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Description of key information

According to the acute toxic class method an LD50 between 50 and 300 mg/kg bw was determined (TOXI-COOP, 2017).

Key value for chemical safety assessment

Acute toxicity: via oral route

Link to relevant study records
Reference
Endpoint:
acute toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-01-31 to 2017-02-18
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)
Version / remarks:
2001-12-17
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Test type:
acute toxic class method
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and batch No. of test material: OUCHI SHINKO CHEMICAL INDUSTRIAL CO., LTD.; 608011
- Expiration date of the lot/batch: 2017-08-31

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Keep to the storeroom with suitable ventilation. Avoid fire, direct sunlight and moisture. Store at room temperature.
Species:
rat
Strain:
Wistar
Sex:
female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Toxi-Coop Zrt. 1103 Budapest, Cserkesz u. 90., Hungary
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 11 weeks old
- Weight at study initiation:
Body weight range at starting (first group): 230-236 g
Body weight range at starting (second group): 242-261 g
Body weight range at starting (third group): 239-244 g
Body weight range at starting (fourth group): 249-258 g
- Fasting period before study: Yes, food was withheld overnight
- Housing: 3 animals/cage
- Diet (e.g. ad libitum): ad libitum, ssniff® SM R/M-Z+H complete diet (ssniff Spezialdiäten GmbH, D-59494 Soest Germany)
- Water (e.g. ad libitum): ad libitum, tap water
- Acclimation period: 26 days for group 1, 27 days for group 2, 28 days for group 3 and 29 days for group 4

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
other: Helianthi annui oleum raffinatum
Remarks:
refined sunflower oil
Details on oral exposure:
VEHICLE
- Concentration in vehicle: 5, 30 and 200 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg bw
- Lot/batch no.: 8001528001

CLASS METHOD
- Rationale for the selection of the starting dose: The starting dose was selected on the basis of the available information about the test item. A full test was performed.
Doses:
5, 50, 300, 2000 mg/kg bw
No. of animals per sex per dose:
3
Control animals:
no
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: once daily; weighing was performed on days 0, 1, 7 and 15
- Necropsy of survivors performed: yes
- Other examinations performed: clinical signs, body weight
Statistics:
Not applicable
Key result
Sex:
female
Dose descriptor:
approximate LD50
Effect level:
>= 50 - <= 300 mg/kg bw
Based on:
test mat.
Mortality:
All female rats dosed at 2000 mg/kg bw died on the treatment day. Two animals (No.: 4257, 4260) died 4 hours after the treatment and animal No.: 4262 died 3 hours after the treatment. All females dosed at 300 mg/kg bw died on Day 1. All deaths might be a consequence of systemic toxic effect of the test item. No mortality occurred at 50 mg/kg bw single oral dose of the test item. All rats in step 3 and step 4 survived until the end of the 14-day observation period.
Clinical signs:
In group 1 treated with 2000 mg/kg bw clinical signs comprised of decreased activity (11 cases out of 11 observations), prone position (4/11), lateral position (8/11), decreased righting reflex (11/11), decreased grip- and limb tone (11/11), decreased body tone (11/11), decreased abdominal tone (11/11), closed eyes (2/11) and increased respiration rate (11/11). These symptoms were detected on the treatment day between 30 minutes and 3 hours after the treatment. All symptoms were regarded as test item related.
In group 2 treated with 300 mg/kg bw clinical signs comprised of decreased activity (15 cases out of 15 observations), tremor (6/15), incoordination (4/15), prone position (2/15), lateral position (9/15), decreased righting reflex (12/15), decreased grip- and limb tone (12/15), decreased body tone (12/15), decreased abdominal tone (12/15), lacrimation (3/15), piloerection (7/15) and increased respiration rate (8/15). These symptoms were detected on the treatment day between 30 minutes and 4 hours after the treatment. All symptoms were regarded as test item related. No treatment related symptoms were observed in the 50 mg/kg bw test item dose groups (group 3 and 4) throughout the 14-day post-treatment period.
Body weight:
The mean body weight and body weight gain data of group 1 (2000 mg/kg bw) and of group 2 (300 mg/kg bw) could not be evaluated, because all rats died. In group 3 and 4 (50 mg/kg bw) the mean body weight and body weight gain of the animals corresponded to their species and age throughout the study.
Gross pathology:
All rats treated with 2000 mg/kg bw and all rats treated with 300 mg/kg bw died spontaneously during the study. All animals treated with 50 mg/kg dose of test item survived until the scheduled necropsy on Day 15.
External necropsy findings as blood around the nose (No.: 4270), lacrimation around the eyes (No.: 4288, 4289) and thin faeces around the anus (No.: 4289) were found in group 2 (300 mg/kg bw). Internal macroscopic changes like pale and yellowish liver and pale kidneys were recorded in animal No.: 4288 of same dose group. Besides, nutmeg like pattern on the liver as internal necropsy finding was observed in animal No.: 4289 of group 2. The external macroscopic changes observed were regarded as test item related. The internal alterations could not be related to the toxic test item effect, but were regarded as individual variation. Moderate hydrometra was detected in two animals (No.: 4278, 4283) of group 4 (50 mg/kg bw). Hydrometra is a physiological finding and connected to the estrous cycle of the animal. No pathological changes were found related to the effect of the test item during the macroscopic examination of animals of group 1 (2000 mg/kg bw) and of group 3 and 4 (50 mg/kg bw).

