4-Hydroxy-3,5-dimethylbenzonitrile

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2003-08-11 to 2003-09-22

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well documented GLP-study performed according to OECD guideline 407 and the EU directive B.7. No deviations were recorded.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2004

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Details on test material:

- Name of test material (as cited in study report): T002251
- Substance type: Brown powder
- Physical state: solid
- Analytical purity: 96%
- Purity test date: no data
- Lot/batch No.: 00410155
- Expiration date of the lot/batch: 2004-05-05
- Stability of test item:stable under storage conditions
- Storage condition of test material: at room temperature in the original container away from direct sunlight.

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: HanBrl: Wist (SPF) from RCC Ltd, Laboratory Animal Services, CH-4414 Füllinsdorf / Switzerland
- Age at delivery: 6 weeks
- Weight at acclimatization: Males: 137 - 153.2 g (mean 145.0g); Females: 116.2-131.6g (mean 124.0g)
- Fasting period before study: no data
- Housing: In groups of five in Makrolon type-4 cages with wire mesh tops and standardized softwood bedding
- Diet: Ad libitum, pelleted standard Provimi Kliba 3443 rat maintenance diet
- Water: Ad libitum, community tap-water from Itingen in water bottles
- Acclimation period: at least 6 days, under test conditions after health examination. only animals without any visible signs of

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3°C
- Humidity (%): 30-70
- Air changes (per hr): 10 -15
- Photoperiod (hrs dark / hrs light): 12/ 12

IN-LIFE DATES: From: 2003-08-25 To: 2003-09-22

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Remarks:

PEG 300

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item formulations were prepared weekly. T002251 was weighed into a glass beaker on tared Mettler Balance and the vehicle added. The mixtures were prepared using a magnetic stirrer and stored at room temperature (17-23°C). Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.


VEHICLE (PEG300)
- Justification for use and choice of vehicle (if other than water): no data
- Amount of vehicle (if gavage): 5mL/kg body weight
- Lot/batch no. (if required): 422989/1 54802033
- Purity: no data

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Concentration, homogeneity and stability (after 2 hours and 7 days) of the dose formulations were determined in samples taken after experimental start. Concentration and homogeneity of the dose formulations were determined in samples taken during week 3 of the treatment. The analyses were performed by RCC Ltd (Environmental Chemistry & Pharmanalytics Division) according to a HPLC method supplied by the Sponsor.

Duration of treatment / exposure:

28 days

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

actual ingested

Doses / concentrations 2

Dose / conc.:

50 mg/kg bw/day (nominal)

Remarks:

actual ingested

Doses / concentrations 3

Dose / conc.:

200 mg/kg bw/day (nominal)

Remarks:

actual ingested

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

actual ingested

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based upon the results of a non-GLP 5-day dose-range-finding study (RCC Study Number 850088) in which T002251 was administrated by gavage to 2 rats per group and sex.

Positive control:

no

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: The animals were for clinical signs once before commencement of administration; twice daily on days 1-3, as well as once daily on daays 4-28.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: The animals were observed in their home cages, outside their home cages in a standard arena and in the hand. These observations were performed in random sequence once before commencement of administration and once weekly (weeks 1-3) thereafter.

BODY WEIGHT: Yes
- Time schedule for examinations: once during pretest weekly thereafter, using an online electronic recording system consisting of a Mettler balance connected to the RCC computer.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- The food consumption was recorded once during the pretest period and weekly thereafter, using an online electronic recording system consisting of a Mettler balance connected to the RCC computer.

