Registration Dossier

Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30 April 1997 - 25 May 1998
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Remarks:
Deviations to the duration of the study and the number of animals per group.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1998
Report Date:
1998

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
yes
Remarks:
Ten-week study / 5 animals per sex per dose
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
- Identification: Amitrole (or Aminotriazole)
- Appearance: Whitish powder
- Storage conditions: At room temperature, away from light
Specific details on test material used for the study:
Details reported in test material information

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
The rat was chosen because it is a rodent species generally accepted by regulatory authorities for this type of study.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River France (Saint Aubin-les-Elbeuf, France)
- Age at study initiation: Approximately 6 weeks old
- Weight at study initiation: A mean body weight of 207 g (range: 191 - 220 g) for the males and 176 g (range: 155 - I 97 g) for the females
- Fasting period before study: No
- Housing: Suspended wire-mesh cages (43.0 x 21.5 x 18.0 cm). A metallic tray, containing autoclaved sawdust (SICSA, Alfortville, France), was placed under each cage.
- Diet (e.g. ad libitum): free access to A04 C pelleted maintenance diet (U.A.R., Villemoisson-sur-Orge, France) distributed weekly (during periods of fasting, food, but not water, was removed)
- Water (e.g. ad libitum): free access to bottles containing tap water, filtered using a 0.22 µm filter
- Acclimation period: 8 days

DETAILS OF FOOD AND WATER QUALITY: Bacteriological and chemical analyses including the detection of possible contaminants (pesticides, heavy metals and nitrosamines) of diet and water are performed regularly by external laboratories. No contaminants were known to have been present in the diet, drinking water or bedding material at levels which may be expected to have interfered with or prejudiced the outcome of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21+/-2°C
- Humidity (%): 50+/-20%
- Air changes (per hr): Approximately 12 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12h/12h (07:00 - 19:00)

IN-LIFE DATES: From: 29 April 1997 To: 18 July 1997

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
The oral route was used since it is a possible route of exposure in man.

Gavage using a glass syringe fitted with a metal gavage tube.
The test substance was administered by gavage, in order to compare the toxicity of the test substance in rodents to that in primates. With monkeys, studies by dietary admixture are not possible owing to the behaviour of the animals who play with the food.
Vehicle:
water
Remarks:
Purified water obtained by reserve osmosis using Milli-Ro plus apparatus (Millipore S.A., St-Quentin en Yvelines, France)
Details on oral exposure:
- PREPARATION OF DOSING SOLUTIONS:
The test substance was administered as a solution in the vehicle.
The test substance was dissolved in the required quantity of vehicle in order to achieve the concentrations of 0.3, 2.4, 20 and 66 mg/ml and then homogenised using a magnetic stirrer.
The test substance preparations were made for up to nine days according to the stability data provided by the Sponsor and were stored at +4 °C, away from light, pending utilisation.

The reference substance was administered as a suspension in the vehicle.
The reference substance was ground to fine powder using a mortar and pestle, suspended in the vehicle in order to achieve the concentrations of 6 or 12 mg/ml and then homogenised using a magnetic stirrer.
The reference substance preparation was made daily.
The preparations were delivered each day to the animal room, protected from light.

