1H-Pyrazole-3-carbonitrile, 4,4'-dithiobis[5-amino-1-[2,6-dichloro-4-(trifluoromethyl)phenyl]-

Specific investigations: other studies

Administrative data

Endpoint:

endocrine system modulation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 Feb 1998 - 02 Feb 2000

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Well documented, acceptable study report; study conducted in compliance with GLP regulations

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2000

Report date:

2000

Materials and methods

Principles of method if other than guideline:

The purpose of the present study was to investigate the effects of the test substance on thyroid function in rats after daily oral administration over a 2-week period, and to compare these effects with those of propylthiouracil, a known inhibitor of thyroid organification in many species, and of phenobarbital, a known hepatic drug metabolizing enzyme inducer which enhances thyroxine (T4) metabolism but not thyroidal synthesis.

Thyroid function was evaluated by investigation of the effects of these compounds on iodide organification using the “Perchlorate Discharge Test” (Atterwill et al., 1987). Any inorganic iodide present in the thyroid glands is released into the blood upon administration of perchlorate.
At 24 hrs after the last dose of the test compound (i.e. Day 15), 12 animals from each treatment group received a single intravenous dose of sodium 125Iodide (ca. 1 µCi) in 0.5 mL 0.9% (w/v) saline solution. This was followed 6 hrs later by an intraperitoneal injection of either potassium perchlorate (10 mg/kg bw in 0.9% saline) to 6 animals of each group or 0.9% saline (as a control to the perchlorate treatment) to the other 6 animals. At 2.5 min after perchlorate or saline administration, blood samples were collected under anaesthesia and thyroids were dissected from the carcass following sacrifice. Both tissues were taken for measurement of total radioactivity.

Atterwill et al. (1987). J. Pharmacol. Methods 18(3): 199-203

GLP compliance:

yes

Type of method:

in vivo

Endpoint addressed:

repeated dose toxicity: oral

Test material

Test material information

Constituent 1

Reference substance name:

5-amino-4-({5-amino-3-cyano-1-[2,6-dichloro-4-(trifluoromethyl)phenyl]-1H-pyrazol-4-yl}disulfanyl)-1-[2,6-dichloro-4-(trifluoromethyl)phenyl]-1H-pyrazole-3-carbonitrile

EC Number:

603-434-4

Cas Number:

130755-46-3

Molecular formula:

C22 H8 Cl4 F6 N8 S2

IUPAC Name:

5-amino-4-({5-amino-3-cyano-1-[2,6-dichloro-4-(trifluoromethyl)phenyl]-1H-pyrazol-4-yl}disulfanyl)-1-[2,6-dichloro-4-(trifluoromethyl)phenyl]-1H-pyrazole-3-carbonitrile

Details on test material:

- Name of test material (as cited in study report): M&B 46307
- Physical state: yellow powder
- Analytical purity: 99.9%
- Lot/batch No.: 95-4023
- Storage condition of test material: room temperature, dark
- Stability of test substance: 16 Apr 1999
- Additional information: intermediate in the synthesis of M&B 46030

Test animals

Species:

rat

Strain:

other: Crl:CD(SD)BR

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Margate, Kent
- Age at study initiation: approx. 5 weeks old
- Weight at study initiation: approx. 115 - 146 g
- Housing: 4 rats/cage
- Diet (ad libitum): pelleted SDS Rat and Mouse No. 1 modified maintenance diet
- Water (ad libitum): tap water
- Acclimation period: 6 and 5 days (between arrival and allocation to groups; between allocation to groups and commencement of treatment)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 - 23
- Humidity (%): 41 - 77
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 0.5% (w/v) aqueous methylcellulose suspension

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

- Constant dosage volume:
- 5 mL/kg bw for the test substance preparation
- 2.5 mL/kg bw for phenobarbital
- 5 mL/kg bw for propylthiouracil

- Preparation of dosing solutions:
- test substance and phenobarbital once weekly (storage at 4°C)
- propylthiouracil once daily

- Stability of formulations:
- test substance formulation assumed stable for one week

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

2 weeks

Frequency of treatment:

daily

Post exposure period:

At 24 hrs after the last dose of the test compound (i.e. Day 15), 12 animals from each treatment group received a single intravenous dose of sodium 125Iodide (ca. 1 µCi) in 0.5 mL 0.9% (w/v) saline solution followed 6 hrs later by an intraperitoneal injection of either potassium perchlorate (10 mg/kg bw in 0.9% saline, at a constant dosage volume of 2 mL/kg bw) to 6 animals of each group or 0.9% saline (as a control to the perchlorate treatment) to the other 6 animals. At 2.5 min after perchlorate or saline administration, blood samples were collected under anaesthesia and thyroids were dissected from the carcass following sacrifice. Both tissues were taken for measurement of total radioactivity.