FOOD EFFICIENCY: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: after 4 weeks, early in the working day to reduce biological variation caused by circadian rhythms
- Anaesthetic used for blood collection: Yes, under light isoflurane anesthesia. Blood samples were drawn from the retro-orbital plexus using a micro-hematocrit glass capillary tube.
- Animals fasted: Yes, for approximately 18 hours before blood sampling but allowed access to water ad libitum.
- How many animals: All.
The following parameters were determined: erythrocyte count, hemoglobin, hematocrit, mean corpuscular volume, red cell volume distribution width, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, hemoglobin concentration distribution width, platelet (thrombocyte) count, reticulocyte count, reticulocyte maturity index, methemoglobin, total leukocyte count, differential leukocyte count, coagulation (thromboplastin time and activated partial thromboplastin time)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: after 4 weeks, early in the working day to reduce biological variation caused by circadian rhythms.
- Anaesthetic used for blood collection: Yes, under light isoflurane anesthesia. Blood samples were drawn from the retro-orbital plexus using a micro-hematocrit glass capillary tube.
- Animals fasted: Yes, for approximately 18 hours before blood sampling but allowed access to water ad libitum.
- How many animals: All
The following parameters were determined: glucose, urea, creatinine, bilirubin total, cholesterol total, triglycerides, phospolipids, aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, glutamate dehydrogenase, creatinine kinase, alkaline phosphatase, gamma-glutamyl-transferase, sodium, potassium, chloride, calcium, phosphorus inorganic, protein total, albumin, globulin, albumin/globulin ratio.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: During week 4, relevant parameters from a modified Irwin screen test
- Dose groups that were examined: all animals
- Battery of functions tested:
- Grip strength: Forelimb and hind limb grip strength measurements were performed using a push-pull strain gauge. The animals were placed with the forepaws inside a triangular grasping ring and with the hind paws outside a triangular grasping ring. Using one hand, the animals were held towards the base of the tail and steadily pulled away or towards the ring until the grip was broken. Each measurement was repeated three times, the means were calculated and recorded
- Locomotor activity: Locomotor (decreased or increased) activity was measured quantitatively with Activity Monitor AM 1052 system. Animals were randomized and monitored during the fourth treatment week for a 60-minute period and the total activity of this time period was recorded. Low beams count was reported in 15-minute intervals as well as the total activity of the measuring period.

OTHER:
MORTALITY:
- observations for mortality/viability were recorded twice daily

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Sacrifice after 4 weeks on 22 September 2003. All animals were weighed and necropsied. Descriptions of all macroscopic abnormalities were recorded. All animals were anesthetized by intraperitoneal injection of sodium pentobarbitone and killed by exsanguination. Samples of the following tissues and organs were collected from all animals at necropsy and fixed in neutral phosphate bufferd 4% formaldehyde solution (unless otherwise indicated)
:adrenal glands, aorta, bone (sternum, femur including joint), bone marrow (femur), brain (cerebrum, cerebellum, pons), cecum, colon, duodenum, epididymides (fixed in Bouin's solution), esophagus, eyes with optic nerve (fixed in Davidson's solution), Harderian gland (fixed in Davidson's soltuion), heart, ileum with Peyer's patches, jejunum with Peyer's patches, kidneys, larynx, lacrimal gland (exorbital), liver, lungs (infused with formalin at necropsy), lymph nodes (mesenteric, mandibular), mammary gland area, nasal cavity, ovaries, pancreas, pituitary gland, prostate gland, rectum, salivary glands (mandibular, sublingual), sciatic nerve, seminal vesicles, skeletal muscle, skin, spinal cord (cervical, midthoracic, lumbar), spleen, stomach, testes (fixed Bouin's solution), thymus, thyroid (incl. parathyroid gland), tongue, trachea, urinary bladder (infused with formalin at necropsy), uterus, vagina, gross lesions.

ORGAN WEIGHT
The following organs weights were recorded on the scheduled dates of necropsy: brain, heart, liver, thymus, kidneys, adrenals, spleen, testes, epididymides and ovaries.
The organ to terminal body wieight ratios as well as organ to brain weight rations were determined. The determination of the terminal body weight was performed immedately prior to necropsy.

