4-amino-m-cresol

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / micronucleus study

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From Sept. 15, 2004 to Jan. 20, 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study well documented, followed methods comparable to current OECD guideline, GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test

Deviations:

no

GLP compliance:

yes

Remarks:

(according to United Kingdom and OECD principles of GLP)

Type of assay:

in vitro mammalian cell micronucleus test

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: crystalline

Details on test material:

- Name of test material: 4-amino-3-methylphenol (Code # A000118)
- TSIN: 23533
- Substance type: Pure active substance
- Physical state: Beige brown crystalline powder
- Stability under test conditions: More than 5 years on storage in dryness and darkness
- Stability in solution: The test solution in DMSO (10%, w/v) confirmed a very good stability, and confirmed degradation in acetone/water 1:1 (10% w/v) and phosphate buffer (0.3% w/v) when tested over a period of 7 days.
- Storage condition of test material: Room temperature in dark
- Solubility: Solubility in different solvents is as follows:
2 weight % in acetonitrile,
0.6 weight% in water,
4 weight % in acetone/water 1:1,
10 weight% in DMSO,
5.5 weight% in ethanol

Method

Species / strain

Species / strain / cell type:

lymphocytes: obtained from blood drawn from the peripheral circulation of two healthy, non-smoking human female volunteers

Details on mammalian cell type (if applicable):

- Type and identity of media: Hepes-buffered RPMI medium
- Properly maintained: Yes, blood was stored refrigerated and pooled prior to use.
- Any known disease in donors: No volunteer was suspected of any virus infection or exposed to high levels of radiation or hazardous chemicals.
- Others: The measured cell cycle time of the donors used falls within the range 13±1.5 hours.

Metabolic activation:

with and without

Metabolic activation system:

Sprague Dawley rat liver induced with Aroclor 1254

Test concentrations with justification for top dose:

Experiment 1(24 hour pretreatment with mitogen (phytohemagglutinin; PHA)):
Without S9 (24 hours treatment + 28 hours recovery): 0, 5, 10, 15, 20, 22.5, 25, 27.50 30, 32.5, 40, and 45 µg/mL
With S9 (3 hours treatment + 45 hours recovery): 0, 50, 100, 125, 150, 175, 200, 225, 250, 275, 300, 350, and 400 µg/mL

Experiment 2 (48 hour pretreatment with mitogen (phytohemagglutinin; PHA)):
Without S9 (24 hours treatment + 28 hours recovery): 0, 5, 10, 20, 30, 35, 40, 45, 50, 55, 60, 65, 70 and 80 µg/mL
With metabolic activation (3 hours treatment + 45 hours recovery): 0, 25, 50, 100, 125, 150, 175, 200, 225, 250, 275, 300 350, and 400 µg/mL

Based on the results of the cytotoxicity assay (concurrent assay), following test doses were selected for micronucleus analysis:

Experiment 1
Without S9 (24 hours treatment + 28 hours recovery): 0, 5, 22.5 and 30 µg/mL
With S9 (3 hours treatment + 45 hours recovery): 0, 50, 100, 125 and 150 µg/mL

Experiment 2
Without S9 (24 hours treatment + 28 hours recovery): 0, 10, 20, 30 and 35 µg/mL
With S9 (3 hours treatment + 45 hours recovery): 0, 50, 100 and 125 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Sterile anhydrous analytical grade dimethyl sulphoxide (DMSO)
- Justification for choice of solvent/vehicle: Preliminary solubility data indicated that The test substance was found to be soluble in DMSO

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

(sterile DMSO was designated as negative control)

Positive controls:

yes

Positive control substance:

other: cyclophosphamide, 4-nitroquinoline-1-oxide, and Vinblastin

Remarks:

Experiment 1: 4-nitroquinoline-1-oxide (2.50 µg/mL), cyclophosphamide (6.25 µg/mL), and Vinblastin (0.08 µg/mL); Experiment 2: 4-nitroquinoline-1-oxide (5.00 µg/mL), cyclophosphamide (12.5 µg/mL), and Vinblastin (0.06 µg/mL).

