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Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
April 11, 1990 - January 1991
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1991
Report date:
1991

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Version / remarks:
1984
Deviations:
yes
Remarks:
(no neurological examinations)
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Version / remarks:
1981
Deviations:
yes
Remarks:
(no neurological examinations)
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Reference substance name:
O,O,O-tris(2(or 4)-C9-10-isoalkylphenyl) phosphorothioate
EC Number:
406-940-1
EC Name:
O,O,O-tris(2(or 4)-C9-10-isoalkylphenyl) phosphorothioate
Cas Number:
126019-82-7
Molecular formula:
C30-54H47-87O3PS
IUPAC Name:
O,O,O-tris(2(or 4)-C9-10-isoalkylphenyl) phosphorothioate
Details on test material:
- Physical state: viscous liquid, insoluble in water; pH 7
- Storage condition of test material: room temperature
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature

Test animals

Species:
rat
Strain:
other: Albino Wistar rat, Hanlbm: WIST, outbred
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: BRL, Biological Research Laboratory Ltd., CH-4414 Fuellinsdorf, Switzerland
- Age at study initiation: 6 wks
- Weight at study initiation: (males) 130-149 g; (females) 112-130 g
- Housing: Individually in Makrolon type-3 cages with standard softwood bedding ("Lignocel", Schill AG, CH-4132 Muttenz, Switzerland)
- Diet: Pelleted standard Kliba No., 343 Batch Nos. 67/90 and 69/90 rat maintenance diet ("Kliba", Klingentalmuehle AG, CH-4303 Kaiseraugst, Switzerland); ad libitum
- Water: ad libitum
- Acclimation period: 8 d

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

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: Destilled Water with 4 % Sodiumcarboxymethylcellulose and 0.2 % Tween 80
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
At all dose levels, both components were weighed into a glass beaker on a tared Mettler balance. The mixture (w/w) was prepared using a homogenizer. Homogeneity of the test article in the vehicle was maintained during treatment using a magnetic stirrer. The mixtures were prepared daily.

VEHICLE
- Justification for use and choice of vehicle: test article is insoluble in water
- Maximal dose volume: 10 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples were analysed for content, homogeneity and stability. Samples were taken from mixtures prepared prior to treatment start. The mean concentration found were in the range from 78.7 % to 95.6 % of the nominal concentration for all dose groups. The homogeneity varied in the range from -4 % to +5 % of the mean concentrations. The test article was stable in distilled water with 4 % CMC and 0.2 % Tween 80 at room temperature for at least two hours. (Since the mixtures were prepared daily, stability over 8 days was not assessed).
Duration of treatment / exposure:
28 days (males), 29 days (females)
Frequency of treatment:
7 days/week
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
200 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: (based upon following studies): Acute Oral Toxicity Study in Rats (LD50 > 2000 mg/kg bw); 5-Day Oral Toxicity (Range Finding) Study
- Post-exposure recovery period in satellite groups: ca 2 wks
- Rationale for selecting satellite groups: for observation of reversibility, persistence, or delayed occurrence of toxic effects
- Satellite dose groups: all dose groups
- No of animals per sex per satellite dose group: 5

Examinations

Observations and examinations performed and frequency:
MORTALITY: Yes
- Time schedule: Mortality was recorded twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Clinical signs were assessed once daily

BODY WEIGHT: Yes
- Time schedule for examinations: The body weight of each animal was recorded weekly throughout the study, using a Mettler balance connected to the RCC Computer.

FOOD CONSUMPTION:
The food consumption was recorded weekly throughout the study, using a Mettler balance connected to the RCC Computer.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: at the end of the treatment and at the end of the recovery
- Dose groups that were examined: all animals during pretest; control group and highest dosage group
- Method: Approximately 10 minutes after the application of a mydriatic solution (Dispersa AG, CH-8442 Hettlingen / Switzerland) the cornea, lens, anterior chamber, vitreous body and ocular fundus of both eyes were examined under dimmed light using a Heine Bifocal Type Miroflex Ophthalmoscope (Eisenhut Vet. AG, CH-4123 Allschwil/Switzerland).

HAEMATOLOGY: Yes
- Time schedule for collection of blood: after 4 wks and after recovery (between 6.30 and 7.45 a.m. to reduce biological variations)
- Anaesthetic used for blood collection: Yes (under light ether anesthesia)
- Animals fasted: Yes (18 h) (food only)
- How many animals: all animals
- Parameters checked: haematocrit, haemoglobin concentrations, erythrocyte count, reticulocytes, total and differential leucocyte count, platelet count, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, nucleated erythrocytes normoblasts, Henz bodies, methemoglobin, cell classification, red cell morphology and red cell morphology cont. Coagulation: thromboplastin time, activated partial thromboplastin time.
- Sampling: Blood samples were drawn from the retroorbital plexus. EDTA-K2 was used as an anticoagulant. Sodium citrate, 3.8 % was used as an anticoagulant for blood samples destined for coagulation analysis.

