Reaction product of: 2,4-diamino-6-[2-(2-methyl-1H-imidazol-1-yl)ethyl]-1,3,5-triazine and cyanuric acid

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 Aug 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

Materials and methods

Test guideline

Test guideline 1

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

Deviations:

yes

Remarks:

instead of the In Vitro Irritancy Score (IVIS) used in TG OECD 437, another scoring system was used: the Curren et al In Vitro Scores of up to 25 are classified as negative; scores greater than 25 are classified as positive potential eye irritants.

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Gautheron, P., Dukic, M., Alix, D. and Sina, J.F. (1992). Bovine corneal opacity and permeability test: An in vitro assay of ocular irritancy. Fundam. Appl. Toxicol. 18:442-449.

Principles of method if other than guideline:

- Principle of test: The test followed the principles established by Gautheron, et al. (1992), the same as are incorporated into OECD TG number 437: Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage. The OECD TG was adopted 26 July 2013.

- Short description of test conditions: Briefly, two endpoints were measured in the assay, which used isolated bovine corneas obtained as a by-product from the meat production industry. These measurements were combined to give an In Vitro Score which was used to determine the potential eye irritancy of the test substance according to the Prediction Model developed by Curren, et al. (1996).

- Parameters analysed / observed: Corneal opacity and permeability.

Curren, R.D., Harbell, J.W., Raabe, H.A.,Sussman, R.G., and Kimmel, T.A. (1996). The utility of a two-test in vitro battery to assess the ocular irritancy of drug intermediates: Developments in Animal and Veterinary Sciences, 27. Proceedings of the 2nd World Congress on Alternatives and Animal use in the Life Sciences, Utrecht, 20-24 October 1996.

GLP compliance:

yes (incl. QA statement)

Remarks:

The Department of Health of the Government of the United kingdom, London, England.

Test material

Test material information

Constituent 1

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Local abattoir as a by-product from freshly slaughtered animals.
- Number of animals: no data
- Characteristics of donor animals: no data
- Storage, temperature and transport conditions of ocular tissue: Eyes were excised after slaughter and were transported on the same day (of slaughter), at ambient temperature, in Hank's Balanced Salt Solution (HBSS) supplemented with antibiotics.
- Time interval prior to initiating testing: Corneas were used within 4 hours of slaughter.
- indication of any existing defects or lesions in ocular tissue samples: The eyes were free of defects such as opacity, scratches, pigmentation, cuts, and neovascularization. Prior to testing, corneal opacity was determined by the amount of light transmitted through the cornea via a opacitometer.
- Indication of any antibiotics used: 1% (w/v) Penicillin/Streptomycin in HBSS.

Test system

Vehicle:

physiological saline

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 0.75 mL
- Concentration (if solution): 20% (w/v) solution in 0.9% (w/v) sodium chloride

VEHICLE
- Amount applied: 0.75 mL
- Concentration (if solution): 0.9% sodium chloride solution
- Lot/batch no.: Sigma, S8776 lot number 125K2365
- Purity: 0.9%

Duration of treatment / exposure:

240 minutes at 32 ± 1 °C

Observation period (in vivo):

Not applicable

Duration of post- treatment incubation (in vitro):

Not applicable

Number of animals or in vitro replicates:

3

Details on study design:

SELECTION AND PREPARATION OF CORNEAS: Sourced from a local abattoir as a by-product from freshly slaughtered animals.

QUALITY CHECK OF THE ISOLATED CORNEAS: The eyes were visually inspected to be free of defects such as opacity, scratches, pigmentation, cuts, and neovascularization. Prior to testing, corneal opacity was determined by the amount of light transmitted through the cornea via a opacitometer.

NUMBER OF REPLICATES: 3

NEGATIVE CONTROL USED: 0.9% sodium chloride

POSITIVE CONTROL USED: Imidazole 20% (w/v) in 0.9% (w/v) sodium chloride

APPLICATION DOSE AND EXPOSURE TIME: 0.75 mL for 240 minutes at 32 ± 1 °C

TREATMENT METHOD: closed chamber

POST-INCUBATION PERIOD: no

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: at least three times

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: a post-treatment opacity was measured and each cornea was visually observed prior to incubation with sodium fluorescein [5 mg/mL in cMEM]
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of [UV/VIS spectrophotometry / microtiter plate reader] (OD490): after 90 minutes at 32 ± 1 °C
- Others: None

SCORING SYSTEM: In Vitro Score according to the Prediction Model developed by Curren, et al (1996).

DECISION CRITERIA: In Vitro Scores up to 25 are classified as negative and scores greater than 25 are classified as positive eye irritants.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

mean out of all 3 eyes

Value:

3

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: throughout the assay,the corneas were examined for opaque spots or other irregularities. The corneas treated with the test material were noted as clear with opaque spots. The corneas treated with the positive control, Imadazole, were very opaque. The negative control treated corneas were clear.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control gave opacity of 0.333 ± 2.309 and permeability of 0.003 ± 0.001, therefore acceptance criteria were satisfied.
- Acceptance criteria met for positive control: The positive control In Vitro Score was 119.3 ± 10.1, therefore the acceptance criteria were satisfied.
- Range of historical values if different from the ones specified in the test guideline: Positive Control, Imidazole assay validity acceptance range: 114.2 to 168.1.

Any other information on results incl. tables

Treatment	Cornea number	Opacity				Permeability (OD)		In Vitro Score
		Pre-trmt	Post-trmt	Post – pre	Corrected change		Corrected
Negative Control	1	5	4	-1		0.004
	2	4	7	3		0.004
	3	4	3	-1		0.002
Test Material	7	5	11	6	5.667	0.006	0.003	5.707
	8	4	8	4	3.667	0.002	-0.001	3.647
	9	5	5	0	-0.333	0.002	-0.001	-0.353
Positive Control	4	4	110	106	105.667	1.477	1.474	127.772
	5	4	93	89	88.667	2.22	2.217	121.917
	6	5	77	72	71.667	2.435	2.432	108.142

OD = optical density

trmt = treatment

Applicant's summary and conclusion

Interpretation of results:

other: CLP/EU GHS criteria are not met, no classification required according to Regulation (EC) No 1272/2008

Conclusions:

The test substance is a negative potential eye irritant.