Three animals died in group 1 treated with 2000 mg/kg dose of the test item. Three animals died in group 2 treated with 300 mg/kg dose. No death occurred after the single 50 mg/kg bw oral dose of Zinc di(benzimidazol-2-yl) disulphide. The observed clinical signs and external necropsy findings observed in the 300 mg/kg bw dose group were regarded as test item related. There was no effect of the test item found in body weights and body weight gains of the surviving animals (50 mg/kg bw) during the study.

Interpretation of results:
Category 3 based on GHS criteria
Conclusions:
Based on the above acute toxic class method conducted in rats, the LD50 must be between 50 and 300 mg/kg bw.
Executive summary:

An acute oral toxicity study was carried out using the class method according to OECD guideline 423 (TOXICOOP, 2017). The method was conducted using a stepwise procedure with the use of 2000 mg/kg bw as the starting dose in three female rats. The starting dose was selected on the basis of the available information about the test item. All female animals died and the test was continued at 300 mg/kg bw dose level on further three female rats. All females died and the test was continued at 50 mg/kg bw dose level on further three female rats. No animal died in the third step at 50 mg/kg bw dose level, therefore three further female rats were treated with the same (50 mg/kg bw) dose. No animal died in the fourth step, too, so the test was finished, because the stopping criteria of Annex 2d of OECD Guideline No. 423 (presented in Appendix 7) were met. Animals were weighed and observed for lethality and toxic symptoms for 14 days after the treatment. Gross pathological examination was carried out in animals that died on treatment day (Day 0) and on Day 1, as well as in animals that survived until the end of the observation period.

Lethality, Clinical symptoms and Body weight:

All three animals treated with 2000 mg/kg bw Zinc di(benzimidazol-2-yl) disulphide died on the treatment day between 3 and 4 hours after the treatment. All three animals treated with 300 mg/kg bw died on Day 1. No lethality was noted at a single oral dose of 50 mg/kg bw. At a dose level of 2000 mg/kg bw, CNS - and emotion symptoms (decreased activity, closed eyes), disturbances of the coordination (prone- and lateral position), decreased righting reflex, decreased muscular tension (grip-, limb-, abdominal- and body tone) and disturbance of the autonomic functions (increased respiration rate) were observed in animals on the treatment day between 30 minutes and 3 hours after the treatment. At a dose level of 300 mg/kg bw, CNS - and emotion symptoms (decreased activity, tremor), disturbances of the coordination (incoordination, prone- and lateral position), decreased righting reflex, decreased muscular tension (grip-, limb-, abdominal- and body tone) and disturbances of the autonomic functions (lacrimation, piloerection, increased respiration rate) were observed in animals on the treatment day between 30 minutes and 4 hours after the treatment. At a dose level of 50 mg/kg bw no clinical symptoms were observed on the day of the treatment and during the 14-day observation period. The general state and behaviour of all experimental animals were normal. The body weight development was normal in all surviving animals.

Gross pathology:

Altogether 6 animals died, and 6 animals were sacrificed according to schedule at the end of the study period. External macroscopic findings as blood around the nose, lacrimation around the eyes and thin faeces around the anus were observed in the 300 mg/kg bw group.

Internal findings included pale and yellowish liver and pale kidneys. A nutmeg like pattern on the liver was recorded in same dose. While the external findings were regarded as test item related, the internal changes could not be related to a toxic test item effect, but were regarded as individual variation. Internal findings as pale liver and hydrometra were detected in 50 mg/kg bw dose. Hydrometra is a physiological finding and connected to the estrous cycle of the animal. All of the animals treated with 2000 mg/kg bw or 50 mg/kg bw proved to be free of treatment related gross pathological changes.

