5-[(2-hydroxyethyl)amino]-o-cresol

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:

From 07 December to 23 June 2005

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)

Version / remarks:

21 July 1997

Deviations:

no

GLP compliance:

yes

Type of assay:

other: in vivo micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

5-[(2-hydroxyethyl)amino]-o-cresol

EC Number:

259-583-7

EC Name:

5-[(2-hydroxyethyl)amino]-o-cresol

Cas Number:

55302-96-0

Molecular formula:

C9H13NO2

IUPAC Name:

5-[(2-hydroxyethyl)amino]-2-methylphenol

Test material form:

solid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Analytical purity: 99.3% (HPLC; Area % without response factor, UV detection)
- Purity test date: August 2004
- Lot/batch No.: 0121442
- Expiration date of the lot/batch: September 2005
- Titre: 99.8% (based upon alkalinity in g/100g)
- Description: beige powder
- Trade name: IMEXINE OAG
- Labeling code: 2948

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
Information on file with the sponsor.
- Stability and homogeneity of the formulated test article was determined in another testing laboratory study. In that study, solutions of 2-methyl-5-hydroxyethylamino-phenol were stable in 0.5% aqueous carboxymethylcellulose for 6 hours in the range of 1-200 mg/mL (room temperature) protected from light in a nitrogen atmosphere .

Test animals

Species:

rat

Strain:

other: Crl:CD (SD)BR

Details on species / strain selection:

Outbred strain that maximizes genetic heterogeneity and therefore tends to eliminate strain-specific response to test articles.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Raleigh, North Carolina
- Age at study initiation: 8 weeks old for the range finding test and 9 weeks old for the main test
- Weight at study initiation: from 234 to 273 g and 196 to 211 g for the males and females, respectively for the range finding test and rom 267 to 316 g and 198 to 230 g for the males and females,
respectively for the main test.
- Assigned to test groups randomly: yes, under following basis: using a computer program
- Housing: animals were housed in sanitary, stainless-steel, hanging, wire cages. The animals were housed, separated by gender, up to two animals per cage during acclimation and individually alter randomization.
- Diet: ad libitum to PMI Certified Rodent Diet #5002. The manufacturer analyzed the diet for nutritional components and environmental contaminants.
- Water: ad libitum to tap water, by an automatic watering system. Water samples are routinely analyzed for specified microorganisms and environmental contaminants.
No contaminants were known to bc present in the diet or water at levels which might interfère with this study.
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-26 (64-79°F)
- Humidity (%): 30-70
- Air changes (per hr): 10 or greater
- Photoperiod (hrs dark / hrs light): 12 / 12
Actual temperature and humidity readings were monitored continuously and averaged twice daily. Any variations to these conditions are maintained in the raw data and had no effect on the outcome of the study .

IN-LIFE DATES: From: 20 January 2005 (Range finding study) and 03 February 2005 (Main test) To: 11 February 2005

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: 0.5% aqueous carboxymethylcellulose saturated with nitrogen gas for at least 15 minutes.
- Justification for choice of solvent/vehicle: based on the results of the stability and homogeneity of the formulated test article study performed in another testing laboratory.

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- All mixing containers were purged with nitrogen gas prior to use . Before preparation, the vehicle was degassed by sonication for at least 15 minutes, then saturated with nitrogen gas. The vehicle was kept under nitrogen atmosphere for at Ieast 15 minutes prior to dosing and during the dosing procedure . The test article was kept under a nitrogen atmosphere during preparation .
- The top stock of the test article, 2-methyl-5-hydroxyethylamino-phenol, was ground to a fine powder using a mortar and pestle, mixed with the required quantity of vehicle, and homogenized thereafter using a magnetic stirrer. The test article was administered as a suspension in the vehiele . Lower concentrations were obtained by dilution with the vehicle . The formulations were held at room temperature, protected from light in a nitrogen atmosphere prior to dosing.
- Dosing volume: 10 ml/kg bw

DOSE RANGE FINDING ASSAY:
- The dose rangefinding assay was performed using the saine treatment regimen used in the micronucleus assay. Both males and females were dosed . Since the Sponsor had relevant acute toxicity data for the test article, the dose rangefinding assay was designed on the basis of information provided by the sponsor. The daily observations of toxic signs and/or mortality data were used to estimate the highest appropriate dose level (maximum tolerated dose) for the micronucleus assay.
- 6 animals were used (3 males and 3 females) dosed with 2000 mg/kg bw of test material (dosing volume 10mL/kg bw and stock concentration of 200 mg/mL).

