[No public or meaningful name is available]

Genetic toxicity: in vivo

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:

From 06 December 2006 to 25 January 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was performed according to international test guidelines and to GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

yes

Remarks:

The relative humidity ranged between 25 - 72% instead of between 30 - 70%. However, this deviation did not affect the validity of the experiment.

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Deviations:

yes

Remarks:

The relative humidity ranged between 25 - 72% instead of between 30 - 70%. However, this deviation did not affect the validity of the experiment.

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Test material form:

other: liquid

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Winkelmann GmbH, D-33178 Borchen, Germany
- Age at study initiation: 8 to 10 weeks
- Weight at study initiation: males: 36.2 +/- 1.3 g; females: 28.6 +/- 1.6g
- Assigned to test groups randomly: yes
- Fasting period before study: no
- Housing: Individually in Makrolon Type I cages, with wire mesh top (EHRET GmbH, D-79302 Emmendingen) and granulated soft wood bedding (Harlan Winkelmann GmbH, D-33178 Borchen).
- Diet: pelleted standard diet (Harlan Winkelmann GmbH, D-33178 Borchen), ad libitum.
- Water: tap water (Gemeindewerke, D-64380 Roßdorf), ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C):22 +/- 3 °C
- Humidity (%): 25 - 72 %
- Air changes (per hr): information not available.
- Photoperiod (hrs dark / hrs light): 12/12.

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

- Vehicle used: corn oil
- Justification for choice of vehicle: The vehicle was chosen to its relative non-toxicity for the animals.

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Volume administered: 10 mL/kg body weight

Duration of treatment / exposure:

one single intraperitoneal injection

Frequency of treatment:

one single intraperitoneal injection

Post exposure period:

The animals of all dose groups were examined for acute toxic symptoms at intervals of around 1 h, 2 - 4 h, 6 h, 24 and 48 h after administration of the test item and sacrificed 24 or 48 hours after treatment.

Doses / concentrations

Remarks:

Doses / Concentrations:
375, 750, 1500 mg/kg bw
Basis:
nominal conc.

No. of animals per sex per dose:

6 animals/sex/dose/sacrifice time

Control animals:

yes, concurrent vehicle

Positive control(s):

yes: CPA (Cyclophosphamide)
- Justification for choice of positive control: reference mutagen
- Route of administration: intraperitoneal injection, once
- Dose: 40 mg/kg b.w.

Examinations

Tissues and cell types examined:

see details below.

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 items. The maximum tolerated dose level is determined to be the dose that causes toxic reactions without having major effects on survival within 48 hours.
In the current study, a preliminary study on acute toxicity was performed with two animals per sex under identical conditions as in the mutagenicity study concerning: animal strain, vehicle, route, frequency, and volume of administration. The animals were treated i.p. with the test item (at doses ranging from 100 to 2000 mg/kg) and examined for acute toxic symptoms at intervals of around 1 h, 2-4 h, 6 h, 24 h, 30 h, and 48 h after administration of the test item.
Based on the preliminary study, the dose levels of 375, 750 and 1500 mg/kg were selected for the main experiment.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
The animals received the test item, the vehicle or the positive control substance once. They were examined for toxic symptoms at regular intervals after administration (see details above) and were sacrificed 24 (375, 750 and 1500 mg/kg dose groups) or 48 hours (additional 1500 mg/kg dose group) after treatment. Sampling of the bone marrow was done at necrospy.


DETAILS OF SLIDE PREPARATION:
The animals were sacrificed using CO2 followed by bleeding. The femora were removed, the epiphyses were cut off and the marrow was flushed out with foetal calf serum using a syringe. The cell suspension was centrifuged at 1500 rpm (390 x g) for 10 minutes and the supernatant was discarded. A small drop of the re-suspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Grünwald (Merck, D-64293 Darmstadt)/Giemsa (Merck, D-64293 Darmstadt). Cover slips were mounted with EUKITT (Kindler, D-79110 Freiburg). At least one slide was made from each bone marrow sample.

METHOD OF ANALYSIS:
Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives. At least 2000 polychromatic erythrocytes (PCE) were analysed per animal for micronuclei*. To describe a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and expressed in polychromatic erythrocytes per 2000 erythrocytes. The analysis was performed with coded slides.
Ten animals (5 males, 5 females) per test group were evaluated as described, the remaining 6th animal of each sex in the respective test group test group being usually evaluated in case an animal dies in its test group spontaneously.

* Originally only 2000 PCEs were scored. However, in order to confirm the obtained data, the number of PCEs to be scored was increased to 6000.

Evaluation criteria:

Acceptance criteria:
The study was considered valid as the following criteria are met:
- the negative controls are in the range of the laboratory historical control data.
- the positive controls are in the range of the laboratory historical control data.
- at least 4 animals per group and sex can be evaluated.
- PCE to erythrocyte ratio should not be less than 20 % of the negative control.

