4,4'-methylenebis(cyclohexylamine)

Endpoint summary

Description of key information

4-4’-Methylenedicyclohexanamine causes serious eye damage in rabbits (DuPont 1985) and showed severe corrosive effect to the skin of rabbits after 165 min exposure (Walker 1991). There is no data on respiratory irritation, but the vapour pressure of the substance is low and inhalation is not expected to be a significant route of exposure.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27.02.1991 to 22.03.1991

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Deviations:

no

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
Source: David Percival Ltd., Moston, Sandbach, Cheshire, U.K.
Strain: New Zealand White rabbit
Age at study initiation: twelve t o sixteen weeks
Sex: male and female
Weight at study initiation: 2.22 - 2.47 kg
Housing: individually in suspended metal cages
Diet(ad libitum):Spillers Rabbit Diet, Dalgety Agriculture Ltd., Almondsbury, Bristol
Water ad libitum

ENVIRONMENTAL CONDITIONS
Temperature: 18- 22 °C
Humidity: 50- 60 %
Air changes/ hour: 15
Photoperiod: 12 hours light/ 12 hours dark

Type of coverage:

semiocclusive

Preparation of test site:

clipped

Vehicle:

unchanged (no vehicle)

Controls:

not required

Amount / concentration applied:

0.5 ml

Duration of treatment / exposure:

3 minutes and approx 2.75 hours (4 hour exposure was terminated early)

Observation period:

1 hour, 24 hours, 48 hours and 72 hours following removal of the test material after the 3 minute exposure.
1 hour following removal of the test material after the 2.75 hour exposure.

Number of animals:

6

Details on study design:

The study was performed to assess the irritancy potential of the test material following single, semi -occluded applications to the intact rabbit skin for exposure periods o f 2 3/4 hours and 3 minutes (Safepharm Standard Method Number OECD 4). The method used followed the recommendations of the OECD Guidelines for Testing of Chemicals (1981) No. 404 "Acute Dermal Irritation/Corrosion" referenced as Method B4 in Commission Directive 84/449/EEC (which constitutes Annex V o f Council Directive 67/548/EEC).
The results may be used as a basis for classification and labelling under Annex VI o f Council Directive 67/548/EEC (as adapted to technical progress by Commission Directive 83/467/EEC).
The test system was chosen because the rabbit has been shown to be a suitable model for this type of study and is recommended in the test method.
The results of the study are believed to be of value in predicting the likely skin irritancy potential of the test material to man.

Approximately twenty-four hours prior to the commencement of the test, each of a group of three rabbits was prepared by closely clipping the fur from the dorsal flank areas using veterinary clippers.
Only animals with a healthy intact epidermis by gross observation were selected for the study. On the day of the test a suitable testsite was selected on the back of each rabbit. A quantity of 0.5 ml of the test material was introduced under a 2.5 cm x 2.5 cm gauze patch and placed i n position on the shorn skin. The patch was secured in position with a strip of surgical adhesive tape (BLENDERM: approximate size 2.5 cm x 4.0 cm).
To prevent the animals from interfering with the patches the trunk of each rabbit was wrapped in an elasticated corset (TUBIGRIP) and the animals were returned to their cages for the duration of the exposure period.
Approximately 2 3/4 hours after application the corset and patches were removed from each animal and any residual test material removed by gentle swabbing with cotton wool soaked in diethyl ether.

An additonal group of three rabbits was exposed to the test material underidentical conditions to the 2 3/4 hour exposure, except that the patches were removed three minutes after application to the skin. Approximately one hour following the removal of the patches, and 24, 48 and 72 hours later (where appropriate), the test sites were examined for evidence of primary irritation and scored according to the following scale from Draize J.H. (1959).

