a positive response at any of the test points is considered non-mutagenic in this system.

Acceptance Criteria
The study was considered valid as the following criteria were met:
- the negative controls are in the range of the laboratory's historical control data (0.30 - 1.5 per thousand; mean = 0.86 ± 0.27 per thousand PCEs with micronuclei).
- the positive controls are in the range of the laboratory's historical control data (10.0 - 27.1 per thousand; mean = 16.53 ± 4.09 per thousand PCEs with micronuclei).
- at least 80 % of animals are evaluable
- the test was performed up to the maximum tolerated dose, clear signs of systemic toxicity demonstrated systemic availablity of the test material

Results and discussion

Test results
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
at the highest dose level
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Systemic availablity as well as the maximum tolerated dose were determined in pre-studies. Both was also shown by the clinical effects in the main study, all animals of the highest dose groups showed reduction of the spontaneous activity, eyelid closure, apathy and abdominal position after the application. There was no significant cytotoxic effect on the PCE/NCE ratio in the bone marrow for tBA as well as for the significantly micronuclei inducing positive control.

Any other information on results incl. tables

males
test group  dose      sampling  PCEs with    PCE / NCE
            mg/kg bw  time (h)  micronuclei 
                                (percentile)
vehicle     0          24        0.50         2000 / 1821
tBA         100       24        0.60         2000 / 1855
tBA         200       24        0.60         2000 / 1760
tBA         400       24        0.70         2000 / 1878
positiv 
control     40         24      19.80         2000 / 1719
vehicle     0          48        0.10         2000 / 1886
tBA         400       48        0.20         2000 / 1886

females
test group  dose      sampling  PCEs with    PCE / NCE
            mg/kg bw  time (h)  micronuclei 
                                (percentile)
vehicle     0          24        0.70         2000 / 1509
tBA         125       24        0.10         2000 / 1542
tBA         250       24        0.90         2000 / 1714
tBA         500       24        0.20         2000 / 1573
positiv 
control     40         24       13.70        2000 / 1627
vehicle     0          48        0.20         2000 / 1752
tBA         500       48        0.40         2000 / 1674

Applicant's summary and conclusion