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Administrative data

Description of key information

Acute oral toxicity: Key study. Test method according to OECD 423, GLP study. The LD50 of the test item was found to be higher than 300 mg/kg bw and lower than 2000 mg/kg by oral route in the rat. The LD50 cut-off of the test item may be considered as 1000 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:
03 September 2019 - 01 October 2019
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)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Test type:
acute toxic class method
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Remarks:
(SPF Caw)
Sex:
female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Elevage JANVIER LABS (53940 Le Genest St Isle – France)
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 8 or 9 weeks
- Weight at study initiation: step 1 (2000 mg/kg bw): 189.3 g (SD = 6.3 g); steps 2 and 3 (300 mg/kg bw): 209.7 g (SD = 18.8 g)
- Fasting period before study: Food was removed on D-1 and then redistributed 4 hours after the test item administration.
- Housing: Rats were housed by group of three in solid-bottomed clear polycarbonate cages with a stainless steel mesh lid. Each cage contains sawdust bedding which was changed at least 2 times a week. Each cage was installed in conventional air conditioned animal husbandry.
- Diet (e.g. ad libitum): Ad libitum. Teklad Global 16% Protein Rodent Diet (ENVIGO 2016).
- Water (e.g. ad libitum): Ad libitum. Drinking water (tap-water from public distribution system). Microbiological and chemical analyses of the water were carried out once every six months by Bureau Veritas – Eurofins (FRANCE).
- Acclimation period: the animals were acclimatized for at least 5 days before treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19ºC to 25ºC
- Humidity (%): 30 to 70%
- Air changes (per hr): ≥ 10 air changes/hour
- Photoperiod (hrs dark / hrs light): 12 hours light (07.00 to 19.00) and 12 hours dark cycle.
Route of administration:
oral: gavage
Vehicle:
DMSO
Details on oral exposure:
VEHICLE
- Concentration in vehicle: 30 mg/mL and 200 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg bw
- Justification for choice of vehicle: Dimethyl sulfoxide (DMSO) was chosen as it produced the most suitable formulation at the requested concentration.

MAXIMUM DOSE VOLUME APPLIED: 10 mL/kg bw

DOSAGE PREPARATION (if unusual): The preparations were stirred using a vortex for 1 minute to obtain black solutions just before the administration.

CLASS METHOD (if applicable)
- Rationale for the selection of the starting dose: Available information suggests that mortality is unlikely at the highest starting dose level (2000 mg/kg body weight). Hence, the study is initiated with the starting dose of 2000 mg/kg body weight.


Doses:
300 and 2000 mg/kg bw
No. of animals per sex per dose:
Step 1 (2000 mg/kg bw): 3 animals; Step 2 (300 mg/kg bw): 3 animals; Step 3 (300 mg/kg bw): 3 animals
Control animals:
yes
Remarks:
(study performed on three animals receiving DMSO under requirements of OECD Guideline 423)
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: At each step, the animals were observed four times on test day 0 (day of administration), i.e. at T0+30 min, T0+1h, T0+3h and T0+4h, and once daily during days 1 to 14 post administration. The body weights were recorded on test day 0 (just before administration) then on D2, D7, and D14.
- Necropsy of survivors performed: yes.
- Clinical signs including body weight: spontaneous activity, Preyer’s reflex (noise), respiratory rate, convulsions, tremors, body temperature, muscle tone, palpebral opening, pupil appearance, salivation, lachrymation, righting reflex, back hair appearance. The body weight evolution of animals treated with the test item was compared with the body weight evolution of the control group.
- Other examinations performed: Macroscopic observations were entered on individual autopsy sheets. Only those organs likely to be modified in cases of acute toxicity were examined. No organ was removed and preserved in view to microscopic examinations.
Key result
Sex:
female
Dose descriptor:
LD50
Effect level:
> 300 - <= 2 000 mg/kg bw
Based on:
test mat.
Key result
Sex:
female
Dose descriptor:
LD50 cut-off
Effect level:
1 000 mg/kg bw
Based on:
test mat.
Mortality:
Two mortalities were noted in animals treated at the dose of 2000 mg/kg bw on Day 2 and Day 6. No mortality was noted in animals treated at the dose of 300 mg/kg bw.
Clinical signs:
Step 1 (2000 mg/kg bw): The mortalities were preceded by a decrease in spontaneous activity (2/2), righting reflex (1/2), muscle tones (1/2), associated with hypothermia (1/2), piloerection (1/2), eyes partly closed (2/2) and an increase of salivation (1/2). Rigor mortis (2/2) was noted before the necropsy. In the surviving animal (1/3), a decrease in spontaneous activity associated with an increase of salivation was noted in the first hours post dose. The animal recovered a normal activity at 48 hours post-dose.
Steps 2 and 3 (300 mg/kg bw): A decrease of spontaneous activity (2/6) was noted in the first hours of the test. The animals recovered a normal activity at 3 hours post-dose.
Body weight:
Step 1 (2000 mg/kg bw): The body weight evolution of the surviving animal revealed an absence of body weight gain at D2 versus D0. Then, the body weight evolution was normal.
Steps 2 and 3 (300 mg/kg bw): The body weight evolution of the animals remained normal throughout the study.
Gross pathology:
Step 1 (2000 mg/kg bw): The macroscopic examination of the animals found dead revealed a thinning of forestomach (2/2), a corpus with red spots (1/2) and a reddish corpus with black spots (1/2). The macroscopic examination of the surviving animal at the end of the study did not reveal treatment related changes.
Steps 2 and 3 (300 mg/kg bw): The macroscopic examination of the animals at the end of the study did not reveal treatment related changes.