Doses / concentrations

Remarks:

Doses / Concentrations:
1000 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

12 animals per dose (3 groups of 4 animals each plus 4 "spare rats")

Control animals:

yes, concurrent vehicle

Details on study design:

Dose formulation:
- 125I-sodium-iodide:
Lot no.: 1021198A
Supplier: NEN Life Science Products

- Potassium perchlorate
Lot no.: BG 03926MF
Supplier: Aldrich Chemical Co Ltd

Examinations

Examinations:

CLINICAL SIGNS
- Individual animals were observed at least twice daily for any signs of behavioural changes, reaction to treatment or ill health. Further checks were made early in each working day and again in the afternoon to look for dead or moribund animals.

BODYWEIGHT
- The weight of each rat was recorded at the time of allocation of animals to groups, on the day of commencement of treatment and twice a week thereafter.

FOOD CONSUMPTION
- The quantity of food consumed by each cage of rats was recorded during the treatment period. Food intake per rat (g/rat/week) was calculated using the total amount of food given to and left by each cage in each group and the number of rats surviving in each cage.

THYROID REMOVAL / ANALYSIS
- Immediately after blood sampling, the animals were sacrificed by cervical dislocation and the thyroid gland from each rat was dissected from the trachea following ventral midline incision and skin reflection. Adhering tissue was removed, and the thyroids were rinsed in ice cold saline solution, blotted dry and weighed into pre-weighed storage tubes prior to measurement of radioactivity by gamma scintillation counting (Cobra II gamma scintillation counter).

Positive control:

1. 200 mg/kg bw/day propylthiouracil (PTU) (direct inhibitor of thyroid iodide organification)
2. 80 mg/kg bw/day phenobarbital (sodium salt) (known hepatic drug metabolizing enzyme inductor)

Results and discussion

Details on results:

1. Propylthiouracil-treatment (200 mg/kg bw/day po) led to pronounced, statistically significant decreases in the amount of 125Iodide incorporated into the thyroid glands and in the thyroid: blood radioactivity concentration ratios (T/B ratios) of rats receiving this agent. These changes were reflected in elevated levels of 125Iodide measured in the whole blood. The weights of thyroids from these animals were increased by ca. 2.2 fold, compared with control animals, thus accounting for a significant reduction of about 50% (saline-treated animals) in the concentration of 125Iodide in whole blood when expressed as a function of thyroid weight.
2. Phenobarbital pre-treatment of rats receiving saline or perchlorate led to statistically significant increases in 125Iodide incorporated in the thyroid glands and in the thyroid: blood radioactivity concentration ratios of rats receiving this agent. Phenobarbital is a known enzyme-inducer which has been shown previously to increase the biliary clearance of conjugated 125Iodide-T4 after repeated doses (75 mg/kg bw/day) during 4 weeks and thus enhance thyroidal follicular activity indirectly via increased levels of circulating TSH (thyroid-stimulating hormone).
3. Similarly as for phenobarbital, test substance pre-treatment of rats administered saline or perchlorate led to statistically significant increases in 125Iodide incorporated in the thyroid glands and in the thyroid: blood radioactivity concentration ratios of rats receiving this agent. On the basis of perchlorate discharge, there was no evidence for direct inhibition of organification by the test substance at the dose level administered and thus a direct effect of the substance on thyroid peroxidase may be excluded. However, the responses to pre-treatment with the test substance paralleled, in large part, with those after pre-treatment with phenobarbital, a known inducer of hepatic drug metabolizing enzymes.

Any other information on results incl. tables

CLINICAL SIGNS AND MORTALITY

- There were no deaths during the treatment period in any treatment group.

- Clinical signs considered to be associated with treatment consisted of:

- Control Group:

No abnormalities were detected.

- Phenobarbital Group:

Unsteady gait was noted immediately post-dosing for all animals throughout the treatment period lasting through to the end of the working day. Collapsed posture was noted for all animals 0.5 hrs post-dose on Days 2, 3 and, in one rat only, Day 4 of the treatment period. The finding was no more apparent (all animals) 1.5 hrs later.

- Propylthiouracil Group:

Salivation (often brown stained) was noted for all animals on occasions during the treatment period. Wet coat was noted in 12/16 animals on Days 2 to 5 only. Paddling of limbs was noted in one animal.