Details on test system and experimental conditions:

METHOD OF APPLICATION: In medium (HEPES-buffered RPMI medium containing 20% (v/v) foetal calf serum and 50 µg/mL gentamycin)

PREPARATION OF TEST SOLUTION: Test material stock solutions were prepared by dissolving the test material in DMSO, with the aid of vortex mixing, to give the top concentrations. The stock solutions were membrane filter-sterilised and subsequent dilutions made using sterile DMSO. The test solutions were protected from light and used within 4 hours of initial formulation.

TEST TREATMENT VOLUME: 0.1 mL per culture.

DURATION
- Treatment with mitogen: Two independent experiments (both experiments with and without metabolic activation) were performed. Prior to test material treatment, cells were exposed to mitogen (phytohemagglutinin (PHA)) for the following durations:
Experiment 1: Approximately 24 hours
Experiment 2: Approximately 48 hours
- Exposure duration: The test material was added to the culture after 24 and 48 hours after culture initiation in Experiment 1 and Experiment 2 respectively. In both the experiments (i.e. experiment 1 and 2), test material exposure duration in the in the absence or presence of S-9 mix was as follows:
In the absence of S9: 20 hours
In the presence of S9: 3 hours
- Recovery period: Test material treatment, in the presence or absence of S9, was followed by recovery period prior to harvest. In both the experiments, the recovery period was as follows:
In the absence of S9: 28 hours
In the presence of S9: 45 hours
- Harvesting time: Experiment 1: 72 hours; Experiment 2: 96 hours
The final 28 hours (approximately) of incubation was in the presence of Cytochalasin B (at a final concentration of 6 µg/mL) in both the experiments.
- Test temperature: Cultures were incubated at 37± 1°C for the designated exposure time.

NUMBER OF REPLICATIONS: Quadruplicate cultures were treated with the vehicle and duplicate cultures were treated with the test material. Additional duplicate cultures were treated with the positive control chemicals.

REMOVAL OF TEST MATERIAL: The test material was removed at following time intervals:
Experiment 1
(i) 20 hours treatment: After 44 hours of culture initiation
(ii) 3 hours treatment: After 27 hours of culture initiation

Experiment 2
(i) 20 hours treatment: After 68 hours of culture initiation
(ii) 3 hours treatment: After 51 hours of culture initiation
For removal of the test chemical, cells were pelleted (approx. 300 x 'g', 10 min), washed twice with pre-warmed, sterile saline, and resuspended in fresh medium (pre-warmed to approximately 37°C) containing foetal calf serum and gentamycin. At the appropriate times, Cytochalasin B, formulated in dimethyl sulphoxide, was either added directly (0.1 mL/culture) or added to post wash-off culture medium to give a final concentration of 6 µg/mL per culture.

HARVESTING METHOD: At the defined sampling time, cultures were centrifuged at approximately 300 x 'g' for 10 min, the supernatant removed and discarded and cells resuspended in 4 mL (hypotonic) 0.075 M KCl at 37°C for 4 min to allow cell swelling to occur. Cells were then fixed by dropping the KCl suspension into fresh, cold methanol/glacial acetic acid (3: 1, v/v). The fixative was changed by centrifugation (approximately 300 x 'g', 10 min) and resuspension. This procedure was repeated several times (centrifuging at approximately 1250 x 'g', 2-3 min) until the cell pellets were clean.

SLIDE PREPARATION: Lymphocytes were kept in fixative in the refrigerator before slides were prepared but slides were not made on the day of harvest to ensure cells were adequately fixed. Cells were pelleted and resuspended in a minimal amount of fresh fixative to give a milky suspension. Several drops of suspension were gently spread onto multiple clean, dry microscope slides.

STAIN: After the slides had dried the cells were stained for 5 minutes in filtered 4% (v/v) Giemsa stain in Gurr’s pH 6.8 buffer. The slides were rinsed, dried and mounted with coverslips.

RATIONALE FOR DOSE SELECTION FOR MICRONUCLEUS ASSAY: Slides were examined, uncoded, for proportions of mononucleate, binucleate and multinucleate cells and the replication index (RI) calculated based on the analysis of 500 cells per replicate (1000 per dose). Analysis of slides from highly cytotoxic concentrations was avoided. Slides from the highest selected concentration or the one at which at least 60% (approximately) reduction in RI occurred and two lower concentrations were taken for microscopic analysis, such that a range of cytotoxicity from maximum to little was covered. Further, For each treatment regime, two vehicle control cultures were analysed for micronuclei.

NUMBER OF CELLS EVALUATED: One thousand binucleate cells from each culture (2000 per concentration) were analysed for micronuclei.

