N-(1,1,3,3-tetramethylbutyl)acrylamide

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 27 March 2012 and 29 March 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do no effect the quality of the relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

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)

Remarks:

Date of inspection: 19 - 21 July 2011 Date of Signature: 31-August-2011

Test material

Test material information

Constituent 1

Reference substance name:

N-(1,1,3,3-tetramethylbutyl)acrylamide (t-OAA), CAS: 4223-03-4.

IUPAC Name:

N-(1,1,3,3-tetramethylbutyl)acrylamide (t-OAA), CAS: 4223-03-4.

Details on test material:

Sponsor's identification : N-(1,1,3,3-tetramethylbutyl)acrylamide (t-OAA), CAS: 4223-03-4.
Description : white solid
Batch number : 1242736
Purity : not supplied
Date received : 08 December 2011
Expiry date : 15 April 2012
Storage conditions : room temperature in the dark

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 ITEM

-The test Item was applied neat.

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

-Concentration (if solution):
The test item 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 mg of the test item.

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

-Type of wrap used:
None used.

REMOVAL OF TEST ITEM
-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 item was compared to the respective untreated negative control and classified according to the following:

Tissue viability <60 = Irritant
Tissue viability ≥60 = Non-Irritant

Results and discussion

In vitro

Results

Irritation parameter:

other: viability of cells

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

The relative mean viability of the test item treated tissues after a 10 Minute exposure period was 92.1%

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 item treated tissues after a 10-Minute exposure period was 92.1%.

It was considered unnecessary to proceed with tissue histopathology.

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 HCE Tissues

Item	OD540of Individual Tissue	Mean OD540	Relative Mean Viability (%)
Negative Control	0.943	0.961	100*
	0.978
Positive Control	0.206	0.182	18.9
	0.157
Test Item	0.900	0.885	92.1
	0.870

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

Applicant's summary and conclusion

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: expert judgment

Conclusions:

According to the study plan followed the test item was considered to be a Non-Irritant.

Executive summary:

Introduction. The purpose of this study was to determine the eye irritation potential of the test item using the SkinEthic reconstructed Human Corneal Epithelium model (HCE, SkinEthic Laboratories,,) 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. The experimental design of the study consists of a test for direct reduction of MTT (3‑[4,5‑dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) by the test item followed by the main test.

For the main test, triplicate SkinEthic tissues were treated with 30 mg of the test item 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 2% w/v Sodium Dodecyl Sulphate (SDS) 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 item was classified according to the following criteria:

If the percentage relative mean tissue viability was ≥60% the test item was considered to be non‑irritant.

If the percentage relative mean tissue viability was <60% the test item was considered to be an irritant.

Results. The relative mean viability of the test item treated tissues after a 10‑Minute exposure period was 92.1%.

It was considered unnecessary to proceed with tissue histopathology.

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

Conclusion. According to the study plan followed the test item was considered to be a Non-Irritant.