[No public or meaningful name is available]

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

9 October 2006 to 30 January 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Test material

Test material information

1

Reference substance name:

-

EC Number:

482-480-5

EC Name:

-

Cas Number:

1187571-02-3

Molecular formula:

(R2C5H4NO2)(0-3)(R3C3H5O3)(0-3)H(0-2)B

IUPAC Name:

Coconut oil reaction products with boric acid and diethanolamine

Test animals

Species:

mouse

Strain:

NMRI

Details on species / strain selection:

The mouse is an animal that has been used for many years as a suitable experimental animal in cytogenetic investigations. There are many data available from such investigations which may be helpful in the interpretation of results from the micronucleus test. In addition, the mouse is an experimental animal in many physiological, pharmacological and toxicological studies. Data from such experiments also may be useful for the design and the performance of the micronucleus test.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Animals
Number of Animals: 72 (36 males/36 females)
Initial Age at Start of
Acclimatisation: 8 - 10 weeks
Acclimatisation: minimum 5 days

Initial body weight at Start of Treatment - males mean value 35.6 g (SD ± 1.8 g), females mean value 29.8 g (SD ± 1.6 g).

According to the suppliers assurance the animals were in healthy condition. The animals were under quarantine in the animal house of RCC - CCR for a minimum of five days after their arrival. During this period the animals did not show any signs of illness or altered behaviour.

The animals were distributed into the test groups at random and identified by cage
number.

Environmental Conditions
The animals were kept conventionally. The experiment was conducted under standard laboratory conditions.

Housing: single
Cage Type: Makrolon Type I, with wire mesh top
Bedding: granulated soft wood bedding
Feed: pelleted standard diet, ad libitum
Water: tap water, ad libitum
Environment:
- temperature 22 ± 3 °C
- relative humidity 30 - 75 %
- artificial light 6.00 a.m. - 6.00 p.m.

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

30% DMSO / 70% corn oil

Duration of treatment / exposure:

The volume administered intraperitoneally (i.p.) was 10 mL/kg b.w.. 24 h and 48 h after a single administration of the test item the bone marrow cells were collected for micronuclei analysis.

Frequency of treatment:

Single administration

Post exposure period:

24 h and 48 h

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

250 mg/kg bw (total dose)

Doses / concentrations 2

Dose / conc.:

500 mg/kg bw (total dose)

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw (total dose)

No. of animals per sex per dose:

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 is usually
evaluated in case an animal dies in its test group spontaneously.

Control animals:

yes, concurrent vehicle

Positive control(s):

CPA; Cyclophosphamide

Solution prepared on day of administration.

Examinations

Tissues and cell types examined:

Bone marrow cells were collected for micronuclei analysis.

Details of tissue and 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 /Giemsa. Cover slips were mounted with EUKITT. At least one slide was made from each bone marrow sample.

Evaluation criteria:

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 is usually evaluated in case an animal dies in its test group spontaneously.

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:

Statistical methods (nonparametric Mann-Whitney test) will be used as an aid in evaluating the results. However, the primary point of consideration is the biological relevance of the results.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The mean number of polychromatic erythrocytes was slightly decreased after treatment with the test substance as compared to the mean value of PCEs of the vehicle control indicating that the test substance had cytotoxic properties in the bone marrow.

In comparison to the corresponding vehicle controls there was no statistically significant or biologically relevant enhancement in the frequency of the detected micronuclei at any preparation interval and dose level after administration of the test item. The mean values of micronuclei observed after treatment with test substance were near to the value of the vehicle control group and within the historical control range.

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

Applicant's summary and conclusion

Conclusions:

During an OECD 474 study and under the experimental conditions reported, the test substance did not induce micronuclei as determined by the micronucleus test in the bone marrow cells of the mouse.

Executive summary:

This study was performed to investigate the potential of the test substance to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse.

The test item was formulated in 30% DMSO / 70% corn oil, which was also used as vehicle control. The volume administered intraperitoneally (i.p.) was 10 mL/kg b.w.. 24 h 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. 2000 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: 250, 500 and 1000 mg/kg b.w..
48 h preparation interval: 1000 mg/kg b.w..

The highest dose (1000 mg/kg b.w.) was estimated by a pre-experiment to be suitable as the high dose. In the main study 1 male died after treatment with this dose.

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 substance exerted cytotoxic effects in the bone marrow.

In comparison to the corresponding vehicle controls there was no biologically relevant or statistically significant enhancement in the frequency of the detected micronuclei at any preparation interval after administration of the test item and with any dose level used.

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 test substance did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse.

Therefore, the test substance is considered to be non-mutagenic in this micronucleus assay.