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

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 30 October 1992 to 10 November 1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1993

Report date:

1993

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

12 May 1981

Deviations:

no

GLP compliance:

yes

Limit test:

no

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
- Titre: 99.3% (by HClO4)
- Purity test date: 30 January 1992
- Lot/batch No.: OP 202
- Expiration date of the lot/batch: no data
- Description: light beige powder
- Trade name: IMEXINE OAG

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature according to the instructions given by the Sponsor.
- Purity control of the test substance was assessed before, on week 4 and after the study.
- The test substance preparations were made on a weekly basis by the the testing laboratory, according to the known stability

Test animals

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl CD (SD) BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River France (76410 Saint-Aubin-lès-Elbeuf, France)
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: approximately 6 weeks old
- Weight at study initiation (mean body weight): 210 g for the males and 175 g for the females
- Fasting period before study: Approximately 24 hours after treatment, overnight fasting of animals was performed for blood samples collection.
- Housing: animals were housed in suspended wire-mesh cages (43.0 x 21.5 x 18.0 cm) and each cage contained 2 rats of the same sex and group. A metallic tray was placed under each cage and contained autoclaved sawdust (Société SICSA, 94142 Alfortville, France). Bacteriological analysis and detection of possible contaminants (pesticides, heavy metals) of the sawdust are performed periodically (Laboratoire Départemental d'Analyses, 27000 Evreux, France; Laboratoire Municipal et Régional de Rouen, 76000 Rouen, France). Bottles and sawdust were changed once a week, feeders, trays, cages and racks once every 4 weeks. Cages were not randomized in the room, but placed in order vertically on the racks. Fortnightly, all the racks were moved around clockwise, rack by rack .
- Diet: ad libitum to AO4 C pelleted diet (U.A.R/, 91360 Villemoisson-sur-Orge, France) except overnight before blood samples collection and during urine collection. The batch was analysed (composition, contaminants) by the supplier.
- Water: ad libitum to bottles containing tap water filtered with a 0.22 micron (Millipore SA, 78140 Vélizy, France). Bacteriological and chemical analyses of the water and detection of possible contaminants (pesticides, heavy metals and nitrosamines) are made periodically (Laboratoire Départemental d'Analyses, 27000 Evreux, France; Laboratoire Municipal et Régional de Rouen, 76000 Rouen, France; Centre de Nutrition Humaine, 54000 Nancy, France).
- Acclimation period: 8-days

DETAILS OF FOOD AND WATER QUALITY: There were no known contaminants in the diet, water or sawdust at levels likely to have influenced the outcome of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2
- Humidity (%): 50 ± 20
- Air changes (per hr): 13 cycles of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 / 12
The temperature and relative humidity were recorded continuously and records retained. The housing conditions were checked monthly (temperature, relative humidity, light/dark cycle and ventilation). Upon their arrival at the testing laboratory, animals were housed in a protected zone. The animal room was disinfected before the arrival of the animals and cleaned regularly thereafter. Microbiological analyses of the air and the surfaces of the walls and floor of the animal room were performed before the beginning of the study.

IN-LIFE DATES: From: 08 December 1992 To: 19 March 1993

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

The test substance was administered by gavage using a glass syringe fitted with a metal probe .
Each animal was given the test substance once a day, at the same approximate daily Lime, 7 days a week over a period of 13 weeks.
The quantity of the test substance was adjusted according to the most recently recorded body weight

Vehicle:

other: hydrogel of carboxymethylcellulose at 0 .5%

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
- The test substance was suspended in the vehicle and homogenized using a magnetic stirrer .
- The test substance preparations were made on a weekly basis by the testing laboratory, according to the known stability.

VEHICLE
- A constant dose volume of 5 ml/kg/day will be used

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- A sample of the test substance was analysed, before and after the in vivo study, to assess the identity and the stability of the test substance .
- Before the beginning of the treatment period, the homogeneity of the test substance suspended in 0.5% aqueous gel of carboxymethylcellulose was check d at lowest and highest concentrations. From each preparations, samples were taken at 3 différent levels (top, middle, bottom) and analysed in duplicate.
- Before the beginning of the treatment period, the stability of suspensions at the same concentrations was checked. Each preparation was sampled the day of preparation, after 3 hours of storage at room temperature and after 4 and 7 days storage at +4°C. Each sample was analysed in duplicate.
On weeks 1, 4, 8 and 12, each preparation (control group included) was checked for achieved concentration of the test substance .