MICRONUCLEUS ASSAY:
- Based on the results of the range finding assay, the following doses were used: 500, 1000 and 2000 mg/kg bw (see Table 1 in "Any other information on materials and methods incl. Tables" for animal assignment to the study groups).
- The high dose, unless non-toxic, should have produced some indication of toxicity, e.g., toxic signs, death, or depression of the ratio of PCEs to normochromatic erythrocytes (NCEs). The use of a high dose, as defined above, increased the likelihood that a weak clastogen could be detected .

BLOOD COLLECTION FOR MEASURING PLASMA LEVELS:
-A satellite group of 6 animais (three rats/sex for the high dose group) were included in the micronucleus study for determinations of plasma concentrations of the test article. Blood samples were taken from ail six satellite animais at approximately 1 and 4 hours after test article administration. Blood (approximately 2 .0 mL, when possible) was collected via jugular vein into glass tubes coated with potassium EDTA and placed on wet ice until centrifuged at approximately 4'C (within 30 minutes of the collection) . Subsequently, a 0 .1 % solution of ascorbic acid was added to the plasma (ratio 13 tL ascorbic acid solution/250 μL plasma) for stabilization. The whole blood was centrifuged to obtain one plasma sample per animal . Plasma was subsequently stored at approximately -70° C until shipment on dry ice to the Bioanalytical Principal Investigator for analysis .
- After blood collection, the satellite pharmacokinetic animals were euthanized by CO2 inhalation followed by the incision of the diaphragm and disposed of without necropsy.

CLINICAL OBSERVATIONS OF ANIMALS
- All animals were examined immediately alter dosing, approximately 1 hour after dosing, and at least daily for the duration of this assay for signs of clinical toxicity and/or mortality .

DISPOSITION OF ANIMALS
- Animals were euthanized by CO2 inhalation followed by incision of the diaphragm, and discarded without necropsy .

Frequency of treatment:

Once

Post exposure period:

24 and 48 (highest dose group only) hours after the treatment

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

500 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

2 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

5 males and 5 females per dose and for Positive and vehicle controls for the 24 hours harvest time
5 males and 5 females for the vehicle control for the 48 hours harvest time
8 males and 8 females for the highest test material dose level (2000 mg/kg bw) for the 48 hours harvest time
3 males and 3 females for the pharmacokinetic evaluation

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide
- Justification for choice of positive control(s):
- Route of administration: oral (gavage)
- Doses / concentrations: 60 mg/kg bw
- 10 animals (5 per sex)

Examinations

Tissues and cell types examined:

Bone marrow

Details of tissue and slide preparation:

Extraction of Bone Marrow
- The hind limb bones (tibias) were removed for marrow extraction from five surviving animals in each treatment and control group. For each animal, the marrow from the bone was flushed into an individuai centrifuge tube containing 3 to 5 mL fetal bovine serum (one tube per animal).

Preparation of Slides
- Following centrifugation to pellet the marrow, the supernatant was removed by aspiration and portions of the pellet were spread on slides and air-dried. The slides were fixed in methanol, stained in acridine orange, and analyzed under fluorescent micrdscopy. For control of bias, all slides were coded prior to analysis.

Slide Analysis
- Slides prepared from the bone marrow collected from five animals per group at the designated harvest timepoints, were scored for micronuclei and the PCE to NCE cell ratio .
- The micronucleus frequency (expressed as percent micronucleated tells) was determined by analyzing the number of micronucleated PCEs from at ieast 2000 PCEs per animal .
- The PCE :NCE ratio was determined by scoring the number of PCEs and NCEs observed while scoring at least 500 erythrocytes per animal .
- The criteria for the identification of micronuclei were those of Schmid (1976) .
- Micronuclei were darkly stained and generally round, although almond- and ring-shaped micronuclei occasionally occurred. Micronuclei were sharp bordered and generally between one-twentieth and one-fifth the size of the PCEs. The unit of scoring was the, micronucleated tell, not the micronucleus ; thus, the occasional tell with more than one micronucleus was counted as one micronucleated PCE, not two (or more) micronuclei .
- The staining procedure permitted the différentiation by coior of PCEs and NCEs (brightorange and ghost-like, dark green, respectively) .
- The historical background frequency of micronucleated cells was expressed as percentage micronucleated tells based on the number of PCEs analyzed. The historical background frequency of micronuclei in the rat strains at this Laboratory is about 0.0 to 0 .4%.

