N,N'-methylenebis[methacrylamide]

Acute Toxicity: oral

Administrative data

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1990-08-17 to 1990-09-20

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1990

Report date:

1990

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

EU Method B.1 (Acute Toxicity (Oral))

Version / remarks:

1984-09-19

GLP compliance:

yes

Test type:

standard acute method

Test material

Test material information

Constituent 1

Reference substance name:

N,N'-methylenebis[methacrylamide]

EC Number:

219-102-3

EC Name:

N,N'-methylenebis[methacrylamide]

Cas Number:

2359-15-1

Molecular formula:

C9H14N2O2

IUPAC Name:

N,N'-methylenebis(2-methylacrylamide)

Test material form:

solid: particulate/powder

Details on test material:

- Designation: N,N’-Methylene-bis-methacrylamide
- Chemical name: 2-Propenamide, N,N’-methylene-bis (2-methyl)
- Characteristics: white powder
- Storage conditions: at room temperature in the dark

Specific details on test material used for the study:

The test item was prepared at various (w/v) concentrations in 1 % w/v aqueous methylcellulose and administered at a volume of 10.0 mL/kg bodyweight.
The test substance was prepared on the day of dosing.
The absorption of the test substance was not determined.

Test animals

Species:

rat

Strain:

Crj: CD(SD)

Remarks:

CD rats (Sprague-Dawley origin Crl. CD (SD) BR VAF plus)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River U.K. Limited, Margate, Kent, England.
- Age at study initiation: 4-6 weeks
- Weight at study initiation: 119-149 g prior to dosing (Day 1)
- Fasting period: access to food was prevented overnight prior to and approximately 4 hours after dosing.
- Housing: The rats were allocated without conscious bias to cages within the treatment groups. They were housed in groups of up to five rats of the same sex in metal cages with wire mesh floors.
- Diet: standard laboratory rodent diet (Biosure LAD 1) ad libitum. The batch of diet used for the study was analysed for certain chemical and microbiological contaminants.
- Water (e.g. ad libitum): domestic quality potable water ad libitum. Results of routine chemical examination of drinking water at source (Grafham Final Water - Huntingdon North supply zone) as conducted by the Anglian Water Services Ltd., are made available to Huntingdon Research Centre.
- Acclimation period: minimum 8 days prior to the start of the study
ENVIRONMENTAL CONDITIONS
- Temperature (°C): mean daily minimum and maximum temperatures of the animal room were 21°C and 24°C, respectively
- Humidity (%): mean daily relative humidity value was 59 %
- Air changes (per hr): rate of air exchange was maintained at approximately 15 air changes/hour
- Photoperiod (hrs dark / hrs light): 12 hours artificial light in each 24-hour period.
- Identification: Each animal was identified by cage number and ear punching. Each cage was identified by a coloured label displaying the dose level, study schedule number, animal mark and the initials of the Study Director and Home Office Licensee.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

methylcellulose

Details on oral exposure:

VEHICLE
1 % (w/v) aqueous methylcellulose
- Concentration in vehicle: 20, 32, 40 and 50 % (w/v)
- Amount of vehicle: 10.0 mL/kg bodyweight
MAXIMUM DOSE VOLUME APPLIED: 5.0 g/kg bodyweight

Doses:

2.0, 3.2, 4.0, 5.0 g/kg bw

No. of animals per sex per dose:

Please see table 1 in section "any other details on materials and methods incl. tables"

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing:
Bodyweight was determined on day 1, 8 and 15 or at death.
Observations: animals surviving treatment were observed once in the morning and again at the end of the experimental day. This latter observation was at approximately 16.30 hours on week days or 11.30 hours on public holidays, including Saturdays and Sundays. Clinical signs were recorded at each observation.
- Necropsy of survivors performed: yes
- Examinations performed: clinical signs, body weight, macroscopic examination

Statistics:

Probit analysis, Chi-Square test

Results and discussion

Preliminary study:

Initially, a group of five male and five female rats were treated at 5.0 g/kg bodyweight. Further groups of five male and/or five females were then dosed, to obtain a dose response curve and permit estimation of a median lethal dose.

Effect levels

Effect levels 1

Key result

Sex:

male

Dose descriptor:

LD50

Effect level:

2 500 mg/kg bw

Based on:

act. ingr.

