Benzamide, 3-amino-4-methoxy-N-methyl-

Endpoint summary

Description of key information

The acute oral median lethal dose (LD₅₀) of the test item in the female Wistar strain rat was approximately 500 mg/kg body weight (LD50 cut-off: 500 mg/kg body weight, Globally Harmonized Classification System: Category 4, >300 - 2000 mg/kg body weight).

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:

2016

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

Qualifier:

according to guideline

Guideline:

EU Method B.1 tris (Acute Oral Toxicity - Acute Toxic Class Method)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test type:

acute toxic class method

Limit test:

yes

Species:

rat

Strain:

Wistar

Sex:

female

Details on test animals or test system and environmental conditions:

Animal Information
Female Wistar (RccHan™:WIST) strain rats were supplied by Envigo RMS (UK) Limited, Oxon, UK. On receipt the animals were randomly allocated to cages. The females were nulliparous and non pregnant. After an acclimatization period of at least 5 days the animals were selected at random and given a number unique within the study by indelible ink marking on the tail and a number written on a cage card. At the start of the study the animals were 8 to 12 weeks of age. The body weight variation did not exceed ±20% of the mean body weight of any previously dosed animals.
Animal Care and Husbandry
The animals were housed in groups of three in suspended solid floor polypropylene cages furnished with woodflakes. With the exception of an overnight fast immediately before dosing and for approximately 3 to 4 hours after dosing, free access to mains drinking water and food (2014C Teklad Global Rodent diet supplied by Envigo RMS (UK) Limited, Oxon, UK) was allowed throughout the study. The diet, drinking water and bedding were routinely analyzed and were considered not to contain any contaminants that would reasonably be expected to affect the purpose or integrity of the study.
The temperature and relative humidity were set to achieve limits of 19 to 25 °C and 30 to 70% respectively. The rate of air exchange was at least fifteen changes per hour and the lighting was controlled by a time switch to give 12 hours continuous light and 12 hours darkness.
The animals were provided with environmental enrichment items which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.

Route of administration:

oral: gavage

Vehicle:

arachis oil

Details on oral exposure:

All animals were dosed once only by gavage

Doses:

300 mg/kg
2000 mg/kg

No. of animals per sex per dose:

3 females per dose, 3 dose groups

Control animals:

no

Details on study design:

In the absence of data regarding the toxicity of the test item, 300 mg/kg was chosen as the starting dose.
Groups of fasted animals were treated as follows:
Dose Level(mg/kg) Concentration(mg/mL) Dose Volume(mL/kg) Number of Rats(Female)
300, 30 10 3
2000, 200 10 3
300, 200 10 3
All animals were dosed once only by gavage, using a metal cannula attached to a graduated syringe. The volume administered to each animal was calculated according to the fasted body weight at the time of dosing. Treatment of animals was sequential. Sufficient time was allowed between each group to confirm the survival of the previously dosed animals.
Clinical observations were made 30 minutes, 1, 2, and 4 hours after dosing and then daily for 14 days. Morbidity and mortality checks were made twice daily, early and late during normal working days, and once daily at weekends and public holidays.
Individual body weights were recorded prior to dosing and 7 and 14 days after treatment.
At the end of the observation period the animals were killed by cervical dislocation. All animals were subjected to gross pathological examination. This consisted of an external examination and opening of the abdominal and thoracic cavities for examination of major organs. The appearance of any macroscopic abnormalities was recorded. No tissues were retained.

Statistics:

None recorded.

Preliminary study:

Not applicable.

Key result

Sex:

female

Dose descriptor:

LD50

Effect level:

ca. 500 mg/kg bw

Based on:

test mat.

Remarks on result:

other: 95% CL not reported

Mortality:

Individual mortality data are given in Appendix 1.
One day after dosing, all animals treated at a dose level of 2000 mg/kg were found dead or killed for humane reasons due to the occurrence of clinical signs of toxicity that exceeded the severity limit set forth in the UK Home Office Project Licence.
There were no deaths noted at a dose level of 300 mg/kg.

