Cyclohexanamine, 4,4’-methylenebis[N-(2,2-dimethyl-3-(4-morpholinyl)propylidene)]-

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2021-10-18 to 2021-12-01

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU method B.48 (Isolated chicken eye test method for identifying occular corrosives and severe irritants)

Version / remarks:

2017-02-14

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

2018-06-25

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

(1E)-N-[4-({4-[(E)-[2,2-dimethyl-3-(morpholin-4-yl)propylidene]amino]cyclohexyl}methyl)cyclohexyl]-2,2-dimethyl-3-(morpholin-4-yl)propan-1-imine

EC Number:

954-590-8

Cas Number:

2522560-40-1

Molecular formula:

C31H56N4O2

IUPAC Name:

(1E)-N-[4-({4-[(E)-[2,2-dimethyl-3-(morpholin-4-yl)propylidene]amino]cyclohexyl}methyl)cyclohexyl]-2,2-dimethyl-3-(morpholin-4-yl)propan-1-imine

Test animals / tissue source

Species:

chicken

Strain:

other: ROSS 308

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: TARAVIS KFT., 9600 Sárvár, Rábasömjéni, utca 129. Hungary
- Number of animals: Number not specified, but eyes used in the assay were from the same groups of eyes collected on one specific day
- Characteristics of donor animals: Approx. 7 weeks old healthy animals, weighting 1.5 – 2.5 kg
- Storage, temperature and transport conditions of ocular tissue: After collection, the heads were wrapped with paper moistened with saline, then placed in a plastic box (4-5 heads/box).The heads were transported to the testing laboratory approx. within 2 hours from collection. The transport temperature was between 19.3 ºC and 20.2 ºC.
- Time interval prior to initiating testing: Approx. 2 hours
- Indication of any existing defects or lesions in ocular tissue samples: Cornea integrity was checked by applying one small drop of fluorescein 2% (w/v) solution onto the cornea surface for a few seconds and subsequently rinsed off with 20 mL isotonic saline. Then the fluorescein-treated cornea was examined with a slit lamp microscope, with the eye in the head, to ensure that the cornea was not damaged.
- Selection and preparation of corneas: After removing the head from the plastic box, it was put on soft paper. The eyelids were carefully cut away with scissors, avoiding damaging the cornea. If the corneas were in good condition, the eyeballs were carefully removed from the orbit. The nictitating membrane was held with a surgical forceps, while cutting the eye muscles with bent scissors without cutting off the optical nerve too short. The procedure avoided pressure on the eye in order to prevent distortion of the cornea and subsequent corneal opacity. Once removed from the orbit, the eye was placed onto damp paper and the nictitating membrane was cut away with other connective tissue. The prepared eyes were kept on the wet papers in a closed box so that the appropriate humidity was maintained. The enucleated eye was placed in a steel clamp, avoiding too much pressure on the eye by the clamp. The clamp with the eyeball was transferred to a chamber of the superfusion apparatus. The clamp holding the eye was positioned in such a way that the entire cornea was supplied with saline solution dripping from a stainless steel tube, at a rate of approximately 3 to 4 drops/minute. The door of the chamber was closed except for manipulations and examinations, to maintain temperature and humidity.
- Quality check of the isolated corneas: The enucleated eyes were examined again in the superfusion apparatus with the slit lamp microscope to ensure that they were in good condition. Eyes with a high baseline fluorescein staining (i.e. > 0.5) or a high corneal opacity score (i.e. > 0.5) were rejected. The cornea thickness was measured using the depth measuring device on the slit lamp microscope. Only corneas were used which did not show a change in cornea thickness by more than ± 5-7 % within approximately 45 to 60 minutes.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 30 μL of test item was applied via a micropipette onto the center of the cornea

Duration of treatment / exposure:

10 seconds

Duration of post- treatment incubation (in vitro):

The control and test item treated eyes stayed in the chambers until measurement were finished (approx. 240 minutes after the post-treatment rinse, minor variations within ± 5 minutes were considered acceptable).

Number of animals or in vitro replicates:

The treatment group and concurrent positive control group consisted of three eyes. The negative control group consisted of one eye.

Details on study design:

EQUILIBRATION AND BASELINE RECORDINGS
At the end of the acclimatization period, a zero reference measurement was recorded for cornea thickness and opacity to serve as a baseline (t=0) for each individual eye. Following the equilibration period, the fluorescein retention was measured. Baseline values were required to evaluate any potential test item related effects after treatment.

