4-amino-m-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:

From Oct. 30, 2001 to May 13, 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study well documented, followed guideline, GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

according to the OECD principles of GLP

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

4-amino-m-cresol

EC Number:

220-621-2

EC Name:

4-amino-m-cresol

Cas Number:

2835-99-6

Molecular formula:

C7H9NO

IUPAC Name:

4-amino-m-cresol

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material: 4-Amino-3-methyl-Phenol
- TSIN: 23533
- Substance type: Pure active substance
- Physical state: Solid (red-brown)
- Stability under test conditions: Test substance is considered to be stable for three years.
- Storage condition of test material: At room temperature

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Winkelmann, Germany
- Age at study initiation: Minimum of 8 weeks (start of treatment)
- Weight at study initiation: The mean body weight of male and female animals was 27.4±1.5 and 23.3±1.5 g, respectively.
- Assigned to test groups randomly: Yes
- Housing: Animals were housed (5 animals of identical sex per cage) in Macrolon Type III (Hereto) type cages with granulated soft wood bedding.
- Diet: Pelleted standard diet (ALTROMIN), ad libitum
- Water: Tap water, ad libitum (Zweckverband Wurmtal, Planegg),
- Quarantine period: Minimum for 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19-25 °C
- Humidity: 55±10%
- Air changes : Not reported
- Photoperiod: 12 hours artificial light (from 6.00 to 18.00 hours)/12 hour dark.

STUDY INITIATION DATE: Oct. 30, 2001
STUDY COMPLETION DATE: May 13, 2002

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

- Vehicle used: 0.9 % NaCl
- Justification for choice of vehicle: The vehicle was chosen due to its non-toxicity.
- Amount of vehicle: 10 mL/kg

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test substance was prepared and diluted with 0.9 % NaCl. No other details on test substance preparation are provided in the study report.

TREATMENT SCHEDULE: The dose volume for all treatment group was 10 mL/kg bw. The treatment schedule was as follows:
Vehicle Control (0.9% NaCl): 10 mL/kg bw (treated for 24 h)
Group I: 20 mg/kg bw (treated for 24 h)
Group II: 100 mg/kg bw (treated for 24 h)
Group III: 200 mg/kg bw (treated for 24 h)
Group IV: 200 mg/kg bw (treated for 48 h)
Positive control (Cyclophosphamide): 40 mg/kg bw (treated for 24 h)

Duration of treatment / exposure:

24 h (20, 100 and 200 mg/ kg bw) and 48 h (200 mg/kg bw)

Frequency of treatment:

Once

Post exposure period:

The animals of all dose groups were examined for acute toxic symptoms at intervals of around 1, 6, 24 and 48 hour after treatment.

Doses / concentrations

Remarks:

Doses / Concentrations:
20, 100 and 200 mg/kg bw
Basis:
nominal conc.

No. of animals per sex per dose:

5/sex/ treatment group

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide (purity-at least 98 %)
- Route of administration: Intraperitoneal  
- Dose: 40 mg/kg bw

Examinations

Tissues and cell types examined:

Tissue: Femoral bone marrow
Cell types: Polychromatic erythrocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: Dose selection was based on the results of preliminary toxicity test 200 mg/kg was determined as maximum applicable level (maximum tolerated dose).

TREATMENT AND SAMPLING TIMES: Animals were dosed once at time 0 h. Bone marrow samples were collected at 24 hour after treatment. For the highest dose level an additional sample was taken at 48 hour after treatment

ISOLATION OF BONE MARROW: Bone marrow was obtained from the femurs immediately following sacrifice by cervical dislocation of the animals. Cells were removed from the femurs by cutting off the epiphyses and by flushing the marrow out with fetal calf serum using a 5 ml syringe. The cell suspension was centrifuged at 200 x g for 10 minutes and the supernatant was discarded.

DETAILS OF SLIDE PREPARATION: A drop of the resuspended cell pellet was spread on a slide as a smear. This was air-dried and stained with May-Grunwald/Giemsa. At least one slide was made from each bone marrow cell sample.