Irritation parameter:

primary dermal irritation index (PDII)

Basis:

mean

Time point:

other: 165 min

Score:

6

Max. score:

12

Reversibility:

no data

Irritation parameter:

erythema score

Remarks:

after 3 min exposure

Basis:

animal #1

Time point:

24/48/72 h

Score:

10

Max. score:

12

Reversibility:

not reversible

Irritation parameter:

erythema score

Remarks:

after 3 min exposure

Basis:

animal #2

Time point:

24/48/72 h

Score:

6

Max. score:

12

Reversibility:

not reversible

Irritation parameter:

erythema score

Remarks:

after 3 min exposure

Basis:

animal #3

Time point:

24/48/72 h

Score:

12

Max. score:

12

Reversibility:

not reversible

Irritation parameter:

edema score

Remarks:

after 3 min exposure

Basis:

animal #1

Time point:

24/48/72 h

Score:

11

Max. score:

12

Reversibility:

not reversible

Irritation parameter:

edema score

Remarks:

after 3 min exposure

Basis:

animal #2

Time point:

24/48/72 h

Score:

10

Max. score:

12

Reversibility:

not reversible

Irritation parameter:

edema score

Remarks:

after 3 min exposure

Basis:

animal #3

Time point:

24/48/72 h

Score:

12

Max. score:

12

Reversibility:

not reversible

Irritant / corrosive response data:

2.75 hour exposure -
Severe dermal necrosis, haemorrhage of the dermal capillaries and loss of skin elasticity were noted at all treated skin sites, one hour after patch removal. The reactions extended beyond the treatment site in two animals and an isolated incident of bleeding was also noted.
An accurate evaluation o f the degree of oedema was not possible due to the other adverse dermal reactions.
All animals were killed for humane reasons and in accordance with Home Office regulations following the one hour observation.

3 minute exposure -
Very slight to well-defined erythema and haemorrhage of the dermal capillaries were noted at all treatment sites one hour after patch removal and persisted at two sites at the 24-hour observation. Well-defined erythema was also noted at one treatment site at the 48- and 72-hour observations. A hardened dark brown/black coloured scab, surrounded by well-defined erythema and with light brown discolouration of the epidermis was also noted at the remaining treatment sites at these times. An accurate evaluation of erythema was not possible at the 48- and 72-hour observations due to the severity of the other dermal reactions.
Very slight oedema was noted at all treatment sites one hour aafter patch removal with moderate to severe oedema noted at all other observation times. Evaluation of oedema was commonly precluded by other adverse dermal reactions.
Green/brown coloured areas of possible dermal necrosis indicative of dermal corrosion were noted at one treated skin site at the 24- and 48-hour observations.
All animals were killed for humane reasons and in accordance with Home Office regulations after the 72-hour observation.

Other effects:

not reported

Conclusions:

The 3 minute exposure did not result in necrosis at the 1-hour observation period but one out of three animals exhibited necrosis at the 24-hour observation. The 2.75 hour exposure resulted in necrosis by the one hour post exposure observation. It was therefore determined that the test substance meets the criteria for classification as a Packing Group II, GHS 1B corrosive, and DSD R35 corrosive.

Executive summary:

In an OECD Guideline 404 study, rabbits were dermally exposed to PACM to assess skin irritancy/corrosivity. 

The test material was regarded as corrosive according to EEC labelling regulations.
The test material also produced a modified primary irritation index of 6.0 (based on values for erythema only).

 

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (corrosive)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Dose was lower, exposure times shorter and washing procedures different than OECD guidelines

Qualifier:

according to guideline

Guideline:

other: Haskell Laboratories protocol, E.I. DuPont DeNemours and Company, Wilmington, DE, USA

Principles of method if other than guideline:

Test material is administered into the conjunctival sac of a New Zealand white rabbit, and after various times, the material is removed by washing with various mixtures. Irritation is observed

GLP compliance:

not specified

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

males, each weighing 2-3 kg.

Vehicle:

unchanged (no vehicle)

Controls:

not required

Amount / concentration applied:

0.01 mL

Duration of treatment / exposure:

1.) 3 rabbits: not washed
2.) 3 rabbits: washed for 1 min, 20 s after dosing
3.) 3 rabbits: washed for 1 min, 2 mins after dosing
4.) 3 rabbits: washed for 15 mins as soon as possible after dosing

Observation period (in vivo):

Observations were made after 1 hr, 4 hrs, 24 hrs, 48 hrs, 72 hrs and weekly up to 60 days

Number of animals or in vitro replicates:

3 per group

Details on study design:

