Rhatany, Krameria triandra, ext.

Acute Toxicity: oral

Administrative data

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03.08.2018 - 11.10.2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The study was performed in compliance with the Principle of Good Laboratory Practice, confirmed by Statement of GLP Compliance.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)

Deviations:

no

Remarks:

The purpose of the study was to evaluate the potential toxic effect of the test item after single oral administration in Wistar rats. The procedure according to OECD Guideline 423 Acute Toxic Class (ATC) method was used.

Principles of method if other than guideline:

none

GLP compliance:

yes (incl. QA statement)

Test type:

acute toxic class method

Limit test:

yes

Test material

Test material information

1

Reference substance name:

Rhatany, Krameria triandra, ext.

EC Number:

283-919-1

EC Name:

Rhatany, Krameria triandra, ext.

Cas Number:

84775-95-1

Molecular formula:

not available

IUPAC Name:

Extractives and their physically modified derivatives such as tinctures, concretes, absolutes, essential oils, oleoresins, terpenes, terpene-free fractions, distillates, residues, etc., obtained from Krameria triandra, Krameriaceae.

Test material form:

liquid: viscous

Specific details on test material used for the study:

Test Item Krameria triandra extract obtained from Rhatany root by hydroalcoholic extraction
Lot Number PES180014
CAS No 84775-95-1
EINECS-No 283-919-1
Purity not applicable, UVCB
Appearance reddish brown viscous substance
Composition Krameria triandra extract obtained from Rhatany root by hydroalcoholic extraction
Homogeneity inhomogenous, warm up to about 60°C and stir
Production Date 01/2018
Expiry Date 01/2019
Storage Fridge (2 - 8°C)
Test Item Handling and Storage According to SOPA-00147-BIO, Test and Reference Items


Vehicle Olive oil
Lot Number L71143
Expiry Date 10/2018
Manufacturer Oleificio Luca, Italy
Storage 20 ± 5 °C
Justification for the Choice of Vehicle Olive oil is a common vehicle in toxicity studies like OECD TG 423

Test animals

Species:

rat

Strain:

Wistar

Sex:

female

Details on test animals or test system and environmental conditions:

Test animals:
Species Wistar rats
Source Slovak Academy of Sciences Dobrá Voda, Slovak Republic
Number and Sex of Animals 6 females
Age at First Dose 8-9 weeks; female animals were non-pregnant and nulliparous
Animal Health The Health condition of animals was examined by a veterinarian before initiation of the study.
Acclimation The animals were acclimated under the conditions identical to the conditions during the experiment 5 days prior to the start of treatment. The acclimation was according to the standard operation procedure.
Housing Condition The animals were housed in plastic cages suspended on stainless steel racks, 3 animals per cage in a room equipped with central air-conditioning. The average room temperature was maintained within the range of 22.38 ± 0.17 °C, relative humidity within 53.58 ± 4.94 %. The light regimen was set to a 12-hour light /12-hour dark cycle. Sanitation was performed according to the standard operation procedures.
Diet The laboratory food ssniff (ssniff Spezialdiäten GmbH, Germany) was available ad libitum. The certificate of analysis is included in the raw data.
Water The animals received tap water for human consumption. Supply of drinking was unlimited. The quality of drinking water is periodically analysed and recorded; certificate of analysis is included in the raw data.
Bedding Lignocel S3/4, Lufa - ITL GmbH, Germany
Animals Identification The animals in the cage were marked by a line (I-III) on the tail with a waterproof marker. Each cage was marked with the study code, ID of animals and date of administration of the test item.
Justification for the Choice of Species Normally females are used for testing according to OECD TG 423 because females are typically the more sensitive gender.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

olive oil

Details on oral exposure:

The test item was administered in a single dose by gavage using a metal stomach tube.

Doses:

Number of Animals and Dose Levels:
The starting dose could be selected from the fixed dose levels of 5, 50, 300, and 2000 mg/kg body weight. A limit dose of 2000 mg/kg body weight was used as a starting dose. One group of 3 females was dosed. One animal died during 24 hours and therefore, in a second step, another 3 females were treated at the same dose.

Dose Preparation:
The required amount of the test item (according to the body weight and dose) was mixed with vehicle (Olive oil) shortly before administration. The dose of 2000 mg/kg was administered in a volume of 5 mL/kg body weight.

Dose Administration:
The test item was administered in a single dose by gavage using a metal stomach tube. Animals were fasted 10-12 h prior to dosing (food but not water was withheld over-night). Following a period of fasting, animals were weighed and the test item administered. After test item administration, food was withheld for further 3-4 hours.