METHOD OF ANALYSIS: All slides, including the controls, were coded before microscopic analysis. Evaluation of the slides was performed using microscopes with 100 x oil immersion objectives. 2000 immature erythrocytes per animal were scored for the incidence of micronucleated immature erythrocytes. To detect an eventually occurring cytotoxic effect of the test substance the ratio between immature and mature erythrocytes was determined. At least 200 immature erythrocytes were counted per animal and the result was expressed as relative PCE (rel. PCE=proportion of polychromatic (immature) erythrocytes among total erythrocytes).

Evaluation criteria:

1) The criteria for determining a positive result was as follows:
- Dose-related increase in the number of micronucleated cells and/or
- Biologically relevant increase in the number of micronucleated cells for at least one of the dose group.

2) The test substance was considered to be negative if there was no biological relevant and/or statistical significant increase in the number of micronucleated cells at any dose level.

3) In the study, both biological relevance and statistical significance were considered together.

Statistics:

- The nonparametric Mann-Whitney test was used.

Results and discussion

Test results

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY:
- Dose range: 200-2000 mg/kg bw
- Clinical signs of toxicity in test animals: In a pre-experiment one female mouse was treated with a dose of 2000 mg/kg bw intraperitoneally and died within 1 hour after start of treatment. In a second step 1000 mg/kg bw were administered to one further female animal which died within 2.5 hour after start of treatment. 500 mg/kg bw were applied to three animals of each sex. Two female animals died within 24 hour. Finally 200 mg/kg bw were tested as the maximum tolerable dose (MTD) that induced signs of toxicity (palpebral closure, lethargy) within the first hour post application. No signs of systemic toxicity were observed at 6, 24 and 48 hour post administration. In the main experiment identical symptoms of toxicity were observed with the MTD. The volume administered was 10 mL/kg bw in the pre-experiments.

- For main study, 200 mg/kg bw was selected as highest dose.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei: In comparison to the corresponding negative controls, there was no statistically significant enhancement (p<0.05) in the percentage of cells with micronuclei at any preparation interval or dose level of the test substance. The values of micronuclei observed after treatment with test substance were 0.33 % for male and 0.29 % for female mice (20 mg/kg bw; 24 hour) and 0.30 % or 0.21 % (100 mg/kg bw; 24 h) respectively. At 200 mg/kg bw (MTD) the values of micronuclei observed were 0.26 % for male and 0.15 % for female mice (24 hour) and 0.20 % or 0.18 % (48 hour) respectively. The values of micronuclei observed in the negative control groups were 0.32 % for male and 0.16 % for female mice.
- Relative PCE (proportion of polychromatic (immature) erythrocytes among total erythrocytes): In all treated groups, the relative PCE frequency was not decreased in any dose group.
- Clinical signs: The highest dose (200 mg/kg bw) induced signs of toxicity (palpebral closure, lethargy) within the first hour after application. No toxic effects were observed at later time points.
- Appropriateness of dose levels and route: The dose and route for definitive test were appropriate. As estimated by a pre-experiment, 200 mg/kg bw was selected as MTD.
- For details on result, refer to ‘Table 1’ under ‘Any other information on results incl. tables’ section.

RESULT WITH POSITIVE CONTROL: Cyclophosphamide (40 mg/kg bw) administered i.p. was used as positive control which showed a significant increase of induced micronucleus frequency (percentage of cells with micronuclei was 0.69 % for male and 0.86 % for female mice). The values for male mice were below the historical control data but a factor of about 2 higher than the values of the concurrent negative control and the historical negative control maximum value. Additionally the value was statistically significant (p = 0.0079) higher than the concurrent negative control. Therefore the low value of the positive control was still considered to be biologically relevant and not to affect the integrity of the study.

