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Administrative data

Endpoint:
short-term repeated dose toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Study period:
08 Feb - 03 March 1989
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1989
Report date:
1989

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)
Version / remarks:
adopted in 1981
Deviations:
yes
Remarks:
Occlusive instead of semi-occlusive conditions
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Ziram
EC Number:
205-288-3
EC Name:
Ziram
Cas Number:
137-30-4
Molecular formula:
C6H12N2S4Zn
IUPAC Name:
ziram

Test animals

Species:
rabbit
Strain:
New Zealand White
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Interfauna U.K. Ltd., Cambridgeshire, UK
- Age at study initiation: 10 - 12 weeks on arrival
- Weight at study initiation: within a weight range of 2.2 - 2.6 kg on arrival
- Housing: All rabbits were housed individually.
- Diet: SQC Rabbit Diet, ad libitum; 10 g of autoclaved hay was given to occasional animals showing a loss of condition.
- Water: Tap water, ad libitum
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.8 - 21.7
- Humidity (%): 45.6 - 50.8
- Air changes (per hr): approximately 19
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Type of coverage:
occlusive
Vehicle:
water
Details on exposure:
TEST SITE
- Area of exposure: mid-dorsal region
- % coverage: approximately 10% of total body surface
- Type of wrap if used: Treatment site was then covered with an impervious bandage consisting of gauze covered with ‘Elastoplast’ elastic adhesive dressing backed with impervious 'Sleek' plaster.
- Time intervals for shavings or clipplings: Hair clipping was carried out approximately 24 h prior to the first application of the test substance.

REMOVAL OF TEST SUBSTANCE
- Washing: with warm tap water
- Time after start of exposure: 6 h

TEST MATERIAL
- Amount(s) applied: 2, 3, and 4 mL for the low, mid and high dose groups; the vehicle control animals received 4 mL of distilled water.
- Constant volume or concentration used: no
- For solids, paste formed: yes, powder was moistened with water
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
21 days for 6 h/day
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control:
no

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily for ill health, behavioural changes or signs of reaction to treatment; twice daily for mortality

DETAILED CLINICAL OBSERVATIONS: No

DERMAL IRRITATION: Yes
- Time schedule for examinations: immediately prior to the first daily application of the test substance and subsequently daily
Local dermal reactions (erythema; edema) resulting from treatment were assessed on a numerical basis according to a modified Draize scoring system.

BODY WEIGHT: Yes
- Time schedule for examinations: prior to dosing and once a week thereafter

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No
Food consumption of all animals was recorded at weekly intervals throughout the study.

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: prior to termination on Day 20; for specified animals procedure was repeated on Day 22
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked: packed cell volume (PCV), haemoglobin (Hb), red blood cell count (RBC), platelet count (Pits), mean corpuscular haemoglobin concentration (MCHC), mean corpuscular volume (MCV), total white blood cell count (WBC), differential white blood cell count (Neutrophils (N), lymphocytes (L), eosinophils (E), basophils (B), monocytes (M)) and thrombotest (TT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: prior to termination on Day 20; for specified animals procedure was repeated on Day 22
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked: glucose, glutamic-pyruvic transaminase (GPT), glutamic-oxaloacetic transaminase (GOT), alkaline phosphatase (AP), total bilirubin, cholesterol (Chol), urea nitrogen (Urea Nitr), total protein, albumin (Alb), globulin (Glob), albumin/globulin ratio (A/G), sodium (Na), potassium (K), calcium (Ca), chloride (CI), inorganic phosphorus (P) and creatinine

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
After 21 days of treatment (Days 22 or 23), all animals were finally killed by i.v. administration of an overdose of pentobarbitone sodium. A complete autopsy was performed on all animals. Adrenals, kidneys, liver, ovaries and testes with epididymides of all animals (males, females) were dissected free of fat and weighed.

