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Toxicological information

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
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Read across to similar substance. Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.
Justification for type of information:
See read-across justification in Section 13.
Cross-reference
Reason / purpose:
other: Target substance
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study conducted on read across material
Justification for type of information:
See read-across justification in Section 13.
Reason / purpose:
read-across source
Dose descriptor:
NOEL
Effect level:
1 233 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Oral administration of the test material, Substance 1658/5, at dose levels of up to 1233 mg/kg/day for twenty-eight consecutive days in the rat, produced no treatment-related changes in the parameters measured.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1990
Report Date:
1990

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
Description: white powder

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: 5-6 weeks
- Weight at study initiation: Males 114 - 151g; Females 113 - 140g
- Fasting period before study: NDA
- Housing: The animals were housed in groups of five by sex in polypropylene grid-floor cages suspended over trays lined with absorbent paper.
- Diet (e.g. ad libitum): ad libitum. A pelleted diet (SQC Rat and Mouse Diet No. 1 Expanded, Special Diet Services Limited, Witham, Essex, U.K.) was used.
- Water (e.g. ad libitum): ad libitum. Mains water was supplied from polycarbonate bottles attached to the cage
The diet and drinking water were considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 24°C
- Humidity (%):40 - 67% relative
- Air changes (per hr): approximately 15
- Photoperiod (hrs dark / hrs light): 12 hours continuous light and 12 hours darkness
Environmental conditions were monitored and recorded daily.
IN-LIFE DATES: From: To: 10 August 1989 – 7 September 1989

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
distilled
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Four groups, each of ten rats (five males and five females) were dosed as follows:

Group Number Dose Level (mg/kg/day) Treatment Volume (mL/kg) Concentration (mg/mL) Treatment Period

1 0 (control) 5 0 28 days
2 50 5 10 28 days
3 250 5 50 28 days
4 1233 5 246.6 28 days

The test material was administered daily, for twenty-eight consecutive days, by gavage using a metal cannula attached to a disposable plastic syringe. Control animals were treated in an identical manner with 5 mL/kg/day of distilled water.
The volume of test and control material administered to each animal was based on the most recent bodyweight and was adjusted at weekly intervals.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
For the purpose of this study the test material was prepared at the appropriate concentrations, as a suspension in distilled water. The stability and homogeneity of the test material formulations were determined by Safepharm Analytical Laboratory. Results show the formulation to be stable for at least nine days. Formulations were therefore prepared weekly and stored at 4°C in the dark.
Samples were taken of each test material formulation and were analysed for concentration of Substance 1658/5 at Safepharm Analytical Laboratory.

METHOD OF ANALYSIS
Summary
Substance 1658/5 is zinc hydroxystannate. The concentration in the test material formulations was determined by atomic absorption spectroscopy, determining the zinc levels in the formulations and comparing them to Substance 1658/5 standards.
Samples
The test material formulations were diluted with 10% hydrochloric acid such that the final solution contained 5 to 10 ppm zinc.
Standards
Standard solutions were prepared in 10% hydrochloric acid at a level of 5 to 10 ppm zinc.
Procedure
The zinc concentrations in the samples and standards were determined by atomic absorption spectroscopy using the following conditions:

Light source: Hollow cathode
Lamp current: 3 mA
Wavelength: 213.9 nm
Slit width: 320 µn
Bandpass: 1 nm
Burner head: Single slot
Flame description: Air-acetylene, oxidising, fuel lean, blue
Photomultiplier voltage: 0.2 - 0.8 V
Homogeneity Determinations
The test material formulations were thoroughly mixed by placing in an ultrasonic bath for 5 minutes and mixing thoroughly with a glass rod. Samples were then taken from three positions within the formulation container; top, middle and bottom, in triplicate. In between each sample the formulation was mixed by inversion for 30 seconds.
Stability Determinations
The test material formulations were sampled and analysed in triplicate. A similar analysis was performed after storage at approximately 4°C in the dark for nine days.
Duration of treatment / exposure:
28 days
Frequency of treatment:
Daily
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 50, 250 and 1233 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
5 males and 5 females
Control animals:
yes, concurrent vehicle
Details on study design:
The rat was selected for this study as it is a readily available rodent species, historically used in safety evaluation studies and is acceptable to appropriate regulatory authorities.
The dose levels were chosen based on the results of a range-finding study. The oral route was selected by the study sponsor as a possible route of human exposure and the results of the study are believed to be of value in predicting the likely toxicity of the test material to man.
The animals were randomly allocated to groups of five by sex and the treatment groups allocated to each cage using random letter tables. The group mean bodyweights were then determined to ensure similarity between the dose groups.
The animals were uniquely identified within the study, by an ear punching system routinely used in the test laboratories. Colour coded cage labels were used to assist recognition of dose groups

Examinations

Observations and examinations performed and frequency:
Clinical Signs
All animals were examined for overt signs of toxicity, ill-health or behavioural change immediately before dosing and one and five hours after dosing during the working week. Animals were observed immediately before dosing and one hour after dosing at weekends. All observations were recorded.
Bodyweight
Individual bodyweights were recorded on the day before the start of treatment (day 0) and on days seven, fourteen, twenty-one and twenty-eight. Bodyweights were also recorded at necropsy.
Food Consumption
Food consumption was recorded for each cage group at weekly intervals throughout the study.
Water Consumption
Water intake was observed daily, for each cage group, by visual inspection of the water bottles.

