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Administrative data

Endpoint:
acute toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2019-01-22 to 2019-08-12
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report date:
2019

Materials and methods

Test guidelineopen allclose all
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
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.1100 (Acute Oral Toxicity)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
(Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany)
Test type:
fixed dose procedure
Limit test:
no

Test material

Constituent 1
Test material form:
liquid

Test animals

Species:
rat
Strain:
Wistar
Sex:
female
Details on test animals or test system and environmental conditions:
TEST SYSTEM
- Species/strain: WISTAR rats Crl: WI(Han)
- Source: Charles River, 97633 Sulzfeld, Germany
- Sex: Female (non-pregnant and nulliparous)
- Number of animals: 5
- Age at the beginning of the study: 8 – 9 weeks
- Body weight on the day of administration: animal no. 1: 157 g; animal no. 2: 170 g; animal no. 3: 196 g; animal no. 4: 178 g; animal no. 5: 169 g
The animals were derived from a controlled full-barrier maintained breeding system (SPF). According to the German Act on Animal Welfare the animals were bred for experimental purposes. This study was performed in an AAALAC-accredited laboratory. According to German animal protection law, the study type has been reviewed and accepted by local authorities. Furthermore, the study has been subjected to Ethical Review Process and was authorised by the Bavarian animal welfare administration.

HUSBANDRY
- Full barrier in an air-conditioned room
- Temperature: 22  3 °C
- Relative humidity: 55  10%
- Artificial light, sequence being 12 hours light, 12 hours dark
- Air change: 10 x / hour
- Free access to Altromin 1324 maintenance diet for rats and mice
- Free access to tap water, sulphur acidified to a pH value of approximately 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals)
- The animals were kept in groups in IVC cages, type III H, polysulphone cages on Altromin saw fibre bedding
- Certificates of food, water and bedding are filed for two years at BSL Munich and afterwards archived at Eurofins Munich
- Adequate acclimatisation period (at least five days) under laboratory conditions

The animals were randomly selected and marked for individual identification by tail painting.
Prior to the administration a detailed clinical observation was made of all animals. Only healthy animals were used.
Prior to the administration food was withheld from the test animals for 16 to 19 hours (access to water was permitted). Following the period of fasting the animals were weighed and the test item was administered. Food was provided again approximately 4 hours post dosing.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Remarks:
Sigma-Aldrich, lot no. MKCG3257, expiry date: 04/04/2019
Details on oral exposure:
ADMINISTRATION
The test item was administered at a single dose by gavage using a feeding tube at a dose volume of 10 mL/kg body weight.

Volume of Test Item Preparation Applied per Animal (no./sex):
1 / Female: Body Weight(*): 157 / Volume(**): 1.6
2 / Female: Body Weight(*): 170 / Volume(**): 1.7
3 / Female: Body Weight(*): 196 / Volume(**): 2.0
4 / Female: Body Weight(*): 178 / Volume(**): 1.8
5 / Female: Body Weight(*): 169 / Volume(**): 1.7

(*) = on Day of Administration (g)
(**) = Test Item Preparation in Vehicle Administrered per Animal (mL)
Doses:
SIGHTING STUDY
The starting dose for the sighting study was 2000 mg/kg body weight. No compound-related mortality was recorded for the first animal dosed.

MAIN TEST
Another four animals were dosed with 2000 mg/kg body weight and no compound-related mortality was recorded. Based on these results and according to the fixed dose method regime no further testing was required.
No. of animals per sex per dose:
- Sighting Study: 1
- Main Study: 4
Control animals:
no
Details on study design:
OBSERVATION PERIOD
All animals were observed for 14 days after dosing for general clinical signs, morbidity and mortality.

WEIGHT ASSESSMENT
The animals were weighed on day 1 (prior to the administration) and on days 8 and 15.

