2-Propen-1-aminium, N,N,N-tripropyl-, salt with N-cyanocyanamide (1:1)

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study (GLP)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD Guideline for Testing of Chemicals No. 437, September 07, 2009 (“Bovine Corneal Opacity and Permeability Test, Method for Identifying Ocular Corrosives and Severe Irritants”)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Details on test material:

- CAS No.: 1312296-85-7
- Purity: 100.4 g/100 g determined by 1H-NMR-analysis.
- Homogeneity: The test substance was homogeneous by visual inspection.
- Storage stability: The stability under storage conditions over the study period was guaranteed by the sponsor, and the sponsor holds this responsibility.
- pH-value: ca. 5 (undiluted test substance, moistened with water); ca. 6 (20% aqueous solution)
- Physical state: Solid

Test animals / tissue source

Species:

other: in vitro study

Strain:

other: in vitro study

Details on test animals or tissues and environmental conditions:

TEST SYSTEM
- The test system (target tissue): isolated bovine cornea.
Bovine eyes are obtained as a by-product of freshly slaughtered cattle (age of the animals: minimum 12 months, maximum 60 months).
- Supplier: Schlachthof Bensheim, Am Schlachthof 7-9, 64625 Bensheim.

Corneas free of defects (opacity, scratches, pigmentation etc.) were dissected with a 2 to 3 mm rim of sclera. Isolated corneas were mounted in specially designed corneal holders that consist of anterior and posterior chambers. Both chambers were filled to excess with pre-warmed Eagles’s MEM (without phenol red) and then equilibrated in a vertical position at about 32 °C for at least 1 hour. After the equilibration period the medium in both chambers was replaced with fresh pre-warmed medium and initial corneal opacity readings were taken for each cornea with an opacitometer. Any corneas that show macroscopic tissue damage or an opacity value < 500 opacity units were discarded. Corneas with opacity values close to the median value of all corneas were selected as negative control (NC). The remaining corneas were then distributed into treatment and positive control groups. Each corneal holder was uniquely identified with a number on the chambers.

Test system

Vehicle:

water

Remarks:

Highly de-ionized water

Controls:

other: Negative control (NC): Highly de-ionized water. Positive control (PC): Imidazole (CAS No. 288-32-4) 20% (w/v) solution in highly deionized water for non-surfactant solid test substances

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 μL of test-substance preparation was applied directly to the epithelial surface of the cornea using a syringe (open chamber method)
- Concentration (if solution): 20% (w/v) suspension in highly de-ionized water

Duration of treatment / exposure:

approx. 4 hours.
The corneas were incubated in a horizontal position at about 32 °C for approximately 4 hours (non-surfactant solids). The test substance, NC and PC were then removed from the anterior chamber using a syringe and the epithelium was washed at least 3 times with Eagle’s MEM (containing phenol red) and once with Eagle’s MEM (without phenol red). Both chambers were then refilled with fresh Eagle’s MEM (without phenol red).

Observation period (in vivo):

Not applicable

Number of animals or in vitro replicates:

Each treatment group (test substance, NC and PC) consisted of 3 corneas

Details on study design:

- Measurement of final corneal opacity: each cornea was observed visually and observations were recorded. Final corneal opacity readings were taken for each cornea with an opacitometer.
- Determination of permeability: the medium in the anterior chamber was replaced by 1 mL sodium fluorescein solution (5 mg/mL for solid test substances) and incubated for 90 ± 5 min in a horizontal position at about 32 °C. The amount of sodium fluorescein that permeated through the corneas into the posterior chamber was measured spectrophotometrically. 3 aliquots per cornea were transferred to a 96-well microtiter plate and the optical density (OD490) was determined.

DATA EVALUATION
- Calculation of the corneal opacity value: The opacity change per cornea was calculated by subtracting the initial from the final opacity. Subsequently, the corrected opacity change should be calculated by subtracting the mean opacity change of the negative control. Due to high opacity values of the negative control this calculation step was not performed in the present study.
- Calculation of permeability value: the OD490 value was firstly calculated by subtracting the mean blank OD490 (blank = Eagle´s MEM w/o phenol red) from the OD490 of each cornea. If the OD490 value of the treated cornea was above 1.5, the OD490 of a 1:5 dilution was used to calculate the OD490 value. The corrected OD490 value was then calculated by subtracting the mean OD490 value of the negative control, and, finally, the mean OD490 value for each test substance could be determined as the mean of all corrected OD490 values per treatment group.
- Calculation of the In Vitro Irritancy Score (IVIS): IVIS was calculated per treated cornea and per treatment group as follows:
* IVIS per cornea = corrected opacity change + 15 * corrected permeability OD change;
* IVIS per treatment group = mean opacity value + 15 * mean permeability OD value.

