4,4'-methylenebis(cyclohexylamine)

Eye irritation

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1985

Report date:

1985

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

4,4'-methylenebis(cyclohexylamine)

EC Number:

217-168-8

EC Name:

4,4'-methylenebis(cyclohexylamine)

Cas Number:

1761-71-3

Molecular formula:

C13H26N2

IUPAC Name:

4,4'-methylenedicyclohexanamine

Details on test material:

4,4'-Methylenedicyclohexanamine > 95% purity. Liquid with freezing/melting point of 13 degrees C.
Isomer content (%):
trans-trans 21.22
cis-cis 14.91
cis-trans 59.67
Impurities:2,4'-Methylenedicyclohexanamine: 4.20%
[4-(p-aminobenzyl)cyclohexylamine] methane (PABC): 0.23%
p-aminodicyclohexylmethane (ADCM): 0.05%
p-PABC: 0.038%

Test animals / tissue source

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

males, each weighing 2-3 kg.

Test system

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.

Results and discussion

In vivo

Results

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.

Any other information on results incl. tables

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

 

Applicant's summary and conclusion

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.