- VEHICLE
For the test substance: The vehicle was purified water obtained by reserve osmosis using Milli-Ro plus apparatus (Millipore S.A., St-Quentin en Yvelines, France).
For the reference substance: The vehicle was 0.5% aqueous methylcellulose (w/w).
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
Ten weeks
Frequency of treatment:
Once daily, seven days a week
Doses / concentrationsopen allclose all
Dose / conc.:
1.5 mg/kg bw/day (nominal)
Remarks:
Days 1 to 56 / Principal / Test substance / Minimal effects observed at the highest dose-level (100 mg/kg/day), therefore animals of the lowest dose-level group received 330 mg/kg/day for the remaining 2 week-treatment period.
Dose / conc.:
330 mg/kg bw/day (nominal)
Remarks:
Days 57 to 72 / Principal / Test substance
Dose / conc.:
12 mg/kg bw/day (nominal)
Remarks:
Days 1 to 72 / Principal / Test substance
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Days 1 to 72 / Principal / Test substance
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Days 1 to 72 / Satellite / Test substance
Dose / conc.:
30 mg/kg bw/day (nominal)
Remarks:
Days 1 to 56 / Principal / Reference substance / Minimal effects observed at 30 mg/kg/day, therefore animals of this group received 60 mg/kg/day for the remaining 2 week-treatment period.
Dose / conc.:
60 mg/kg bw/day (nominal)
Remarks:
Days 57 to 72 / Principal / Reference substance
No. of animals per sex per dose:
5 animals per sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose-levels were selected by the Study Monitor on the basis of results of toxicological studies performed by dietary admixture on the same species.
- Rationale for animal assignment (if not random): Allocated according to a computerised stratification procedure.
- Rationale for selecting satellite groups: None
- Post-exposure recovery period in satellite groups: None
- Section schedule rationale (if not random): None
Positive control:
6-propyl-2-thiouracil (P.T.U.) or 4-Hydroxy-2-mercapto-6-propyl· pyrimidine 99%

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice a day including weekends and public holidays

DETAILED CLINICAL OBSERVATIONS: Yes (principal groups)
- Time schedule: at least once a day, at approximately the same time each day

BODY WEIGHT: Yes
- Time schedule for examinations:
- Principal groups: for each animal once before allocation of the animals to groups, on the first day of treatment and then once a week until the end of the study.
- Satellite group: for each animal once before allocation of the animals into groups and on the first day of treatment.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes (principal groups)
- The quantity of food consumed by the animals of each cage were recorded once a week, over a seven-day period, until the end of the study.
- Food intake per animal and per day was calculated by subtracting the amount of food left in each food-hooper, from that given.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: No data

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on days 21 and 42.
- Anaesthetic used for blood collection: Yes (not specified)
- Animals fasted: Yes
- How many animals: all animals of groups 1, 2, 3 and 4
- Parameters checked in table 1 [see section "Any other informaton on materials and methods incl. tables"] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on days 21 and 42
- Animals fasted: Yes
- How many animals: all animals of groups 1, 2, 3 and 4
- Parameters checked in table 1 [see section "Any other informaton on materials and methods incl. tables"] were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

OTHER: Thyroid function evaluation (T3, T4, TSH and rT3)
- Time schedule for collection of blood: on days 7, 21 and 42.
- Anaesthetic used for blood collection: Yes (not specified)
- Animals fasted: Yes
- How many animals: all surviving animals of groups 1, 2, 3, 4 and 5
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- On completion of the treatment period, after at least 14 hours fasting, all surviving animals were killed by carbon dioxide asphyxiation and exsanguination.

HISTOPATHOLOGY: Yes
- For all animals, including the one that died during the study, macroscopic lesions, thyroids with parathyroids and pituitary gland were preserved in 10% buffered formalin.
- The thyroids, parathyroids, and pituitary gland were embedded in paraffin wax, sectioned at approximately four microns in thickness and stained with hematoxylin-eosin for microscopic examination.

ORGAN WEIGHTS: Yes
Pituitary gland, Prostate, Thyroid (including parathyroid)
Other examinations:
None
Statistics:
Statistical analysis of body weight, food consumption, haematology, blood biochemistry, urinalysis and organ weight data follows the sequences reported in section "Attached background material".

