Extract of fava d'anta, obtained from the fruits of Dimorphandra mollis (Leguminosae) by solvent extraction

Acute Toxicity: oral

Administrative data

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25.03.2021-19.04.2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

Extract of fava d'anta, obtained from the fruits of Dimorphandra mollis (Leguminosae) by solvent extraction

EC Number:

953-265-8

Molecular formula:

Not applicable

IUPAC Name:

Extract of fava d'anta, obtained from the fruits of Dimorphandra mollis (Leguminosae) by solvent extraction

Test material form:

solid: particulate/powder

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: not specified
- Females nulliparous and non-pregnant: yes
- Rationale for use of males (if applicable)
- Age at study initiation: 10-11 weeks
- Weight at study initiation: 164.87 g to 179.29 g
- Fasting period before study: 16 to 18 hours
- Housing: Three animals were housed in standard polypropylene cage (Size: L 430 x B 285 x H 150 mm) with stainless steel mesh top grill having facilities for holding pelleted feed and drinking water in water bottle fitted with stainless steel sipper tube. Clean sterilized paddy husk was provided as bedding material.
- Diet: Altromin Maintenance Diet for rats and mice manufactured by Altromin Spezialfutter GmbH & Co. KG was provided ad libitum
- Water: It was provided ad libitum throughout the experimental period. Deep bore-well water passed through Reverse osmosis unit was provided in plastic water bottles with stainless steel sipper tubes.The contaminant analysis test reports for the water are included
- Acclimation period: Healthy young adult animals used for Step-I, Step-I confirmation, Step-II and Step- II confirmation were acclimatized for five, seven, nine and eleven days respectively
- Method of randomisation in assigning animals to test and control groups: Randomization was done during acclimatization period.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.6°C to 22.9°C
- Humidity (%): 47% to 66%
- Air changes (per hr): 12 to 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours fluorescent light and 12 hours dark

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

VEHICLE
- Concentration in vehicle: 30 and 200 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg bw
- Justification for choice of vehicle: Solubility of the test item in water
- Lot/batch no. (if required): 435

MAXIMUM DOSE VOLUME APPLIED: 10 mL/kg bw

DOSAGE PREPARATION (if unusual): The required quantity of test item was weighed, grind well using mortar and pestle by adding little volume of vehicle and transferred into the measuring cylinder. Again a small quantity of vehicle was added to mortar, mixed well and transferred into the measuring cylinder. The rinsing procedure was repeated to ensure complete transfer of the test item formulation into a measuring cylinder. Finally, the volume was added to the required mark to get a desired concentration. Additionally, to maintain the uniformity of the formulation prepared, the formulations were kept under continuous stirring conditions using magnetic stirrer during dosing

CLASS METHOD
- Rationale for the selection of the starting dose: As there is no previous information, a starting dose of 300 mg/kg b.w. was administered following the sequential procedure presented in the OECD 423 test guideline Annex 2c.

Doses:

300 and 2000 mg/kg b.w.

No. of animals per sex per dose:

3

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: At each step, all the animals were observed for clinical signs of toxicity and mortality at 20 to 30 mins, 1 hr (±10 mins), 2 hrs (±10 mins), 3 hrs (±10 mins) and 4 hrs (±10 mins) post dosing on day 1 and once daily thereafter for clinical signs of toxicity and twice daily for mortality during the 14 days observation period. Individual animal body weights were recorded at receipt, on day 1 (before test item administration), on days 8 and 15 during the observation period.
- Necropsy of survivors performed: yes
- Clinical signs including body weight: Observations included changes in skin, fur, eyes and mucous membranes and also respiratory, circulatory, autonomic and central nervous systems and somatomotor activity and behaviour pattern.
- Other examinations performed: clinical signs, body weight,organ weights, histopathology, other:
At the end of observation period, all the animals were humanely sacrificed by carbon dioxide asphyxiation, subjected to necropsy and a complete gross pathological examination and the observations were recorded. Histopathological examination was not carried out as no gross pathological changes were observed during necropsy

Results and discussion

Effect levels

Key result

Sex:

female

Dose descriptor:

LD50

Effect level:

5 000 mg/kg bw

Based on:

test mat.

Mortality:

No mortality was observed at Step-I, Step-I confirmation (300 mg/kg b.w.) as well as in Step-II and Step-II confirmation (2000 mg/kg b.w.).

Clinical signs:

other: No clinical signs of toxicity were observed at Step-I, Step-I confirmation (300 mg/kg b.w.) as well as in Step-II and Step-II confirmation (2000 mg/kg b.w.).

Gross pathology:

No gross pathological changes were observed in Step-I, Step-I confirmation dosed at 300 mg/kg body weight, Step-II and Step-II confirmation animals dosed at 2000 mg/kg body weight.

Any other information on results incl. tables

Table 1. Individual animal clinical signs of toxicity and mortality record.

Study Steps & Dose (mg/kg body weight)

Animal No.