Any other information on results incl. tables

Table 1. Results on micronuclei assay after treatment with 4-amino-3-methylphenol (study # 71284)

Treatment (Intraperitoneal)	Dose	Sampling Time (h)	Sex	% of cells with micronuclei	Mean relative PCE	p-value
Vehicle control (0.9% NaCl)	10 mL/kg bw	24	Female	0.16	0.56	-
			Male	0.32	0.63	-
Positive control (Cyclophosphamide)	40 mg/kg bw	24	Female	086^S(+)	0.49	0.0079
			Male	0.69^S(+)	0.64	0.0079
Test substance (Group I)	20 mg/kg bw	24	Female	0.29^ns	0.52	0.1508
			Male	0.33^ns	0.56	0.8413
Test substance (Group II)	100 mg/kg bw	24	Female	0.21^ns	0.58	0.4206
			Male	0.30^ns	0.65	0.6905
Test substance (Group III)	200 mg/kg bw	24	Female	0.15^ns	0.59	1
			Male	0.26^ns	0.6	0.3095
Test substance (Group IV)	200 mg/kg bw	48	Female	0.18^ns	0.5	0.8413
			Male	0.20^S(-)	0.48	0.0317

S(+): Significantly positive

S(-): Significantly reduced frequency of micronuclei as compared to the negative control group.

ns: not significant

Applicant's summary and conclusion

Conclusions:

4-amino-3-methylphenol was non-mutagenic in the micronucleus test with mouse bone marrow cells when administered intraperitoneally at 20, 100 and 200 mg/kg bw.

Executive summary:

The purpose of this study was to assess the mutagenic potential of 4-amino-3-methylphenolin the micronucleus assay by following methods according to the OECD guideline 474 (Mammalian Erythrocyte Micronucleus Test).

Male and female NMRI mice (source: Harlan Winkelmann, Germany) weighing 27.4±1.5 g (males) and 23.3±1.5 g (females),were used in the study. The animals were housed (5 animals of identical sex per cage) in Macrolon Type III (Hereto) type cages with granulated soft wood bedding.Animals were maintained under standard laboratory conditions (temperature:19-25 °C, humidity:55±10%). 

Each treatment group contained 5 animals/sex. The test substance was prepared with 0.9% NaCl solution. The doses for the definitive test were selected on the basis of a pre-experiment. In a pre-experiment one female mouse was treated with a dose of 2000 mg/kg bw intraperitoneally and died within 1 hour after start of treatment. In a second step 1000 mg/kg bw was administered to another female animal which died within 2.5 hour after start of treatment. 500 mg/kg bw was applied to three animals of each sex. Two female animals died within 24 hours. Finally 200 mg/kg bw was tested as the maximum tolerable dose (MTD) that induced signs of toxicity (palpebral closure, lethargy) within the first hour post application. No signs of systemic toxicity were observed at 6, 24 and 48 hour post administration. In the main experiment identical symptoms of toxicity were observed with the MTD. The following dose levels of the test substance were investigated in the main experiment:

24 h preparation interval: 20, 100 and 200 mg/kg bw

48 h preparation interval: 200 mg/kg bw

After treatment, animals were sacrificed and the bone marrow tissues were isolated fromboth femora. Slides were prepared,stained with May-Gruenwald-Giemsa and were examined under a microscope. 2000 immature erythrocytes per animal were scored for the incidence of micronucleated immature erythrocytes. At least 200 immature erythrocytes were counted per animal and the result wasexpressed as relative PCE (rel. PCE=proportion of polychromatic (immature) erythrocytes among total erythrocytes).

 

In comparison to the corresponding negative controls, there was no statistically significant enhancement (p<0.05) in the percentage of cells with micronuclei at any preparation interval or dose level of the test substance.

Cyclophosphamide (40 mg/kg bw) administered i.p. was used as a positive control and showed a significant increase of induced micronucleus frequency (percentage of cells with micronuclei was 0.69 % for male and 0.86 % for female mice).

Based on above,4-amino-3-methylphenol was non-mutagenic in the micronucleus test with bone marrow cells of the mouse when administered intraperitoneally at 20, 100 and 200 mg/kg bw. 

This mammalian erythrocyte micronucleus test is classified as acceptable, and satisfies the guideline requirements of the OECD 474 method.