3-Isocyanatomethyl-3,5,5-trimethylcyclohexyl isocyanate, oligomers reaction product with 3-(cyclohexylamino)propane-1-sulfonic acid and N,N-dimethylcyclohexanamine

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

February 2015

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Principles of method if other than guideline:

Test system HET-CAM: Assessment of the eye irritation potential of the test item by observing its effect on the extra-embryonal blood vessel system (e.g. haemorrhage, vessel lysis, coagulation) of the fertile 8 days incubated chicken egg.

The protocol for this study is executed according to the ICCVAM Test Method Evaluation Report Appendix B3: ICCVAM-Recommended Protocol for Future Studies Using the Hen's Egg Test–Chorioallantoic Membrane (HET-CAM) Test Method, September 2010.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Test material form:

liquid - solid: mixture of

Specific details on test material used for the study:

- The substance in the test item is dissolved in approx. 30 % solvent (Substance content approx. 70 %).

Test animals / tissue source

Details on test animals or tissues and environmental conditions:

The test system used for this study is the fertile Lohmann Brown hen egg, incubated for 8 days before application of test item.

SOURCE OF FERTILIZED CHICKEN EGGS
- Source: Brinkschulte Josef GmbH & Co.KG, 48308 Senden, Germany
- Indication of any existing defects or lesions in eggs: After 7 days of incubation, all eggs were candled in order to discard those that were defect and to mark the air bubble.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

300 µL per chicken egg

Duration of treatment / exposure:

5 min.

Duration of post- treatment incubation (in vitro):

not applicable

Number of animals or in vitro replicates:

4 eggs

Details on study design:

SELECTION AND PREPARATION OF EGGS
After 7 days in an incubator (37.5 °C, relative humidity 63%) with an automatic rotating device, all eggs were candled in order to discard those that were defect and to mark the air bubble. The eggs were replaced into the incubator with the large end upward but not rotated, thus ensuring accessibility to the CAM region. At day 8 of incubation the sections marked for the air bubble were sawed out of the shell. The inner membrane was moistened with NaCl 0.9 % and carefully removed with forceps. Only eggs with normally developed embryos and blood vessel systems were used for testing.

NUMBER OF REPLICATES
4 eggs each were used for the test substance, negative and positive controls.

APPLICATION DOSE AND EXPOSURE TIME / TREATMENT METHOD
For the positive (1% sodium dodecyl sulfate) and negative control 300 µl were applied per egg. Due to the viscosity of the test item a different kind of application has been selected. A piece of mesh was immersed in 300 µl of the unchanged test item and placed on the CAM. The size of mesh corresponding to approximately half of the CAM surface.

METHODS FOR MEASURED ENDPOINTS:
Observations of effects to the blood vessels, albumen or embryo over a period of 300 seconds after substance application are determined for each single egg. The time to the appearance of each of the observations mentioned above has been monitored and recorded. If no effect appeared during the observation period of 300 seconds (observation = 0) the result was assigned as negative for the related endpoint, and the factor set to 0 for this endpoint when calculating the Irritation Score (IS).

Formula for calculation of IS: IS = 5 x (301 - H [sec]/300) + 7 x (301 - L [sec]/300) + 9 x (301 - C [sec]/ 300)

where H= observed start of haemorrhage reactions in seconds, L= observed start of vessel lysis, strong haemorrhage in seconds and C= observed start of blood - coagulation, albumen - coagulation in seconds

Assessment of Irritation Scores (IS) according to Luepke, N.P., Fd. Chem. Toxic 23, 287-291, 1985:
Score                
0 - 0.9               no irritation
1 - 4.9               slight irritation
5 - 8.9               moderate irritation
9 - 21                strong irritation

DECISION CRITERIA:
A test substance is considered to cause severe eye irritation when the Irritation Score is equal to or above 9.

Results and discussion

In vitro

Results

Irritation parameter:

other: Irritation Score

Run / experiment:

Maximum Value 21; mean of 4 eggs

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Any other information on results incl. tables

Results with the test item:

Egg	Effect		Effects detected after [sec]	IS
1	H		> 300
	L		> 300
	C		> 300	0
2	H		> 300
	L		> 300
	C		> 300	0
3	H		> 300
	L		> 300
	C		> 300	0
4	H		> 300
	L		> 300
	C		> 300	0

H= observed start of haemorrhage reactions in seconds, L= observed start of vessel lysis, strong haemorrhage in seconds and C= observed start of blood - coagulation, albumen - coagulation in seconds

Applicant's summary and conclusion

Interpretation of results:

other: negative

Executive summary:

The HET-CAM (Hen's Egg Test on the Chorioallantoic Membrane) was conducted according to a protocol of the ICCVAM Test Method Evaluation Report 2010 in order to assess the eye irritation potential of the substance. In this test eight days incubated fertile hen eggs were opened and 300 µL of the test item was applied onto the chorioallantoic membrane. The test item contained the substance at 70 % (= "active ingredient") in 30 % solvent. Due to the viscosity of the test item, instead of pipetting the test item directly, a piece of mesh soaked with 300 µL was applied onto the chorioallantoic membrane. The effects occuring during the first 300 seconds after application were measured and used to calculate the irritation score. Based on this irritation score (0 of maximal 21) the test item was identified as non irritant to the chorioallantoic membrane.