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
No clinical signs were observed in animals given 12 mg/kg/day of AMITROLE throughout the study or in those which received 1.5 mg/kg bw/day of AMITROLE for the first 8 weeks and then 330 mg/kg/ bw/day for the last 2 weeks.
The noteworthy clinical signs observed during the study were as follows:
- at 100 mg/kg/day of AMITROLE: piloerection (5/5 males and 2/5 females), pallor of extremities (4/5 males and 2/5 females) and hypokinesia (1/5 males only) were observed from week 7 to 10, up to the end of the study.
- in animals which received the reference substance (PTU) at 30 mg/kg/day for the first 8 weeks and then 60 mg/kg/day for the last 2 weeks, the most frequent clinical signs were round back (1/5 males and 5/5 females), piloerection (5/5 males and 4/5 females), pallor of extremities (1/5 males and 3/5 females) and ptyalism (4/5 males and 2/5 females). These clinical signs were noted most frequently from week 4 to 11, up to the end of the study.

All the above mentioned clinical signs were considered to be related to treatment with the test or reference substances.
Due to their low incidence, other clinical signs such as chromorhinorrhea, observed in one animal given PTU at 30/60 mg/kg bw/day, were considered to be unrelated to treatment with the reference substance.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One control female (R22901) was found dead in week 6 (day 40). Prior to death, signs of poor clinical condition (abdominal and loud breathing, chromodacryorrhea, emaciation, eyes half­ closed, hypokinesia and lacrimation were observed). Ptyalism was also noted. At necropsy, whitish/serous contents were seen in the thoracic cavity, the pericardium was thickened and showed adhesions to the heart. Furthermore, the liver showed an accentuated lobular pattern.
The lesions in the heart/pericardium were considered likely factors contributing to death.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
No effect on body weight gain was observed in males and females treated with AMITROLE at 1.5 mg/kg/day for eight weeks.
Slightly higher body weight gain was observed in males treated with the test substance at 12 mg/kg/day (+10%). Similar body weight gain to that of control animals was observed in animals treated with AMITROLE at 330 mg/kg/day for two weeks.
On the contrary, markedly lower body weight gains were observed in animals given the test substance at 100 mg/kg/day (-50% and -20% in males and females respectively) and in those which received the reference substance at 30 mg/kg/day (-81% and -77% in males and females respectively). The increase in dose-level on that group to 60 mg/kg/day, from the beginning of week 8 to the end of the treatment period, led to slight body weight loss in males and females (-1 g and -2 g respectively).

All the above-mentioned effects on body weight and body weight gain were considered to be related to treatment with the test or reference substance. Lower body weight gain was observed in animals treated with the test substance at 100 mg/kg/day when compared with those given the reference substance at 30 mg/kg/day.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
On the completion of the study, and when compared to the mean control values, slightly to moderately lower food consumption was observed in males and females given 100 mg/kg/day of AMITROLE (-25% and -6% respectively). Decrease in food consumption was more pronounced in males and females which received 30 mg/kg/day and then 60 mg/kg/day of PTU (-39% and -29% respectively).

These effects were related to treatment with the test or reference substance and attributed to their anti-thyroidal properties.
Food efficiency:
not examined
Description (incidence and severity):
Not applicable
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not specified
Haematological findings:
no effects observed
Description (incidence and severity):
No changes related to treatment with the test or reference substances were observed on day 21 or 42 at any dose-level.

The differences seen during the course of the study (statistically significant or not) were biologically irrelevant and not dose-related. Furthermore, since all individual values were within the range of historical background data. Consequently they were considered to be unrelated to the treatment with the test or reference substance.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
This investigation was performed on animals of all groups on days 21 and 42, before the increase in dose-level.

When compared to the mean control values the following changes were seen in animals given 100 mg/kg/day of AMITROLE:
- moderately higher mean cholesterol levels on days 21 and 42 in both genders which were maximum on day 42 in males (2.1-fold increase). Since this change was dose-related and since 8/10 individual values obtained on day 42 were outside the range of historical background data, a relationship to treatment cannot be ruled out.
. Moderately lower mean triglyceride levels on days 21 and 42 in both genders which were maximum in males on day 42 (-74%). Although no clear relationship to the dose could be established and great variability of this parameter is usually observed in that species, 5/10 individual values obtained on day 42 were outside range of historical background data, and consequently a relationship to the treatment cannot be ruled out.

