5-amino-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:

From Oct. 04, 2005 to Nov. 01, 2005

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:

2005

Report date:

2005

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

(according to German and OECD principles of GLP)

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

5-amino-o-cresol

EC Number:

220-618-6

EC Name:

5-amino-o-cresol

Cas Number:

2835-95-2

Molecular formula:

C7H9NO

IUPAC Name:

5-amino-2-methylphenol

Constituent 2

Reference substance name:

4-Amino-2-Hydroxytoluene

IUPAC Name:

4-Amino-2-Hydroxytoluene

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material: 4-Amino-2-Hydroxytoluene (Haarpurpur)
- TSIN: 23032; test substance code: A000177
- Substance type: Pure active substance
- Physical state: Beige Solid
- Storage condition of test material: At room temperature, protected from light and moisture.
- Stability under test conditions: Stable in PEG 400 for at least 7 days
- Stability under storage conditions: The substance is considered to be stable for more than 6 years, if stored dry and protected from light at room temperature.
- Solubility: 7.3 weight% in acetonitrile, 0.6 weight% in water, 6 weight% in acetone/water 1:1, 10 weight% in DMSO, 6.4 weight% in ethanol; > 5 weight % in PEG 400

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Winkelmann GmbH, 0-33178 Borchen
- Age at study initiation: Approximately 9-11 weeks
- Weight at study initiation: 35.2 ± 2.1 g
- Assigned to test groups randomly: Yes, The animals were distributed into the test groups at random
- Fasting period before study: No
- Housing: Individually in Makrolon Type 1 with wire mesh top with granulated soft wood bedding.
- Diet: Pelleted standard diet (Harlan Winkelmann GmbH, 0-33178 Borchen); ad libitum
- Water: Tap water; ad libitum
- Acclimation period: Minimum of 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3°C
- Humidity: 30-70%
- Air changes: Not reported
- Photoperiod: 12 h artificial light / 12 h dark cycle per day
Experiment start date: Oct. 04, 2005
Experiment completion date: Nov. 01. 2005

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

-Vehicle: PEG 400
- Justification for choice of solvent/vehicle: The vehicle was chosen due to its non-toxicity for the animals. The test substance was freely soluble in the vehicle at the administered doses.
- Concentration of test material in vehicle: 0, 6.25, 12.5 and 25.0 mg/mL
-Amount of vehicle: 20 mL/kg bw
-Lot/batch no.: Merck-VWR 64293 Darmstadt

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test substance was prepared and diluted with vehicle (PEG-400)

TREATMENT SCHEDULE: The dose volume for all treatment group was 20 mL/kg bw. The treatment schedule was as follows
Vehicle control (PEG 400): 20 mL/kg bw (sampling time was 24 h)
Group 1 (low dose group): 125 mg/kg bw (sampling time was 24 h)
Group 2(mid dose group): 250 mg/kg bw (sampling time was 24 h)
Group 3 (high dose group): 500 mg/kg bw (sampling time was 24 h)
Group 4 (high dose group): 500 mg/kg bw (sampling time was 48 h)
Positive control (cyclophosphamide): 40 mg/kg bw dissolved in deionized water (sampling time was 24 h)

Duration of treatment / exposure:

24 h (0, 125, 250 and 500 mg/ kg bw) and 48 h (500 mg/kg bw)

Frequency of treatment:

Singe oral administration

Post exposure period:

None

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 125, 250, 500 mg/kg bw
Basis:
nominal conc.

No. of animals per sex per dose:

6 male/test group/bone marrow harvest time

Control animals:

yes, concurrent vehicle

Positive control(s):

CPA; Cyclophosphamide (Sigma-Aldrich 82041)
- Justification for choice of positive control: In accordance with the OECD guideline
- Route of administration: Oral gavage
- Doses / concentrations: 40 mg/kg bw (Dose volume: 10 mL/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: As estimated by pre-experiments 500 mg/kg bw was found to be suitable. At the next higher dose (750 mg/kg b.w.) one female died, thus implicating that the performance of the main experiment using doses above 500 mg/kg bw could greatly affect the survival rates.
For details on the five pre-experiments, refer to 'Any other information on material and methods' section.

TREATMENT AND SAMPLING TIMES: Animals were dosed once at time 0 h. Bone marrow samples were collected at 24 h after treatment. For the highest dose level an additional sample was taken at 48 h 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 syringe. The cell suspension was centrifuged at 1500 rpm (390 g) for 10 minutes and the supernatant was discarded.

DETAILS OF SLIDE PREPARATION: A small drop of the resuspended cell pellet was spread on a slide as a smear. This was air-dried and stained with May-Grünwald/Giemsa. Cover slips were mounted with EUKITT. 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. Slides preparations from 5 males per test group were analyzed as described above while the sixth animal was utilized in the case of a spontaneous death in the group.

Evaluation criteria:

- The test was to be classified as mutagenic if it induced either a dose-related increase or a clear increase in the number of micronucleated polychromatic erythrocytes in a single dose group.
- Statistical comparison was used; however the primary consideration was the biological relevance of the results. A test substance that failed to produce a biologically relevant increase in the number of micronucleated polychromatic erythrocytes was to be considered non-mutagenic in this system.

