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

Endpoint summary

Description of key information

Acute oral toxicity. Key study. Method according to OECD 423, GLP study. The LD50 value of the test item is 5000 mg/ kg body weight by oral route in rat.

Acute inhalation toxicity. Data waiving (study scientifically not necessary): According to REACH Annex VIII, column 2: In addition to the oral route, for substances other than gases, the information mentioned under 8.5.2 to 8.5.3 shall be provided for at least one other route. The information is provided for dermal route.

Acute dermal toxicity. Key study. Method according to OECD 402, GLP study. The LD50 value of the test item is >2000 mg/ kg body weight by dermal route in rat.

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:

25.03.2021-19.04.2021

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

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

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

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.

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

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

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LD50

Value:

5 000 mg/kg bw

Quality of whole database:

The study has a Klimisch score of 1.

Acute toxicity: via inhalation route

Link to relevant study records

Reference

Endpoint:

acute toxicity: inhalation

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Justification for type of information:

JUSTIFICATION FOR DATA WAIVING
According to REACH Annex VIII, column 2: In addition to the oral route (Annex VII, 8.5.1.), for substances other than gases, the information mentioned under 8.5.2 to 8.5.3 shall be provided for at least one other route. The information is provided for dermal route.

Clinical signs:

other:

Endpoint conclusion

Endpoint conclusion:

no study available

Acute toxicity: via dermal route

Link to relevant study records

Reference

Endpoint:

acute toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25.03.2021-19.04-21

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 402 (Acute Dermal 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

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: not specified
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 10-11 weeks
- Weight at study initiation: 202.52 g to 224.30 g
- Housing: Maximum of three animals were housed in a standard polypropylene cage (430x285x 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. For range finding study animals were housed individually during and after treatment. For main study, during treatment, the animals were housed individually; and after patch removal, animals were housed together. Clean sterilized paddy husk was provided as bedding material. Paper shredding was provided as enrichment.
- 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: 5 (200 mg/kg body weight), 7 (1000 mg/kg body weight) and 9 days (2000 mg/kg body weight) for range finding study, and 11 days for the main study animals
- 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 cycle

Type of coverage:

semiocclusive

Vehicle:

water

Remarks:

Mysore Research Chemical Laboratories, Batch. No 435

Details on dermal exposure:

TEST SITE
- Area of exposure: dorso-lateral of the trunk
- % coverage: 10%
- Type of wrap if used: cotton gauze dressing and non-irritating adhesive tape

REMOVAL OF TEST SUBSTANCE
- Washing (if done): with distilled water and dried with absorbent cotton
- Time after start of exposure: 24h

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 46.4 to 472.9 mg in Range Finding Study, 431.4 to 435.6 mg in Main Study
- Constant volume or concentration used: yes/no
- For solids, paste formed: yes

VEHICLE
- Amount(s) applied (volume or weight with unit):0.5 mL
- Lot/batch no. (if required): 435

Duration of exposure:

72 hours

Doses:

- 200 mg/kg bw (Range Finding)
- 1000 mg/kg bw (Range Finding)
- 2000 mg/kg bw (Range Finding and Main Study)

No. of animals per sex per dose:

Range Finding: 1 female per dose (3 females in total)
Main Study: 2 females per dose (2000 mg/kg bw)

Control animals:

not required

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: All the animals were observed for clinical signs of toxicity and mortality at 20 to 30 mins, 1 hr , 2 hrs , 4 hrs and 6 hrs (±10 mins) on treatment day 1 and thereafter once daily for clinical signs of toxicity and twice daily for mortality during the 14 days observation period. The body weights were recorded at receipt, on day 1 before test item application, on days 8 and 15.
- Necropsy of survivors performed: yes
- Clinical signs including body weight: Observations included changes in skin, fur, eyes and mucous membranes along with changes in respiratory, circulatory, autonomic and central nervous system, somatomotor activity and behaviour pattern. Individual animal body weights were recorded at receipt, on day 1 before test item application and on days 8 and 15 during the experimental period.
- Other examinations performed: all the animals were subjected to necropsy and a complete gross pathological examination. Histopathological examination was not carried out as there were no gross pathological findings in any of the animals.