95% CL:

>= 2 000 - <= 3 300

Effect levels 2

Key result

Sex:

female

Dose descriptor:

LD50

Effect level:

4 300 mg/kg bw

Based on:

act. ingr.

95% CL:

>= 3 700 - <= 5 100

Mortality:

Deaths following a single oral dose of the test item occurred among male rats dosed at 3.2 g/kg bodyweight and above and among female rats dosed at 4.0 g/kg bodyweight and above. Deaths occurred on Days 2 and 3.

Clinical signs:

- abnormal body carriage (hunched posture)
- abnormal gait (waddling)
- lethargy
- decreased respiratory rate
- pallor of the extremities in all rats
- ptosis in a majority of rats
- increased sensitivity to disturbance in two females dosed at 5.0 g/kg.

Body weight:

Slightly lower bodyweight gains were recorded for a majority of rats on Day 8; remaining rats achieved anticipated gains during this period. All rats achieved anticipated bodyweight gains during the second week of the study.

Gross pathology:

Terminal autopsy revealed no macroscopic abnormalities.

Any other information on results incl. tables

DETAILS ON RESULTS

1. Groups of five male and/or five female rats were dosed at 2.0, 3.2, 4.0 and 5.0 g/kg bodyweight to determine the acute toxicity of the test substance.

2. Mortality (Table 2)

There were deaths following a single oral dose of the test item among male rats dosed at 3.2 g/kg bodyweight and above and among female rats dosed at 4.0 g/kg bodyweight and above. Deaths occurred on Days 2 and 3. Bodyweight losses were recorded for all decedents. Post mortem examination of rats that died during the study revealed no macroscopic abnormalities.

3. Clinical signs (Table 3)

Pilo-erection was observed in all rats within five minutes of dosing and throughout the remainder of Day 1. This sign was accompanied on Day 1and/or at later intervals by abnormal body carriage (hunched posture), abnormal gait (waddling), lethargy, decreased respiratory rate and pallor of the extremities in all rats; ptosis in a majority of rats; increased sensitivity to disturbance in two females dosed at 5.0 g/kg.

Recovery of surviving rats, as judged by external appearance and behaviour, was complete by Day 3 (2.0, 4.0 and females at 3.2 g/kg) or Day 7 (females at 5.0 g/kg).

4. Bodyweight (Table 4)

Slightly low bodyweight gains were recorded for a majority of rats on Day 8; remaining rats achieved anticipated gains during this period. All rats achieved anticipated bodyweight gains during the second week of the study.

5. Post mortem examination: Terminal autopsy revealed no macroscopic abnormalities.

6. Estimation of LD₅₀ values

When probit analysis was carried out by fitting two parallel lines the values were:

Males: 2.5 (2.0 to 3.3) g/kg bodyweight

Females: 4.3 (3.7 to 5.1) g/kg bodyweight

The slope of the parallel probit lines was 14.3 with a standard error of 4.4 using log Transformation of dose. The heterogeneity factor was not significant.

In the mortality response curves, the difference between the lines for male and female rats was statistically significant (P <0.001). Hence, there is a difference in the susceptibility of each sex to the test compound. A combined LD₅₀ value was not therefore given since, as this is the mean value of the male and female LD₅₀ estimations, 50 % mortality would be expected, but would not actually occur, at this dose level. The chi-square test for parallelism gave no evidence of non-parallelism.

Table 2: time and number of deaths of rats dosed orally. The hour/day indicated is the time that the animal was observed to die or found dead. a = First observation; b = Second observation.

Sex	Dose (g/kg)	Number of deaths in a group of 5	Day
			1							2		3		4 to 15
			Hours after dosing
			0.5	1	2	3	4	5	6	a	b	a	b	a	b
males	2.0	0
	3.2	5								4		1
	5.0	5								4		1
females	3.2	0
	4.0	3								3
	5.0	3								2		1

Table 3: signs of reaction to treatment observed in rats dosed orally.