CLINICAL EXAMINATION
A careful clinical examination was made several times on the day of dosing (at least once during the first 30 minutes and with special attention given during the first 4 hours post-dose). Thereafter, the animals were observed for clinical signs once daily until the end of the observation period.
Cageside observations included changes in the skin and fur, eyes and mucous membranes. Also respiratory, circulatory, autonomic and central nervous systems and somatomotor activity and behaviour pattern were examined. Particular attention was directed to observations of tremor, convulsions, salivation, diarrhoea, lethargy, sleep and coma.

PATHOLOGY
At the end of the observation period the animals were sacrificed with an overdosage of pentobarbital injected intraperitoneally at a dosage of 250-400 mg/kg bw.
All animals were subjected to gross necropsy and examined macroscopically for gross pathological changes. In the absence of gross pathological changes no tissues were preserved for a possible histopathological evaluation.

EVALUATION
Results were interpreted according to OECD Guideline 420, Annex 3. Individual reactions of each animal were recorded at each time of observation. Toxic response data were recorded by dose level. Nature, severity and duration of clinical observations were described. The body weight changes were summarised in a tabular form. Necropsy findings were described.
Statistics:
According to OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.

Results and discussion

Preliminary study:
SIGHTING STUDY
The starting dose for the sighting study was 2000 mg/kg body weight. No compound-related mortality was recorded for the first animal dosed.
Effect levels
Key result
Sex:
female
Dose descriptor:
LD50 cut-off
Effect level:
> 5 000 mg/kg bw
Based on:
test mat.
Mortality:
All animals survived until the end of the study without showing any signs of toxicity.
Clinical signs:
Sighting study Animal 1 / female
Observations: during the whole time of the observation period nsf (no specific findings)

Main study Animals 2, 3, 4, 5 / female
Observations: during the whole time of the observation period nsf (no specific findings)

Based on these results and according to the fixed dose procedure regime no further testing was required.
Therefore, according to OECD Guideline 420 a sufficient estimation of the acute oral toxicity of the test item is provided.
Body weight:
None of the animals showed weight loss during the observation period
Gross pathology:
No specific gross pathological changes were recorded for any animal.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Under the conditions of the present study, a single oral application of the test item to rats at a dose of 2000 mg/kg body weight was not associated with signs of toxicity or mortality.
The median lethal dose of the test item after a single oral administration to female rats, observed over a period of 14 days is:
LD50 cut-off (rat): ˃ 5000 mg/kg bw
Executive summary:

Five female WISTAR Crl: WI(Han) rats, were treated with the test item by oral gavage administration at a dosage of 2000 mg/kg body weight. The test item was dissolved in the vehicle corn oil at a concentration of 0.2 g/mL and administered at a dose volume of 10 mL/kg.

All animals used in the study after their entrance at BSL were allowed to acclimatise to the laboratory conditions for at least 5 days. The animals were observed on delivery, on inclusion in the study and before administration for mortality/morbidity and other clinical signs. All animals were examined for clinical signs several times on the day of dosing and once daily until the end of the observation period. Their body weights were recorded on day 1 (prior to the administration) and on days 8 and 15. All animals were necropsied and examined macroscopically.

All animals survived until the end of the study without showing any signs of toxicity.

Throughout the 14-day observation period, the weight gain of the surviving animals was within the normal range of variation for this strain.

At necropsy, no treatment-related macroscopic findings were observed in any animal.

LD50cut-off (rat): ˃ 5000mg/kg bw

Species/strain:  WISTAR Crl: WI(Han) rats

Vehicle:  corn oil

Number of animals: 1 animal sighting study / 4 animals main study

Method: OECD 420, EC 440/2008, Method B.1 tris, OPPTS 870.1100

 

Conclusion

Under the conditions of the present study, a single oral application of the test item

to rats at a dose of 2000 mg/kg body weight was not associated with signs of toxicity or mortality.

The median lethal dose of the test item after a single oral administration to female rats, observed over a period of 14 days is:

LD50cut-off (rat): ˃ 5000mg/kg bw