Preliminary Test:
10 mg of the test material were sprinkled onto the cornea of each eye of a rabbit, a second rabbit receives 0,1 mL of a 10 % solution (pH= 11) of the test material in propylene glycol in each eye. The left eye of each rabbit was washed with tap water 20 s after contact, the right eye was not washed.
Results indicated that 10 mg of the test item, as solid or as a solution in propylene glycol, produced severe and extensive damage to the cornea, iris and conjunctivae in the unwashed eyes. One week after treatment the eye that received the solution of the test item showed a severe and presumably irreversible injury. Similar treatments followed by prompt washing produced a temporary mild to moderate irritation of the cornea, iris and conjunctivae.

Main study (first procedure):
The test item used in the preliminary study was a white solid (MP 46°C), the "new" test item is a clear liquid (MP 13°C), because of different ratios of geometric isomers.
First 0,1 mL of the test item were used. The test reveales, that the liquid test item is a much more severe eye irritant than the solid test item.
0,1 mL of the test material were placed into the right conjuntival sac of each rabbit.
1.) 2 rabbits: not washed
2.) 2 rabbits: washed for 1 min, 20 s after dosing
3.) 2 rabbits: washed for 1 min, 2 mins after dosing
One day after treatment none of the treated eyes reacted to light, corrosive effects of all visible eye tissue were observed and the animals were losing weight. They were sacrificed two days after treatment when it became obvious the treated eye could not possible heal and the animals might not live through the two-month observation period.

Main study (second/ final procedure):
A reduced volume of 0,01 mL was used for the second study. The Test material (0.01 mL) was placed in the conjunctival sac and washed with tap water:
1.) 3 rabbits: not washed
2.) 3 rabbits: washed for 1 min, 20 s after dosing
3.) 3 rabbits: washed for 1 min, 2 mins after dosing
4.) 3 rabbits: washed for 15 mins as soon as possible after dosing
Observations of the cornea, iris and conjunctiva were made after 1 hr, 4 hrs, 24 hrs, 48 hrs, 72 hrs and weekly up to 60 days. Flour-i-strip opthalmic applicators and a biomicroscope were used at examinations after the day of treatment. Dr. Lionel F. Rubin (Veterinary Opthalmologist, University of Pennsylvania College of Veterinary Medicine) was consulted several weeks after treatment to observe some of the unusual ocular effects.
Seventy days post-exposure, three rabbits' eyes were chosen for histopathologic examination. The rabbits were sacrificed by air ambolism injected into ear vein. The eyes were quickly removed, fixed, washed and hardened using a procedure outlined by Saunders and Rubin. Trimming, processing, embedding, cutting ans staining were done by usual Haskell Laboratory techniques. The eye sections were sent to Dr. Rubin for description and commentary.

Irritation parameter:

other: qualitative assessment

Basis:

mean

Time point:

other: 60 days

Reversibility:

other: Each group had one rabbit whose eye was normal or nearly normal at 60 days, one whose eye was left with partial pannus and one in which one or both eyes had severe permanent ocular effects.

Remarks on result:

other: Dose of 0.01 ml is 1/10th "standard" dose of 0.1 ml.

Irritant / corrosive response data:

Pupillary constriction at 1-4 h, pupillary dilation through day 7, iridial congestion, with injection at 28 days, clouds of precipitate in anterior chamber, diffuse fibrous opacities of the lens, swelling and endothelial relucency by 7 days, corneal vascularization by 14 days, with subsequent healing of the ulceration by 35 days. The conjunctiva showed extreme redness and swelling within 1 h, with moderate to severe discharge (sometimes bloody and purulent).

Other effects:

Histopathology revealed dense corneal scarring, inflammation and lenticular opacity in selected animals. Vascular scarring of cornea can occur. In severe cases, crystals form and clots, with protein and cells present in the anterior chamber, and the lens capsule can rupture.