Sex

Time of Dosing (AM)

Clinical Signs of Toxicity and Mortality on Day 1

Clinical Signs of Toxicity and Mortality on Day

20 to 30 min

1 hr (±10 min)

2hrs (±10 min)

3hrs (±10 min)

4hrs (±10 min)

2

3

4

5

6

7

8

9

10

11

12

13

14

15

Step-I & 300

Rf3961

F

10:17

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

Rf3962

F

10:18

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

Rf3963

F

10:19

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

Step-I Confirmation & 300

Rf3964

F

10:08

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

Rf3965

F

10:09

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

Rf3966

F

10:10

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

Step-II & 2000

Rf3967

F

10:09

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

Rf3968

F

10:10

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

Rf3969

F

10:11

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

Step-II Confirmation & 2000

Rf3970

F

10:26

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

Rf3971

F

10:27

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

Rf3972

F

10:28

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

 

Table 2. Individual animal body weight (g) and percent change in body weight with respect to day 1.

Study Steps & Dose (mg/kg body weight)	Animal No.	Sex	Volume Administered (mL)	Body Weight (g) on Day			Percent Change in Body Weight with Respect to Day
				1	8	15	1 to 8	1 to 15
Step-I & 300	Rf3961	F	1.8	178.92	191.26	204.96	6.90	14.55
	Rf3962	F	1.7	170.27	184.13	201.22	8.14	18.18
	Rf3963	F	1.7	168.93	181.44	199.85	7.41	18.30
	Mean			172.71	185.61	202.01	7.48	17.01
	±SD			5.42	5.07	2.65	0.62	2.13
Step-I Confirmation & 300	Rf3964	F	1.7	174.36	187.24	202.13	7.39	15.93
	Rf3965	F	1.8	176.49	189.13	202.56	7.16	14.77
	Rf3966	F	1.8	175.73	188.28	203.02	7.14	15.53
	Mean			175.53	188.22	202.57	7.23	15.41
	±SD			1.08	0.95	0.45	0.14	0.59
Step-II & 2000	Rf3967	F	1.7	166.61	180.32	194.26	8.23	16.60
	Rf3968	F	1.8	178.33	191.13	204.31	7.18	14.57
	Rf3969	F	1.6	164.78	177.56	191.06	7.76	15.95
	Mean			169.91	183.00	196.54	7.72	15.70
	±SD			7.35	7.17	6.91	0.53	1.04
Step-II Confirmation & 2000	Rf3970	F	1.8	182.64	196.13	209.13	7.39	14.50
	Rf3971	F	1.9	191.45	205.21	218.22	7.19	13.98
	Rf3972	F	1.8	181.05	194.36	207.26	7.35	14.48
	Mean			185.05	198.57	211.54	7.31	14.32
	±SD			5.60	5.82	5.86	0.11	0.29

 

Table 3. Individual animal gross pathology findings

Study Steps & Dose (mg/kg body weight)	Animal No.	Sex	Fate	Gross Pathology Findings
				External	Internal
Step-I & 300	Rf3961	F	TS	NAD	NAD
	Rf3962	F	TS	NAD	NAD
	Rf3963	F	TS	NAD	NAD
Step-I Confirmation & 300	Rf3964	F	TS	NAD	NAD
	Rf3965	F	TS	NAD	NAD
	Rf3966	F	TS	NAD	NAD
Step-II & 2000	Rf3967	F	TS	NAD	NAD
	Rf3968	F	TS	NAD	NAD
	Rf3969	F	TS	NAD	NAD
Step-II Confirmation & 2000	Rf3970	F	TS	NAD	NAD
	Rf3971	F	TS	NAD	NAD
	Rf3972	F	TS	NAD	NAD

NAD: No Abnormality Detected; F: Female; TS: Terminal Sacrifice

Applicant's summary and conclusion

Interpretation of results:

other: Not classified (CLP Regulations EC no. 1272/2008)

Conclusions:

The LD50 value of the test item is 5000 mg/ kg body weight by oral route in rat.

Executive summary:

The acute oral toxicity of the test item has been tested in accordance with OECD Test Guideline 423, following GLP. 12 female Sprague-Dawley rats were administered by oral gavage the test item diluted in water. A first test (Step-I) using three female rats was performed using a concentration of 300 mg/kg b.w., following the procedure described in the OECD 423 test guideline Annex 2c. As no clinical signs of toxicity nor mortality were observed, an additional test (Step-I confirmation) using the same concentration and the same amount and type of animals was performed, obtaining the same results.  After this, a second step test (Step-II) with three female rats was carried out using 2000 mg/kg b.w. Again, no clinical signs of toxicity nor mortality were observed, so a final confirmation test (Step-II confirmation) was carried out with three more female rats leading to the same results. No adverse changes were observed in body weight and percent change in body weight with respect to day 1 and no gross pathological changes were observed in Step-I, Step-I confirmation (300 mg/kg b.w.), Step-II and Step-II confirmation animals (2000 mg/kg b.w.). Therefore, the test item has a LD50 value of 5000 mg/kg b.w. when administered as a single dose by oral gavage. As such, the test item is not classified acccording to CLP Regulations EC no. 1272/2008