No changes related to treatment with the reference substance were observed on days 21 and 42. The other changes observed during the course of the study wether statistically significant or not were slight (i.e. changes in urea and creatinine levels in females given l 00 mg/kg/day), and all individual values were within the range of our historical background data. Consequently they were considered to be unrelated to the treatment with the test or reference substance.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Higher mean absolute and relative thyroid weights were seen in males receiving 100 mg/kg/day (x 5.6 abs., x 8 rel.) or 1.5/330 mg/kg/day (x 3.1 abs., x 3.4 mg/kg/day rel.) and in females receiving 100 mg/kg/day (x 4.6 abs., x 5.4 rel.) or 1.5/330 mg/kg/day (x 3.8 abs.,x 3.6 rel.) of the test substance and in the males (x 3.8 abs., x 7.6 rel.) and females (x 5 abs., x 7.3 rel.) receiving 60 mg/kg/day of the reference substance.
Slight effect on thyroid weight was also noted in males (+29% abs., +19% rel.) and females (+42% abs., +41% rel.) given the test substance at 12 mg/kg/day.

The above higher thyroid weights corresponded with the microscopic findings indicative of hyperactivity (see below) and were considered to be related to treatment with the test or reference substance.

Mean relative pituitary weights were higher in males (+66%) receiving 30/60 mg/kg/day of the reference substance whereas mean absolute pituitary weights were lower in females (-27%) in this group. Higher relative pituitary gland weight was also noted in males given the test substance at 100 mg/kg/day (+41 %).
The higher pituitary weights corresponded with the vacuolated cells seen at microscopic examination (see below) and were considered to be related to treatent with the test or reference substance.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Enlarged thyroid glands were noted in all males and females given the test substance at 100 or 330 mg/kg/day and in those which received PTU at 30 and then 60 mg/kg/day. This corresponded with the microscopic findings described below and was considered to be treatment-related.

All other macroscopic findings were those which are commonly found in the laboratory rat of this strain and age and their incidence (1/5 to 2/5 maximum) was considered of no toxicological importance.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Morphological changes, indicative of thyroid hyperactivity (follicular cell hypertrophy, small follicles and increased vascularity) were observed in all animals given the test substance with increasing incidence and severity corresponding to the increased dose and in those which received PTU at 30/60 mg/kg/day.

The above mentioned findings were associated with signs of hyperactivity of the pituitary gland characterised by an increased incidence of vacuolated cells. In animals receiving the reference substance the incidence and severity of this lesion was slightly higher.

The above findings in the thyroids and pituitary were considered to be a result of the action of the test substance.
Histopathological findings: neoplastic:
not examined
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Thyroid function evaluation:
This investigation was performed in animals of all groups on days 7, 21 and 42 before the increase in dose-level.
When compared to the mean control values, the main changes noted in animals treated with AMITROLE were as follows:
- At 1.5 mg/kg/day:
A moderate increase in TSH levels was observed on day 42 in males only.
- At 12 mg/kg/day:
Unexpected and transient increase in T4 levels was observed on day 21 (+69% and +28% in males and females respectively).
This was also noted for T3 levels in males and females on day 21 (+57% and +22% respectively). Since these changes were not associated with any signs of hyperthyroidism, their toxicological significance remains uncertain.
Increase in TSH levels (x2.6 for both sexes) observed in males and females on day 42 was moderate.
- At 100 mg/kg/day:
Slightly lower T3 levels in males and females after seven days of treatment (-24% and -22% respectively). This effect was related to the time of exposure and reached its maximum on day 42 (-84% and -80% in males and females respectively).
Moderately lower T4 levels in males and females after 21 days of treatment (-57% and -54% respectively). A time-related effect was noted, the maximum being observed on day 42 of the study (-90% and -89% in males and females respectively).
Moderately to markedly higher TSH levels (maximum was observed on day 42 in the males given 100 mg/kg/day of AMITROLE: 16.9.fold increase) were noted in animals of both sexes at all observation times. This marked biological change correlated with morphological changes indicative of hyperactivity of the pituitary gland seen at microscopic examination (increased incidence and severity of vacuolated cells) and was attributed to the inhibition of thyroidal hormone synthesis caused by the test substance.
When compared to the effects of the reference substance (PTU), thyroidal inhibition induced by the test substance seems to be delayed and somewhat less marked. Markedly lower T3 levels were observed in both sexes after seven days administration of 30 mg/kg/day of PTU (-97% and -75% in males and females respectively) and moderately lower T4 levels (·47% and ·68% in males and females respectively) were also noted. These effects were more pronounced on days 21 and 42. These changes were considered to be related to the administration of the referenee substance. Such a thyroid function inhibition led rapidly to a marked pituitary response, although maximal levels of TSH were observed on the occasion of the last sampling (x 20.5 and x 95.4 on day 42 in males and females respectively).