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

once a day, 7 days a week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

No. of animals per sex per dose:

10 males and 10 females per dose group including the vehicle control group.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: determined in agreement with the sponsor following the results of a previously conducted 2 week toxicity study, in which the test substance was administered at 100, 300 or 900 mg/kg/day. In this study, the 900 mg/kg/day dose level was set as the No Observable Adverse Effect Level and 1000 mg/kg/day was therefore chosen as the maximal dose that can be administered by this route .
- Rationale for animal assignment: Groups were formed in order to obtain the same approximate mean body weight and standard deviation. The stratified body weight procedure and assigment to the treatment groups were performed using a random computer selection.
- Plasma levels of the test substance: The plasma levels of the test substance will be determined during the study : 1 millilitre of blood will be taken into a tube containing lithium heparinate from the orbital sinus under light ether anaesthesia . on week 13, 2 hours alter the last administration in the first 4
animals of each sex, in each treated group . Blood will be centrifuged and plasma will be kept frozen in individual tubes at -20 °C pending possible future analysis by the testing laboratory.

Positive control:

No.

Examinations

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: clinical signs were recorded for each animal, at least once a day, at the same approximate daily time. Animals were checked at least twice a day for mortality or signs of morbidity, including weekends and Public Holidays

BODY WEIGHT: Yes
- Time schedule for examinations: Body weight was recorded for each animal, once before allocation of the animals into groups, on the first day of treatment, then once a week until the end of the study.

FOOD CONSUMPTION: Yes
- The quantity of food consumed by the animals of each cage was recorded on a weekly basis, over a 7-day period until the end of the study.
- Food intake per animal and per day was calculated using the amount of food given and left in each feeder, divided by 2.

FOOD EFFICIENCY: Yes
- Efficiency of food utilization was estimated by calculation of the food conversion ratios .
- Food conversion ratios were calculated on a weekly basis for each sex and each group, using body weight gain and food consumption means during the maximal growth period (first 13 weeks of the study).
FCR = FC/ BWG
FCR: food conversion ratio
FC: mean food consumption (g/animal/week)
BWG: mean body weight gain (g/animal/week)

OPHTHALMOSCOPIC EXAMINATION: Yes
- Ophthalmological examinations were performed on ail animais of each sex of the control and high dose level groups once before the beginning of the treatment period and on week 13.
- These examinations included visual reflexes (retinal and corneal) and the examination of the appendages, optic media and fundus by indirect ophthalmoscopy (Ail Pupil, Keeler, Windsor Berks, England). Prior to examination, the pupils of the animais were dilated using Mydriaticum® (Merck-Sharp & Dohme-Chibret, 75008 Paris, France).

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Approximately 24 hours after treatment, blood samples were taken from the orbital sinus of the overnight fasted animals under light ether anaesthesia. The samples were collected into tubes
containing the appropriate anticoagulant.
- Anaesthetic used for blood collection: Yes (light ether)
- Animals fasted: Yes
- How many animals: all surviving animals on week 13.
- Parameters checked: Leucocytes (WBC); Erythrocytes (RBC); Haemoglobin (HB); Packed Cell Volume (PCV); Mean Cell Volume (MCV); Mean Cell Haemoglobin (MCH); Mean Cell Haemoglobin Concentration (MCHC); Thrombocytes (PLAT); Differential White Cell Count with cell morphology (neutrophils (N), eosinophils (E), basophils (B), lymphocytes (L), monocytes (M)). For the last parameter, blood smears were prepared for ail sampled animals. In the absence of abnormalities, a differential white cell count was only determined in animais of the control and high dose level groups. Prothrombin Time (PT); Activated Partial Thromboplastin Time (APTT); Fibrinogen (FIB).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Approximately 24 hours after treatment, blood samples were taken from the orbital sinus of the overnight fasted animals under light ether anaesthesia. The samples were collected into tubes
containing the appropriate anticoagulant.
- Animals fasted: Yes
- How many animals: all surviving animals on week 13.
- Parameters checked: Sodium (Na+); Potassium (K+); Chloride (CT); Calcium (Ca++); Inorganic phosphorus (I.PHOS); Glucose (GLUC); Urea (UREA); Creatinine (CREAT); Total Bilirubin (TOT.BIL); Total Proteins (PROT); Albumin (ALB); Albumin/globulin ratio (A/G); Cholesterol (CHOL); Triglycérides (TRIG); Alkaline phosphatase (ALP); Aspartate aminotransferase (ASAT); Alanine aminotransferase (ALAT).