Table 1: Body weight and weight gain in grams. Step 1 (2000 mg/kg bw)

Females D0 D2 D2-D0 D7 D7-D0 D14 D14-D0
Rf4203 187 161 -26      
Rf4204 184          
Rf4205 197 190 -7 220 23 241 44
MEAN 189.3 175.5 -16.5 220 23.0 241.0 44.0
S.D. 6.8 20.5 13.4        

†: Rf4204 found dead on D2

†: Rf4203 found dead on D6

Table 2: Body weight and weight gain in grams. Steps 2 & 3 (300 mg/kg bw)

Females D0 D2 D2-D0 D7 D7-D0 D14 D14-D0
Rf4220 225 232 7 246 21 266 41
Rf4221 234 244 10 273 39 268 34
Rf4222 216 217 1 226 10 242 26
Rf4286 186 196 10 212 26 230 44
Rf4287 192 206 14 228 36 255 63
Rf4288 205 217 12 239 34 260 55
MEAN 209.7 218.7 9.0 237.3 27.7 253.5 43.8
S.D. 18.8 17.3 4.6 21.0 10.9 14.8 13.5
Interpretation of results:
other: Category 4 (CLP Regulation EC no. 1272/2008)
Conclusions:
The LD50 of the test item was found to be higher than 300 mg/kg bw and lower than 2000 mg/kg by oral route in the rat. The LD50 cut-off of the test item may be considered as 1000 mg/kg bw.

Executive summary:

The acute oral toxicity of the test item has been tested in accordance with OECD Test Guideline 423, following GLP. 9 female Sprague-Dawley rats divided in 3 groups were administered sequentially with test item by oral gavage. The first set of 3 female rats was given a single dose of 2000 mg/kg body weight. Two mortalities were observed at this dose level, so a second set of 6 female rats was treated at 300 mg/kg bw. The mortalities were preceded by a decrease in spontaneous activity (2/2), righting reflex (1/2), muscle tones (1/2), associated with hypothermia (1/2), piloerection (1/2), eyes partly closed (2/2) and an increase of salivation (1/2). Rigor mortis (2/2) was noted before the necropsy. The macroscopic examination of the animals revealed a thinning of forestomach (2/2), a corpus with red spots (1/2) and a reddish corpus with black spots (1/2). In the surviving animal (1/3), a decrease in spontaneous activity associated with an increase of salivation was noted in the first hours post dose. The animal recovered a normal activity at 48 hours post-dose. The body weight evolution of the animal revealed an absence of body weight gain at D2 versus D0. Then, the body weight evolution was normal. The macroscopic examination of the animal at the end of the study did not reveal treatment related changes. In the 300 mg/kg bw dose level no mortalities were found. A decrease of spontaneous activity (2/6) was noted in the first hours of the test. The animals recovered a normal activity at 3 hours post-dose. The body weight evolution of the animals remained normal throughout the study and the macroscopic examination at the end of the study did not reveal treatment related changes. Based on these results, the LD50 of the test item is higher than 300 mg/kg bw and lower than 2000 mg/kg by oral route in the rat. As per OECD Test Guideline 423, the LD50 cut-off of the test item may be considered as 1000 mg/kg bw.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LD50
Value:
1 000 mg/kg bw
Quality of whole database:
Key study with Klimisch score = 1

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

Justification for classification or non-classification

Based on the available data, the substance is classified as category 4 for acute toxicity (oral) according to CLP Regulation (EC) no. 1272/2008.