4,4'-methylenebis(cyclohexylamine)

Endpoint summary

Description of key information

From reliable experimental studies, the LD50 for 4,4'-Methylenedicyclohexanamine has been established for oral and dermal routes of administration (Biosearch 1987). There are only insufficient data available so that no reliable LC50 for inhalation could be derived. However, due to the corrosive properties of the test material, no acute inhalation toxicity study has to be performed (see Waiver for acute inhalation toxicity).

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:

1987-08-03 to 1987-09-22

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPP 81-1 (Acute Oral Toxicity)

Version / remarks:

1984

GLP compliance:

yes

Test type:

standard acute method

Limit test:

yes

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS:
- Source: Buckshire Corp., Perkasie, PA 18944
- Strain: Sprague-Dawley rat
- Sex: male and female
- Weight: 200- 300 g
- Housing: The animals were housed and mainained in accordance with standards set forth in the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 86-23)
- Acclimation period: at least 5 days prior to dosing, stainless steel elevated wire mesh flooring, 3-5 rats/ cage by sex
- Diet (ad libitum): Wayne Rodent-Blox
- Water (ad libitum): tap water
- Fasting period before study: the animals were deprived of food but not water over night prior to dosing

ENVIRONMENTAL CONDITIONS
- Temperature: 70- 80 °F
- Relative humidity: 30- 80 %
- Light: 12 hour light/ dark cycle

Route of administration:

oral: gavage

Vehicle:

other: 10% ethanol and corn oil

Details on oral exposure:

The test item was dosed as an 10 % (w/v) suspension in 10 % ethanol and corn oil.

Doses:

- males: 100, 250, 500, 562, 708, 1000 mg/kg
- females: 100, 250, 398, 500, 631 mg/kg

No. of animals per sex per dose:

5

Control animals:

no

Details on study design:

The laboratory rat was the system of choice since it is required in the federal guideline for this study and in addition has a long history of use for this kind of study.
Following the administration, the animals were allowed food and water ad libitum for the 14 days observation period during which the rats were observed for signs of toxicity and mortality. Animals were observed frequently on the day of dosing, twice per day on week days and once per day on weekends and holidays.
Individual weights were recorded on the day of dosing, weekly thereafter and prior to sacrifice. The animals were euthanized at the conclusion of the observation period. Gross necropsies were performed on all animals.

Statistics:

The LD 50 and its 95 % confidence interval were calculated employing the Litchfield & Wilcoxon Method (Litchfield, J.T. and Wilcoxon F., "A Simplified Method of Evaluating Dose-effect Experiments", J. Pharmacol. Exp. Ther. 96:99-115, 1949).

Sex:

male

Dose descriptor:

LD50

Effect level:

480 mg/kg bw

Based on:

test mat.

95% CL:

> 360 - < 650

Sex:

female

Dose descriptor:

LD50

Effect level:

350 mg/kg bw

Based on:

test mat.

95% CL:

> 260 - < 470

Key result

Sex:

male/female

Dose descriptor:

LD50

Effect level:

380 mg/kg bw

Based on:

test mat.

95% CL:

> 270 - < 550

Mortality:

MALES:
No deaths occured in the dose groups 100 mg/ kg bw and 250 mg/ kg bw.
1/5 animals died in the 500 mg/ kg bw dose group on day 1.
2/5 animals died in the 562 mg/ kg bw dose group on day 1.
5/5 animals died in the 708 mg/ kg bw and 1000 mg/ kg bw dose groups.

FEMALES:
No deaths occured in the dose groups 100 mg/ kg bw and 250 mg/ kg bw.
3/5 animals died on day 1 in the 398 mg/ kg bw dose group.
4/5 animals died in the 500 mg/ kg bw dose group, 3 on day 1 and 1 on day 2.
5/5 animals dies in the 631 mg/ kg dose group, 4 on day 1 and 1 on day 7.

Clinical signs:

other: Males: 100 and 250 mg/kg : ruffled appearance on day 1 500 mg/kg: ruffled appearance and (dark) nasal staining on the first 3 days 562 mg/kg: ruffled appearance, piloerection, genital staining, convulsions, stained body, dark nasal staining, muzzle stai

Gross pathology:

No treatment-related macroscopic findings were noted in the surviving animals. In the animals found dead, following observations were made:
FEMALES:
398 g/ kg bw, animal 1: spongy hemorrhagic lungs and gas in the stomach, animal 2: ocular discharge, dark nasal discharge, hemorrhagic lungs and dark liquid in the lower gastrointestinal tract, animal 3: oral and nasal discharge, spongy hemorrhagic lungs and injected stomach vessels.