URINALYSIS: Yes
- Time schedule for collection of urine: animalswere deprived of food and placed in metabolism cages. The urine was collected in a tube containing thymol crystals during an overnight period of about 18 hours.
- Animals fasted: Yes
- How many animals: all surviving animals on week 13.
- Parameters checked: Quantitative parameters: Volume (VOLUME); pH (pH); Specific gravity (SP.GRAV)/ Semi-quantitative parameters: Proteins (PROT); Glucose (GLUC); Ketones (CETO); Bilirubin (BILL); Nitrites (NITR); Blood (BLOOD); Urobilinogen (UROB); Cytology: leucocytes (WBC); erythrocytes (RBC); cylinders (CYLIN); magnesium ammonium phosphate crystals (AMM.PH.); calcium phosphate crystals (CAL.PH); calcium oxalate crystals (CAL .OX.); cells (CELL)/ Qualitative parameters: Appearance (APP)

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

Sacrifice and pathology:

SACRIFICE: On completion of the treatment period, after about 18 hours fasting, all animals were asphyxiated by carbon dioxide and sacrificed by exsanguination. The animals were weighed before necropsy (except for the animals found dead or sacrificed prematurely) .

GROSS PATHOLOGY: Yes
- Organ weights: In all animals sacrificed at the end of the treatment period, the following organs were weighed wet as soon as possible after dissection: adrenals; heart; kidneys; liver; ovaries; spleen; testes; thymus.
Paired organs were weighed separately.
- A complete macroscopic examination was performed on all animals. All gross observations were recorded individually. Photograph was taken from the uterus of one female animal.
- Preservation of tissues: In all animals, all the macroscopic lesions and the following tissues were preserved in 10 % buffered formalin (except for the eyes and pituitary gland, which were fixed in formolsublimate for the animals sacrificed at the end ot the treatment period): adrenals; aorta*; brain including medulla/ pons, cerebellar and cerebral cortex*; caecum*; colon*; duodenum*; eyes with Harderian glands*; femoral bone with articulation*; heart; ileum*; jejunumum*; kidneys; liver; lungs with bronchi*; lymph nodes; mandibular and mesenteric*; mammary glands*; oesophagus*; ovaries; pancreas*; pituitary gland*; prostate*; rectum*; salivary glands* (sublingual and submaxillary); sciatic nerve*; seminal vesicle*; skeletal muscle*; skin*; spinal cord; cervical, thoracic and lumbar*; spleen; sternum (with bone marrow)*; stomach; testes and epididymides; thymus*; thyroids with parathyroids; tongue*; trachea*; urinary bladder*; uterus*; horns and cervix; vagina*.
Tissues marked by * were preserved in fixative for possible future microscopic examination.

HISTOPATHOLOGY: Yes
- All tissues required for microscopic examination were embedded in paraplast, sectioned at approximately 4 microns in thickness and stained with hemalum-eosin.
- Microscopic examination was performed on:
. all macroscopic lesions and tissues listed above (except those marked by *) in all animals of the high dose level and control groups sacrificed at the end of the treatment period.
. all macroscopic lesions and lungs, liver and kidneys of all the animals of the low and intermediate dose level groups.

Other examinations:

None.