No. of animals per sex per dose:

6

Control animals:

no

Details on study design:

Number and Sex of Animals 6 females
Age at First Dose 8-9 weeks; female animals were non-pregnant and nulliparous
Animal Health The Health condition of animals was examined by a veterinarian before initiation of the study.
Acclimation The animals were acclimated under the conditions identical to the conditions during the experiment 5 days prior to the start of treatment. The acclimation was according to the standard operation procedure.
Housing Condition The animals were housed in plastic cages suspended on stainless steel racks, 3 animals per cage in a room equipped with central air-conditioning. The average room temperature was maintained within the range of 22.38 ± 0.17 °C, relative humidity within 53.58 ± 4.94 %. The light regimen was set to a 12-hour light /12-hour dark cycle. Sanitation was performed according to the standard operation procedures.
Diet The laboratory food ssniff (ssniff Spezialdiäten GmbH, Germany) was available ad libitum. The certificate of analysis is included in the raw data.
Water The animals received tap water for human consumption. Supply of drinking was unlimited. The quality of drinking water is periodically analysed and recorded; certificate of analysis is included in the raw data.
Bedding Lignocel S3/4, Lufa - ITL GmbH, Germany
Animals Identification The animals in the cage were marked by a line (I-III) on the tail with a waterproof marker. Each cage was marked with the study code, ID of animals and date of administration of the test item.
Justification for the Choice of Species Normally females are used for testing according to OECD TG 423 because females are typically the more sensitive gender.

Instruments, Material:
Balance AE-163 Mettler Instruments, Switzerland 04/2019
Balance PS 2100.R2 Radwag Wagi Elektroniczne, Poland 10/2018 (in compliance with SOPG-00042-GEN)
IVC Touch Slim Plus Tecniplast, Italy 04/2019
Others Mini-shaker Heidolph Reax top; Automatic pipette – type ThermoScientific, Finpipette; metal stomach tube; auxiliary materials

Clinical Observation:
Animals were observed individually immediately after administration of the test item and 0.5, 1, 2, and 4 hours later. Each animal was inspected daily for the next 14 days.
Observations included: changes in skin, fur, eyes, mucous membranes, respiratory, circulatory, autonomic and central nervous systems, somatomotor activity, and behavioural pattern. Particular attention was given to potential neurologic endpoints such as tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.

Body Weight:
Individual weights of animals were measured immediately prior to administration of the test item and weekly thereafter. Weight differences after first and second weeks after administration were calculated and recorded.

Necropsy:
All test animals were subjected to gross necropsy and the results were recorded for each animal.

Statistics:

Administration Results:
5/6 females survived the limit dose of 2000 mg/kg body weight.

Results and discussion

Effect levels

Key result

Sex:

female

Dose descriptor:

LD50 cut-off

Effect level:

> 2 000 mg/kg bw

Based on:

test mat. (total fraction)

Mortality:

Administration Results:
5/6 females survived the limit dose of 2000 mg/kg body weight.

Clinical signs:

other: Clinical Observations: Test item-related mortality was observed in animal No 3 within 24 hours. The animal died probably during the night hours, because cadaver was found in a state of autolysis a day after administration of the test item. The rest animal

Gross pathology:

All test animals were subjected to gross necropsy and the results were recorded for each animal.
All animals were necropsied. During necropsy, no macroscopic findings were observed. Animal No 3 could not be necropsied because of autolysed cadaver.

Other findings:

Piloerection and lethargy were observed during the observation period.

Any other information on results incl. tables

Clinical Observation

Observation	Time After Administration
	Hour					Day
	I	0.5	1	2	4	1	2	3	4	5	6	7	8	9	10	11	12	13	14
Skin and Hair	-	-	-	3*	3*	-	-	-	-	-	-	-	-	-	-	-	-	-	-
Eyes	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
Mucosa	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
Respiratory System	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
Circulatory System	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
CNS	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
Somatomotoric Activity	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
Tremor	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
Spasms	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
Salivation	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
Diarrhoea	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
Lethargy	-	-	-	3	3	-	-	-	-	-	-	-	-	-	-	-	-	-	-
Sleep	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
Coma	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
Death	-	-	-	-	-	3	-	-	-	-	-	-	-	-	-	-	-	-	-

 -No observed signs, I-Immediately, *-piloerection

Applicant's summary and conclusion

Interpretation of results:

Category 5 based on GHS criteria

Conclusions:

Based on Annex 2d Test Procedure with a Starting Dose of 2000 mg/kg body weight of OECD Guideline 423 it can be concluded that the test item “Krameria triandra extract obtained from Rhatany root by hydroalcoholic extraction” is according to GHS criteria classified in Category 5 with a LD50 cut off value 2500 mg/kg body weight, after single oral administration to Wistar rats.

Executive summary:

The purpose of the study was to evaluate the potential toxic effect of the test item “Krameria triandra extract obtained from Rhatany root by hydroalcoholic extraction”when administered as a single oral dose to Wistar rats.

The procedure according to OECD Guideline 423 Acute Toxic Class(ATC)method was used.

A limit dose of 2000 mg/kg body weight was used as a starting dose.The test itemadministeredto 6 females at a limit dose caused death of one animal.The body weights of all survived animals increased during the study. No body weight losses were observed between the first and second week after administration.During necropsy, no macroscopic findings were observed.

The LD₅₀of the test item “Krameria triandra extract obtained from Rhatany root by hydroalcoholic extraction”higher than 2000 mg/kg body weight and lower than 5000 mg/kg body weight after single oral administration to Wistar rats.

Based on Annex 2d Test Procedure with a Starting Dose of 2000 mg/kg body weight of OECD Guideline 423 it can be concluded that thetest item “Krameria triandra extract obtained from Rhatany root by hydroalcoholic extraction”is according to GHScriteriaclassified in Category 5 with a LD₅₀cutoff value2500 mg/kg body weight,after single oral administration to Wistar rats.