4-chloro-o-cresol

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:

1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study is guideline and in accordance with GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1997

Report date:

1997

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

4-chloro-o-cresol

EC Number:

216-381-3

EC Name:

4-chloro-o-cresol

Cas Number:

1570-64-5

Molecular formula:

C7H7ClO

IUPAC Name:

4-chloro-2-methylphenol

Constituent 2

Reference substance name:

PCOC

IUPAC Name:

PCOC

Details on test material:

- Physical state: solid
- Analytical purity:98.0% w/w
- Purity test date:11/12/96
- Lot/batch No.:NML/96/15

Test animals

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River UK Limited , Margate , Kent, England.
- Weight at study initiation: males 28 - 30 gams, females 22-24 grams.
- Assigned to test groups randomly: yes
- Fasting period before study: not specified
- Housing: plastic disposable cages in a controlled environment.
- Diet : ad libitum
- Water: ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 22°C
- Humidity (%): 38 -64%
- Air changes (per hr): approximately 20
- Photoperiod (hrs dark / hrs light): 12 hours light/12 hours dark.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: aqueous 0.5% gum tragacanth
- Justification for choice of solvent/vehicle: PCOC has low solubility in water and was incompatible with methylcellulose the originally intended test vehicle. The test material was prepared as a suspension.
- Concentration of test material in vehicle: 5, 10 and 20 mg/ml
- Amount of vehicle (if gavage or dermal): 20ml/kg body weight.
- Lot/batch no. (if required): PT70780/20, L95158/20
- Purity: 0.5%

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Dosing solutions were prepared fresh on the morning of the test.

DIET PREPARATION
The aminals were dosed by gavage therefore no dietary preparation was necessary.

Duration of treatment / exposure:

Single dose.

Post exposure period:

Examination of test groups was maintained over 24 and 48 hours.

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
100 mg/kg
Basis:
actual ingested

Doses / concentrations 2

Remarks:

Doses / Concentrations:
200mg/kg
Basis:
actual ingested

Doses / concentrations 3

Remarks:

Doses / Concentrations:
400 mg/kg
Basis:
actual ingested

No. of animals per sex per dose:

At dose rates of 100 and 200 mg/kg, 5male and 5 female animals were used.
At 400mg/kg dose level, 10 animals of each sex were dosed. An additional 2 males and 1 female were dosed with 400mg/kg in order to replace any animals that may die.

Control animals:

yes, concurrent vehicle

Positive control(s):

mitomycin C
- Route of administration: gavage
- Doses / concentrations: Concentration of 0.6 mg/ml in the test solution, delivered at a dose of 12mg/kg bodyweight.

Examinations

Tissues and cell types examined:

Immature and mature erythocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: The doses were selected based on the results of a preliminary toxicity test.

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields):

DETAILS OF SLIDE PREPARATION:
The animals were killed by cervical dislocation and both femurs dissected out. The femurs were cleared of tissue and the proximal epiphysis removed. The bone marrow of both femurs was flushed out and pooled with a total volume of 2 mls of pre-filtered foetal calf serum using a 2ml disposable syringe fitted with a 21-gauge needle. The cells were centrifuged and the supernatant discarded. The cells were re-suspended in a small volume of fresh serum, a small drop of the cell suspension was transferred to a glass microscope slide and a smear prepared in the conventional manner. At least 3 smears were made for each animal. The prepared smears were fixed in methanol, after air dying the smears were stained for 10 minutes in 10% Giemsa. Following rinsing in distilled water and differentiation in buffered distilled water (pH6.8) the smears were air dried and mounted with cover slips using DPX.

METHOD OF ANALYSIS:
The smears were examined by light microscopy for the incidence of micro nucleated cells per 1000 polychromatic erythrocytes per animal. Usually only 1 smear per animal was examined.

Evaluation criteria:

The number of micro nucleated immature and mature erythrocytes were evaluated. The proportion of immature erythrocytes was then defined.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

not examined

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY PHASE I
- Dose range: 432 - 2000 mg/kg
- Clinical signs of toxicity in test animals: All 4 animals at dose level levels 1200 and 2000 mg/kg died. At 720 mg/kg 1 animal died. No deaths were observed at the 432mg/kg dose level.

RESULTS OF RANGE-FINDING STUDY PHASE II
- Dose range: 243 - 576 mg/kg
- Clinical signs of toxicity in test animals: At 576mg/kg, 3 deaths were observed out of 4 animals tested. No deaths were observed at the lower dose levels.

RESULTS OF DEFINITIVE STUDY
One female died in the high dose group, this female was replaced by one from the concurrently treated satellite group.
Severe lethargy was observed for animals in the high dose group directly after treatment, resulting in the one death. The remaining animals recovered within 90 minutes.
No adverse clinical signs were obtained for the vehicle control or the positive control group.

The test substance did not cause any significant increase in the number of micro nucleated immature erythrocytes at either 24 or 48 hours.
The positive control, Mitomycin C, caused very significant increases in the frequency of micro nucleated immature erythrocytes.

The test substance did not cause any substantial increase in the incidence of micro nucleated mature erythrocytes at either sampling time.
The test substance failed to cause a decrease in the proportion of immature erythrocytes.

Applicant's summary and conclusion

Conclusions:

Interpretation of results: negative
As the test substance did not cause any substantial increase in the incidence of micro nucleated immature erythrocytes or decrease in the proportion of immature erythrocytes, it is concluded that PCOC did not show any evidence of causing chromosome damage or bone marrow cell toxicity when administered orally by intragastric gavage in this in vivo test procedure.

Executive summary:

A guideline study was performed to GLP to evaluate the potential effect of PCOC (4 -chloro-2 -methyl phenol) on bone marrow cells in mice. Mice were treated with a single oral dose at levels of 100, 200 and 400 mg/kg body weight. The dose levels being defined by a preliminary toxicity test.

The test substance, negative control (vehicle - aqueous 0.5% gum tragacanth) and positive control (Mitomycin C) were administered by intragastric gavage.

The positive control was administered at 12 mg/kg bodyweight.

Bone marrow smears were obtained from 5 male and 5 female animals in the negative control group, at each dose level and the positive control group, 24 hours after dosing.

In addition bone marrow smears were obtained 48 hours after dosing from the negative control and the high-level dose group.

One smear from each animal was examined for the presence of micronuclei in 1000 immature erythrocytes. The proportion of immature erythrocytes was assessed by the examination of at least 1000 erythrocytes from each animal.

No evidence of increased frequency of micro nucleated immature erythrocytes was observed after either 24 or 48 hours.

No significant decrease in the proportion of immature erythrocytes after treatment of the animals was observed.

The positive control produced large highly significant increases in the frequency of micro nucleated immature erythrocytes.

It was concluded that PCOC did not show any evidence of causing chromosome damage or bone marrow cell toxicity in this test.