Clinical signs:

other: Individual clinical observations are given in Appendix 2 and Appendix 3. Signs of systemic toxicity noted in two animals treated at a dose level of 2000 mg/kg were hunched posture, lethargy, pilo-erection, ataxia and exophthalmos. Additional signs of sys

Gross pathology:

Individual necropsy findings are given in Appendix 6 and Appendix 7.
Dark liver and dark kidneys were noted at necropsy of animals treated at a dose level of 2000 mg/kg that were found dead 1 day after dosing. Abnormally red lungs were also noted in one of these animals. No abnormalities were noted at necropsy of the animal treated at a dose level of 2000 mg/kg that was humanely killed 1 day after dosing.
No abnormalities were noted at necropsy of animals treated at a dose level of 300 mg/kg.

Other findings:

None

Appendix1     Mortality Data

			Deaths	During	Day of	Dosing (Hours)	Deaths	During	Period	After	Dosing	(Days)			Deaths
Dose Level mg/kg	Sex	Number of Animals Treated	½	1	2	4	1	2	3	4	5	6	7	8-14
300	Female	3	0	0	0	0	0	0	0	0	0	0	0	0	0/3
	Female	3	0	0	0	0	0	0	0	0	0	0	0	0	0/3
2000	Female	3	0	0	0	0	3*								3/3

*=    One animal killed for humane reasons due to the occurrence of clinical signs of toxicity that exceeded the severity limit set forth in the UK Home Office Project Licence

Appendix2     Individual Clinical Observations -300 mg/kg

Dose Level mg/kg	Animal Number and Sex	Effects	Noted	After	Dosing (Hours)	Effects	Noted	During	Period	After	Dosing	(Days)
		½	1	2	4	1	2	3	4	5	6	7	8	9	10	11	12	13	14
300	1-0 Female	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	1-1 Female	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	1-2 Female	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	3-0 Female	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	3-1 Female	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	3-2 Female	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0

Appendix3     Individual Clinical Observations -2000 mg/kg

		Effects Noted	During Period	After Dosing
Dose Level mg/kg	Animal Number and Sex	30 Minutes	1 Hour	2 Hours	4 Hours	1 Day
2000	2-0 Female	0	0	0	0	HLPBAÅX·
	2-1 Female	0	0	0	0	HLPBAÅ (HoRdE)X*
	2-2 Female	0	0	0	0	X

0=   No signs of systemic toxicity

A =  Ataxia

B =  Exophthalmos

E =  Pallor of the extremities

H =  Hunched posture

Ho =      Hypothermia

L =  Lethargy

P =   Pilo‑erection

Rd =Decreased respiratory rate

Å=  Cheeks swollen and bulging

( ) =Observations noted prior to humane killing

X =  Animal dead

X·=Animal found dead at mortality inspection

*=    Animal killed for humane reasons due to the occurrence of clinical signs of toxicity that exceeded the severity limit set forth in the UK Home Office Project Licence

0=   No signs of systemic toxicity

Appendix4     Individual Body Weights and Body Weight Changes -300 mg/kg

		Body	Weight (g)	at Day	Body Weight Gain (g)	During Week
Dose Level mg/kg	Animal Number and Sex	0	7	14	1		2
300	1-0 Female	163	178	196	15		18
	1-1 Female	166	181	197	15		16
	1-2 Female	153	168	189	15		21
	3-0 Female	162	174	182	12		8
	3-1 Female	166	176	189	10		13
	3-2 Female	162	174	191	12		17

Appendix5     Individual Body Weights and Body Weight Changes -2000 mg/kg

		Body	Weight (g)	at Day	Body Weight (g)	Body Weight Gain (g)	During Week
Dose Level mg/kg	Animal Number and Sex	0	7	14	at Death	1	2
2000	2-0 Female	181	-	-	180	-	-
	2-1 Female	172	-	-	168	-	-
	2-2 Female	156	-	-	166	-	-