Key result

Sex:

female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Mortality:

No treatment related mortality was observed in both range finding study and main study animals.

Clinical signs:

other: No treatment related clinical signs of toxicity were observed in both range finding study and main study animals.

Gross pathology:

No treatment related gross pathological changes were in both range finding study and main study animals.

Table 1. Clinical signs of toxicity and mortality record

Phase of the Experiment

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 days

20-30 mins

1 hr (±10 mins)

2 hrs (±10 mins)

4 hrs (±10 mins)

6 hrs (±10 mins)

2

3

4

5

6

7

8

9

10

11

12

13

14

15

Range Finding Study

200

Rf3976

F

10:15

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

1000

Rf3977

F

10:17

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

2000

Rf3978

F

10:20

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

Main Study

2000

Rf3979

F

10:32

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

Rf3980

F

10:34

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

 N: Normal; F: Female; mins: minutes; hr/hrs: hour/hours

 

Table 2. Individual animal local skin reactions

Phase of the Experiment	Animal No	Sex	Dose (mg/kg body weight)	Days
				3 (24 hours)		4 (48 hours)		5 (72 hours)
				ER	ED	ER	ED	ER	ED
Range finding Study	Rf3976	Female	200	0	0	0	0	0	0
	Rf3977	Female	1000	0	0	0	0	0	0
	Rf3978	Female	2000	0	0	0	0	0	0
Main Study	Rf3979	Female	2000	0	0	0	0	0	0
	Rf3980	Female	2000	0	0	0	0	0	0

ER: Erythema; ED: Edema; 0: No erythema/edema

 

Table 3. Body weight (g) and percent change in body weight with respect to day 1

Phase of the Experiment	Dose (mg/kg body weight)	Animal No.	Sex	Quantity Administered (mg)	Body Weight (g) on Days				Percent Change in Body Weight with Respect to Day
					1	8	15		1 to 8	1 to 15
Range Finding Study	200	Rf3976	F	46.4	231.93	246.12	261.28		6.12	12.65
	1000	Rf3977	F	232.7	232.68	246.21	260.02		5.81	11.75
	2000	Rf3978	F	472.9	236.47	249.83	264.21		5.65	11.73
Main Study	2000	Rf3979	F	431.4	215.72	230.31	245.20		6.76	13.67
		Rf3980	F	435.6	217.78	233.14	248.12		7.05	13.93
		Mean			216.75	231.73	246.66		6.91	13.80
		±SD			1.46	2.00	2.06		0.20	0.19
		n			2	2	2		2	2

F: Female; SD: Standard Deviation; n: number of animals.

 

Table 4. Gross pathology findings

Phase of the Experiment	Dose (mg/kg body weight)	Animal No.	Sex	Fate	Gross Pathology Findings
					External	Internal
Range finding Study	200	Rf3976	F	TS	NAD	NAD
	1000	Rf3977	F	TS	NAD	NAD
	2000	Rf3978	F	TS	NAD	NAD
Main Study	2000	Rf3979	F	TS	NAD	NAD
		Rf3980	F	TS	NAD	NAD

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

Interpretation of results:

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

Conclusions:

The LD50 value of the test item is >2000 mg/ kg body weight by dermal route in rat.

Executive summary:

The acute dermal toxicity of the test item has been performed in accordance with OECD Test Guideline 402, following GLP. A thin paste of the test item and water was applied to a total of 5 female Sprague-Dawley rats in two differents studies. A range finding study using three female rats was performed using a concentration of 200, 1000 and 2000 mg/kg b.w., respectively. As no clinical signs of toxicity nor mortality were observed at any of these concentrations, the main test was carried out using 2 more female rats at a concentration of 2000 mg/kg b.w. Again, no clinical signs of toxicity nor mortality were observed. 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 any of the studies performed. Therefore, the test item has a LD50 value of >2000 mg/kg b.w. As such, the test item is not classified acccording to CLP Regulations EC no. 1272/2008.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LD50

Value:

> 2 000 mg/kg bw

Quality of whole database:

The study has a Klimisch score of 1.

Justification for classification or non-classification

Based on the available information, the substance is not classified for acute toxicity in accordance with CLP Regulation (EC) No 1272/2008.