Evaluation criteria:

ASSAY ACCEPTANCE CRITERIA

- Acceptable Controls: The vehicle control group mean must lie within the historical control range and will usually be less than 0.4% micronucleated PCEs. There must be a statistically significant elevation of the mean of the positive control group relative to the vehicle control group, and the positive control response must be consistent with historical positive control data .
- Acceptable High Dose: Generally the high dose should reach the limit dose or produce some indication of toxicity, e.g., toxic signs and/or mortality in the test article dosed animals and/or a reduction in the PCE :NCE ratio. If there are solubility constraints, the highest dose tested will be the solubility limit or higher doses if a well-dispersed suspension is obtained that does not seule out rapidly .
- Assay Evaluation Criteria: The criteria for a positive response was the detection of a statistically signifcant increase in micronucleated PCEs for at least one dose level, and a statistically signif tant doserelated response. A test article that did not induce both of these responses was considered negative. Statistical significance was not the only determinant of a positive response; the biological relevance of the results in the final evaluation was also considered.

Statistics:

• Assay data analysis was performed using an analysis of variance (Winer, 1971) on untransformed proportions of tells with micronuclei per animal and on untransformed PCE :NCE ratios when the variances were homogeneous. Ranked proportions were used for heterogeneous variances.

• If the analysis of variance was statistically significant (p <_ 0 .05), Dunnett's t-test (Dunnett, 1955 ; 1964) was used to determine which dose groups, if any, were statistically significantly différent from the vehicle control. Analyses were performed separately for each sampling time.

For each sex, the 500, 1000, and 2000 mg/kg dose groups, as well as the positive control group, were compared with the vehicle control group at the 5%, one-tailed probability level .

Results and discussion

Test results

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Remarks:

clinical signs reported: clear oral discharge, squinted eyes, hunched posture and/or slight hypoactivity were noted for test article treated groups

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Survival and clinical observations: There were no deaths observed following single dosing at 2000 mg/kg. Clinical observations included squinted eyes and/or red eye crust.
- Based on these results, the top dose selected for the micronucleus assay was 2000 mg/kg, the limit dose for this assay based on OECD guidelines.

RESULTS OF DEFINITIVE STUDY
- Survival and clinical observations: The test article, 2-methyl-5-hydroxyethylamino-phenol, did not induce mortality at any concentration. All animals in the vehicle and positive control groups appeared normal after dosing and remained healthy until the appropriate harvest timepoint. Clear oral discharge, squinted eyes, hunched posture and/or slight hypoactivity were noted for test article treated groups .
- Induction of micronuclei (for Micronucleus assay): 2-methyl-5-hydroxyethylamino-phenol did not induce increases in micronucleated PCEs at any test article dose examined (500, 1000 and 2000 mg/kg).
- Ratio of PCE/NCE (for Micronucleus assay): 2-methyl-5-hydroxyethylamino-phenol did not induce decreases in the PCE/NCE ratios at any test article dose examined (500, 1000 and 2000 mg/kg) (see Table 2 in "Any other information on results incl. tables").
- The results of plasma analysis eonfirmed the systemic exposure of the test animals alter oral administration of the test article at 2000 mg/kg (3 .9 µg/mL maximum for males and 5.8 μg/mL maximum for females) (see Table 2 in "Any other information on results incl. tables").
- As expected, the positive control article induced a significant increase in micronucleated PCE, which demonstrated sensitivity of the assay. Indeed, the positive control, cyclophosphamide, induced a statistically significant increase in micronucleated PCEs as compared to that of the vehicle control, with a mean and standard error of 3.11 f 0.20% for the males and 2 .58 ± 0.11% for the females (see Table 2 in "Any other information on results incl. tables").
- The vehicle control group had less than the published value (0 .0 to 0 .4%, Salamone and Mavournin, 1994) of micronucleated PCEs. The vehicle control group mean in the definitive micronucleus assay was within the Laboratory 2003 historical vehicle control group mean of 0.085 ± 0 .009% and the historical vehicle control individual mean of 0.085 ± 0 .007% for males at the 24-hour bone marrow sampling time. Additionally, the vehicle control group mean in the definitive micronucleus assay was within the Laboratory 2003 historical vehicle control group mean of 0 .073 ± 0.008% and the historical vehicle control individual mean of 0 .073 ± 0 .007% for males at the 48-hour bone marrow sampling time . The vehicle control group mean in the definitive micronucleus assay was within the Laboratory 2003 historical vehicle control group mean of 0.068 ± 0 .0014% and the historical vehicle control individual mean of 0.068 ± 0.011% for females at the 24-hour bone marrow sampling time. Additionally, the vehicle control group mean in the definitive micronucleus assay was similar to the Laboratory 2003 historical vehicle control group mean of 0.040 ± 0 .010% and the historical vehicle control individual mean of 0.040 ± 0 .000% for females at the 48-hour bone marrow sampling time. However, this mean is based only on 5 animals (1 trial). (see Table 2 in "Any other information on results incl. tables").