Opthalmoscopic and biomicroscopic findings

Rabbit No.	Dose	Treatment	Ocular Effects at Day ()
11834	0,1 mL	not washed	(1-2) Corrosive: all rabbits sacrificed 2 days post-treatment
11835	0,1 mL	not washed
11836	0,1 mL	washed 1 min, 20 s after dosing
11837	0,1 mL	washed 1 min, 20 s after dosing
11838	0,1 mL	washed 1 min, 2 mins after dosing
11839	0,1 mL	washed 1 min, 2 mins after dosing
11840	0,01 mL	not washed	(63) Eyelids irregular, 1/2 cornea pannus, lens opacities
11814	0,01 mL	not washed	(63) right eye: total pannus, corneal ulcer; left eye: lens opacities growth on iris
11902	0,01 mL	not washed	(16) small spot of corneal opacity, almost normal 21 days
11826	0,01 mL	washed 1 min, 20 s after dosing	(16) normal
11828	0,01 mL	washed 1 min, 20 s after dosing	(63) 2/3 - 1/2 corneal pannus with 2 cysts
11903	0,01 mL	washed 1 min, 20 s after dosing	(62) pannus, degradation of the iris, lens opacities, flattening of cornea (?)
11812	0,01 mL	washed 1 min, 2 mins after dosing	(16) normal
11815	0,01 mL	washed 1 min, 2 mins after dosing	(63) < 1/2 pannus with 1 cyst on cornea
11846	0,01 mL	washed 1 min, 2 mins after dosing	(62) 3/4 corneal pannus
11841	0,01 mL	washed 15 mins ASAP after dosing	(62) total pannus with corneal ulcer
11899	0,01 mL	washed 15 mins ASAP after dosing	(62) 1/2 corneal pannus, scattered lens opacities
11848	0,01 mL	washed 15 mins ASAP after dosing	(62) 1/4 very slight corneal opacity, scattered lens opacities
11833	-	washed 15 mins ASAP after dosing	(3) normal

 

Conclusions:

4,4'-Methylenedicyclohexanamine causes severe damage to the eye.

Executive summary:

At the original dose of 0,1 mL, the test item is irreversibly corrosive to rabbit eyes. Even at a dose one- tenth that normally used to test toxicity in rabbit eyes, the test item proved to be a severe eye irritant. No washing procedure tried appeard to be better than not washing the eyes. There were possible systemic effects in 10/12 rabbits with pupil dilation or constriction. In all groups there was considerable variability in the rate and extend of healing, although in all treated eyes there was severe initial irritation. 

Each group had one rabbit whose eye was normal or nearly normal at 60 days, one whose eye was left with partial pannus and one in which one or both eyes hab severe permanent ocular effects. 

Several rabbits' eyes developed lens opacities. Histopathology revealed a few abnormalities not found grossly. However some gross observations were missed upon examination of the sections, perhaps because the area of interest was not in the plane of section or because lenticular abnormalities are difficulr to preserve and see histopathologically.

When the eyes which are trated with the test item are washed with tap water (room temperature or colder) the compound tends to "gum up" and become opaque. This gum adheres tenaciously and wherever it sticks, the injury is greatest. Mechanical removal (with Q-tips ®) or washing with a substance other than water may reduce the severity of eye injury.

The possibility of delayed ocular effects due to the exposure to the test item does exist, it is a very severe eye irritant. All possible measures should be taken to avoid eye exposure.  

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

There is conflicting evidence in the literature regarding the degree of skin irritation caused by this substance. The weight of the evidence and the most reliable study showed the material to be corrosive.

Personal protective equipment is recommended for workers handling this substance.

Justification for selection of skin irritation / corrosion endpoint:
Most reliable study.

Justification for selection of eye irritation endpoint:
similar to but not according to guideline methods. 0.1 ml of liquid PACM resulted in clear eye corrosion. 100 mg of solid PACM resulted in eye corrosion.

Effects on skin irritation/corrosion: corrosive

Effects on eye irritation: corrosive

Justification for classification or non-classification

4-4’-Methylenedicyclohexanamine is classified as Category 1B, corrosive to the skin and causing serious damage to the eye according to CLP regulation 1272/2008.

Because of the low vapour pressure of this substance (0.05 Pa at 20 degrees C, measured. See section 4.6 in IUCLID), it is not anticipated to be volatile and able to be inhaled by workers. It is thus not classified as a respiratory irritant.