CLINICAL BIOCHEMISTRY: Yes
- Time schedule for collection of blood: after 4 wks and after recovery (between 6.30 and 7.45 a.m. to reduce biological variations)
- Anaesthetic used for blood collection: Yes (under light ether anesthesia)
- Animals fasted: Yes (18 h) (food only)
- How many animals: all animals
- Parameters checked: glucose, urea, creatinine, bilirubin, cholesterol, triglycerides, aspartat aminotransferase, alanine aminotransferase, lactate dehydrogenase, alkaline phosphatase, gamma-glutamyl-transferase, calcium, phosphorus, sodium, potassium, chloride, albumin, protein (total), globulin, albumin/globulin ratio
Blood samples were drawn from the retroorbital plexus.

URINALYSIS: Yes
- Time schedule for collection of urine: after 4 wks and after recovery
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes (18 h)
- Parameters checked: volume (18 h), specific gravity, color, appearance, pH, protein, glucose, ketone, bilirubin, blood, urobilinogen, urine sediment
Sacrifice and pathology:
All animals were necropsied and descriptions of all macroscopic abnormalities were recorded.
- Necropsy dates: At the end of the treatment period and at the end of the recovery period.
- Organ weights: adrenal glands, heart, kidneys, liver, ovaries, spleen, testes, thyroid gland
Other examinations:
- Histopathology: Tissue samples of adrenal glands, heart, kidneys, liver, lungs, ovaries, spleen, testes and thyroid gland from rats of the control and high doses and all gross lesions from all animals were prepared. Histologic alterations were described according to their distribution, severity and morphologic character.
Statistics:
The following statistical methods were used to analyze the body weights, food consumption, organ weights and clinical laboratory data: Univariate one-way analysis of variance was used to assess the significance of intergroup differences. If the variables could be assumed to follow a normal distribution, the Dunnett-test (many to one t-test) based on a pooled variance estimate was applied for the comparison between the treated groups and the control groups. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Individual values, means, standard deviations and statistics were rounded off before printing. For example, test statistics were calculated on the basis of exact values for means and pooled variances and then rounded off to two decimal places. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
No signs of clinical toxicity occurred.
Mortality:
no mortality observed
Description (incidence):
No premature mortalities occurred.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Mean body weight and body weight gain values recorded in both sexes in all treated groups were similar to those of the controls throughout the study.

Bodyweight gain in the 4th week (N=10) (%):
- control: 117 (males), 58.2 (females)
- 50 mg/kg bw: 115.2 (males), 55.7 (females)
- 200 mg/kg bw: 113.9 (males), 54.8 (females)
- 1000 mg/kg bw: 116.1 (males), 62.0 (females)

Bodyweight gain of recovery animals (N=5) after ca. 14 d recovery (%):
- control: 142.6 (males), 74.9 (females)
- 50 mg/kg bw: 150.8 (males), 65.9 (females)
- 200 mg/kg bw: 142.8 (males), 67.5 (females)
- 1000 mg/kg bw: 137.7 (males), 71.3 (females)
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Mean values of food consumption and food consumption to body weight ratio recorded in both sexes in all treated groups were comparable to those of the controls throughout the study. Statistical analysis of food consumption and of food consumption to body weight ratio revealed occasional differences from control values scattered in males of the low and the high dose groups. Since no consistent pattern emerged, these variations were considered to be incidental and of no toxicological relevance for the study.

Mean food consumption after 4 weeks (g/animal/day) N=10
- Males: 22.0 (control), 22.6 -23.0 (test groups)
- Females: 15.9 (control), 15.4-16.2 (test groups)

Mean food consumption of recovery animals (after ca. 14 d recovery) (g/animal/day) N=10
- Males: 23.60 (control), 23.3-24.5 (test groups)
- Females: 17.3 (control), 16.9-17.9 (test groups)
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
No treatment-related ocular changes were observed by ophthalmoscopic examination performed towards the end of the treatment and recovery periods.
Haematological findings:
no effects observed
Description (incidence and severity):
The assessment of hematological data indicated no changes of toxicological significance at termination of the treatment nor at the end of the treatment free recovery period. All statistical differences in the results of the hematology parameters were considered to be incidental and of normal biological variation for rats of this strain and age (see attachment 1 for historical reference values for untreated Wistar Han. rats).