Any other information on results incl. tables

Table 2. Micronucleus Assay- Summary table of results

Treatment	Dose	Harvest time	% Micronucleated PCEs (mean of 2000 per animal ± S.E.)		Ratio PCE : NCE (mean ± S.E.)
			Males	Females	Males	Females
Vehicle Control	0.5% CMC 10mL/kg	24 hours	0.05 ± 0.02	0.09 ± 0.03	0.99 ± 0.05	0.95 ± 0.02
		48 hours	0.08 ± 0.01	0.06 ± 0.02	0.96 ± 0.02	1.01 ± 0.03
Positive Control	CP 60 mg	24 hours	3.11 ± 0.20*	2.58 ± 0.11 *	0.77 ± 0.07**	0.90 ± 0.02
Test material	500 mg/kg	24 hours	0.04 ± 0 .02	0.04 ± 0.01	0.98 ± 0.03	1.01 ± 0.02
	1000 mg/kg	24 hours	0.04 ± 0.02	0.09 ± 0.02	0.98 ± 0.01	0.97 ± 0.01
	2000 mg/kg	24 hours	0.04 ± 0.02	0.05 ± 0.02	0.97 ± 0.02	0.97 ± 0.01
		48 hours	0.09 ± 0.03	0.06 ± 0.02	0.95  ± 0.02	1.02 ± 0.03

* Significantly greater than the corresponding vehicle control, p<= 0 .01 .

** Significantly less than the corresponding vehicle control, p<= 0 .05 .

0.5% CMC = 0.5% aqueous carboxymethylcellulose

CP = Cyclophosphamide

PCE = Polychromatic erythrocyte

NCE=Normochromatic erythrocyte

Applicant's summary and conclusion

Conclusions:

Under the experimental conditions of this study, the test article, 2-methyl-5-hydroxyethylamino-phenol, was evaluated as negative in the rat bone marrow micronucleus assay (no induction of chromosome aberrations or damage to the mitotic apparatus) when tested up to the testing limit of 2000 mg/kg.

Executive summary:

This GLP-compliant study was performed to assess the potential of 2-Methyl-5-hydroxyethylaminophenol to induce in vivo clastogenic activity and/or disruption of the mitotic apparatus by detecting micronuclei in polychromatic erythrocytes (PCE) in Crl :CD (SD)BR rat bone marrow, according to OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test) (dated 21st July 1997).

Material and methods

Based on the results of the dose rangefinding assay (no toxic effects), the top dose selected for the micronucleus assay was 2000 mg/kg, which is the limit dose for this assay based on OECD guidelines. In the main micronucleus assay, the test article was formulated in 0.5% aqueous carboxymethylcellulose and administered by oral gavage to male and female rats once as follows:

- 5 males and 5 females per test material dose (500, 1000 and 2000 mg/kg bw) and for Positive and vehicle controls for the 24 hours harvest time

- 5 males and 5 females for the vehicle control for the 48 hours harvest time

- 8 males and 8 females for the highest test material dose level (2000 mg/kg bw) for the 48 hours harvest time

- 3 males and 3 females for the pharmacokinetic evaluation

Bone marrow was extracted and at least 2000 PCEs per animal were analyzed for the frequency of micronuclei. Cytotoxicity was assessed by scoring the number of PCEs and normochromatic erythrocytes (NCEs) in at least the first 500 total erythrocytes for each animal.

Results

The test article, 2-methyl-5-hydroxyethylamino-phenol, did not induce mortality at any dose level. Clinical observations included squinted eyes, slight hypoactivity, hunched posture and/or clear oral discharge. 2-methyl-5-hydroxyethylamino-phenol did not induce increases in micronucleated PCEs or decreases in the PCE/NCE ratios at any test article dose examined (500, 1000 and 2000 mg/kg). The results of plasma analysis confirmed the systemic exposure of the test animals after oral administration of the test article at 2000 mg/kg.

As expected, the positive control article did induce a significant increase in micronucleated PCE, which demonstrated sensitivity of the assay. All vehicle control groups had mean values for micronucleated PCEs which were within the available testing laboratory historical control ranges or were similar to the historical control mean.

Conclusion

Under the experimental conditions of this study, the test article, 2-methyl-5-hydroxyethylamino-phenol, was evaluated as negative in the rat bone marrow micronucleus assay (no induction of chromosome aberrations or damage to the mitotic apparatus) when tested up to the testing limit of 2000 mg/kg.