Statistics:

The following sequence was used for the statistical analysis of body weight, food consumption, haematology, blood biochemistry and organ weight data:
- The normality of the distribution of the values in each group was checked by Kolmogorov-Smirnov's test (1948).
- If the distribution was normal, the homogeneity of variances between the groups was assessed by Bartlett's test (1937) (more than 2 groups) or Fisher's test (1934) (2 groups).
- If no significant heterogeneity of the variances was established, the comparison between treated and control groups was performed by Dunnett's test (1955).
- If the variances were heterogeneous, the comparison between treated and control groups was performed by Dunn's test (1964) (more than 2 groups) or by Mann Whitney's test (1947) (2 groups).
- If the distribution of values in the groups was not normal, the analysis was repeated after logarithmic transformation of the values (except for organ weights).
- If this logarithmic transformation failed to normalise the distribution of the values, comparison of treated and control groups was performed by Dunn's test using original values.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

- No cinical signs of toxicological importance were noted in the animals given 50 or 220 mg/kg/day.
- In the animals given 1000 mg/kg/day, loud breathing was noted in 5/10 males and 4/10 females, hypokinesia was noted in 2/10 females. Ptyalism was also noted in all males and females given 1000 mg/kg/day, accompanied by regurgitation in one male and one female. These findings were considered to be treatment-related .
- Findings in relation with the staining properties of the test substance were noted as follows: brown tail and/or fur in 6/10 males given 50 mg/kg/day and all males and females given 220 or 1000 mg/kg/day, yellow or orange coloured urine in all treated animals.

Mortality:

no mortality observed

Description (incidence):

No deaths were noted in any group.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- The mean body weight gain of the males and females given 50 or 220 mg/kg/day and of the males given 1000 mg/kg/day was similar to that of respective controls.
- The mean body weight gain of the females given 1000 mg/kg/day was very slightly lower than that of controls: over the 13 weeks of the treatment period, it was 145 g vs . 166 g (i.e. -13%). Although not statistically significant, this différence, which was constant from week 9 approximately, was considered to be treatment-related .

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

The mean food consumption of the treated animals of both sexes was similar to that of respective controls.

Food efficiency:

no effects observed

Description (incidence and severity):

The efficiency of food utilization, estimated by the food conversion ratios, was similar between the control and treated groups .

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No treatment-related abnormalities were noted, neither before, not at the end of the treatment period.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

On week 13, the differences noted between control and treated animals in some haematological parameters, namely, total white and red blood cell count, haemoglobin concentration, mean cell volume, mean cell haemoglobin concentration and platelet count, were considered to be of no toxicological importance, as they were minimal, not dose-related and the individual values were within the range of our background data .

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

On week 13, when the values obtained in treated animals were compared with respective controls, minor differences were noted in sodium, chloride, glucose, urea and total bilirubin levels. These differences were not considered of toxicological importance, although they were statistically significant, as they were minor, not dose-related and the individual values were within the range of our background data.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

- Proteinuria was noted with a higher incidence in males and females given 1000 mg/kg/day (see Table 1 in "Any other information on results incl. Tables").
- This was associated with a higher specific gravity and low pH values in the same animals. Although not associated with microscopic changes in the kidneys, these abnormalities were considered to be treatment-related .
- No other qualitative or quantitative abnormalities were noted in the other urine parameters.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

- Although they were statistically significant, the minor differences noted between treated and control animals in the absolute and/or relative weight of some organs, namely, heart, kidneys and liver were not considered of toxicological importance as they were minor and not dose-related.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

- Brownish colour of the body extremities and hair was noted in 6 females of the low dose group ; in 7 males and all females of the intermediate dose; all animals of both sexes of the top dose group. This was considered to be treatment-related and in relationship to the staining properties of the
test substance .
- The other macroscopic findings encountered were those which are commonly recorded in the rat of this strain and age and consequently were not considered of toxicological importance.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

- Chronic tubulo-interstitial nephropathy was noted in one female given 1000 mg/kg/day. This finding can occur spontaneously in untreated rats of this strain and age, consequently the reported weak incidence was not considered treatment-related .
- The other microscopic findings encountered were those which are commonly recorded in the rat of this strain and age. Moreover, their incidence, severity and morphological characteristics were comparatively similar in both control and treated animals and consequently were not considered to be of toxicological importance .

