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Description of key information

For the acute oral toxicity study with the test item in rats the determined LD50 is between 300 and 2000 mg/kg bw.

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:
2015-11-11 to 2016-01-13
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)
Version / remarks:
2001
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.1 tris (Acute Oral Toxicity - Acute Toxic Class Method)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.1100 (Acute Oral Toxicity)
Version / remarks:
2002
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test type:
acute toxic class method
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Toxi-Coop Zrt. 1103 Budapest, Cserkesz u. 90
- Females nulliparous and non-pregnant: yes
- Age at study initiation: Young adult rats, 11 weeks old in group 1, group 2 and group 3
- Weight at study initiation: first step: 232-237 g; second step: 217-233 g; third step: 235-246 g
- Fasting period before study: The day before treatment the animals were fasted. The food but not water was withheld overnight. Animals were weighed before the application and the food was given back 3 hours after the treatment.
- Housing: 3 animals/sex/cage, type II polypropylene/polycarbonate; rat type cages with a solid floor, stainless steel wire covers and self-feeding baskets. Environmental conditions were maintained by an air-conditioning system. Temperature and relative humidity were verified and recorded daily during the study.
- Diet: The animals received ssniff® SM R/M-Z+H complete diet produced by ssniff Spezialdiäten GmbH, D-59494 Soest Germany, ad libitum. The food is periodically analysed and is considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study. Copies of the relevant Certificates of Analysis are maintained in Toxi-Coop Zrt.’s archive.
- Water: Animals received tap water from watering bottles ad libitum. The drinking water is periodically analysed and is considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study. Copies of the relevant Certificates of Analysis are maintained in Toxi-Coop Zrt.’s archive.
- Acclimation period: 26 days in first step, 27 days in second step and 28 days in third step

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity: 30 - 70 %
- Air changes: above 10 air exchanges/hour by central air-condition system.
- Photoperiod: Artificial light, from 6 am. to 6 pm.

IN-LIFE DATES: From: 2015-10-22 (first, second and third group) To: 2015-11-17 (first group); 2015-12-03 (second group); 2015-12-04 (third group)
Route of administration:
oral: gavage
Vehicle:
other: Helianthi annui oleum raffinatum
Details on oral exposure:
VEHICLE
- Concentration in vehicle: 200 and 30 mg/mL
- Amount of vehicle: treatment volume of 10 mL/kg bw
- Lot/batch no.: 1506-4604
- Expiry date: 02.06.2016
- Manufactured: Parma Produkt Kft.

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.
Doses:
2000 mg/kg bw; 300 mg/kg bw
No. of animals per sex per dose:
3 females
Control animals:
no
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: The body weight were recorded on day 0 (shortly before the treatment), on day 7 and on day 15 on all animals with a precision of 1 g, respectively.
- Necropsy of survivors performed: yes
- Other examinations performed:
- Clinical observations: Animals were observed individually after dosing once during the first 30 minutes, then 1 h, 2 h, 3 h, 4 h, after the treatment and once per day for 14 days thereafter. Individual observations were performed on the skin and fur, eyes and mucous membranes and also respiratory, circulatory, autonomic and central nervous system, somatomotor activity and behaviour pattern. Particular attention was directed to observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.
- Mortality: Inspection for signs of morbidity and mortality were made twice daily at the beginning and end of the working day.
- Gross Pathology: All animals were subjected to gross pathology. All surviving animals were exsanguinated under isoflurane anaesthesia. After examination of the external appearance the cranial, thoracic and abdominal cavities were opened and the appearance of the tissues and organs were observed. All gross pathological changes were recorded for each animal on the post mortem record sheets.
Key result
Sex:
female
Dose descriptor:
LD50
Effect level:
> 300 - < 2 000 mg/kg bw
Based on:
test mat.
Mortality:
All female rats dosed at 2000 mg/kg bw with the test item died on the treatment day 30 minutes after the treatment. All deaths might be a consequence of systemic toxic effect of the test item. No mortality occurred at 300 mg/kg single oral dose of the test item. All rats in step 2 and step 3 survived until the end of the 14-day observation period.
Clinical signs:
In group 1 treated with 2000 mg/kg bw dose clinical signs comprised of clonic convulsion (1 case out of 1 observation). This symptom (score +2) was observed in animal No.: 286 on the treatment day 30 minutes after the treatment and it was regarded as a test item related effect.

In group 2 treated with 300 mg/kg bw dose clinical signs comprised of decreased activity (7 cases out of 57 observations), vocalisation (11/57), irritability (15/57), tremor (15/57), incoordination (15/57) and decreased body temperature (3/57). Decreased activity (score -1; -2), vocalisation (score +2), irritability (score +1; +2), tremor (score +1; +2), incoordination (score +1; +2), and decreased body temperature (-1) were observed in all animals. These symptoms were observed on the treatment day between 30 minutes and 4 hours after the treatment and they were regarded as test item related effects.