Signs	No. of rats in a group of five showing signs
	Dose (g/kg)
	2.0	3.2		4.0	5.0
	male	male	female	female	male	female
Pilo-erection	5	5	5	5	5	5
Abnormal body carriage (hunched posture)	5	5	5	5	5	5
Abnormal gait (waddling)	5	5	5	5	5	5
Lethargy	5	5	5	5	5	5
Decreased respiratory rate	5	5	5	5	5	5
Ptosis	0	5	5	1	5	5
Pallor of the extremities	5	5	5	5	5	5
Increased sensitivity to disturbance	0	0	0	0	0	2

Table 4: Individual and group mean bodyweights (g) of male rats dosed orally.

Sex	Dose (g/kg)	Animal number & ear mark	Bodyweight (g) at:
			Day 1	Day 8	Day 15	Death
male	2.0	21 RP	119	183	240	-
		22 LP	125	190	246	-
		23 RPLP	127	211	278	-
		24 RIRO	130	192	242	-
		25 LILO	126	207	267	-
	mean		125	197	255	-
	3.2	11 RP	122	-	-	116
		12 LP	130	-	-	117
		13 RPLP	128	-	-	117
		14 RIRO	132	-	-	125
		15 LILO	131	-	-	122
	mean		129	-	-
	5.0	1 RP	136	-	-	118
		2 LP	131	-	-	113
		3 RPLP	149	-	-	134
		4 RIRO	149	-	-	131
		5 LILO	140	-	-	113
	mean		141	-	-

Table 5: Individual and group mean bodyweights (g) of female rats dosed orally.

Sex	Dose (g/kg)	Animal number & ear mark	Bodyweight (g) at:
			Day 1	Day 8	Day 15	Death
female	2.0	16 RP	133	172	204	-
		17 LP	132	174	207	-
		18 RPLP	145	201	233	-
		19 RIRO	128	167	207	-
		20 LILO	129	172	212	-
	mean		133	177	213
	3.2	26 RP	127	169	195	-
		27 LP	124	-	-	109
		28 RPLP	130	183	216	-
		29 RIRO	122	-	-	109
		30 LILO	121	-	-	112
	mean		125	176	206
	5.0	6 RP	120	154	186	-
		7 LP	131	-	-	109
		8 RPLP	121	-	-	118
		9 RIRO	131	-	-	117
		10 LILO	134	163	213	-
	mean		127	159	200

 

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The acute median lethal oral doses (LD50) and their 95 % confidence limits to rats of the test item were estimated to be:
Males only: 2.5 (2.0 to 3.3) g/kg bodyweight
Females only: 4.3 (3.7 to 5.1) g/kg bodyweight

Executive summary:

In an acute oral toxicity study according to EU method B.1, version 1984-09-19, groups of five male and/or five female fasted rats were dosed with N,N’-methylenebis[methacrylamide] at 2.0, 3.2, 4.0 and 5.0 g/kg bodyweight. The rats were in the weight range of 119 to 149 g prior to dosing and approximately four to six weeks of age. The test item was dissolved in 1 % w/v aqueous methylcellulose and administered via gavage. The rats were observed for 14 days.

Deaths occurred following a single oral dose of N,N’-methylenebis[methacrylamide] among male rats dosed at 3.2 g/kg bodyweight and above and among female rats dosed at 4.0 g/kg bodyweight and above. Deaths occurred on Days 2 and 3.

Pilo-erection was observed in all rats within five minutes of dosing and throughout the remainder of Day 1. This sign was accompanied on Day 1 and/or at later intervals by abnormal body carriage (hunched posture), abnormal gait (waddling), lethargy, decreased respiratory rate and pallor of the extremities in all rats; ptosis in a majority of rats and increased sensitivity to disturbance in two females dosed at 5.0 g/kg.

Recovery of surviving rats, as judged by external appearance and behaviour, was complete by Day 3.

Slightly lower bodyweight gains were recorded for a majority of rats on Day 8; the remaining rats achieved anticipated gains during this period. All rats achieved anticipated bodyweight gains during the second week of the study.

Terminal autopsy revealed no macroscopic abnormalities.

The acute median lethal oral doses (LD₅₀) and their 95 % confidence limits to rats of N,N’-methylenebis[methacrylamide] were estimated to be:

Oral LD₅₀ (rat, males): 2.5 (2.0 to 3.3) g/kg bodyweight

Oral LD₅₀ (rat, females): 4.3 (3.7 to 5.1) g/kg bodyweight