HISTOPATHOLOGY: Yes
Histopathological evaluation of the following tissues was carried out in animals (males, females) of the high dose group and control group: kidneys, liver, skin and abnormal tissues.
Tissues were preserved in 10% buffered formalin.
Fixed tissue samples required for microscopic examination were embedded in paraffin wax (m. p. 56 °C), sections were cut at 4 µm and stained with haematoxylin and eosin.
Statistics:
All analyses were carried out separately for male and female rabbits.
The following tests were used for food and water consumption, bodyweight, relative organ weight and clinical pathology data:
- If the data consisted predominantly of one particular value (relative frequency of the mode exceeds 75%), the proportion of animals with values different from the mode was analysed by appropriate methods. Otherwise:
- Bartlett’s test was applied to test for heterogeneity of variance between treatments. Where significant (at the 1% level) heterogeneity was found, a logarithmic transformation was tried to see if a more stable variance structure could be obtained.
- If no significant heterogeneity was detected (or if a satisfactory transformation was found), a one-way analysis of variance was carried out. If significant heterogeneity of variance was present, and could not be removed by a transformation, the Kruskal-Wallis analysis of ranks was used.
- Analyses of variance were followed by a Student’s ‘t’ test and Williams’ test for a dose-related response, although only the one thought most appropriate for the response pattern observed has been reported. The Kruskal-Wallis analyses were followed by the non-parametric equivalents of the ‘t’ test and Williams’ test (Shirleys’ test).
Where appropriate for organ weight data, analysis of covariance was used in place of analysis of variance.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
No clinical signs of reaction to treatment were observed.
Dermal irritation:
no effects observed
Description (incidence and severity):
No dermal reactions to treatment were observed amongst rabbits of the control and test groups. Infrequent, transient cases of staining (light brown) of the dose site were recorded amongst animals of the high dose group (1000 mg/kg bw/day).
Mortality:
no mortality observed
Description (incidence):
There were no test substance-related effects on mortality.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weight losses or reduced body weight gain were recorded for female rabbits of the high-dose group during the dosing period. Compared to the female controls, differences were statistically significant for all three weeks. No other significant differences in body weight between animals receiving the test substance and the controls were noted.
For details, please refer to attachment 1 under "Overall remarks, attachments".
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
A statistically significant reduction in food consumption during study Week 1 was recorded for female rabbits in the high-dose group in comparison with the female controls. Food consumption for these animals was also reduced (but did not achieve statistical significance) during study weeks 2 and 3. For all other treated animals food consumption was similar to that of the control animals.
For details, please refer to attachment 2 under "Overall remarks, attachments".
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
A treatment-related lower lymphocyte count was observed for animals treated with 1000 mg/kg bw/day, reaching statistical significance in female rabbits, but not in male rabbits. There were no other statistically significant differences between rabbits treated with the test substance and rabbits of the respective control group.
For details, please refer to attachment 3 under "Overall remarks, attachments".
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Increases in the liver enzymes GPT and GOT were observed in males and females treated with 300 and 1000 mg/kg bw/day. The increase was statistically significant for GPT in females treated with 1000 mg/kg bw/day and for GOT in females treated with 300 and 1000 mg/kg bw/day. Females treated with 1000 mg/kg bw/day also showed statistically significantly increased levels of bilirubin. Statistically significantly increased levels of cholesterol were observed for male and female animals treated with 1000 mg/kg bw/day.
Overall, a clear effect on biochemical parameters was observed amongst rabbits of the high-dose groups. Amongst females of the mid-dose group, the occurrence of a real effect on liver enzymes was further supported by individual data. Individual values above the laboratory control range were observed for GOT in 3/5 females and for GPT in 1/5 females (Laboratory 99 percentiles for female rabbits: GOT, 42 mU/mL; GPT, 99 mU/mL).
For details, please refer to attachment 3 and 6 under "Overall remarks, attachments".
Endocrine findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There were no statistically significant differences between animals treated with the test substance and the controls. For details, please refer to attachment 4 under "Overall remarks, attachments".
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
The macroscopic examination performed at termination revealed no changes that were attributable to treatment with the test substance.
An increased incidence of irregular cortical scarring of the kidneys was recorded amongst rabbits of all groups. However, this was not considered to be related to treatment with the test substance.
For details, please refer to attachment 5 under "Overall remarks, attachments".
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
In microscopic pathology, no changes were revealed that were attributable to treatment with the test substance.
One female rabbit of the high-dose group showed localised area of ulceration and minimal acanthosis was considered most probably due to physical trauma, since similar changes were not observed throughout this treated site.
Microscopic changes seen in other tissues examined were considered to be spontaneous in origin and therefore of no toxicological importance.
For details, please refer to attachment 5 under "Overall remarks, attachments".
Histopathological findings: neoplastic:
not examined

Effect levels

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
general systemic toxicity
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed at this dose level
Key result
Dose descriptor:
LOAEL
Remarks:
general systemic toxicity
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical biochemistry
other: additionally, changes in body weight (females), food consumption (females) and haematological parameters (males and females) were observed at 1000 mg/kg bw/day.
Key result
Dose descriptor:
NOAEL
Remarks:
local effects
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No dermal irritation observed at the highest dose level tested.
Remarks on result:
other: corresponding to a NOAEL for local effects of 11.81 mg/cm².
Remarks:
The local NOAEL of 11.81 mg/cm² was calculated retrospectively according to Derelanko, M. J., The toxicologist’s pocket handbook, CRC Press, 2008 and is based on a body surface area of 2540 cm² for a 3 kg rabbit. The local NOAEL of 1000 mg/kg bw/day corresponds to an amount of 3000 mg test substance for a rabbit (3 kg) and divided by 10% of the body surface area (254 cm²), this results in a local NOAEL of 11.81 mg/cm².

Target system / organ toxicity

Key result
Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
The study was conducted similar to OECD guideline 410 and under GLP. Under the conditions of the test, the test substance caused no dermal irritation when applied dermally for three weeks to the intact skin of male and female rabbits up to and including 1000 mg/kg bw/day. Treatment-related disturbances in body weight, food consumption and various haematological and biochemical parameters were observed for rabbits receiving the test substance at 1000 mg/kg bw/day although, in the main, statistical significance was only achieved for females. Amongst animals receiving 300 mg/kg bw/day, females showed increased enzyme levels, in particular GOT. Thus, 1000 mg/kg bw/day was regarded as the NOAEL for local skin effects and the NOAEL for systemic effects was 100 mg/kg bw/day for males and females.