Appendix6     Individual Necropsy Findings-300 mg/kg

Dose Level mg/kg	Animal Number and Sex	Time of Death	Macroscopic Observations
300	1-0 Female	Killed Day 14	No abnormalities detected
	1-1 Female	Killed Day 14	No abnormalities detected
	1-2 Female	Killed Day 14	No abnormalities detected
	3-0 Female	Killed Day 14	No abnormalities detected
	3-1 Female	Killed Day 14	No abnormalities detected
	3-2 Female	Killed Day 14	No abnormalities detected

Appendix7     Individual Necropsy Findings -2000 mg/kg

Dose Level mg/kg	Animal Number and Sex	Time of Death	Macroscopic Observations
2000	2-0 Female	Found dead Day 1	Liver: dark Kidneys: dark
	2-1 Female	Humanely killed Day 1	No abnormalities detected
	2-2 Female	Found dead Day 1	Lungs: abnormally red Liver: dark Kidneys: dark

Interpretation of results:

Category 4 based on GHS criteria

Conclusions:

The acute oral median lethal dose (LD50) of the test item in the female Wistar strain rat was approximately 500 mg/kg body weight (LD50 cut-off: 500 mg/kg body weight, Globally Harmonized Classification System: Category 4, >300 - 2000 mg/kg body weight).

Executive summary:

Introduction

The study was performed to assess the acute oral toxicity of the test item in the Wistar strain rat.

Methods

A group of three fasted females was treated with the test item at a dose level of 300 mg/kg body weight. Based on the results from this dose level, further groups of fasted females were treated at dose levels of 2000 and 300mg/kg body weight. Dosing was performed sequentially.

The test item was administered orally as a suspension in arachis oil BP. Clinical signs and body weight development were monitored during the study. All animals were subjected to gross necropsy.

Results

Mortality. One day after dosing, all animals treated at a dose level of 2000 mg/kg were found dead or killed for humane reasons due to the occurrence of clinical signs of toxicity that exceeded the severity limit set forth in the UK Home Office Project Licence. There were no deaths noted at a dose level of 300 mg/kg.

Clinical Observations. Signs of systemic toxicity noted at a dose level of 2000 mg/kg were hunched posture, lethargy, pilo‑erection, ataxia, exophthalmos, decreased respiratory rate, hypothermia and pallor of the extremities. The cheeks of two animals treated at a dose level of 2000 mg/kg were swollen and bulging. No signs of systemic toxicity were noted prior to death in one animal treated at a dose level of 2000 mg/kg. No signs of systemic toxicity were noted at a dose level of 300 mg/kg.

Body Weight. Surviving animals showed expected gains in body weight.

Necropsy. Abnormalities noted at necropsy of animals treated at a dose level of 2000 mg/kg, that were found dead, were abnormally red lungs,dark liver and dark kidneys. No abnormalities were noted at necropsy of the animal treated at a dose level of 2000 mg/kg that was humanely killed during the study and animals treated at a dose level of 300 mg/kg.

Conclusion

The acute oral median lethal dose (LD₅₀) of the test item in the female Wistar strain rat was approximately 500 mg/kg body weight (LD50 cut-off: 500 mg/kg body weight, Globally Harmonized Classification System: Category 4, >300 - 2000 mg/kg body weight).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LD50

Value:

500 mg/kg bw

Quality of whole database:

reliable without restriction

Acute toxicity: via inhalation route

Endpoint conclusion

Endpoint conclusion:

no study available

Acute toxicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Category 4

The acute oral median lethal dose (LD₅₀) of the test item in the female Wistar strain rat was approximately 500 mg/kg body weight (LD50 cut-off: 500 mg/kg body weight, Globally Harmonized Classification System: Category 4, >300 - 2000 mg/kg body weight).