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Details on results:

CHEMICAL ANALYSIS OF THE PREPARATIONS
- Controls performed before and after the in vivo study assessed the identity and the stability of the test substance since the percentage of purity was the same after 13 weeks of study .
- The results of analyses revealed a good homogeneity of preparations for lowest and highest concentrations.
- The results of analyses revealed a good stability (3 hours at room temperature and 7 days at +4°C) for investigated suspensions under the conditions of preparation.
- Throughout the study, a satisfactory concordance between obtained and nominal concentrations was found.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical signs

urinalysis

Target system / organ toxicity

Critical effects observed:

no

Any other information on results incl. tables

Table 1. Incidence of proteinuria

Dose (mg/kg/day)	0		50		220		1000
	Male	Female	Male	Female	Male	Female	Male	Female
Grade 1	1	1	3	0	4	1	5	2
Grade 2	0	0	0	0	0	0	2	1
Grade 3	0	1	0	0	0	0	0	1

Applicant's summary and conclusion

Conclusions:

The test substance, when administered daily by oral route for 13 weeks to Sprague-Dawley rats at 50, 220 or 1000 mg/kg/day induced at all dose levels findings in relationship with the staining properties of the test substance. The 50 and 220 mg/kg/day dose levels did not show signs of toxicity. At 1000 mg/kg/day were noted mainly ptyalism in all animals, loud breathing, a very slightly lower (not statistically significant) body weight gain (females) and a higher incidence of proteinuria.
Under the experimental conditions of this study, the No Observable Adverse Effect Level (NOAEL) was defined as 220 mg/kg/day.

Executive summary:

This GLP-compliant study was performed to assess the potential toxicity of 2-Methyl-5-hydroxyethylaminophenol when administered daily to Sprague-Dawley rats for 13-weeks, according to OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents) (dated 12th May 1981).

Material and Methods

Groups of 20 animals (10 males and 10 females) each received daily the test substance at doses of 0 (control group receiving the vehicle alone: hydrogel of methylcellulose at 0.5%), 50, 220 or 1000 mg/kg/day by oral route (gavage) for 13 weeks.

Clinical signs and mortality were recorded daily (at least once and twice respectively). Food consumption and body weight were recorded on a weekly basis. Ophthalmological examinations were performed in the animals of the control and high dose groups, before the beginning of the treatment period and on week 13 Haematological, blood biochemical examinations and urinalysis were performed in all animals on week 13. At the end of the treatment period, all animals were sacrificed. Designated organs were weighed; the animals were examined macroscopically, and representative tissue specimens were submitted to a microscopic examination.

Results

No mortalities occurred during the study.

Loud breathing was noted in 5/10 males and 4/10 females given 1000 mg/kg/day; this was accompanied by hypokinesia in 2 females and by regurgitation in one male and one female. In the same group, ptyalism was noted in all animals.

Findings in relation with the staining properties of the test substance were noted in most treated animals: brown tail) and/or fur, yellow or orange coloured urine.

Mean food consumption of the treated animals of both sexes was similar to that of respective controls. The body weight gain of the females given 1000 mg/kg/day was very slightly lower (not statistically significant) than that of respective controls.  The efficiency of food utilization was similar between control and treated groups.

No ophthalmological abnormalities were noted.

No treatment-related findings were observed for haematology, blood biochemistry, and organ weights examinations.

For urinalysis, when compared to the controls, proteinuria was noted with higher incidence in the animals of both sexes given 1000 mg/kg/day, and this was accompanied by a higher specific gravity and lower pH values.

For macroscopic and microscopic examinations, no findings of toxicological significance were noted (except for the findings in relation with the staining properties of the test substance: brownish colour of the body extremities and hair).

Conclusion

The test substance, when administered daily by oral route for 13 weeks to Sprague-Dawley rats at 50, 220 or 1000 mg/kg/day induced at all dose levels findings in relationship with the staining properties of the test substance. The 50 and 220 mg/kg/day dose levels did not show signs of toxicity. At 1000 mg/kg/day were noted mainly ptyalism in all animals, loud breathing, a very slightly lower (not statistically significant) body weight gain (females) and a higher incidence of proteinuria.

Under the experimental conditions of this study, the No Observable Adverse Effect Level (NOAEL) was defined as 220 mg/kg/day.