500 mg/ kg bw, 2 animals found dead on day 1: hemorrhagic lungs, injected stomach vessels and a hemorrhagic stomach lining, animal 3: dark kidneys, injected stomach vessels, dark red swollen areas on the stomach lining and dark red liquid in the large intestines, animal 4: red nasal and oral discharge, a pale liver, injected stomach vessels, gas in the stomach, dark tarry material in the large intestines and yellowish material in the small intestines.

631 mg/ kg bw, 3 animals found dead on the initial day of dosing: injected stomach vessels, an irritated stomach lining and dark liquid in the stomach and lower gastrointestinal tract, animal 4: injected stomach vessels and dark liquid in the stomach and lower gastrointestinal tract, animal 5: pale liver, dark kidneys, dark liquid in the stomach and lower gastrointestinal tract.

MALES:
500 mg/ kg bw, animal 1: hemorrhagic lungs, injected stomach vessels and hemorrhagic areas on the stomach lining.

562 mg/ kg bw, animal 1: nasal discharge, pale lungs, injected stomach vessels, yellow material in the stomach and dark liquid in the lower gastrointestinal tract, animal 2: stained muzzle, 50 % of the stomach lining appeared irritated, liquid in the stomach and lower gastrointestinal tract, animal 3: bloated stomach containing food, adhesions between the stomach and abdominal wall, a possible blockage of the upper gastrointestinal tract.

708 mg/ kg bw, 5/5 animals: injected stomach vessels, an irritated stomach lining and dark liquid in the stomach and lower gastrointestinal tract.

*)

Other findings:

*) 1000 mg/ kg, 5/5 animals: pale lungs and kidneys, injected stomach vessels, orrotation of 30 % of the stomach lining and dark liquid in the lower gastrointestinal tract

Conclusions:

The LD50 of 4-4’-methylenedicyclohexanamine by oral gavage to Sprague-Dawley rats is 380 mg/kg bw.

Executive summary:

The test material was tested in an acute oral toxicity study with 55 male and female albino rats.

The animals were deprived of food but not water overnight prior to dosing. Each animal was weighted and dosed by directly administration of the test material, as prepared, into the stomach by gavage. During the 14 day observation period, the animals were observed for signs of toxicity and mortality and individual weights were recorded. All males which died were dead on day 1. The deaths occured in the two highest dose groups. All deaths of the females occured on day 1, except for 1 female given 500 mg/kg on day 2 and 1 female given 631 mg/kg on day 7. Clinical signs in all dose groups were ruffled appearance. In the higher dose groups of male animals also piloerection, genital staining, convulsions, stained body, dark nasal staining, muzzle staining and/or lethargy during the observation period occured. In the hihgest dose groups the male animals were comatose and died on day one. Females showed gasping immediately after dosing, ruffled appearance, lethargy and/or piloerection between days 1 and 8. No treatment-related macroscopic findings were noted in the surviving animals. In animals found dead the following observations were made: signs of irritated stomach, dark liquid in the stomach and lower gastrointestinal tract, pale lungs and kidneys, pale liver and/or hemorrhagic lungs.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LD50

Value:

380 mg/kg bw

Quality of whole database:

adequate
Klimisch 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:

the study does not need to be conducted because the substance is classified as corrosive to the skin

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:

31.10.1986 to 06.02.1986

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPP 81-2 (Acute Dermal Toxicity)

Version / remarks:

1978

GLP compliance:

not specified

Test type:

standard acute method

Limit test:

no

Species:

rabbit

Strain:

New Zealand White

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
-Source: Ace Animals Inc., Boyertown, PA
-Strain: New Zeeland white rabbit
-Weight at study initiation: 2-3 kg
-Caging: Stainless steel with elevated wire mesh flooring, 1 rabbit/cage.
The animals were individually housed and maintained in accordance with standards set forth in the Guide for the Care and Use of Laboratory Animals (DHEW Publication No. 80-23). The rabbits were acclimated to the laboratory for at least 5 days prior to dosing.
The animals were individually identified by an ear tag. Each cage was identified with a cage card.

ENVIRONMENTAL CONDITIONS:
-Temperature: 60°F - 75°F
-Relative Humidity %: 55 -+ 25
-Light: 12 hour light/dark cycle
-Diet/ water: Wayne 15% Rabbit Ration and tap water were provided ad libitum. Based on our current knowledge, no contaminants are known to be in this diet or water which might be expected to interfere with the objectives of the study.