HISTOPATHOLOGY: Yes
Histotechnique: Following organ and tissues samples were processed and cut at an approximate thickness of 2 to 4 µm and stained with hematoxylin and eosin: adrenal glands, bone marrow (femur), brain (3 levels), cecum, colon, duodenum, epididymides (fixed in Bouin's solution), , heart, ileum with Peyer's patches, jejunum with Peyer's patches, kidneys, liver, lungs (infused with formalin at necropsy), lymph nodes (mesenteric, mandibular), ovaries, prostate gland, rectum, sciatic nerve, seminal vesicles, spinal cord (cervical, midthoracic, lumbar), spleen, stomach, testes (fixed Bouin's solution), thymus, thyroid (incl. parathyroid gland), trachea, urinary bladder (infused with formalin at necropsy), uterus, vagina, gross lesions.
Slides of all organs and tissues which were collected at scheduled sacrifice from the animals of control and high-dose groups were examined by a pathologist. As test item-related morphological changes were detected in the organs of high-dose animals, the same organs (liver and spleen) form animals of the mid- and low-dose groups were examined.

Statistics:

To analyze the grip strength, locomotor activity, body weight, organ weights and ratios, as well as:
- The Dunnett-test (many to one t-test) based on a pooled variance estimate were applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
- FIsher's exact test were applied to the macroscopic findings.
For clinical laboratory data:
- quantitative data were analyzed by one-way analysis of variance (ANOVA) when the variances are considered homogeneous according to Bartlett. Alternatively, if the variances are considered to be heterogeneous (p<=0.05), a non-parametric Kruskal-Wallis test was used. Treated groups were compared to the control groups using Dunnett's test if the ANOVA was significant at the 5% level and by Dunn's test in the case of a significant Kruskal-Wallis test (p<= 0.05)

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Soft feces was noted during daily observations in control-and test item-treated animals form treatment day 5 onwards. This clinical sign was considered to be a typical passive effect resulting from the use of PEG300 as vehicle, and is without toxicological relevance. No abnormal effects were noted during weekly behavioral observations.

Mortality:

no mortality observed

Description (incidence):

All animals survived until scheduled necorpsy.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The mean body weights of control and test item-treated rats were similar.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

The mean daily food consumption of the test item-treated rats was similar to or exceeded that of the controls.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Significantly decreased hemoglobin levels were noted in males treated with 200 mg/kg/day (p<0.05) and 1000 mg/kg/day (p<0.05), and increased red cell distriubution width was noted in males at 1000 mg/kg/day (p<0.05), when compared with the controls. The mean absolute and relative reticulocyte counts were significantly increased at 1000 mg/kg/day in males (p<0.05) and females (p<0.01) when compared with controls, and slight (but not significant) "left-shifts" in the reticulocyte maturity indices were noted. Although these changes remained within the range of the historical data, they correlated with microscopic changes seen in the spleen and were considered to be test item-related.
Females treated with 1000 mg/kg/day had significantly elevated mean platelet count (p<0.01) and significantly prolonged mean activated partial thromboplastin time (p<0.01) when compared with controls. These changes remained within the range of the historical control cata, and were considered to be incidental.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

No differences of toxicological relevance were noted at any dose level.
Males treated with 1000 mg/kg/day had marginally but significantly reduced potassium values (p<0.05), but this finding remained within the range of the historical control values and was considered to be of no toxicological relevance.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

No abnormal effects were noted during the functional observational battery, including mean gip strength and locomotor activity.
- Grip strength: No test item-related differences in mean fore- or hindlimb grip strength were noted at any dose level. The mean forelimb grip strength of males treated with 1000 mg/kg/day was significantly higher (p<0.01) than that of the control males. In the absence of a similar finding in the mean hindlimb strength of these animals or changes in the females at this dose level, this finding was considered to be incidential and of no toxicological relevance.
- Locomotor activity: No test item-related differences in mean locomotor activity were noted at any dose level. Significantly elevated (p< 0.05) mean locomotor activity was noted in:
- males treated with 50 mg/kg/day from 45-60 minutes
- females treated with 50 mg/kg/day from 30-45 minutes
- females treated with 1000 mg/kg/day from 15-30 minutes
compared with the respective controls. These transient differences were considered to be unrelated to the treatment with the test item.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Significantly elevated mean absolute and relative liver weights were noted in males and females treated with 1000 mg/kg/day (p<0.01). Significantly elevated kidney-to-body weights were noted in males treated with 1000 mg/kg/day (p<0.05) and significantly elevated mean absolute and relative spleen weights were noted in females treated with 1000 mg/kg/day when compared to the controls. Although not siginificant, a trend for slightly elevated mean absolute and relative spleen weights were noted in females treated with 200 mg/kg/day.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