Statistics:

Statistical methods (nonparametric Mann-Whitney test) were used as an aid in evaluating the results.

Results and discussion

Test results

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 2000, 1500, 500, 1000, and 750 mg/kg bw in pre-experiment 1, 2, 3, 4 and 5 respectively
- Clinical signs of toxicity in test animals: No obvious gender-specific difference in the sensitivity against the test material was observed. For animal welfare reasons and in line with OECD guideline 474 it was therefore decided to test only males in the main experiment. The clinical signs observed in five pre-experiments were as follows:
First pre-experiment (2000 mg/kg bw): Reduction of spontaneous activity, abdominal position, ruffled fur, apathy, change in urine color and mortality
Second pre-experiment (1500 mg/kg bw): Reduction of spontaneous activity, abdominal position, eyelid closure, ruffled fur, apathy, tremor, change in urine color and mortality
Third pre-experiment (500 mg/ kg bw): Reduction of spontaneous activity, ruffled fur, and change in urine color
Fourth and fifth pre-experiment (1000 and 750 mg/kg bw respectively): Reduction of spontaneous activity, abdominal position, ruffled fur, apathy, change in urine color and mortality
Details are provided in the study report.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei: 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 harvest time (24 and 48 h) and dose level after administration of the test substance.
- Ratio of PCE/NCE: The mean number of polychromatic erythrocytes was not decreased after treatment with the test substance as compared to the control
- Clinical signs: The animals treated with 500 and 250 mg/kg bw expressed toxic reactions such as reduction of spontaneous activity, ruffled fur and orange colored urine. The urine of the treated animals had taken the colour of the test substance, indicating the systemic distribution of the test substance and thus its bioavailability. No toxic reactions were observed in animals treated with 125 mg/kg bw or vehicle treated animals. Details are provided in the study report.
- Statistical evaluation: 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 harvest time or dose level of the test substance.

RESULT WITH POSITIVE CONTROL: Cyclophosphamide (40 mg/kg bw) administered orally was used as positive control which showed a significant increase of induced micronucleus frequency (percentage of cells with micronuclei was 3.31% as compared to 0.09 for vehicle control group).

Any other information on results incl. tables

Table 1: Summary of Micronucleus Test Results (study # 83851)

Test group	Dose mg/kg bw	Sampling time (h)	PCEs with micronuclei (%)	Range	PCE per 2000 erythrocytes
Vehicle	0	24	0.09	1-4	1193
test substance	125	24	0.11	1-4	1136
test substance	250	24	0.07	0-3	1207
test substance	500	24	0.07	0-3	1187
Positive control	40	24	3.31*	46 -79	1087
test substance	500	48	0.09	0-4	1122

* Statistically significant

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
Under the test conditions reported, 4-Amino-2-Hydroxytoluene (Haarpurpur) did not induce micronuclei in bone marrow polychromatic erythrocytes of the mouse.

Executive summary:

The in-vivo micronucleus test of 4-Amino-2-Hydroxytoluene (Haarpurpur) was determined following OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test).

A total of 36 NMRI male mice (source: Harlan Winkelmann), age: minimum 9-11 weeks, mean weight 35.2 g, were used in the study. The animals were housed in groups of 5 in Macrolon Type 1 cages provided with granulated soft wood bedding. The animals were maintained under standard laboratory conditions (temperature: 22 ± 3°C; humidity: 30-70% and 12 h light/12 h dark cycle per day). The animals were acclimated for a minimum of 5 days before treatment and fed on pelleted standard diet; ad libitum.

As estimated by pre-experiments (conducted to determine toxicity of test substance), 500 mg/kg bw was found to be suitable. At the next higher dose (750 mg/kg b.w.) one female died, thus implicating that the performance of the main experiment using doses above 500 mg/kg bw could greatly affect the survival rates.

During the main study, animals were randomly divided into experimental groups containing 6 males/ bone marrow harvest time as follows: 

24 h harvest time: 0, 125, 250 and 500 mg/kg bw

48 h harvest time: 500 mg/kg bw

The reference mutagen, Cyclophospamide (40 mg/kg bw) was used as a positive control.

Bone marrow was obtained from the femurs immediately following sacrifice. At least one slide was made from each bone marrow sample. 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 animals treated with 250 and 500 mg/kg bw expressed toxic reactions such as reduction of spontaneous activity, ruffled fur and orange colored urine. The urine of the treated animals had taken the colour of the test substance, indicating the systemic distribution of the test substance and thus its bioavailability. No toxic reactions were observed in animals treated with 125 mg/kg bw or vehicle treated animals.

 

The mean number of polychromatic erythrocytes was not decreased after treatment with the test item as compared to the mean value of PCEs of the vehicle control.

 

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 material. The mean values of micronuclei observed after treatment with 4-Amino-2-Hydroxytoluene were below or close to the value of the vehicle control group.

 

The positive control substance (Cyclophosphamide (40 mg/kg)), showed significantly higher percentage of cells with micronuclei (percentage of cells with micronuclei was 3.31% as compared to 0.09 for vehicle control group).

 

In conclusion, under the test conditions reported, 4-amino-2-hydroxytoluene did not induce micronuclei in bone marrow polychromatic erythrocytes of the mouse.

 

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