Type of coverage:

semiocclusive

Vehicle:

other: none, test substance applied as powder

Details on dermal exposure:

The Test Article was dosed as supplied at the following levels:
Males Females
0.20 g/kg 0.20 g/kg
0.40 g/kg 1.58 g/kg
1.58 g/kg 2.24 g/kg
6.31 g/kg 3.16 g/kg
6.31 g/kg
All rabbits were weighed and the correct amount of Test Article was applied to approximately 10% of the body surface on each animal. The treated area was covered with a large porous gauze patch and wrapped with an impervious material to ensure that the animals would not ingest the Test Article. The dressings were removed after 24 hours and any excess material removed, where practical, using water or an appropriate solvent.

Duration of exposure:

24 hours

Doses:

- Males: 200, 400, 1580, 6310 mg/kg
- Females: 200, 1580, 2240, 3160, 6310 mg/kg

No. of animals per sex per dose:

5

Control animals:

no

Details on study design:

The Test Article was dosed as supplied at the mentioned levels. All rabbits were weighed and the correct amount of Test Article was applied to approximately 10% of the body surface on each animal. The treated area was covered with a large porous gauze patch and wrapped with an impervious material to ensure that the animals would not ingest the Test Article. The dressings were removed after 24 hours and any excess material removed, where practical, using water or an appropriate solvent.
The animals were observed for a 14 day period for signs of toxicity (systemic and topical) and for mortalities. Animals were observed frequently during the first day of dosing, and twice per day (morning and afternoon) on weekdays. On weekends and holidays, animals were observed once per day. Individual weights were recorded on the day of dosing, weekly thereafter, and prior to sacrifice. The animals were euthanized at the conclusion of the observation period. Gross necropsies were performed on all animals.

Statistics:

Statistical method: The LD50 was calculated employing the Thompson Moving Average Method as
modified by Well, (Biometries, September, 1952, Vol. 8, No. 3, pp 249-263).

Key result

Sex:

male/female

Dose descriptor:

LD50

Effect level:

2 110 mg/kg bw

Based on:

test mat.

95% CL:

> 1 380 - < 3 220

Sex:

male

Dose descriptor:

LD50

Effect level:

1 390 mg/kg bw

Based on:

test mat.

95% CL:

> 710 - < 2 750

Sex:

female

Dose descriptor:

LD50

Effect level:

2 150 mg/kg bw

Based on:

test mat.

95% CL:

> 1 820 - < 2 550

Mortality:

- Males: 200, 400, 1580, 6310 mg/kg: 0/5, 0/5, 3/5, 5/5. All deaths within 4 days post-dose
- Females: 200, 1580, 2240, 3160, 6310 mg/kg: 0/5, 0/5, 3/5, 5/5, 5/5. All deaths within 3 days post-dose

Clinical signs:

other: Males: - Mild erythema, oedema and/or signs of necrosis were noted after unwrapping at 24 hours. - Throughout the observation period surviving males showed slight irritation (200 mg/kg) or necrosis (400, 1580, 6310 mg/kg). Incidental findings were dried

Gross pathology:

Males:
-0,2 g/ kg: no gross abnormalities
-0,4 g/ kg: Small pustula on the liver of 1 animal
-1,58 g/ kg: Hemorrhagic lungs and injected stomach vessels for the animals found dead at 48 hours and 4 days. A nasal discharge, hemorrhagic lungs and injected stomach vessels for the 1/5 animals found dead at 72 hours. Hemorrhagic lungs were noted for one of the animals necropsied at the conclusion of the 14 days. No gross abnormalities were noted for the remaining 1/5 animals.
-6,31 g/ kg: A nasal discharge, a pale mottled liver and pale kidneys for one of the animals found dead at 48 hours. Hemorrhagic lungs, a pale liver and pale kidneys were noted for a second animal found dead at 48 hours, Hemorrhagic lungs, a pale mottled liver and pale kidneys were noted for the third animal found dead at 48 hours. A nasal discharge, a mottled liver and pale kidneys were noted for the animal euthanized at 72 hours.

Females:
-0,2 g/ kg: no gross abnormalities
-1,58 g/ kg: Pitted kidneys for 1 animal
-2,24 g/ kg: Gas in the gastrointestinal tract, for 1 animal found dead at 4 days. Clear fluid filled nodules on the kidneys noted for 1/5 animals, no gross abnormalities for the 2/5 animals necropsied at the conclusion of the 14 day observation period.
3,16 g/ kg: Clear fluid in the abdominal cavity for 1/5 animals found dead at 24 hours. Hemorrhagic lungs were noted for the other animal found dead at 24 hours. A nasal discharge, pale spongy lungs, pale kidneys and a pale liver with white nodules were noted for the animal found dead at 48 hours. Hemorrhagic lungs, a pale mottled liver and pale mottled kidneys
were noted for 1/5 animals found dead on day 3. Hemorrhagic lungs, a pale liver and pale kidneys were noted for the remaining animal found dead on day 3.
6,31 g/ kg: A pale liver and pale mottled kidneys were noted for the 1/5 moribund animals euthanized at 24 hours. A pale mottled liver and pale kidneys were noted for the 1/5 animals found dead at 48 hours.

