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

Acute Toxicity: oral

Administrative data

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016 - 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

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

Test material

Test material information

Constituent 1

Reference substance name:

4-methoxy-N-methyl-3-nitrobenzamide

EC Number:

812-957-9

Cas Number:

333350-60-0

Molecular formula:

C9H10N2O4

IUPAC Name:

4-methoxy-N-methyl-3-nitrobenzamide

Test material form:

solid: particulate/powder

Details on test material:

Batch: Op. 2/16
Purity: 99.5%
Expiry date: 14.07.2018

Test animals

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.

Administration / exposure

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.

Results and discussion

Preliminary study:

Not applicable.

Effect levels

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:

All animals treated at a dose level of 2000 mg/kg were killed for humane reasons, 4 hours after dosing, due to the occurrence of clinical signs of toxicity that reached 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: Signs of systemic toxicity noted at a dose level of 2000 mg/kg were ataxia, hunched posture, lethargy, tiptoe gait, decreased and increased respiratory rate, labored respiration, ptosis, occasional body tremors and prostration. Hunched posture was noted

Gross pathology:

Abnormalities noted at necropsy of animals treated at a dose level of 2000 mg/kg were patchy pallor of the liver, white substance present in the stomach and epithelial sloughing of the gastric mucosa and non-glandular epithelium of the stomach.
No abnormalities were noted at necropsy of animals treated at a dose level of 300 mg/kg.

Other findings:

None

Any other information on results incl. tables

Appendix1     Mortality Data

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

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

Appendix2     Individual Clinical Observations -300 mg/kg

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

0=   No signs of systemic toxicity

A =  Ataxia

H =  Hunched posture

L =  Lethargy

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
2000	2-0 Female	HA	HWt	HLWtRi	RdRlPtPrToX*
	2-1 Female	HA	HWt	HLWtRi	RdRlPtPrToX*
	2-2 Female	HA	HWt	HLWtRi	RdRlPtPrToX*

A=   Ataxia

H =  Hunched posture

L =  Lethargy

Pr = Prostration

Pt = Ptosis

Rd =Decreased respiratory rate

Ri = Increased respiratory rate

Rl = Labored respiration

To =Occasional body tremors

Wt =Tiptoe gait

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

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	162	178	198	16		20
	1-1 Female	166	179	187	13		8
	1-2 Female	164	172	188	8		16
	3-0 Female	188	207	221	19		14
	3-1 Female	163	182	196	19		14
	3-2 Female	179	194	214	15		20

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	166	-	-	163	-	-
	2-1 Female	159	-	-	158	-	-
	2-2 Female	169	-	-	164	-	-

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	Humanely killed Day 0	Liver: patchy pallor Stomach: white substance present Gastric mucosa: epithelial sloughing Non-glandular epithelium of the stomach: epithelial sloughing
	2-1 Female	Humanely killed Day 0	Liver: patchy pallor Stomach: white substance present Gastric mucosa: epithelial sloughing Non-glandular epithelium of the stomach: epithelial sloughing
	2-2 Female	Humanely killed Day 0	Liver: patchy pallor Stomach: white substance present Gastric mucosa: epithelial sloughing Non-glandular epithelium of the stomach: epithelial sloughing

Applicant's summary and conclusion

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. Allanimals treated at a dose level of 2000 mg/kg were killed for humane reasons, 4 hours after dosing, due to the occurrence of clinical signs of toxicity that reached 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 ataxia, hunched posture, lethargy, tiptoe gait, decreased and increased respiratory rate, labored respiration, ptosis, occasional body tremors and prostration. Signs of systemic toxicity noted at a dose level of 300 mg/kg were hunched posture, ataxia and lethargy. All animals treated at a dose level of 300 mg/kg appeared normal 1 day after dosing.

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 were patchy pallor of the liver, white substance present in the stomach and epithelial sloughing of the gastric mucosa and non-glandular epithelium of the stomach. No abnormalities were noted at necropsy of 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).