Graphs with thyroid hormone levels are reported in secttion "Attached background material".

Effect levels

open allclose all
Key result
Dose descriptor:
LOAEL
Effect level:
<= 12 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: TSH levels observed on day 42 (increase)
Key result
Dose descriptor:
LOAEL
Effect level:
<= 1.5 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: TSH levels observed on day 42 (increase)

Target system / organ toxicity

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
1.5 mg/kg bw/day (nominal)
System:
endocrine system
Organ:
pituitary gland
thyroid gland
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes

Any other information on results incl. tables

Summaries of the results are reported in section "Attached background material".

Results of the toxicokinetic of the substance:

The absorption and elimination of the substance were evaluated by HPLC in serum from satellite animals which received a single administration of AMITROLE at the dose-level of 100 mg/kg.

Absorption of the test substance was observed at all time points in both sexes and comparable Cmax were noted 2 hours after the administration of the test substance (394 and 341µg/mL in males and females respectively). Extent of absorption, as measured by AUC0 -24h was somewhat greater in the males than in the females (725 and 668 µ.g/h respectively).

Elimination, estimated by apparent tmax was not influenced by gender (2 hours for both sexes).

Applicant's summary and conclusion

Conclusions:
Under the test conditions, the Lowest Observed Adverse Effect Level (LOAEL) was lower than or equal to 12 mg/kg bw/day in females and lower than or equal to 1.5 mg/kg bw/day in males according to observed effects on thyroid and pituitary glands, including changes in thyroidal hormone levels, especially the TSH levels observed on day 42.
Executive summary:

This study evaluated the potential toxicity of the test substance, AMITROLE, administered daily by oral route (gavage) for ten weeks in rats when compared to that of an anti-thyroidal reference substance, 6-propyl-2-thiouracil (PTU). The study was performed according to a protocol similar to the OECD guideline No. 408 and under GLP compliance.

Three principal groups, each of five male and five female Sprague-Dawley rats received the test substance, AMITROLE, daily, by oral route (gavage), at 1.5, 12 and 100 mg/kg bw/day (groups 2, 3 and 4). Two principal additional groups, each of five males and five females, were given the vehicle alone (group 1: purified water) or 6-propyl-2-thiouracil (PTU, group 5) at 30 mg/kg bw/day under the same conditions, and acted as control and reference groups respectively. From day 57 (first day of week 8) of the study, animals of group 2, which initially received 1.5 mg/kg bw/day of AMITROLE, were given the test substance at 330 mg/kg bw/day and the dose-level was also increased in the reference group (group 5, PTU) to 60 mg/kg bw/day for a new treatment period of 15 days.

Satellite animals (nine males and nine females) were given the test substance at 100 mg/kg bw/day of under the same conditions for the determination of plasma levels of the test substance at designated time observations (15, 30 min and 1, 2, 4, 8 and 24 hours) after the first dosing. After the last sampling, all satellite animals were killed and discarded without necropsy.