In group 3 treated with 300 mg/kg bw dose clinical signs comprised of decreased activity (5 cases out of 57 observations), vocalisation (12/57), irritability (12/57), tremor (15/57), incoordination (15/57) and decreased body temperature (3/57). Decreased activity (score -1; -2), vocalisation (score +1; +2), irritability (score +1; +2), tremor (score +1; +2), incoordination (score +1; +2), and decreased body temperature (-1) were observed in all animals. These symptoms were observed on the treatment day between 30 minutes and 4 hours after the treatment and they were regarded as test item related effects.
Body weight:
The body weight and body weight gain data of group 1 (2000 mg/kg) could not be evaluated, because all rats died in this group. In group 2 and 3 (300 mg/kg) 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 dose of the test item spontaneously died during the study. All animals treated with 300 mg/kg dose of test item survived until the scheduled necropsy on Day 15. Slight hydrometra was found in female No.: 259 of the group 3 (300 mg/kg bw). Hydrometra is physiological finding and connected to the cycle of the animal. No pathological changes were found related to the test item during the macroscopic examination of animals of group 1 (2000 mg/kg bw), group 2 (300 mg/kg bw) and group 3 (300 mg/kg bw).
Interpretation of results:
Category 4 based on GHS criteria
Conclusions:
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 in rats the determined LD50 is between 300 and 2000 mg/kg bw.
Executive summary:

An acute oral toxicity study was carried out using the class method according to OECD guideline 423. 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. Since three female animals died the test was continued at 300 mg/kg bw dose level on further three female rats. No animal died in the second step at 300 mg/kg bw dose level, three further female rats were treated with the same (300 mg/kg bw) dose. No animal died in the third step, too, so the test was finished, the stopping criteria of Annex 2d of OECD Guideline No. 423 were met. Animals were weighed, observed for lethality and toxic symptoms for 14 days after the treatment. Gross pathological examination was carried out in animals died on the treatment day, as well as 15th day after the treatment in surviving animals. All of three animals treated with 2000 mg/kg bw of the test item died on the treatment day 30 minutes after the treatment. No lethality was noted at single oral dose of 300 mg/kg bw. In the first step at a dose level of 2000 mg/kg bw, CNS - and emotion symptom as clonic convulsion was observed in one animal on the treatment day 30 minutes after the treatment. In the second step at a dose level of 300 mg/kg bw, CNS - and emotion symptoms (decreased activity, vocalisation, irritability, tremor), disturbance of the coordination (incoordination) and of the autonomic functions (decreased body temperature) were observed in animals on the treatment day between 30 minutes and 4 hours after the treatment. In the third step at a dose level of 300 mg/kg bw, CNS - and emotion symptoms (decreased activity, vocalisation, irritability, tremor), disturbance of the coordination (incoordination) and of the autonomic functions (decreased body temperature) were observed in animals on the treatment day between 30 minutes and 4 hours after the treatment. The body weight development was normal in all surviving animals. Altogether 3 animals died, and 6 animals were sacrificed scheduled during the study. All organs of the animals treated with 2000 mg/kg bw or 300 mg/kg bw dose proved to be free of treatment related gross pathological changes.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Quality of whole database:
GLP and guideline study

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

Acute oral toxicity:

An acute oral toxicity study was carried out using the class method according to OECD guideline 423. 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. Since three female animals died the test was continued at 300 mg/kg bw dose level on further three female rats. No animal died in the second step at 300 mg/kg bw dose level, three further female rats were treated with the same (300 mg/kg bw) dose. No animal died in the third step, too, so the test was finished, the stopping criteria of Annex 2d of OECD Guideline No. 423 were met. Animals were weighed, observed for lethality and toxic symptoms for 14 days after the treatment. Gross pathological examination was carried out in animals died on the treatment day, as well as 15th day after the treatment in surviving animals. All of three animals treated with 2000 mg/kg bw of the test item died on the treatment day 30 minutes after the treatment. No lethality was noted at single oral dose of 300 mg/kg bw. In the first step at a dose level of 2000 mg/kg bw, CNS - and emotion symptom as clonic convulsion was observed in one animal on the treatment day 30 minutes after the treatment. In the second step at a dose level of 300 mg/kg bw, CNS - and emotion symptoms (decreased activity, vocalisation, irritability, tremor), disturbance of the coordination (incoordination) and of the autonomic functions (decreased body temperature) were observed in animals on the treatment day between 30 minutes and 4 hours after the treatment. In the third step at a dose level of 300 mg/kg bw, CNS - and emotion symptoms (decreased activity, vocalisation, irritability, tremor), disturbance of the coordination (incoordination) and of the autonomic functions (decreased body temperature) were observed in animals on the treatment day between 30 minutes and 4 hours after the treatment. The body weight development was normal in all surviving animals. Altogether 3 animals died, and 6 animals were sacrificed scheduled during the study. All organs of the animals treated with 2000 mg/kg bw or 300 mg/kg bw dose proved to be free of treatment related gross pathological changes. 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 in rats the determined LD50 is between 300 and 2000 mg/kg bw.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on acute toxicity, the test item is classified and labelled as acute toxic Category 4 (H302: "Harmful if swallowed") according to Regulation (EC) No 1272/2008 (CLP), as amended for the eighth time in Regulation (EU) No 2016/918.