- 50 mg/kg bw: increased platelets (ca. 116% compared to controls in recovery males (p> 0.05)), reduced reticulocytes concentration (ca. 62.5% compared to controls in recovery males), reduced white blood cell count in test group and recovery females (75% compared to controls, respectively)

- 200 mg/kg bw: increased platelets (ca. 123% compared to controls in recovery males (p> 0.01)), reduced white blood cell count recovery females (77% compared to controls)

- 1000 mg/kg bw: Increased Methaemoglobin in test group females (140 % compared to controls)
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
These findings reflect metabolic changes and were not considered to be of toxicological significance. In addition, the findings were only slightly different from those of the controls and within limits of the historical control data, nor were the changes supported by any morphological findings.
At termination of the treatment-free recovery period these findings were no longer observed and were comparable to those of the controls.
All the statistical differences in the results of the clinical biochemistry parameters were considered to be incidental and of normal biological variation
for rats of this strain and age (see attachment 1 for historical reference values for untreated Wistar Han. rats)

- Slightly increased glucose concentration for both sexes of medium and high dose groups (118-122% compared to controls)
- Slightly increased total cholesterol concentration for females of high dose group (127.2% compared to controls)
- Slightly decreased calcium concentration for males of medium and high dose groups (96.7% and 95% compared to controls, respectively)
- Slightly decreased phosphorus concentration for females of medium dose group (83% compared to controls) and in both sexes of high dose group (ca. 90% compared to controls)
- Slightly increased potassium concentration for males (109% compared to controls) and females (113% compared to controls) of high dose group
- Slightly increased chloride concentration for females of high dose group (101.5% compared to control)
Urinalysis findings:
no effects observed
Description (incidence and severity):
Urinalysis data indicated no treatment-related changes after 4 weeks of treatment nor at the end of the treatment-free recovery period. Variations in urinalysis parameters were considered to be incidental and of normal biological variation for rats of this strain and age.
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
In all treated animals of both sexes, all mean organ weights and mean organ to body weight ratios were comparable to those of the controls, at the end of the treatment and at the end of the treatment-free recovery period.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No treatment-related macroscopic alterations were observed at necropsy, either at the end of the treatment or at the end of the recovery period. The following incidental changes were observed at the end of the treatment period:

- right eye damaged during blood collection in one male
- reddish coloration of urinary bladder in one male
- mandibular lymph node enlarged in one male
- (reddish) foci on thymus in 1 male and 3 females
- uterus horns enlarged in one female

The following incidental change was observed at the end of the recovery period:
- black-brown foci on stomach mucosa in one female
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No treatment-related findings were recorded. The small number of findings were considered to be of a spontaneous nature consistent with the age and strain of animal.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed

Effect levels

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed
Key result
Dose descriptor:
NOEL
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical biochemistry

Target system / organ toxicity

Key result
Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
Based upon these results, the "no-adverse-effect-level" for the test article when administered daily by gavage to rats during four consecutive weeks was 1000 mg/kg bw in both sexes. Under the same conditions, the "no-observable-effect level" was 50 mg/kg bw in both sexes.
Executive summary:

In this subacute 4-week toxicity study, the test article was administered daily (7 days per week) by gavage to albino, SPF-bred, Wistar rats during 28 consecutive days for males and 29 days for females. The study design included one main group and one recovery group, for a total of 10 male and 10 female rats at each dose level. From the 10 rats per sex in each dosage group, 5 were designated for sacrifice at the end of the treatment, and 5 were designated for sacrifice after a 2-week, treatment-free (recovery) period. Dose levels were 0 (control), 50, 200 and 100 mg/kg body weight. No mortality occurred throughout the study. No treatment-related ocular changes were observed by ophthalmoscopic examination performed towards the end of the treatment and recovery periods. Mean body weight and mean body weight gain values recorded in both sexes in all treated groups were similar to those of the controls throughout the study. Mean values of food consumption and of food consumption to body weight ratio recorded in both sexes in all treated groups were comparable to those of the controls throughout the study. The assessment of hematological data indicated no changes of toxicological significance at termination of the treatment nor at the end of the treatmentfree recovery period. For clinical biochemical slight metabolic changes were noted which were not considered to be of toxicological significance. In addition, the findings were only slightly different from those of the controls and within limits of the historical control data. At termination of the treatment-free recovery period these findings were no longer observed and were comparable to those of the controls. Urinalysis data indicated no treatment-related changes after 4 weeks of treatment nor at the end of the treatment-free recovery period. In all treated animals of both sexes, all mean organ weights and mean organ to body weight ratios were comparable to those of the controls, at the end of the treatment and at the end of the treatment-free recovery period. No treatment-related macroscopic alterations were observed at necropsy, either at the end of the treatment or at the end of the recovery period. There were no treatment-related histological findings recorded. Based upon these results, the "no-adverse-effect-level" for the test article when administered daily by gavage to rats during four consecutive weeks was 1000 mg/kg bw in both sexes. Under the same conditions, the "no-observable-effect level" was 50 mg/kg bw in both sexes.