The animals were checked daily for mortality and clinical signs. The food consumption and body weight of the animals were measured once a week. Haematology and blood biochemistry investigations were performed on all surviving animals on days 21 and 42. Additional blood samples were taken for thyroid function evaluation on days 7, 21 and 42, on all surviving animals.

At the end of the treatment period, the animals were killed and a macroscopic post-mortem examination was perfonned. Thyroids with parathyroids and the pituitary gland were weighed (all surviving animals) and submitted to a microscopic examination (animals of control and high dose-level groups).

No mortality treatment related is observed during the study.

No clinical signs were observed in animals given 12 mg/kg bw/day of AMITROLE throughout the study or in those which received 1.5 mg/kg bw/day of AMITROLE for the first 8 weeks and then 330 mg/kg/ bw/day for the last 2 weeks.

Piloerection, pallor of extremities and hypokinesia (1/5 males only) were observed from week 7 to 10, up to the end of the study in the group treated with 100 mg/kg bw/day of AMITROLE. In animals which received PTU at 30 mg/kg/day for the first 8 weeks and then 60 mg/kg bw/day for the last 2 weeks, the most frequent clinical signs in both sexes were round back, piloerection, pallor of extremities and ptyalism. These clinical signs were noted most frequently from week 4 to 11, up to the end of the study.

All the above mentioned clinical signs were considered to be related to treatment with the test or reference substances.

 

No effect on body weight gain was observed in males and females treated with AMITROLE at 1.5 mg/kg bw/day for eight weeks.

Slightly higher body weight gain was observed in males treated with the test substance at 12 mg/kg bw/day (+10%). Similar body weight gain to that of control animals was observed in animals treated with AMITROLE at 330 mg/kg bw/day for two weeks.

Markedly lower body weight and body weight gain were observed in males and females given the test substance at 100 mg/kg bw/day or the reference substance 30 mg/kg bw/day. Lower body weight gain was observed in animals treated with the test substance at 100 mg/kg bw/day when compared with those given the reference substance at 30 mg/kg bw/day.

On the completion of the study, and when compared to the mean control values, slightly to moderately lower food consumption was observed in males and females given 100 mg/kg bw/day of AMITROLE and those which received 30 mg/kg bw/day and then 60 mg/kg bw/day of PTU

These effects were related to treatment with AMITROLE or the reference substance and attributed to their anti-thyroidal properties.

No haemological changes were observed and no clinical biochemistry effects were considered non treatment-related.

Higher mean absolute and relative thyroid weights were observed in males receiving 100 mg/kg bw/day or 1.5/330 mg/kg bw/day and in females receiving 100 mg/kg bw/day or 1.5/330 mg/kg bw/day of the test substance and in both sexes receiving 60 mg/kg bw/day of the reference substance. Slight effect on thyroid weight was also noted in males and females given the test substance at 12 mg/kg bw/day.

The higher thyroid weights corresponded with the microscopic findings indicative of hyperactivity and were considered to be related to treatment with the test or the reference substance.

Mean relative pituitary weights were higher in males (+66%) receiving 30/60 mg/kg/day of the reference substance whereas mean absolute pituitary weights were lower in females in this group. Higher relative pituitary gland weight was also noted in males given the test substance at 100 mg/kg bw/day (+41 %).

The higher pituitary weights corresponded with the vacuolated cells seen at microscopic examination and were considered to be related to treatent with the test substance or reference substance.

Enlarged thyroid glands were noted in all males and females given the test substance at 100 or 330 mg/kg/day and in those which received PTU at 30 and then 60 mg/kg/day. This corresponded with the microscopic findings described below and was considered to be treatment-related.

All other macroscopic findings were those which are commonly found in the laboratory rat of this strain and age and their incidence (1/5 to 2/5 maximum) was considered of no toxicological importance.

Enlarged thyroid glands were noted in all males and females given the test substance at 100 or 330 mg/kg bw/day and in those which received PTU at 30 and then 60 mg/kg bw/day. This corresponded with the microscopic findings described below and was considered to be treatment-related.