The test material used in this test may be different from the material used currently. The currently available substance is a colorless liquid, whereas this substance is described as a powder. The difference has been explained by different proportions of isomers.

 

Mortality

 

Table 1

Male Rabbits	Dose Levels [g/kg]
	0.2	0.4	1.58	6.31
Day 1	0	0	0	1
Day 2	0	0	1	3
Day 3	0	0	1	1
Day 4	0	0	1	−
Day 5	0	0	0	−
Day 6	0	0	0	−
Day 7	0	0	0	−
Day 8	0	0	0	−
Day 9	0	0	0	−
Day 10	0	0	0	−
Day 11	0	0	0	−
Day 12	0	0	0	−
Day 13	0	0	0	−
Day 14	0	0	0	−
Total Dead	0	0	3	5
Total Alive	5	5	2	0
Average body weights, kg: initial	2,54	2,35	2,87	2,78
Average body weights, kg: 7 th day	2,56	2,33	2,69	-
Average body weights, kg: final	2,63	2,62	2,99	-

 

Table 2

Female Rabbits	Dose Levels [g/kg]
	0.20	1.58	2.24	3.16	6.31
Day 1	0	0	0	2	4
Day 2	0	0	2	1	1
Day 3	0	0	0	2	−
Day 4	0	0	1	−	−
Day 5	0	0	0	−	−
Day 6	0	0	0	−	−
Day 7	0	0	0	−	−
Day 8	0	0	0	−	−
Day 9	0	0	0	−	−
Day 10	0	0	0	−	−
Day 11	0	0	0	−	−
Day 12	0	0	0	−	−
Day 13	0	0	0	−	−
Day 14	0	0	0	−	−
Total Dead	0	0	3	5	5
Total Alive	5	5	2	0	0
Average body weights, kg: initial	2,20	2,87	2,27	2,72	2,46
Average body weights, kg: 7 th day	2,72	2,85	1,80	-	-
Average body weights, kg: final	2,91	22,95	2,02	-	-

Conclusions:

The Test Article, when dosed as supplied and studied in male and female albino rabbits combined, has an acute dermal LD50 of 2.11 g/kg with Confidence Limits from 1.38 to 3.22 g/kg.

Executive summary:

Object of Test: To determine the acute dermal toxicity of the Test Article.

Reference: The methods employed in the testing of this Test Article were similar to those proposed in 40 CFR, Section 163.81-2, Federal Register, August 22, 1978 and subsequently modified in accordance with the revised EPA Pesticide Assessment Guidelines of November, 1982.

Justification for Test System: The laboratory albino rabbit was the system of choice since it is required in the federal guideline for this study and in addition has a long history of use for this type of study.

The solid material was tested in new Zealand white rabbits according to guideline EPA OPP 81-2 (Acute Dermal Toxicity). From dose groups of 0.4 g/kg upwards, signs of necrosis and loss of body weight were seen in male rabbits. In the two highest dose groups some animals died on day 1-4 (see Table 1).

Females died in the three highest dose groups mainly within the first 4 days after application (see Table 2).

The compound has an acute dermal LD50 of 2.11 g/kg with Confidence Limits from 1.38 to 3.22 g/kg.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LD50

Value:

2 110 mg/kg bw

Quality of whole database:

adequate

Additional information

No reliable data exist for acute inhalation toxicity.

Justification for selection of acute toxicity – oral endpoint
Modern GLP study according to EPA guideline

Justification for selection of acute toxicity – inhalation endpoint
No further acute toxicity testing is necessary because the substance is corrosive.

Justification for selection of acute toxicity – dermal endpoint
performed according to EPA guideline protocol.

Justification for classification or non-classification

4,4'-Methylenedicyclohexanamine is classified as an acute Category 4 for oral exposure, as the LD50 of 380 mg/kg falls between 300 and 2000 mg/kg according to CLP regulation 1272/2008. This substance is not classified as an acute dermal toxicant as the LD50 of 2110 exceeds the threshold of 2000 mg/kg bw. No reliable information is available for acute inhalation toxicity.