ACCEPTANCE CRITERIA
- A study is considered acceptable if the positive control gives an IVIS that falls within two standard deviations of the current historical mean.
- The negative control responses should result in opacity and permeability values that are less than the established upper limits.
- Since the IVIS per treatment group is determined from the mean of three single corneas, the variability between the corneas treated per test substance should be acceptably low. If no clear prediction is possible, e.g. different predictions are obtained for single corneas, the test will be repeated.

EVALUATION CRITERIA:
- A risk of serious damage to the eyes is predicted if the IVIS per treatment group is greater than 55;
- No risk of serious damage to the eyes is predicted if the IVIS per treatment group is ≤ 55.

POSITIVE CONTROL
Imidazole (CAS No. 288-32-4) 20% (w/v) solution in highly deionized water for non-surfactant solid test substances.

HISTORICAL CONTROL DATA
Historical control values of negative and positive controls, gathered over an appropriate time period, were available. These data demonstrate the reproducibility of results and robustness of the procedures. They are used to derive suitable acceptance criteria for the test system.

Results and discussion

In vivo

Irritant / corrosive response data:

Based on the observed results and applying the evaluation criteria it was concluded, that the test item does not cause serious eye damage in the Bovine Corneal Opacity and Permeability Test (BCOP Test) under the test conditions chosen.

Any other information on results incl. tables

Table 1: Summary of opacity scores, Permeability scores and in vitro irritancy scores (IVIS)

 

Test item	Cornea number	Opacity score		Permeability score		In vitro irritancy score (IVIS)
		Corrected opacity change	Group mean ± SD	Mean Corrected OD490	Group mean ± SD	Per cornea	Group mean ± SD
Negative control	1	NA	12.8 ± 5.9	NA	- 0.006 ± 0.004	13.6	12.7 ± 5.9
	2	NA		NA		6.5
	3	NA		NA		18.2
Test substance	7	NA	4.3 ± 1.3	0.005	0.003 ± 0.004	5.8	4.4 ± 1.3
	8	NA		0.005		4.1
	9	NA		0.000		3.2
Positive control substance	4	NA	58.7 ± 1.7	3.181	2.887 ± 0.257	104.7	102.0 ± 2.4
	5	NA		2.774		100.1
	6	NA		2.706		101.1
NA: not applicable; SD: standard deviation

 

- All data were in the range of the historical control data.

Applicant's summary and conclusion

Interpretation of results:

other: no risk of serious eye damage

Conclusions:

The test item did not cause serious eye damage in the Bovine Corneal Opacity and Permeability Test (BCOP Test) under the test conditions chosen.

Executive summary:

The study was performed according to OECD guideline 439 in compliance with GLP.

The potential of the test item to cause serious damage to the eyes was assessed by a single topical application of 750 μL of a 20% test-substance preparation to the epithelial surface of isolated bovine corneas. Three corneas were treated with the test-substance preparation for an exposure period of 4 hours. Corneal opacity was measured quantitatively as the amount of light transmission through the cornea. Permeability was measured quantitatively as the amount of sodium fluorescein dye that passes across the full thickness of the cornea. Both measurements were used to calculate an In Vitro Irritancy Score of the test substance relative to the control corneas.

The BCOP test showed the following results:

Test item	Mean Opacity Value	Mean Permeability Value	In Vitro Irritancy Score
Negative control (NC)	12.8	- 0.006	12.7
Test substance	4.3	0.003	4.4
Positive control (PC)	58.7	2.887	102.0

Due to high opacity values of the negative control the opacity of the NC was not subtracted from the values of the test substance and the PC.

Conclusion: Based on the observed results and applying the evaluation criteria it was concluded, that the test item did not cause serious eye damage in the Bovine Corneal Opacity and Permeability Test (BCOP Test) under the test conditions chosen.