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

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study (OECD 408); GLP compliant.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1997

Report date:

1997

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPP 82-1 (90-Day Oral Toxicity)

GLP compliance:

yes

Limit test:

no

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): Disulfide / MB 46307 / RPA 98028 Intermediate of Fipronil (MB 46030).
- Physical state: yellow powder.
- Analytical purity: 97.2 %.
- Purity test date: 23-May-1995.
- Lot/batch No.: 95-4023.
- Storage condition of test material: The test substance was stored in an air-tight, light-resistant container at room temperature.

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Iffa-Credo, 69210 St, Germain-sur-L'Arbresle, France.
- Age at study initiation: 6 to 7 weeks.
- Weight at study initiation: weight range from 162 to 260 g for the males and from 163 to 197 g for the females.
- Housing: individually in suspended stainless steel wire mesh cages.
- Diet: Certified rodent diet A04C P1 (UAR, Villemoisson-sur-Orge, France), ad libitum.
- Water: filtered and softened water from the municipal water supply were available ad libitum.
- Acclimation period: 8 days prior to the treatment.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20°C - 24°C.
- Humidity (%): 40% - 70%.
- Air changes (per hr): 10 to 15 air changes per hour (average, not monitored).
- Photoperiod (hrs dark / hrs light): 12-hour light, 12-hour dark cycles.

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): approximately every three weeks

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

High Performance Liquid Chromatography

Duration of treatment / exposure:

90 days

Frequency of treatment:

continuously in food

Doses / concentrations

Remarks:

Doses / Concentrations:
12, 36, 360 and 1200 ppm; (males: 0.77, 2.30, 22.55 and 73.63 mg/kg/day, females: 0.92, 2.78, 28.47 and 93.28 mg/kg/day)
Basis:
actual ingested

No. of animals per sex per dose:

10

Control animals:

yes, plain diet

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were checked for clinical signs, moribundity and mortality twice daily (once daily on week-ends or public holidays).


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least weekly during the treatment period


BODY WEIGHT: Yes
- Time schedule for examinations: once during the acclimatization period, on day 1, then weekly and before necropsy.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
weekly mean achieved dosage intake in mg/kg/day for each week and for weeks 1 to 12 was calculated.


OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: During the acclimatization period and during week 12
- Dose groups that were examined: control and top dose group


HAEMATOLOGY: Yes
- Time schedule for collection of blood: days 85, 86 or 90
- Anaesthetic used for blood collection: Yes, anesthetized by inhalation of isoflurane
- Animals fasted: Yes
- How many animals: all
- Parameters examined: Red blood cell count, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, white blood cell count and differential count evaluation and platelet count.



CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: days 85, 86 or 90
- Animals fasted: Yes
- How many animals: all
- Parameters examined: Total bilirubin, glucose, urea, total protein, albumin, total cholesterol, triglycerides and inorganic phosphate concentrations, aspartate aminotransferase, alanine arminotransferase, alkaline phosphatase, chloride, sodium, potassium, calcium and creatinine.


URINALYSIS: Yes
- Time schedule for collection of urine: overnight before necropsy
- Metabolism cages used for collection of urine: No data
- Animals fasted: Yes, overnight
- Parameters examined: Volume, pH, refractive index, glucose, bilirubin, ketones, occult blood, protein, urobilinogen, red blood cells, white blood cells, epithelial cells, bacteria, casts, crystals.


NEUROBEHAVIOURAL EXAMINATION: No data

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, The necropsy included the examination of all major organs, tissues and body cavities.
HISTOPATHOLOGY: Yes, adrenal gland, aorta, articular surface, bone (sternum), bone marrow (sternum), brain, epididymis, esophagus, eye and opticnerve, harderian gland, heart, intestine (duodenum, jejunum, ileum, cecum, colon, rectum), kidney, larynx, liver, lung, lymphnodes (submaxillary, mesenteric), mammary gland, ovary, pancreas, pituitary gland, prostate, sciatic nerve, seminal vesicle, skeletal muscle, skin, spinal cord (cervical, thoracic, lumbar) spleen, stomach, submaxillary (salivary) gland, testis, thymus, thyroid, tongue, trachea, urinary bladder, uterus, vagina.

Other examinations:

Adrenals, brain, epididymis, heart, kidneys, liver, ovaries, pituitary gland, prostate, spleen, testes, thymus, thyroid gland (with parathyroid) and uterus were weighed. Paired organs were weighed together.

Statistics:

Statistical analyses were performed using SAS programs.

