1,6-Hexanediamine, N1,N6-bis(1,2,2-trimethylpropyl)-

Eye irritation

Administrative data

Endpoint:

eye irritation

Remarks:

in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

26 August 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Followed established Guideline and GLP requirements

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

In vitro test in step wise procedure of Guideline 405

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: OECD No. 437 (2009) "Bovine Corneal Opacity and Permeability Assay"

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

(3,3-dimethylbutan-2-yl)({6-[(3,3-dimethylbutan-2-yl)amino]hexyl})amine

EC Number:

682-872-8

Cas Number:

957787-76-7

Molecular formula:

C18H40N2

IUPAC Name:

(3,3-dimethylbutan-2-yl)({6-[(3,3-dimethylbutan-2-yl)amino]hexyl})amine

Details on test material:

- Name of test material (as cited in study report): SD10
- Physical state: Clear, colorless liquid
- Analytical purity: 96.9%
- Lot/batch No.: 9147-192-3a
- Expiration date of the lot/batch: 11 April 2012
- Storage condition of test material: room temperature in dark, under nitrogen

Test animals / tissue source

Species:

other: bovine corneas

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: local abbatoir
- Age at study initiation: adult cattle, eyes obtained from freshly slaughtered animals

ENVIRONMENTAL CONDITIONS
- Temperature (°C): eyes transported in Hanks' Balanced Salt Solution supplemented by Penicillin/Streptomycin and placed on ice packs. Eyes refrigerated on arrival to lab, and used within 24 hours.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.75 mL test item or control item applied to each cornea.
- Concentration (if solution): Negative control was 0.9% sodium chloride solution.

Duration of treatment / exposure:

After test item or control item applied to cornea in the BCOP holder and the holder tilted to assure uniform applicaiton to the cornea, the holder containing the cornea was incubated at 32 +/- 1 degrees C for 10 minutes. The cornea was then rinsed three times with complete Minimum esssential medium (MEM) containing phenol red before a final rinse with complete MEM.

Observation period (in vivo):

A pre-treatment opacity reading of each cornea was taken before the exposure period. A post-treatment opacity reading was taken and each cornea observed visually after the rinsing with complete MEM. The holders were then incubated at 32 +/- 1 degrees C for 120 minutes +/- 10 minutes. After that incubation, a final opacity reading was taken and each cornea visually observed.Following the final opacity reading, the medium was removed from the anterior chamber and replaced with 1 ml sodium fluorescein (4 mg/ml). The holders were incubated for 90 minutes at 32 +/- 1 degrees C. Medium from the posterior chamber was used for optical density reading (a sample of 360 ul represented each cornea.

Number of animals or in vitro replicates:

Three corneas each for test item, negative control and positive control

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): Yes, after 10 minutes, the corneas were rinsed with MEM plus phenol red three times before a final rinse with complete MEM
- Time after start of exposure: 10 minutes of exposure

SCORING SYSTEM: Opacity reading using calibrated opacitometer; permeability measured by optical density at 492 nm;

TOOL USED TO ASSESS SCORE: Calibrated opacitometer; 96 well plate for measuring optical density at 429 nm.

Results and discussion

In vivo

Results

Irritation parameter:

other: In Vitro Irritation Score (IVIS)

Basis:

other: mean value for treated cornea group

Score:

ca. 66

Remarks on result:

other: The IVIS score was greater then 55, thus the test item is a severe irritant or corrosive

Irritant / corrosive response data:

See tables in results section for individual corneal and mean treatment values for opacity, permeability, corrected values of each, and the resulting IVIS scores.

Any other information on results incl. tables

Individual and Mean Corneal Opacity Measurements

		Opacity
Treatment	Cornea Number	Pretreatment/ Postreatment/Post Incubation	Post incubation - Pretreatment	Corrected Value
Negative Control	1	1 1 2	1
	2	1 1 1	0
	3	2 2 3	1
	Mean		0.7
Positive Control	4	2 28 28	26	25.3
	5	1 27 27	26	25.3
	6	2 25 25	23	22.3
	Mean			24.3
Test Item	7	3 5 9	6	5.3
	8	1 10 13	12	11.3
	9	1 4 18	17	16.3
	Mean			11.0

Individual and Mean Permeability Measurements and Resulting IVIS

		Permeability (Optical Density, OD)
Treatment	Cornea Number		Corrected Value	In vitro Irritancy Score (IVIS)
Negative Control	1	0.057
	2	0.127
	3	0.056
	Mean	0.080		1.9
Positive Control	4	2.860	2.780
	5	2.650	2.570
	6	3.350	3.270
	Mean		2.873	67.4
Test Item	7	4.545	4.465
	8	3.665	3.585
	9	3.025	2.945
	Mean		3.665	66.0

Corneal Epithelium Post Treatment and Post Incubation

		Observation
Treatment	Cornea Number	Post Treatment	Post Incubation
Negative Control	1	Clear	Clear
	2	Clear	Clear
	3	Clear	Clear
Positive Control	4	Cloudy	Cloudy
	5	Cloudy	Cloudy
	6	Cloudy	Cloudy
Test Item	7	Clear	Cloudy
	8	Cloudy	Cloudy
	9	Clear	Cloudy

Applicant's summary and conclusion

Interpretation of results:

highly irritating

Remarks:

Migrated information Criteria used for interpretation of results: other: Criteria in OECD No. 437

Conclusions:

Following assessment of all endpoint data in the Bovine Corneal Opacity and Permeability Assay, the test item was considered to have the potential to cause severe ocular irritancy in vivo. The in vivo eye irritation study is therefore not needed.

According to CLP (Reg. 1907/2006 and amendments) the substance is classified as causing irreversible eye effects (category 1) based on this in vitro result.

(Reg. 1907/2006 and amendments) the substance is classified as posing the risk of serious damage to eyes (R41).

Executive summary:

Following assessment of all endpoint data in the Bovine Corneal Opacity and Permeability Assay, the test item was considered to have the potential to cause severe ocular irritancy in vivo. The in vivo eye irritation study is therefore not needed. The In Vitro Irritancy Score, calculated on changes in opacity and permeability (optical density), was 66.0 for the test item compared to 67.4 for the positive control item (ethanol). According to the OECD Guideline No. 437, an IVIS of 55 or greater is interpreted as the test item has the potential for severe ocular irritancy in vivo.

According to CLP (Reg. 1907/2006 and amendments) the substance is classified as causing irreversible eye effects (category 1) based on this in vitro result.

(Reg. 1907/2006 and amendments) the substance is classified as posing the risk of serious damage to eyes (R41).