SLIDE ANALYSIS FOR MICRONUCLEI: Slides from the selected treatments and from solvent and and positive controls were coded using randomly generated letters. The number of cells containing micronuclei and the number of micronuclei per cell on each slide was noted. Slide analysis was performed by competent analysts trained in the applicable Covance Laboratories Harrogate (CLEH) standard operating procedures.

DETERMINATION OF CYTOTOXICITY: Cytotoxicity (%) was expressed as (100 - Relative Rl).
The Replication Index (RI), which indicates the relative number of nuclei comparedto controls was determined using the formulae below:
RI = [number binucleate cells + 2(number multinucleate cells)]/Total number of cells in treated cultures
Relative Rl (expressed in terms of percentage) for each treated culture was calculatedas follows:
Relative RI (%) = [RI of treated cultures/RI of vehicle controls] x 100
A selection of random fields was observed from enough treatments to determine whether chemically induced cell cycle delay or cytotoxicity has occurred.

Evaluation criteria:

A test substance was considered as clearly positive in the assay if:
- A statistically significant increase in the proportion of cells with micronuclei occurred at one or more concentrations.
- The incidence of micronucleated cells at such data points exceeded the normal range.

Statistics:

After completion of scoring and decoding of slides, the numbers of binucleate cells with micronuclei in each culture were obtained:
- The proportions of micronucleated cells in each replicate were used to establish acceptable homogeneity between replicates by means of a binomial dispersion test.
- The proportion of cells with micronuclei for each treatment condition was compared to the proportion in solvent controls by using Fischer’s exact test.
- Probability values of p< or = 0.05 were accepted as significant.
- Additionally, the number of micronuclei per binucleate cell were obtained and recorded.

Results and discussion

Test results

Test results 1

Species / strain:

lymphocytes: obtained peripheral blood of circulation human female volunteers

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

(in Experiment 1 (24 hour pretreatment with PHA): 20 + 28 hours treatment)

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(24, 35 and 60% at 5, 22.5 and 30 µg/mL)

Vehicle controls validity:

other: yes (the negative control culture in Experiment 1 (20+28 hours, S-9 absent) was not within the historical control range but fell close to historical control, this observation was assumed to be a chance event)

Positive controls validity:

valid

Test results 2

Species / strain:

lymphocytes: obtained peripheral blood of circulation human female volunteers

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

(in Experiment 2 (48 hour pretreatment with PHA): 20 + 28 hours treatment)

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(15, 48 and 48% at 20, 30 and 35 µg/mL)

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

lymphocytes: obtained peripheral blood of circulation human female volunteers

Metabolic activation:

with

Genotoxicity:

ambiguous

Remarks:

(Frequencies of micronuclei binucleate cells (MNBN) were significantly higher than the concurrent vehicle control cultures in Experiment 1: 3 + 45 hours treatment. However, the overall mean MNBN frequency fell within historical control range.)

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(6, 31, 50 and 67% at 50, 100, 125 and 150 µg/mL)

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

lymphocytes: obtained peripheral blood of circulation human female volunteers

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

(in Experiment 2 (48 hour pretreatment with PHA): 3 + 45 hours treatment)

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(24, 41 and 63% at 50, 100 and 125 µg/mL)

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH and osmolality: Post-treatment media were measured in the absence and presence of S-9 (final concentration of 1232 µg/mL for osmolality and pH, and also 615.8 and 307.9 µg/mL for measurement of pH). These measurements indicated that the test substance had no marked effect on osmolality (greater than a shift of 50 mOsm/kg) or pH (shift of greater than 1 pH unit) as compared to concurrent vehicle controls.
- Precipitation: A 100 fold dilution of test substance at a final concentration of 1232 µg/mL did not result in visible precipitation.

RANGE-FINDING/SCREENING STUDIES: Concentrations for the main experiment were selected based on the cytotoxicity range-finding experiment. Duplicate cultures were treated with the vehicle, and single cultures were treated with the test material at appropriate concentrations (0.1 mL per culture). Positive control treatments were not included. Test material concentration were as follows:

Experiment 1 (24 h PHA prior to treatment)
With (3+45 hour treatment) and without S9 (20+28 hour treatment) mix: 0, 4.812, 9.625, 19.25, 38.5, 77, 154, 308, 615 and 1232 µg/mL.