Results and discussion

Results of examinations

Details on results:

CLINICAL SIGNS AND MORTALITY
There were three mortalities during the study. At 360 ppm, one male with paralysis of the hindquarters was found moribund on Day 58 and another male was found dead on Day 75. At 1200 ppm, one male was found dead on Day 73.

Signs of aggressivity and irritability to touch were observed at physical examination with an increased incidence starting at 36 ppm.

BODY WEIGHT AND WEIGHT GAIN
The body weight evolution of all treated males and females was unaffected by treatment.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
During Week 6, abnormally elevated mean food consumption were noted in males in control and treated at all dose levels and in females in the control, 36 ppm and 1200 ppm groups. This was due to some individual animals scattered in these groups for which the daily food consumption was higher than this usually observed in rats of this age and strain. During Week 13, due to an abnormal number of higher mean food consumption than this usually observed in rats of this age and strain, food consumption data of this week were dismissed.

OPHTHALMOSCOPIC EXAMINATION
No treatment-related ophthalmological abnormalities were observed after 12 weeks of treatment at 1200 ppm.

HAEMATOLOGY
In hematology, prothrombin time was increased in a dose-related manner in males only, at 36, 360 and 1200 ppm (+41%, +114% and +116%, respectively). Platelets were also increased (+25%) in males and females at 1200 ppm. No other toxicologically meaningful changes were noted in hematology.

CLINICAL CHEMISTRY
In clinical chemistry, the major changes were an increase in protein concentration in males and females at 360 and 1200 ppm (7-12 %), an increase in total cholesterol in males at 1200 ppm (+46%) and in females at 360 (+40%) and 1200 ppm (+36%). An increase in Urea concentrations in males at 360 (+30%) and 1200 ppm (+31%) and in females at only 1200 ppm (+31%) was also noted.

URINALYSIS
No meaningful changes were noted in the parameters assayed.

ORGAN WEIGHTS
There was a statistically significantly higher mean absolute and/or relative liver weight in both sexes at 1200 and 360 ppm and in females at 36 ppm, when compared with controls.
Statistically significantly higher mean absolute and/or mean relative thyroid weights were seen exclusively in male rats at 1200 and 360 ppm only, when compared with controls.
All other changes were not considered to be of toxicological importance.

GROSS PATHOLOGY
Unscheduled deaths: At the necropsy, in the three dead animals, there were macroscopic and histological evidentes of hemorrhages.

ScheduIed sacrifice: An increased incidence of prominent liver lobulation was seen in the majority of males and females at 36, 360 and 1200 ppm, when compared with controls. Dark liver color was noted in the majority of males at 1200 ppm and a proportion of males from all other treatment groups and in females at 360 and 1200 ppm, when compared with controls. Dark kidney color was seen in a proportion of males from all treatment groups, but not in controls. There was a slightly increased incidence of this change in females at 360 and 1200 ppm, when compared with controls.

HISTOPATHOLOGY: NON-NEOPLASTIC
Unscheduled deaths: In each of the unscheduled deaths hemorrhage was a likely contributory factor to death.

ScheduIed sacrifice: Hepatocyte hypertrophy with ground glass cytoplasm was seen in the majority of females at 360 and 1200 ppm and in a proportion of males at 1200 ppm and a single male at 360 ppm, when compared with controls. In females this hypertrophy ranged from centrilobular to generalized in distribution, In the males the distribution was generalized. These changes are consistent with the statistically significantly higher absolute and relative liver weights recorded in these dose groups. Thyroid follicular cell hypertrophy of a very minor degree was seen in both sexes at 36, 360 and 1200 ppm.

Effect levels

1

Dose descriptor:

NOEL

Effect level:

0.77 mg/kg bw/day (nominal)

Sex:

male

Basis for effect level:

other: clinical signs; mortality; haematology; clinical chemistry; organ weights

2

Dose descriptor:

NOEL

Effect level:

0.92 mg/kg bw/day (nominal)

Sex:

female

Basis for effect level:

other: clinical signs; mortality; haematology; clinical chemistry; organ weights

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

It can be considered that the No Observed Effect Level (NOEL) of Disulfide / MB 46307/RPA 098028 is 12 ppm in males and 12 ppm in females (approximately 0.77 mg/kg/day and 0.92 mg/kg/day respectively).

Because of the diversity of the observed liver effects starting at 360 ppm (= 22.5 mg/kg bw/day for males and 28.47 mg/kg bw/day for females), which included mortality, the following classification is considered to be warranted: R48/22; STOT RE 2 (target organ: liver)