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Description of key information

Acute oral toxicity: Key study: Test method according OECD Guideline 420. GLP study. The oral-LD50 for the test substance in female rats was determined to be higher than 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:
21/02/2018 - 20/03/2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 420 (Acute Oral Toxicity - Fixed Dose Method)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.1 bis (Acute Oral Toxicity - Fixed Dose Procedure)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Test type:
fixed dose procedure
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 (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 9 - 10 weeks old
- Weight at study initiation: mean bw 231.4 g.
- Fasting period before study: Food was removed on D1 and then redistributed 4 hours after the test item administration.
- Housing: Healthy female 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: ad libitum (Teklad Global 16% Protein Rodent Diet, ENVIGO, 2016)
- Water: ad libitum tap water. Microbiological and chemical analyses of the water were carried out once every six months by Bureau Veritas (Eurofins, FRANCE).
- Acclimation period: at least 5 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25
- Humidity (%): 30-70
- Air changes (per hr): at least 10/h
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 21/02/2018 (step 1), 28/02/2018 (step 2), 06/03/2018 (step 3) To: 07/03/2018 (step 1), 14/03/2018 (step 2), 20/03/2018 (step 3).
Route of administration:
oral: gavage
Vehicle:
olive oil
Details on oral exposure:
VEHICLE
- Justification for choice of vehicle: Olive oil was chosen as it produced the most suitable formulation at the requested concentrations.

MAXIMUM DOSE VOLUME APPLIED: 1.61 mL/kg bw

CLASS METHOD (if applicable)
- Rationale for the selection of the starting dose: The starting dose for the sighting study was selected from the fixed dose levels of 5, 50, 300, and 2000 mg/kg bw as a dose expected to produce evident toxicity. Since no data were available, the sighting study commenced with the administration of the test item at a dose of 300 mg/kg bw to one female rat. Since no evident toxicity was produced, the test item at a single dose of 2000 mg/kg bw was administered to the second animal. On the grounds of the sighting study results (no evidence toxicity), the dose of 2000 mg/kg bw was selected to be used in the main study.
Doses:
Sighting study: 300 and 2000 mg/kg bw
Main study: 2000 mg/kg bw
No. of animals per sex per dose:
1 female for the preliminary experiment at 300 mg/kg bw.
5 females (included the one from preliminary) for the main experiment at 2000 mg/kg bw.
Control animals:
no
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: Systematic examinations were carried out to identify any behavioural or toxic effects on the major physiological functions at 30 min, 1h, 3h, 4h on day 1 and day 2 and daily during 14 days following the administration of the test item. The animals were weighed on day D0 (just before administering the test item) then on D2, D7, and on D14.
- Necropsy of survivors performed: yes. Macroscopic observations were entered on individual autopsy sheets. Only those organs likely to be modified in cases of acute toxicity were examined.
- Other examinations performed: clinical signs and mortality were recorded daily, body weight evolution of treated animals was compared with the historical control group.
Preliminary study:
No evident toxicity was observed in dose 300 mg/kg bw and the animal survived the experiment.
Key result
Sex:
female
Dose descriptor:
LD50
Effect level:
> 2 000 mg/kg bw
Based on:
test mat.
Mortality:
No mortalities were recorded, all animals survived the experiment.
Clinical signs:
At 2000 mg/kg bw, a decrease of spontaneous activity (5/5) and muscle tones (5/5), Preyer’s reflex (5/5), righting reflex (5/5), associated with myosis (2/5), piloerection (2/5) and an increase of salivation (1/5) were noted at 24 hours post-dose. The animals recovered a normal activity between days 2 and 4.
Body weight:
The body weight evolution of the animals revealed an absence of body weight gain at day 2 versus day 0. Then, the body weight evolution was normal.
Gross pathology:
The macroscopic examination of the animals at the end of the study did not reveal treatment related changes.

Table 1. Body weights and weight gain of the animals (grams)

Dose: 300 mg/kg body weight (STEP 1)

FEMALES

D0

D2

D2-D0

D7

D7-D0

D14

D14-D0

Rf 2329

240

255

15

265

25

269

29

MEAN

240.0

255.0

15.0

265.0

25.0

269.0

29.0

Dose: 2000 mg/kg body weight (STEP 2 and STEP 3)

FEMALES

D0

D2

D2-D0

D7

D7-D0

D14

D14-D0

Rf 2328

262

262

0

271

9

288

26

Rf 2341

227

215

-12

241

14

284

57

Rf 2342

219

224

5

247

28

267

48

Rf 2343

233

230

-3

242

9

270

37

Rf 2344

216

206

-10

231

15

262

46

MEAN

231.4

227.4

-4

246.4

15.0

274.2

42.8

Standard deviation

18.4

21.4

7.0

14.9

7.8

11.2

11.8

Table 2. Clinical signs, overall list

Dose

(mg/kg bw)

Time

Mortality

Step 1.

Rf 2329

300

30 min

0

N

1 h

0

N

3 h

0

N

4 h

0

N

D1-D14

0

N

Dose

(mg/kg bw)

Time

Mortality

Step 2.

Step 3. Animal Rf.