Experiment 2 (48 h PHA prior to treatment)
With (3+45 hour treatment) and without S9 (20+28 hour treatment) mix: 0, 4.812, 9.625, 19.25, 38.5, 77, 154, 308, 615 and 1232 µg/mL.

Slides (from the cytotoxicity range-finding experiment) were initially examined, uncoded, for proportions of mono-, bi- and multinucleate cells, to a minimum of 200 cells per concentration. From these data the replication index (RI) was determined and a suitable range of concentrations was selected for the main experiment based on these toxicity data. Micronucleus analysis was not conducted on slides generated from the rangefinder treatments.
Based on the results of cytotoxicity range-finding experiment, 400 µg/mL was determined as the highest dose level for the main experiment.

COMPARISON WITH HISTORICAL CONTROL DATA: Yes, concurrent vehicle control values were compared to the historical vehicle control ranges for human lymphocyte micronucleus assay (Dec. 2003 to Oct. 2004).

ADDITIONAL INFORMATION ON CYTOTOXICITY: During the main study, the replication index (RI) was determined. Cytotoxicity (%) was expressed as (100 - Relative Rl). Based on the above, the test material dose levels for micronucleus analysis were selected by evaluating the effect of test material on the replication index. Micronuclei were analysed at three or four dose levels.

Experiment 1: The highest concentrations chosen for analysis, 30.00 µg/mL in the absence of S-9 and 150.0 µg/mL in the presence of S-9, induced approximately 60% and 67% reduction in RI respectively.

Experiment 2: The highest concentrations chosen for analysis, 35.00 µg/mL in the absence of S-9 and 125.0 µg/mL in the presence of S-9, induced approximately 48% and 63% reduction in RI respectively.

MAIN STUDY RESULT :
(i) Experiment 1 (24 hour PHA:
Without metabolic activation: Treatment of cells with test material in the absence of metabolic activation (20+28 hour treatment) resulted in frequencies of micronucleated binucleate (MNBN) cells that were not significantly different from (P≤0.05) concurrent vehicle control cultures for all concentrations analysed. The MNBN cell frequencies of all cultures exposed to the test material fell within the historical vehicle control range.
With metabolic activation: Treatment of cells with the test material (3+45 hour treatment) resulted in frequencies of MNBN cells that were significantly higher (P≤0.05) than the concurrent vehicle control cultures, for the three highest concentrations. Although two single cultures, exposed to 100 µg/mL and 150 µg/mL of test material exceeded the historical vehicle control range, the overall mean MNBN frequency fell within the historical control range on both occasions. These increases are small and, taken in isolation, were of questionable biological relevance.

(ii) Experiment 2 (48 hour PHA treatment):
Without metabolic activation: The frequencies of MNBN cells in the solvent control cultures were relatively low, and the frequencies of MNBN cells in cultures treated with the test material exceeded the concurrent negative control frequencies. Single cultures exposed to 10, 20 and 30 µg/mL and both cultures exposed to 35 µg/mL of test material exceeded the historical control range. Most of the increases were small, the mean frequencies of MNBN cells after exposure to 10, 20 and 30 µg/mL being only slightly higher than the historical vehicle control range. These data met the criteria for a positive control. There was heterogeneity between cultures, but this was considered to be attributable to the effects of test material, as there was good concordance between duplicate negative or positive control cultures.

With metabolic activation: The frequencies of MNBN cells in the solvent control cultures were relatively low and the cultures treated with the test material showed frequencies MNBN cells that significantly exceeded these values. One culture exposed to 100 and both cultures exposed to 125 µg/mL of test material exceeded the historical vehicle control range. There was a concentration related effect. These data fulfilled the criteria for a positive response.

RESULTS OF CONTROL GROUPS:
(i) Vehicle control:
Appropriate negative (solvent) control cultures were included in the test system under each treatment condition. The proportion of micronucleated binucleate cells (MNBN) in all but one of these cultures fell within historical solvent control ranges. The single exception was one negative control culture in Experiment 1 (20+28h, S-9 absent) which fell close to this range, and this is assumed to be a chance event. The inclusion or exclusion of this culture does not affect the outcome of the statistical analysis or the interpretation of the data in any way.