Laboratory Investigations
Haematological and blood chemical investigations were performed on all animals from each test and control group at the end of the study (day 28). Blood samples were obtained by orbital sinus puncture under ether anaesthesia (diethyl ether, BDH Chemicals Limited, Poole, U.K.). Animals were not fasted prior to sampling.
Haematology
The following parameters were measured on blood collected into tubes containing lithium heparin anti-coagulant:
Haemoglobin (Hb)
Haematocrit (Hct)
Erythrocyte count (RBC)
Total leucocyte count (WBC)
Erythrocyte indices - mean cell haemoglobin (MCH)
- mean cell volume (MCV)
- mean cell haemoglobin concentration (MCHC)

Samples were withdrawn into tubes containing potassium EDTA anticoagulant for platelet counts (PLT) and differential leucocyte counts. Clotting time (CT) was assessed by Hepato Quick time using samples collected into sodium citrate solution (0.11 mol/L).
Blood Chemistry
The following parameters were measured on plasma from blood collected into tubes containing lithium heparin anti-coagulant:
Blood urea
Total protein
Albumin
Albumin/globulin ratio (by calculation)
Sodium
Potassium
Chloride
Calcium
Inorganic phosphorus
Creatinine
Alkaline phosphatase (AP)
Alanine aminotransferase (ALAT)
Aspartate aminotransferase (ASAT)
Glucose
Total bilirubin
Sacrifice and pathology:
Pathology
On completion of the dosing period all animals were killed by intravenous administration of sodium pentobarbitone solution (Sagatal, 60 mg/mL; May and Baker Limited, Dagenham, Essex, U.K.) followed by exsanguination.
All animals were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.
Organ Weights
The following organs from animals that were killed at the end of the study, dissected free from fat, were weighed before fixation.
Adrenals
Kidneys
Brain
Pituitary
Liver
Gonads
Spleen
Heart
Histopathology

Samples of the following tissues were removed from all animals and preserved in 10% buffered formalin.

Adrenals
Aorta (thoracic)
Bone & Bone Marrow (femur)
Brain
Caecum
Colon
Duodenum
Eyes
Heart
Ileum
Jejunum
Kidneys
Liver
Lungs
Lymph nodes (cervical and mesenteric)
Gross lesions
Muscle (skeletal)
Oesophagus
Ovaries
Pancreas
Pituitary
Rectum
Sciatic nerve
Skin (hind limb)
Spleen
Stomach
Testes
Thymus
Thyroid/parathyroid
Trachea
Urinary bladder

All tissues were despatched to Experimental Pathology Services, Willow Court, Netherwood Road, Rotherwas, Hereford, U.K. for processing. The following preserved tissues from all high dose and control group animals (groups 1 and 4) were prepared as paraffin blocks, sectioned at nominal thickness of 5 µm and stained with haematoxylin and eosin for subsequent microscopic examination.
Adrenals
Heart
Kidneys
Liver
Spleen

Macroscopically observed lesions were also processed in addition to the above.
Statistics:
Data were processed to give group mean values and standard deviations where appropriate.
Relative organ weights, haematological and blood chemical data were analysed by one way analysis of variance incorporating 'F-max' test for homogeneity of variance. Data showing heterogenous variances were analysed using Kruskall Wallis non-parametric analysis of variance and Mann Whitney U-Test.
Probability values were calculated as:
p < 0.001 ***
p < 0.01 **
p < 0.05 *
p≥ 0.05 (not significant)

