Reaction mass of 2-methylpentane and Hexanol, branched and linear and diisopropyl ether

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was performed between 23 March 2010 and 25 March 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

no guideline available

Principles of method if other than guideline:

The protocol followed was considered to be a reliable alternative to the in vivo rabbit Draize eye irritation test in a pre-validation study. This study, using human derived keratinocytes which form a corneal epithelial tissue reconstruct, has been recommended by ECVAM for inclusion in a formal international validation study designed to offer a stand alone replacement to the in vivo test. Validation is expected to commence in 2010.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction Products of C3 alcohols and C3 alkenes obtained as by-products from the manufacture of propan-2-ol by hydration of propylene

EC Number:

701-241-0

Molecular formula:

A complex and variable combination of hydrocarbons having carbon numbers predominantly in the C3, C6 & C9 chain length and oxygenated organic molecules, predominantly diisopropyl ether and hexanol (branched and linear). See diagram

IUPAC Name:

Reaction Products of C3 alcohols and C3 alkenes obtained as by-products from the manufacture of propan-2-ol by hydration of propylene

Test animals / tissue source

Species:

other: Reconstructed Human Corneal Model

Strain:

other: Reconstructed Human Corneal Model

Details on test animals or tissues and environmental conditions:

Not applicable

Test system

Vehicle:

other: No vehicle used

Controls:

no

Amount / concentration applied:

TEST MATERIAL

-The test Material was applied neat.

-Amounts(s) applied (volume or weight with unit):
Triplicate tissues were treated with 30 µl of the test material.

-Concentration (if solution):
The test material was used as supplied.

VEHICLE
No vehicle used.

Duration of treatment / exposure:

10 Minutes.

Observation period (in vivo):

Not applicable

Number of animals or in vitro replicates:

Not applicable

Details on study design:

TEST SITE
-Area of exposure:
Triplicate tissues were treated with 30 µl of the test material.

-% coverage:
The test material was applied topically to the corresponding tissues ensuring uniform covering.

-Type of wrap used:
None used.

REMOVAL OF TEST SUBSTANCE
-Washing (if done):
At the end of the relevant exposure period, each tissue insert was rinsed using a wash bottle containing Dulbecco’s Phosphate Buffered Saline (DPBS). Rinsing was achieved by filling and emptying each tissue insert using a constant soft stream of DPBS to gently remove any residual test material. Excess DPBS was removed by blotting the bottom of the insert with absorbent paper.

-Time after start of exposure:
10 Minutes post exposure.

SCORING SYSTEM:
The relative mean tissue viability (percentage of the negative control) was calculated as follows:

mean OD540 of test material / mean OD540 of negative control x 100 = relative mean tissue viability (percentage of negative control)

The mean tissue viability for the test material was compared to the respective untreated negative control and classified according to the following:

Tissue viability <60 = Irritant (I)
Tissue viability =60 = Non-Irritant (NI)

Results and discussion

In vitro

Results

Irritation parameter:

other: percentage viability of cells

Value:

45.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

In vivo

Irritant / corrosive response data:

The relative mean viability of the test material treated tissues was 45.8% after a 10-minute exposure.

Any other information on results incl. tables

RESULTS

Assessment of Direct Test Material Reduction of MTT

The test material was not able to directly reduce MTT.

Assessment of Eye Irritation Potential

The mean OD₅₄₀ values and mean viabilities for each treatment group are given in Table 1.

The relative mean viability of the test material treated tissues after a 10 minute exposure was 45.8%.

It was considered unnecessary to proceed with tissue histopathology.

Qualitative Evaluation of Tissue Viability (MTT Uptake Visual Assessment)

The qualitative evaluation of tissue viability is presented in Table 2.

The test material and negative control material treated tissues appeared blue which was considered to be indicative of viable tissue. The positive control material treated tissues appeared blue/white which was considered to be indicative of semi-viable tissue.

Assay Acceptance Criterion

The quality criterion required for the acceptance of results in the test was satisfied.

Table 1          Assessment of Eye Irritation Potential – Viability of RHC Tissues

Material	Mean OD540 of Individual Tissues	Mean OD540 of Duplicate Tissues ±SD	Viability (%)
Negative Control	0.878	0.888 ± 0.014	100*
	0.898
Positive Control	0.029	0.029 ± 0.000	3.3
	0.029
Test Material	0.420	0.407 ± 0.018	45.8
	0.394

*=      The mean viability of the negative control tissues is set at 100%

Table 2          Qualitative Evaluation of Tissue Viability (MTT uptake visual evaluation)

Material	Score
	Tissue 1	Tissue 2
Negative Control	-	-
Positive Control	++	++
Test Material	-	-

MTT Visual Scoring Scheme of SkinEthic Tissues

-     =  Blue tissue (viable)

+    =  Blue/White tissue (semi viable)

++  =  Tissue completely white (dead)

Applicant's summary and conclusion

Interpretation of results:

Category 2 (irritating to eyes) based on GHS criteria

Conclusions:

According to the protocol followed the test material was considered to be an Irritant (I).

Executive summary:

Introduction. 

The purpose of this study was to determine the eye irritation potential of the test material using the Skin Ethic Reconstituted Human Corneal model (HCE, SkinEthic Laboratories, France) after a treatment period of 10 minutes. The test is based on the hypothesis that irritant chemicals are able to penetrate the corneal epithelial tissue and are sufficiently cytotoxic to cause cell death.

Methods. 

For the main test, triplicate SkinEthic tissues were treated with 30 µl of the test material for 10 minutes. Triplicate tissues treated with 30 µl of Solution A served as the negative control and triplicate tissues treated with 30 µl of 1% w/v Sodium Dodecyl Sulphate served as the positive control.

At the end of the exposure period each SkinEthic tissue was rinsed. The rinsed tissues (two per group) were taken for MTT loading. The remaining tissues were retained for possible histopathology. Following MTT loading the reduced MTT was extracted from the tissues.

After extraction the absorbency of triplicate aliquots of the extracted MTT solution for each SkinEthic tissue was measured. The optical density was measured at 540 nm (OD₅₄₀). Data are presented in the form of percentage viability (MTT conversion relative to negative controls).

The test material was classified according to the following criteria:

i)                   If the percentage relative mean tissue viability was =60% the test material was considered to be non-irritant (NI).ii)  If the percentage relative mean tissue viability was <60% the test material was considered to be an irritant (I).

Results. 

The relative mean viability of the test material treated tissues after a 10 minute exposure was 45.8%.

It was considered unnecessary to proceed with tissue histopathology.

Quality criteria. 

The quality criteria required for acceptance of results in the test were satisfied.

Conclusion.  According to the protocol followed the test material was considered to be an Irritant (I).