Evaluation of results:
- A test item is classified as mutagenic if it induces either a dose-related increase or a clear increase in the number of micronucleated polychromatic erythrocytes in a single dose group.
- A test item that fails to produce a biological relevant increase in the number of micronucleated polychromatic erythrocytes is considered non-mutagenic in this system.

Statistics:

nonparametric Mann-Whitney test

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

ambiguous

Remarks:

see details below

Toxicity:

yes

Remarks:

see details below

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Doses producing toxicity:
A pre-study was conducted with 2 mice per sex per dose to determine the Maximum Tolerated Dose. Doses of 100, 1000, 2000, 1500 and 1750 mg/kg b.w. i.p. were consecutively tested. The maximum tolerated dose was determined to be 1500 mg/kg for i.p. administration in mice (animals expressed toxic reactions but without mortality) and was thus considered to be suitable for the main experiment. Higher doses induced mortality (1 male and 1 female at 48 hrs at 1750 mg/kg, and 2 males and 2 females within 2 hrs at 2000 mg/kg).

Results from the main experiment:
During the main experiment, the main clinical signs observed following treatment were reduction of spontaneous activity (all animals in the high
dose group), abdominal position (only in some animals of the high dose group), eyelid closure and ruffled fur.

The mean number of PCE per 2000 erythrocytes was slightly decreased after treatment with the high dose as compared to the mean value in the vehicle control group, showing some cytoxicity in the bone marrow cells:

- vehicle control, 24 h: 1115

- 375 mg/kg, 24 h: 1069

- 750 mg/kg, 24 h: 1076

- 1500 mg/kg, 24 h: 935

- positive control (CPA), 24h: 1130

- 1500 mg/kg, 48 h: 1071


In comparison to the vehicle control group (0.115% of PCE with micronuclei), there was a dose-dependent increase in the frequency of the detected micronuclei (0.145%, 0.160%, and 0.210%) after administration of the notified substance. The increase was statistically significant only at 1500 mg/kg dose. The increase at that dose also exceeded the upper limit of the historical control range (0.16%).

The positive control group (40 mg/kg ip cyclophosphamide) showed a statistically significant increase of induced micronucleus frequency (2.68%).

Results of the vehicle control group were within the historical control ranges.

Due to the slight but statistically significant increase of PCEs with micronuclei at the highest dose, the result of this test is considered equivocal.

Any other information on results incl. tables

Summary of Micronucleus Test Results:

test group	dose (mg/kg b.w.)	sampling time (h)	PCEs with micronuclei (%)	range	PCE per 2000 erythocytes
vehicle	0	24	0.115	1 -4	1115
test item	375	24	0.145	2 - 4	1069
	750	24	0.160	1 - 6	1076
	1500	24	0.210*	2 - 9	935
	1500	48	0.095	0 - 4	1071
Positive control	40	24	2.680*	23 - 81	1130

 

* : p < 0.05

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): ambiguous
Under the experimental conditions, the results of this study performed with Dimethyl 2-methyl glutarate are inconclusive.

Executive summary:

This study was performed to investigate the potential of Dimethyl 2 -methyl glutarate to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse.

The test item was formulated in corn oil, which was also used as vehicle control. The volume administered intraperitoneally (i.p.) was 10 mL/kg b.w.. Twenty four and 48 h after a single administration of the test item the bone marrow cells were collected for micronuclei analysis.

Ten animals (5 males, 5 females) per test group were evaluated for the occurrence of micronuclei. 6000 polychromatic erythrocytes (PCEs) per animal were scored for micronuclei.

To describe a cytotoxic effect due to the treatment with the test item the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and reported as the number of PCEs per 2000 erythrocytes.

The following dose levels of the test item were investigated:

24 h preparation interval: 375, 750, and 1500 mg/kg b.w..
48 h preparation interval: 1500 mg/kg b.w..

The highest dose (1500 mg/kg) was estimated by pre-experiments to be suitable.

After treatment with the test item the number of PCEs was slightly decreased as compared to the mean value of PCEs of the vehicle control thus indicating that the test item exerted slight cytotoxic effects in the bone marrow.

In comparison to the corresponding vehicle controls there was a statistically significant enhancement in the frequency of the detected micronuclei after administration of the test item. This enhancement, however, could not be reproduced in follow up studies. Therefore, the biological relevance of the observed effect is questionable.

40 mg/kg b.w. cyclophosphamide administered i.p. was used as positive control which showed a substantial increase of induced micronucleus frequency.

In conclusion, it can be stated that under the experimental conditions reported, the results of this study performed with Dimethyl 2-methyl glutarate are inconclusive.