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
No clinically observable signs of toxicity were noted during the study.
Mortality:
no mortality observed
Description (incidence):
No clinically observable signs of toxicity were noted during the study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
All animals showed normal gains in bodyweight throughout the study period. Bodyweight gain in test animals was comparable with that seen in controls.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There was no apparent effect on food consumption during the study.
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
Food efficiency in test animals was comparable with that in controls.
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Visual inspection of water bottles revealed no overt intergroup differences.
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
There were no statistically or toxicologically significant changes in the haematological parameters measured in test animals compared with controls.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
There were no blood chemical changes which could be considered toxicologically significant.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There were no changes in the organ weights measured which could be considered attributable to treatment with the test material
Gross pathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No treatment-related changes were observed.
Histopathological findings: neoplastic:
no effects observed
Details on results:
Mortality Data
There were no treatment-related deaths but one female from the intermediate dose group was killed in extremis on day 28 due to an eye injury caused during blood sampling.
Blood Chemistry,
Alanine aminotransferase levels showed a slight but statistically significant reduction in intermediate and high dose males. All values were within the normal range for rats of this strain and age and in any event a reduced level of this enzyme is unlikely to be indicative of any adverse effect.
The only other statistically significant difference between test and control groups was confined to intermediate dose males and as such showed no dose-related response.
Necropsy.
The intermediate dose female killed on day 28 showed ocular damage and thickening of the gastric epithelium. The gastric change was probably autolytic in nature and was not apparent in any of the animals killed on day 29. No treatment-related macroscopic abnormalities were detected.
Organ Weights
There were no changes in the organ weights measured which could be considered attributable to treatment with the test material. High and intermediate dose females showed a statistically significant reduction in pituitary weights relative to bodyweight compared with controls. These differences were slight and probably represent normal variation.
The other minor statistically significant difference between test and control groups was confined to the low dose group and, as such, showed no dose-related response.
Histopathology
All findings were entered into the ROELEE84 pathology computerisation system for tabulation and report production.
No treatment-related changes were observed.
All morphological changes were those commonly observed in laboratory maintained rats of the age and strain employed and, since there were no differences in incidence or severity between control and treatment groups, all were considered to be without toxicological significance.

Effect levels

Dose descriptor:
NOEL
Effect level:
1 233 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Oral administration of the test material, Substance 1658/5, at dose levels of up to 1233 mg/kg/day for twenty-eight consecutive days in the rat, produced no treatment-related changes in the parameters measured.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

REFERENCES

1. Drabkin, D.L. and Austin, J.H., (1932) J. Biol. Chem., 98, 719

2. Gutmann, I., and Bergmeyer N.U. "Methods of Enzymatic Analysis", 2nd Ed. P1791. Verlag Chemie Weinheim and Academic Press Inc.

3. Weichselbaum, T.E. (1946), Amer. J. Clin, Path. 16. 40.

4. Doumas, B.T. et al. (1971) Clin. Chem. Acta. 31. 87.

5. Ray Sarker, B.C., and Chauhan U.P.S. (1967). Anal. Biochem. 20. 155.

6. Mod. acc. to Popper, H., et al (1937). Biochem. Z. 291, 354. Seeling, H.P., and Wust, H., (1969) ArzH. Lab. 15. 34.

7. Wahlefeld, A.W., et al, (1972) Scand. J. Clin. Lab. Invest. 29 (Suppl. 126 Abstr. 11. 12.)

8. Anon. (1970) Z. Klin, Chem. n. Klin. Biochem. 8,658 (1972) ibid 10. 182

9. Bergmeyer, H.U. and Horder, M., (1980) Clin. Chem. Acta. 105, 147.

10. Bergmeyer, H.U., et al. (1978). Clin. Chem. 24. 58.

11. Trinder, P. (1969) Ann. Clin. Biochem. 6. 24.

12. Persijn, J.P., and Van der SUK, W., {1976). J. Clin. Chem. Clin. Biochem. 14. 421. Szasz, G., Persijn, J.P., et al. (1974). Z. Klin. Chem. u. Klin. Biochem. 12. 228.

13. Casarett and Doull "Toxicology 3rd Ed. 1986 Collier Macmillan Pub.

14. Coles, E.H., Veterinary Chemical Pathology 3rd Ed. W.B. Saunders Co. Pub.

15. Eastham, R.D., Biochemical Values in Clinical Medicine, 5th Ed. John Wright & Sons Ltd. Pub.

16. Evelyn, K.A., and Malloy, H.T. (1938). Journal of Biological Chemistry 126,655

17. Reinhold J.S., rtd., Methods in Clinical Chemistry, Vol. I Academic Press, Inc., P. 88, 1953.

Applicant's summary and conclusion

Conclusions:
Oral administration of the test material, at dose levels of up to 1233 mg/kg/day for twenty-eight consecutive days in the rat, produced no treatment-related changes in the parameters measured.
1233 mg/kg/day was, therefore, considered to be the NOEL.
Executive summary:

Oral administration of the test material, Zinc Hydroxystannate, at dose levels of up to 1233 mg/kg/day for twenty-eight consecutive days in the rat, produced no treatment-related changes in the parameters measured.

1233 mg/kg/day was therefore considered to be the NOEL.

The study was designed to comply with Method B7, Annex V of the EEC Commission Directive 84/449/EEC and follows the recommendations of the OECD Guidelines for Testing of Chemicals No. 407 "Repeated Dose Oral Toxicity - Rodent 28-day or 14-day study".