(ii) Positive control:
4-Nitroquinoline I-oxide (NQO) and Vinblastine (VIN) were employed as clastogenic and aneugenic positive control chemicals respectively in the absence of liver S-9. Cyclophosphamide (CPA) was employed as a clastogenic positive control chemical in the presence of liver S-9. Cells receiving positive control induced statistically significant increases in the proportion of cells with micronuclei. The experiments reported were considered to be valid.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Results of Experiment 1 (24 hour PHA) (study # 70145)

Treatment period+ recovery period	Concentration (µg/mL)	Cytotoxicity (%)	Mean MNBN cell frequency (%)	Historical Control range (%)	Statistical significance
20+28 hours -S9	Vehicle**	-	1.15	0.3-1.7	-
	5.0	24	1.30		NS
	22.5	35	1.30		NS
	30.0	60	1.20		NS
	*Vinblastine, 0.08	ND	5.30		P ≤ 0.001
	*NQO,2.50	ND	3.10		P ≤ 0.001
3+45 hours +S9	Vehicle**	-	0.70	0.3-1.7	-
	50	6	1.10		NS
	100	31	1.55		P ≤ 0.01
	125	50	1.45		P ≤ 0.05
	150	67	1.65		P ≤ 0.01
	*CPA,6.25	ND	18.80		P ≤ 0.001

 

Table 2: Results of Experiment 2 (48 hour PHA) (study # 70145) 

Treatment period+ recovery period	Concentration (µg/mL)	Cytotoxicity (%)	Mean MNBN cell frequency (%)	Historical Control range (%)	Statistical significance
20+28 hours -S9	Vehicle**	-	0.30	0.0-1.1	-
	10	0	1.25		P ≤ 0.001
	20	15	1.20		P ≤ 0.001
	30	48	1.15		P ≤ 0.001
	35	48	1.85		P ≤ 0.001
	*Vinblastine, 0.06	ND	4.25		P ≤ 0.001
	*NQO,5.0	ND	4.55		P ≤ 0.001
3+45 hours +S9	Vehicle**	-	0.10	0.0-1.0	-
	50	24	0.95		P ≤ 0.001
	100	41	1.55		P ≤ 0.001
	125	63	2.15		P ≤ 0.001
	*CPA,12.5	ND	12.05		P ≤ 0.001

* Positive control

** Vehicle control was DMSO only

ND = Not determined

NS = Not significant

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative without metabolic activation (in Experiment 1 (24 hour pretreatment with PHA): 20 hours treatment period + 28 hours recovery period)
positive without metabolic activation (in Experiment 2 (48 hour pretreatment with PHA): 20 hours treatment period + 28 hours recovery period)
ambiguous with metabolic activation (Frequencies of micronuclei binucleate cells (MNBN) were significantly higher than the concurrent vehicle control cultures in Experiment 1:. However, the overall mean MNBN frequency fell within historical control range.)
positive with metabolic activation (in Experiment 2 (48 hour pretreatment with PHA): 3 hours treatment period + 45 hours recovery period)

4-amino-3-methylphenol (Oxyrot) induced micronuclei in cultured human peripheral blood lymphocytes following stimulation with mitogen (Phytohaemagglutinin [PHA]) (commenced 48 hours prior to treatment with Oxyrot) in the absence and presence of S9.

Ambiguous results were obtained following treatment of cultures with Oxyrot in the presence of S9 when mitogen (PHA) stimulation commenced 24 hours prior to treatment.

Negative results were obtained following treatment of cultures with Oxyrot in the absence of S9 when mitogen (PHA) stimulation commenced 24 hours prior to treatment.

Executive summary:

The in-vitro micronucleus test of 4-amino-3-methylphenol (Oxyrot), following methods comparable to the OECD guideline 487 (In vitromicronucleus test) was conducted using human lymphocyte cultures.

The purpose of this in vitro chromosome aberration test was to evaluate the clastogenic and aneugenic potential of the test substance by examining its ability to induce micronuclei in the lymphocytes of human donors, cultured in vitro and treated in the absence and presence of a rat liver metabolizing system (S-9).

The test substance was tested in an in vitro micronucleus assay using duplicate human lymphocyte cultures prepared from the pooled blood of two female donors Two independent experiments were performed in this study. The test substance was dissolved in sterile anhydrous analytical grade dimethyl sulphoxide (DMSO) and the highest dose level used in the main experiment, 400.0 µg/mL, was determined following a preliminary cytotoxicity range-finding experiment.