Rf 2328

2341

2342

2343

2344

2000

30 min

0

N

N

N

N

N

1 h

0

N

SIGNS

N

N

N

3 h

0

N

SIGNS

N

N

N

4 h

0

N

SIGNS

N

N

SIGNS

D1

0

SIGNS

SIGNS

SIGNS

SIGNS

SIGNS

D2

0

N

SIGNS

N

SIGNS

SIGNS

D3

0

N

N

N

SIGNS

N

D4-D14

0

N

N

N

N

N

 

Remarks

1h

Rf2341: decreased spontaneous activity.

3h

Rf2341: decreased spontaneous activity; decreased muscle tone.

4h

Rf2341, 2344: decreased spontaneous activity; decreased muscle tone, piloerection.

D1

Rf2328, 2343: decreased spontaneous activity, preyer’s reflex, muscle tone and righting reflex, piloerection.

Rf2341: none spontaneous activity, preyer’s reflex, muscle tone and righting reflex.

Rf2342: decreased spontaneous activity and muscle tone; none preyer’s reflex and righting reflex.

Rf2344: decreased spontaneous activity, muscle tone and righting reflex.

D2

Rf2341: decreased spontaneous activity, preyer’s reflex and righting reflex. None muscle tone

Rf2343: None spontaneous activity, muscle tone and righting reflex; Decreased preyer’s reflex, piloerection.

Rf2344: decreased spontaneous activity, preyer’s reflex, muscle tone and righting reflex. Increased salivation.

D3

Rf2343: Decreased spontaneous activity, preyer’s reflex and righting reflex; None muscle tone. Pupil myosis. Increased lachrymation and piloerection.

Table 3. Necropsy findings, overall list

Observations

Step 1

Animal

Rf2329

Step 2

Animal

Rf2328

Step 3

Animals

Rf2341-2344

General Appearance

Normal

Normal

Normal

Oesophagus

NtR

NtR

NtR

Stomach

NtR

NtR

NtR

Duodenum

NtR

NtR

NtR

Jejunum

NtR

NtR

NtR

Ileon

NtR

NtR

NtR

Caecum

NtR

NtR

NtR

Colon

NtR

NtR

NtR

Rectum

NtR

NtR

NtR

Spleen

NtR

NtR

NtR

Liver

NtR

NtR

NtR

Thymus

NtR

NtR

NtR

Trachea

NtR

NtR

NtR

Lungs

NtR

NtR

NtR

Heart

NtR

NtR

NtR

Kidneys

NtR

NtR

NtR

Urinary Bladder

NtR

NtR

NtR

Overies

NtR

NtR

NtR

Uterus

NtR

NtR

NtR

Adrenals

NtR

NtR

NtR

Pancreas

NtR

NtR

NtR

Particulars

None

None

None

Interpretation of results:
other: Not classified (CLP Regulation EC no. 1272/2008)
Conclusions:
The oral-LD50 for the test substance in female rats was greater than 2000 mg/kg bw.
Executive summary:

An acute toxicity study was conducted with the test item according to the fixed dose procedure specified in OECD guideline 420, under GLP. Since no data was available, the preliminary experiment started with the administration of the test item suspended in corn oil at a dose of 300 mg/kg b.w. to one animal. Since no evident toxicity was observed at 300 mg/kg bw, the test item at a single dose of 2000 mg/kg b.w. was administered to the second animal. On the grounds of the preliminary experiment results, the dose of 2000 mg/kg bw was selected to be used in the main experiment and it was applied to four further rats. The observation period consisted of 14 days and included detailed clinical observations, body weight gain records and gross examinations at termination. No mortality was observed throughout the study. In the main test, decrease of spontaneous activity (5/5) and muscle tones (5/5), Preyer’s reflex (5/5), righting reflex (5/5), associated with myosis (2/5), piloerection (2/5) and an increase of salivation (1/5) were noted at 24 hours post-dose. The animals recovered a normal activity between days 2 and 4. The body weight evolution of the animals revealed an absence of body weight gain at day 2 versus day 0. Then, the body weight evolution was normal. The macroscopic examination of the animals at the end of the study did not reveal treatment related changes. Based on the test results, the acute oral LD50 was determined to be greater than 2000 mg/kg bw in female rats.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
LD50
Value:
2 000 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

Acute oral toxicity: Key study: An acute toxicity study was conducted with the test item according to the fixed dose procedure according to OECD guideline 420, under GLP. The preliminary experiment started with the administration of the test item suspended in corn oil at a dose of 300 mg/kg b.w. to one animal. Since no evident toxicity was observed at 300 mg/kg bw, the test item at a single dose of 2000 mg/kg b.w. was administered to the second animal. Based on results of these preliminary studies, the dose of 2000 mg/kg bw was selected to be used in the main experiment with 4 further rats. No mortality was observed throughout the study. Decrease of spontaneous activity (5/5) and muscle tones (5/5), Preyer’s reflex (5/5), righting reflex (5/5), associated with myosis (2/5), piloerection (2/5) and an increase of salivation (1/5) were noted at 24 hours post-dose. The animals recovered a normal activity between days 2 and 4. The body weight evolution was normal. The macroscopic examination of the animals at the end of the study did not reveal treatment related changes. Based on these results, the acute oral LD50 was determined to be greater than 2000 mg/kg bw in female rats.

Justification for classification or non-classification

Based on the available data, the substance is not classified for acute toxicity according to CLP Regulation (EC) no. 1272/2008.