- Test Substance Group:

No abnormalities were detected.

BODYWEIGHT

- Bodyweight gain of the animals receiving 200 mg/kg bw/day propylthiouracil was statistically significantly lower (p < 0.001) than for controls, while gains for animals receiving the test substance or phenobarbital were similar to those of control animals.

FOOD CONSUMPTION

- There was a statistically significant (p < 0.001) reduction in food intake for animals receiving 200 mg/kg bw/day propylthiouracil in comparison with the controls, this being consistent with reduced weight gain in this group. Despite of a small, but statistically significant difference (p < 0.005) between the control and the phenobarbital group, food intake for all other treated animals was considered similar to that of controls.

METABOLIC STUDIES

- Thyroid weights:

- An analysis of variance demonstrated that for thyroid weight, there was evidence of an interaction between treatment and subgroup (saline/perchlorate treated) (p = 0.004). When comparisons were made between the control and treated groups, the Propylthiouracil treated group was found to have a significantly higher group mean value than the controls for both the Saline and Perchlorate subgroups (p < 0.001). When further comparisons between the Saline and Perchlorate subgroups were made for each treatment group separately, the only difference found between the two subgroups was for the Propylthiouracil treated groups (p < 0.001).

- Radioactivity in thyroids:

- An analysis of variance of concentration of radioactivity in thyroids demonstrated that for thyroid radioactivity (% dose/g thyroid), both the test substance and Phenobarbital treated groups were found to have significantly higher group mean values than the controls (p <= 0.002) in contrast to the Propylthiouracil treated group which had significantly lower group mean values than the controls (p < 0.001). There was no evidence of a difference between the Saline and Perchlorate subgroups (p = 0.76).

- Concentrations of radioactivity in whole blood:

- An analysis of variance demonstrated that for blood radioactivity, the Phenobarbital treated group was found to have significantly lower group mean values than the controls (p = 0.047) in contrast to the Propylthiouracil treated group which had significantly higher group mean values than the controls (p < 0.001). The Perchlorate treated subgroups were generally found to have significantly higher group means than the Saline treated subgroups (p = 0.004).

- Thyroid : blood radioactivity concentration ratios:

- Both the test substance and Phenobarbital treated groups were found to have significantly higher group mean values than the controls (p < 0.001) in contrast to the Propylthiouracil treated group which had significantly lower group mean values than the controls (p < 0.001). there was no evidence of a difference between the Saline and Perchlorate subgroups.

- Blood radioactivity concentration : thyroid weight ratios:

- There was evidence of an interaction between treatment and subgroup (p = 0.048). When comparisons were made between the control and treated groups for the Perchlorate subgroup, all treated groups were found to have significantly lower group mean values than the controls (p <= 0.016). Only the Propylthiouracil treated group was found to have a significantly lower group mean value than the controls for the Saline subgroup (p < 0.001). When further comparisons between the Saline and Perchlorate subgroups were made for each treatment group separately, both the Phenobarbital and Propylthiouracil treated groups were found to have higher mean values for Perchlorate than for Saline (p <= 0.003). A similar difference between the subgroups was seen for the controls although this did not attain formal statistical significance (p = 0.0052).

OVERVIEW:

Pre-treatment of rats with the test substance did not affect the thyroid weight and blood radioactivity concentration, but increased the thyroid radioactivity content and concentration. The thyroid : blood radioactivity concentration ratio was also increased, whereas the blood radioactivity concentration : thyroid weight ratio was decreased, when compared with those of saline pre-treated control animals. These changes paralleled, in large part, those of animals pre-treated with phenobarbital.

Pre-treatment with propylthiouracil, considered to be an inhibitor of the iodine organification enzyme thyroid peroxidase, increased thyroid weight and the blood radioactivity concentration. It decreased the thyroid radioactivity content and concentration, the thyroid : blood radioactivity concentration ratio and the blood radioactivity concentration : thyroid weight ratio.

Injection of perchlorate resulted in no statistically significant differences (from those in saline-injected rats of each group) in control animals or in animals pre-treated with phenobarbital or the test substance, except for an increase in the blood radioactivity concentration : thyroid weight ratio of phenobarbital pre-treated rats. However, perchlorate injection was associated with decreases in thyroid weight and the thyroid radioactivity content of propylthiouracil pre-treated rats and an increase in the blood radioactivity concentration : thyroid weight ratio of these animals, compared to saline treated controls.