Evaluation:

The method used is not intended to allow for the calculation of a precise LD50 value. However for this acute oral toxicity study with the test item Zinc di(benzimidazol-2-yl) disulphide in rats the determined LD50 is between 50 and 300 mg/kg bw. The test item is ranked into the following class according to current EU Regulation on classification, labeling and packaging (CLP) (EC) No 1272/2008:

Dose (mg/kg bw)

Mortality (dead/treated)

LD50 (mg/kg bw)

CLP category

50

0/6

between 50 and 300

3

Endpoint conclusion
Endpoint conclusion:
no study available

Acute toxicity: via inhalation route

Endpoint conclusion
Endpoint conclusion:
no study available

Acute toxicity: via dermal route

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

An acute oral toxicity study was carried out using the class method according to OECD guideline 423 (TOXICOOP, 2017). The method was conducted using a stepwise procedure with the use of 2000 mg/kg bw as the starting dose in three female rats. The starting dose was selected on the basis of the available information about the test item. All female animals died and the test was continued at 300 mg/kg bw dose level on further three female rats. All females died and the test was continued at 50 mg/kg bw dose level on further three female rats. No animal died in the third step at 50 mg/kg bw dose level, therefore three further female rats were treated with the same (50 mg/kg bw) dose. No animal died in the fourth step, too, so the test was finished, because the stopping criteria of Annex 2d of OECD Guideline No. 423 were met. Animals were weighed and observed for lethality and toxic symptoms for 14 days after the treatment. Gross pathological examination was carried out in animals that died on treatment day (Day 0) and on Day 1, as well as in animals that survived until the end of the observation period.

Lethality, Clinical symptoms and Body weight:

All three animals treated with 2000 mg/kg bw Zinc di(benzimidazol-2-yl) disulphide died on the treatment day between 3 and 4 hours after the treatment. All three animals treated with 300 mg/kg bw died on Day 1. No lethality was noted at a single oral dose of 50 mg/kg bw. At a dose level of 2000 mg/kg bw, CNS - and emotion symptoms (decreased activity, closed eyes), disturbances of the coordination (prone- and lateral position), decreased righting reflex, decreased muscular tension (grip-, limb-, abdominal- and body tone) and disturbance of the autonomic functions (increased respiration rate) were observed in animals on the treatment day between 30 minutes and 3 hours after the treatment. At a dose level of 300 mg/kg bw, CNS - and emotion symptoms (decreased activity, tremor), disturbances of the coordination (incoordination, prone- and lateral position), decreased righting reflex, decreased muscular tension (grip-, limb-, abdominal- and body tone) and disturbances of the autonomic functions (lacrimation, piloerection, increased respiration rate) were observed in animals on the treatment day between 30 minutes and 4 hours after the treatment. At a dose level of 50 mg/kg bw no clinical symptoms were observed on the day of the treatment and during the 14-day observation period. The general state and behaviour of all experimental animals was normal. The body weight development was normal in all surviving animals.

Gross pathology:

Altogether 6 animals died, and 6 animals were sacrificed according to schedule at the end of the study period. External macroscopic findings as blood around the nose, lacrimation around the eyes and thin faeces around the anus were observed in the 300 mg/kg bw group.

Internal findings included pale and yellowish liver and pale kidneys. A nutmeg like pattern on the liver was recorded in same dose. While the external findings were regarded as test item related, the internal changes could not be related to a toxic test item effect, but were regarded as individual variation. Internal findings as pale liver and hydrometra were detected in 50 mg/kg bw dose. Hydrometra is a physiological finding and connected to the estrous cycle of the animal. All of the animals treated with 2000 mg/kg bw or 50 mg/kg bw proved to be free of treatment related gross pathological changes.

Evaluation:

The method used is not intended to allow for the calculation of a precise LD50 value. However for this acute oral toxicity study with the test item Zinc di(benzimidazol-2-yl) disulphide in rats the determined LD50 is between 50 and 300 mg/kg bw. The test item is ranked into the following class according to current EU Regulation on classification, labeling and packaging (CLP) (EC) No 1272/2008:

Dose (mg/kg bw)

Mortality (dead/treated)

LD50 (mg/kg bw)

CLP category

50

0/6

between 50 and 300

3

Justification for classification or non-classification

Based on the available experimental data, the test item is considered to be classified as acutely toxic cat. 3, H301 (Toxic if swallowed) under Regulation (EC) No 1272/2008.