4-chlororesorcinol

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:

26.07.- 09.12.2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

yes

Remarks:

The relative humidity ranged between 30 - 78 % and not between 30 –70 % as described in the study plan. Due to the repetition of the blood sampling experiment the number of male animals used for this study was increased by 9.

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

4-chlororesorcinol

EC Number:

202-462-0

EC Name:

4-chlororesorcinol

Cas Number:

95-88-5

Molecular formula:

C6H5ClO2

IUPAC Name:

4-chlorobenzene-1,3-diol

Test animals

Species:

mouse

Strain:

NMRI

Details on species / strain selection:

The mouse is an animal which has been used for many years as 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:

Number of animals: 90 (54 males/36 females)
Initial age at start of acclimatization: males: 5 - 8 weeks, females: 7 - 10 weeks
Acclimatisation: minimum 5 days
Initial Body Weight at Start of Treatment: males mean value 35.0 g (SD ± 2.1 g), females mean value 32.4 g (SD ± 2.0 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.

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

Housing: single
Cage type: Makrolon Type I, with wire mesh top (EHRET GmbH, D-79302 Emmendingen)
Bedding: granulated soft wood bedding (Harlan Winkelmann GmbH, D-33178 Borchen)
Feed: pelleted standard diet, ad libitum (Harlan Winkelmann GmbH, D-33178 Borchen)
Water: tap water, ad libitum, (Gemeindewerke, D-64380 Roßdorf) temperature 22 ± 3°C; relative humidity 30 - 78 %; artificial light 6.00 a.m. - 6.00 p.m.

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

deionised water

Details on exposure:

On the day of the experiment, the test item was formulated in deionised water. The vehicle was chosen to its relative non-toxicity for the animals. All animals received a single standard volume of 10 mL/kg body weight i.p..

Duration of treatment / exposure:

single administration

Frequency of treatment:

single administration

Post exposure period:

24 hours (all dose groups) and 48 hours (only high dose group)

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

vehicle control

Doses / concentrations 2

Dose / conc.:

25 mg/kg bw/day (nominal)

Remarks:

24 h preparation interval

Doses / concentrations 3

Dose / conc.:

50 mg/kg bw/day (nominal)

Remarks:

24 h preparation interval

Doses / concentrations 4

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

24 h preparation interval

Doses / concentrations 5

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

48 h preparation interval

No. of animals per sex per dose:

low and mid dose: 6
high dose: 12

Control animals:

yes, concurrent vehicle

Positive control(s):

CPA; Cyclophosphamide
Dissolved in: deionised water
Dosing: 40 mg/kg
Route and frequency of administration: i.p., once
Volume administered: 10 mL/kg b.w.

Examinations

Tissues and cell types examined:

Blood cells - erythrocytes

Evaluation criteria:

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. 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.
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.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Applicant's summary and conclusion

Conclusions:

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse.
Therefore, A 012 is considered to be non-mutagenic in this micronucleus assay.

Executive summary:

This study was performed to investigate the potential of A 012 to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse.

The test item was formulated in deionised water, 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. At least 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 total 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: 25, 50, 100 mg/kg b.w.

48 h preparation interval: 100 mg/kg b.w.

As estimated by pre-experiments 100 mg A 012 per kg b.w. was the highest applicable dose without significant effects on the survival rates, but with clear signs of toxicity. At a higher dose (150 mg/kg) all treated animals had to be euthanised after 10 minutes due to the severity of the induced toxic effects.

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 A 012 had some cytotoxic effects in the bone marrow. The bioavailability of the test item was, however, confirmed by chemical analysis of the blood of the treated animals.

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.