At the end of the treatment period, no test item-related gross lesions were observed. The macroscopic findings recorded (reduced size of testes in one male treated with 1000 mg/kg/day) were considered to be within the range of normal background lesions, which may be seen in rats of this strain and age in subacute studies and were considered incidental, reflecting the usual individual variability.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

There were a number of findings which distinguished test item-treated animals from controls.
Liver:
- Hepatocellular hypertrophy of minimal severity and centrilobular distribution was present in four males and three femals treated with 1000 mg/kg/day. This finding was not evident in any control male or control female. This finding was considered an adaptive change of no adverse character.
- Microvesiculart fatty change of minimal and peribular distribution was observed in four males treated with 1000 mg/kg/day versus one male in the control group. This finding was considered of no adverse character. In females, no clear test item-related increase in incidence of fatty change was observed.
Spleen:
- Extramedullary hematopoiesis showed slightly increased incidence and severity in animals of both sexes treated with 1000 mg/kg/day. There were five males with average grade 1.8 versus three control males with average greade of 1.3 and five females with average grade of 1.8 versus two control females with average grade of 1.5. This finding was considered of no adverse character.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Details on results:

Analysis of dose preparations:
Concentration, homogeneity and stability (after 2 hours and 7 days) of the dose formulations were determined in samples taken after experimental start.
Concentration and homogeneity were determined in samples taken during week 3 of the treatment. The analyses were performed by RCC Ltd according to a HPLC method supplied by the Sponsor.

Effect levels

1

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

haematology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

2

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

haematology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Target system / organ toxicity

1

Critical effects observed:

yes

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

System:

hepatobiliary

Organ:

liver

spleen

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

2

Critical effects observed:

yes

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

System:

urinary

Organ:

kidney

Treatment related:

no

Dose response relationship:

yes

Relevant for humans:

not specified

Any other information on results incl. tables

Results of 5 -Day Range-Finding Oral Toxicity (Gavage) Study in the Wistar Rat:

Based on the results of this five-day dose range-finding study (RCC 850088) dose levels of 50, 200 or 1000 mg/kg/ body weight/day are proposed for the subsequent 28-day study with T002251.

analysis of dose formulations:

The chemical analysis determined concentration, homogeneity and stability of T002251 in PEG300.

- date of preparation 2003 -08 -11: The mean concentrations of the homogeneity samples taken during pretest preparations were found to be 99.9%, 106.8% and 104.5% of the nominal concentrations of dose group 2 (10 mg/ml), dose group 3 (40 mg/ml), and dose group 4 (200 mg/ml), respectively. The individual concentrations varied in the range from -1% to +1% of the mean concentrations. Therefore, the test item was found to be homogeneously distributed in the vehicle. T002251 is stable in the vehicle under storage conditions for seven days.

- date of preparation 2003 -09 -08: The mean concentrations of the homogeneity samples were found to be 97.6%, 100.9% and 98.3% of the nominal concentrations of dose group 2 (10 mg/ml), dose group 3 (40 mg/ml), and dose group 4 (200 mg/ml), respectively. The individual concentrations varied in the range from -2% to +3% of the mean concentrations. Therefore, the test item was found to be homogeneously distributed in the vehicle.

Applicant's summary and conclusion

Conclusions:

Based on the results of this study, the no-observed-effect-level (NOEL) of T002251 was considered to be 50 mg/kg body weight/day, whereas the no-adverse-effect-level (NOAEL) was considered to be 200 mg/kg body weight/day.