NUMBER OF REPLICATES
Three replicate eyes (positive control and treatment group) and one replicate eye (negative control)

OBSERVATION PERIOD
The control and test item treated eyes were evaluated pre-treatment and at approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse. Minor variations within ± 5 minutes were considered acceptable.

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: The time of application was monitored. After an exposure period of 10 seconds the cornea surface was rinsed thoroughly with 20 mL isotonic saline at ambient temperature, taking care not to damage the cornea but attempting to remove the entire residual test item if possible. The cornea surface of negative and positive control treated corneas was also rinsed thoroughly after an exposure period of 10 seconds with 20 mL saline solution at ambient temperature, while taking care not to damage the cornea. The eye in the holder was then returned to its chamber. The time while the eye was out of the chamber was limited to a minimum.

METHODS FOR MEASURED ENDPOINTS
- Corneal opacity: Slit-lamp microscope
- Damage to epithelium based on fluorescein retention: Slit-lamp microscope
- Swelling: Depth measuring device on the slit lamp microscope (Haag-Streit BQ 900) with the slit-width set at 9½, equaling 0.095 mm
- Macroscopic morphological damage to the surface: Slit-lamp microscope

SCORING SYSTEM
- Mean corneal swelling (%)
- Mean maximum opacity score
- Mean fluorescein retention score at 30 minutes post-treatment

DECISION CRITERIA: The decision criteria as indicated in the TG were used.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

fluorescein retention score

Run / experiment:

Mean of three replicate eyes

Value:

1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation parameter:

corneal swelling 

Run / experiment:

Mean of three replicate eyes

Value:

2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 3

Irritation parameter:

cornea opacity score

Run / experiment:

Mean of three replicate eyes

Value:

4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: No

DEMONSTRATION OF TECHNICAL PROFICIENCY: Prior to routine use of the method, the laboratory demonstrated the technical proficiency in a separate study (Study Code: 392.549.3229) using the ten Proficiency Chemicals according to OECD Test Guideline No. 438.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Remarks:

The final classification is chosen based on the results from both OECD 438 and OECD 492 guidelines and is described in the endpoint summary.

Conclusions:

In this ICET according to OECD guideline 438, the overall ICE classes of the test item were once I, once II and once IV. According to the guideline OECD 438, the test item is categorized as “Non prediction can be made”.

Executive summary:

The purpose of this Isolated Chicken Eye Test (ICET) according to OECD guideline 438 and GLP was to evaluate the potential ocular corrosivity or severe irritancy of the test item its ability to induce toxicity in enucleated chicken eyes. The test item was applied in a single dose onto the cornea of isolated chicken eyes in order to potentially classify the test compound as either 1: causing "serious eye damage" [category 1 of the Globally Harmonised System for the Classification and Labelling of chemicals (GHS)], or not requiring classification for eye irritation or serious eye damage according to the GHS. The ICET does not fully replace the in vivo rabbit eye test (OECD 405); however, the ICET is used as part of a tiered testing strategy for regulatory purposes. The test item SIKA Hardener MD (SIKA Härter MD), the positive control (5% solution of Benzalkonium chloride), and the negative control (NaCl, 9 g/L saline) were applied in a volume of 30 μL/eye, in such a way that the test and control items evenly cover the whole cornea surface of each tested eye. Three test item treated eyes, three positive control eyes and one negative control eye were used in this study. After an exposure period of 10 seconds from the end of the application the cornea surface was rinsed thoroughly with ~20 mL saline solution at ambient temperature and this procedure was repeated for each eye. In this ICET, the test item did not cause ocular corrosion or severe irritation in the enucleated chicken eyes. The overall ICE classes of the test item were once I (based on the fluorescein retention of 0.0) and once II (based on the cornea swelling of 17 % within 240 minutes) and once IV (based on the corneal opacity score of 2.7). The positive control was classed as corrosive/severely irritating, UN GHS Classification: Category 1 and the negative control had no significant effects on the chicken eye in this study. So, the positive and negative controls showed the expected results. The experiment was considered to be valid. In this ICET, the overall ICE classes of the test item were once I, once II and once IV. According to the guideline OECD 438, the test item is categorized as “Non prediction can be made”.