Morphological changes, indicative of thyroid hyperactivity (follicular cell hypertrophy, small follicles and increased vascularity) were observed in all animals given the test substance with increasing incidence and severity corresponding to the increased dose and in those which received PTU at 30/60 mg/kg bw/day. TheThese findings were associated with signs of hyperactivity of the pituitary gland characterised by an increased incidence of vacuolated cells. In animals receiving the reference substance the incidence and severity of this lesion was slightly higher.

 

Thyroid function was evaluated in animals of all groups on days 7, 21 and 42 before the increase in dose-level. When compared to the mean control values, the main changes noted in animals treated with AMITROLE were as follows:

 - At 1.5 mg/kg bw/day:

A moderate increase in TSH levels was observed on day 42 in males only.

 - At 12 mg/kg bw/day:

Unexpected and transient increase in T4 levels was observed on day 21 (+69% and +28% in males and females respectively). This was also noted for T3 levels in both sexes on day 21 (+57% and +22% respectively). Since these changes were not associated with any signs of hyperthyroidism, their toxicological significance remains uncertain. Increase in TSH levels observed in males and females on day 42 was moderate.

 - At 100 mg/kg bw/day:

Slightly lower T3 levels in males and females after seven days of treatment (-24% and -22% respectively). This effect was related to the time of exposure and reached its maximum on day 42 (-84% and -80% in males and females respectively).

Moderately lower T4 levels in males and females after 21 days of treatment (-57% and -54% respectively). A time-related effect was noted, the maximum being observed on day 42 of the study (-90% and -89% in males and females respectively).

Moderately to markedly higher TSH levels (maximum was observed on day 42 in the males with 16.9.fold increase) were noted in animals of both sexes at all observation times. This marked biological change correlated with morphological changes indicative of hyperactivity of the pituitary gland seen at microscopic examination (increased incidence and severity of vacuolated cells) and was attributed to the inhibition of thyroidal hormone synthesis caused by the test substance.

When compared to the effects of the reference substance (PTU), thyroidal inhibition induced by the test substance seems to be delayed and somewhat less marked. Markedly lower T3 levels were observed in both sexes after seven days administration of 30 mg/kg bw/day of PTU (-97% and -75% in males and females respectively) and moderately lower T4 levels (·47% and ·68% in males and females respectively) were also noted. These effects were more pronounced on days 21 and 42. These changes were considered to be related to the administration of the reference substance. Such a thyroid function inhibition led rapidly to a marked pituitary response, although maximal levels of TSH were observed on the occasion of the last sampling (x 20.5 and x 95.4 on day 42 in males and females respectively).

The absorption and elimination of the substance were evaluated by HPLC in serum from satellite animals which received a single administration of AMITROLE at the dose-level of 100 mg/kg bw.

Absorption of the test substance was observed at all time points in both sexes and comparable Cmaxwere noted 2 hours after the administration of the test substance (394 and 341 µg/mL inmalesand females respectively). Extent ofabsorption,asmeasured by AUC0 -24h was some what greater in the males than in the females (725 and 668 µg/h respectively).Elimination, estimated by apparent tmaxwas not influenced by gender (2 hours for both sexes).

The test substance (up to 12 mg/kg bw/day) and the reference substance (up to 30 mg/kg bw/day) were clinically well-tolerated. The effects observed in animals given the test substance at 100 mg/kg bw/day or those which received PTU at 30 and then 60 mg/kg w/day were related to the anti-thyroidal properties of the test or reference substances. When compared to the reference substance, the treatment with the test substance resulted in a less marked inhibition of thyroid function.

Under the test conditions, the Lowest Observed Adverse Effect Level (LOAEL) was lower than or equal to 12 mg/kg bw/day in females and lower than or equal to 1.5 mg/kg bw/day in males according to observed effects on thyroid and pituitary glands, including changes in thyroidal hormone levels, especially the TSH levels observed on day 42.