Registration Dossier

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:
24 November to 10 December 1992
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Proprietary GLP and guideline-compliant study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Report Date:
1993

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Trimethylolpropandiallylether (chemical name 1-butanol, 2,2-Bis[(2-propenyloxy)methyl], manufactured by Bayer AG, sample no. DZA-VER 9204435-B, product no. 020834. The substance was a clear liquid stored in a refrigerator. The content of the substance (confirmed by analytical result on 7 May 1992) was trimethylolpropandiallylether (80.3%), trimethylolpropanmonoallylether (10.6%), trimethylolpropantriallylether (7.3%).
The batch used was analysed prior to study intitiation and approved for use during the test period. A stability test in the vehicle did not detect a relevant change in the percent active ingredient.

Test animals

Species:
mouse
Strain:
other: Bor: NMRI (SPF Han)
Sex:
male/female
Details on test animals and environmental conditions:
The animals were young adult male and virgin female mice, strain Bor: NMRI (SPF Han), bred and supplied by F. Winkelmann, Borcehn. They initially weighed 28-41 g, and were approximately 8-12 weeks of age.

The breed's state of health was regularly spot checked for the major specific pathogens. On the day of arrival, the health of the animals was checked before acclimatising them to the housing conditions for a period of at least one week. Only healthy animals were used in the study.

The females were housed in groups of up to 3 in Makrolon type I cages. Males were singly housed in type I cages. Bedding of soft wood granules (spot-checked for contaminants at regular intervals), type S 8/15 (J. Rettenmaier & Sohne) was used. Individuals were identified by picric acid marks.
The room temperature was maintained at 22-23°C, and 46-52% mean relative humidity, artificial light was provided for 12 hours per day, and there were approximately 10 air changes per hour.

Tap water and feed (Altromin 1324 Standard Diet) were provided ad libitum. The nutritive composition and contaminant content of the diet were checked regularly, along with water quality.
Mice were randomly divided into treatment groups by a randomisation plan, produced by the Insititue of Biometrics, Bayer AG.

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
Corn oil
Details on exposure:
The test substance was dissolved in corn oil and injected i.p. The injection volume was 5 ml/kg bw.
Duration of treatment / exposure:
Mice were scarificed at 16, 24 or 48 hours following administration.
Frequency of treatment:
Single i.p. injection.
Post exposure period:
A post exposure period was not included, mice were sacrificed at varying times following administration.
Doses / concentrations
Remarks:
Doses / Concentrations:
1250 mg/kg bw
Basis:

No. of animals per sex per dose:
5 mice/sex/dose
Control animals:
yes, concurrent vehicle
Positive control(s):
The positive control was Cyclophosphamide, in the form of Endoxan 100 mg injection vials of dry sbstance (Asta), batch 091520. Cyclophosphamide was dissolved in deionised water and injected i.p. The dose was 20 mg/kg bw, and the injection volume was 10 ml/kg bw.

Examinations

Tissues and cell types examined:
Bone marrow - erythrocytes.
Details of tissue and slide preparation:
Schmid's method was used to produce the smears. At least one intact femur was prepared from each sacrificed animal (not pretreated with a spindle inhibitor. A tube was filled with foetal calf serum, a small amount of serum was drawn from the tube into a suitable syringe with a thin cannula. Te cannula was pushed into the open end of the marrow cavity of the femur. The femur was then completely immersed in the calf serum, the contents were then flushed several times and the bone marrow was passed in the serum as a fine suspension. If necessary the flushing was repeated from the other end of femur. The tube was then centrifuged at approximately 1000 rpm for 5 minutes. The supernatant was removed, leaving a small amount remaining. The sediment was mixed to produce a homogenous suspension. One drop of the viscous suspension was placed on a clean slide and spread. The labelled slides were dried overnight. If fresh smears needed to be stained, they were dried with heat for a short period.
The smears were stained automatically with an Ames Haematek Slide Stainer. The slides with then 'destained' with methanol, rinsed with deionised water, and left to dry.
Following this treatment, the smears were then transferred to a holder. A cuvette was filled with xylene, and the holder was immersed into the cuvette for approximately 10 minutes. The slides were removed singly and covered. A small amount of covering agent was taken from a bottle and applied to the coated side of the slide. A cover glass was then placed in position. Slides were not evaluated until the covering agent had dried.
Evaluation criteria:
Coded slides were evaluated using a light micropscope. 1000 polychromatic erythrocytes were counted per animal. The incidence of cells with micronuclei was established by scanning the slides in a meandering pattern. The number of normochromatic erythrocytes showing micronuclei was also established.
A test was considered positive in there was a relevant and significant increase in the number of micronucleated polychromatic erythrocytes compared to the negative control.
Statistics:
Wilcoxon's non-parametric rank sum test, and one-sided chi-square test.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls valid:
yes
Negative controls valid:
not examined
Positive controls valid:
yes
Additional information on results:
There were no differences in the results between males and females, therefore both sexes were evaluated together. No biologically important or statistically significant variations existed between the negative control and treated groups, with respect to the incidence of micronucleated polychromatic erythrocytes, micronucleated normoerythrocytes. The ratio of polychromatic to normochromatic erythrocytes was altered, in a biologically relevant manner, in the treated groups compared to the negative control. The ratio was not altered in the positive controls. The positive control caused a clear increase in the number of micronucleated polychromatic erythrocytes.

Any other information on results incl. tables

After a single i.p. dose of 1250 mg/kg bw, treated mice showed apathy, anaesthethised-like state, roughened fur, staggering gait, sternal recumbency, spasm, shivering, difficulting breathing and breathing noises. Symptoms were present until sacrifice. Feeding behaviour was normal (no data included to support this). There were no mortalities. The number of micronucleated PCEs was comparable in all treated groups and control: an appropriate response was seen to the postive control compound (CPA).

Table 1. Summary of Results

Treatment

Sacrifice (hours after treatment)

No. evaluated polychromatic erythrocytes (n=10 mice)

No. normochromatic erythrocytes per 1000 polychromatic erythrocytes

Micronucleated cells per 1000

Normochromatic erythrocytes

Polychromatic erythrocytes

Negative Control (corn oil)

24

10000

573 ± 143

1.2 ± 1.1

1.7 ± 1.5

TMPDE

16

10000

1207 ± 402

1.2 ± 1.0

2.6 ± 2.2

24

10000

790 ± 245

1.6 ± 2.1

1.7 ±1.4

48

10000

782 ± 186

1.5 ± 0.9

1.5 ± 1.2

Positive Control (cyclophosphamide)

24

10000

483 ± 135

1.6 ± 2.1

17.9 ± 6.6*

Data shown are mean ± 1 standard deviation

TMPDE = trimethylolpropandiallylether (1250 mg/kg bw i.p.)

* P < 0.01

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
There was no evidence of a clastogenic effect following a single i.p. administration of 1250 mg/kg bw in mice.
Executive summary:

A mouse micronucleus test was employed to assess the clastogenic potential of trimethylolpropandiallylether, in male and female mice, according to OECD method 474. The mice received a single intraperitoneal injection of the test substance at a dose level of 1250 mg/kg bw in corn oil. Vehicle controls and positive controls (cyclophosphamide) were included. The treated mice were sacrificed at 16, 24 and 48 hours after administration. All treated mice survived to sacrifice, however all showed signs of toxicity. There was an altered ratio between polychromatic and normochromatic erythrocytes. No evidence of a clastogenic effect was found. The response to the posive control (CPA) confirmed the sensitivity of the assay.