The test concentrations used in the main study were as follows:

Experiment 1 (24 hour pretreatment with mitogen (phytohemagglutinin; PHA)):

Without S9 (20 hours treatment + 28 hours recovery): 0, 5, 10, 15, 20, 22.5, 25, 27.50 30, 32.5, 40, and 45 µg/mL

With S9 (3 hours treatment + 45 hours recovery): 0, 50, 100, 125, 150, 175, 200, 225, 250, 275, 300, 350, and 400 µg/mL

Experiment 2 (48 hour pretreatment with mitogen (phytohemagglutinin; PHA)):

Without S9 (20 hours treatment + 28 hours recovery): 0, 5, 10, 20, 30, 35, 40, 45, 50, 55, 60, 65, 70 and 80 µg/mL

With metabolic activation (3 hours treatment + 45 hours recovery): 0, 25, 50, 100, 125, 150, 175, 200, 225, 250, 275, 300 350, and 400 µg/mL

Based on the effect ofthe test substance on the replication index (RI) during the main study, the following test doses were selected for micronucleus analysis:

Experiment 1

Without S9 (20 hours treatment + 28 hours recovery): 0, 5, 22.5 and 30 µg/mL

With S9 (3 hours treatment + 45 hours recovery): 0, 50, 100, 125 and 150 µg/mL

Experiment 2

Without S9 (20 hours treatment + 28 hours recovery): 0, 10, 20, 30 and 35 µg/mL

With S9 (3 hours treatment + 45 hours recovery): 0, 50, 100 and 125 µg/mL

4-Nitroquinoline I-oxide (NQO), Cyclophosphamide (CPA) and Vinblastine (VIN) were employed as positive controls.Quadruplicate cultures were treated with the vehicle (DMSO) and duplicate cultures were treated with the test material. Additional duplicate cultures were treated with the positive control chemicals.

The proportion of micronucleated binucleate cells (MNBN) in all vehicle controls except one fell within the historical solvent control ranges. The single exception was one negative control culture in Experiment 1 (20+28 h, S-9 absent) which fell close to this range, however this was assumed to be a chance event. The inclusion or exclusion of this culture did not affect the outcome of the statistical analysis or the interpretation of the data in any way. All positive control substances induced statistically significant increases in the proportion of cells with micronuclei.

During Experiment 1 (24 hour pretreatment with PHA):

With metabolic activation:Ambiguous results were obtained following treatment of cultures in the presence of S9. Statistically higher frequencies ofmicronuclei binucleate (MNBN) were observed for the 3 highest concentrations in treatment cultures, when compared with concurrent vehicle control. The single cultures exposed to 100 and 150 µg/mL of test material exceeded the historical vehicle control range. However, the overall mean micronuclei binucleate (MNBN) frequency fell within the historical control range.These increases were small and, taken in isolation, were of questionable biological relevance.

Without metabolic activation:Negative results were obtained following treatments in the absence of S-9 following 24 hours pre-treatment with PHA. The frequencies of MNBN cells in the cultures treated with the test material were not significantly different from the concurrent vehicle control cultures for all concentrations analysed. The MNBN cell frequencies of all cultures exposed to the test material fell within the historical vehicle control range.

During Experiment 2 (48 hour pretreatment with PHA):

With and without metabolic activation: The test material induced micronuclei in cultured human peripheral blood lymphocytes following treatment in the absence and presence of S9. The frequencies of MNBN cells in the solvent control cultures were relatively low (0.30%) and the cultures treated with the test material showed frequencies of MNBN cells that significantly exceeded the vehicle control values.

Based on above, 4-amino-3-methylphenol (Oxyrot) induced micronuclei in cultured human peripheral blood lymphocytes following stimulation with mitogen (Phytohaemagglutinin [PHA]) (commenced 48 hours prior to treatment with Oxyrot) in the absence and presence of S9.

Ambiguous results were obtained following treatment of cultures with Oxyrot in the presence of S9 when mitogen (PHA) stimulation commenced 24 hours prior to treatment.

Negative results were obtained following treatment of cultures with Oxyrot in the absence of S9 when mitogen (PHA) stimulation commenced 24 hours prior to treatment.

This mammalian gene mutation test is classified as acceptable, and satisfies the guideline requirements of the OECD 487 method.