Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
The study was performed between 22 June 2010 and 25 June 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. certificate)
Remarks:
Date of inspection: 15-09-2009 Date of Signature: 26-11-2009

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Details on test material:
Sponsor's identification : Bis (2-hydroxyethyl) hydrogenated tallow alkylamine (CAS Number 90367-28-5)
Description : pale brown solid block
Batch number : S-001017
Date received : 08 July 2009
Expiry date : 09 July 2017
Storage conditions : approximately 4°C in the dark under nitrogen

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 mg of the test material. To improve test material tissue contact 30 µl of PBS was applied to the tissue surface and aspirated prior to the application of the test material.

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

VEHICLE
No vehicle used.
Duration of treatment / exposure:
60 Minutes and 16 post exposure incubation.
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 material for an exposure period of 60 minutes. To improve test material tissue contact 30 µl of PBS was applied to the tissue surface and aspirated prior to the application of the test material.

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

-Type of wrap used:
None used.

REMOVAL OF TEST SUBSTANCE
-Washing (if done):
At the end of the exposure period, each tissue insert was removed from the well using forceps and rinsed using a wash bottle containing Phosphate Buffered Saline Dulbeccos (PBS) without Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert at least ten times using a constant stream of PBS.

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

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

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

Tissue viability ≤ 50 = Irritant
Tissue viability > 50 = Non-Irritant

Results and discussion

In vitro

Results
Irritation parameter:
other: Viability of tissue %
Value:
50.2
Negative controls validity:
valid
Positive controls validity:
valid

Any other information on results incl. tables

RESULTS

Assessment of Direct Test Material Reduction of MTT

An assessment found the test material was able to directly reduce MTT. Therefore, an additional procedure using freeze-killed tissues was performed during the determination of ocular irritation potential. However the results obtained showed that no degree of interference due to direct reduction of MTT occurred indicating the test material was adequately rinsed from the tissues. It was therefore considered unnecessary to use the results of the freeze-killed tissues for quantitative correction of results or for reporting purposes.

Assessment of Eye Irritation Potential

The mean OD540 values and the relative mean tissue viability of the test material, and negative and positive control treatment groups are given in Table 1.

The relative mean viability of the test material treated tissues after the 60 -Minute exposure period and a 16-Hour post exposure incubation period was 50.2

Qualitative Evaluation of Tissue Viability (MTT Uptake Visual Assessment)

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

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

Mean OD 540

Viability (%)

Negative Control

0.917

0.934

100*

0.944

0.942

Positive Control

0.028

0.023

2.5

0.020

0.022

Test Material

0.457

0.469

50.2

0.507

0.442


*=      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

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

Conclusions:
According to the protocol followed the test material was considered to be a Non-Irritant (NI). However the viability of 50.2 is extremely close to the classification cut off of 50% for eye irritation and the in-vitro skin irritation the data indicates less sensitivity for this chemistry than in-vivo. Therefore Ethanol, 2,2’-iminobis-,N-(hydrogenated tallow alkyl) derivs. CAS No 90367-28-5 (registered as 2,2’-(C16-18 (evennumbered), alkyl imino) diethanol CAS No 1218787-30-4) will be classified as EU CLP(GHS) Category 2 for eye irritation with Hazard statement H319 Causes serious eye irritation.
Executive summary:

Introduction. 

The purpose of this study was to determine the eye irritation potential of the test material, using the SkinEthic Reconstituted Human Corneal Epithelial (HCE) model. The principle of the assay is based on the measurement of cytotoxicity in reconstituted human corneal epithelium cultures after topical exposure to the test material by means of the colourimetric MTT reduction assay. 

The endpoint, cytotoxicity in the MTT assay, expressed as percent viability of treated cultures in comparison to negative controls, was evaluated.

The test material was classified based on MTT viability analysis according to the following prediction model after a 60-Minute exposure period and 16-Hour post exposure incubation period:

i)  The test material was considered to be non-irritant to the eye if the tissue viability was >50%
ii) The test material was considered to be irritant to the eye if the tissue viability was ≤50%.

Results. 

The relative mean viability of the test material treated tissues after the 60‑Minute exposure period and a 16‑Hour post exposure incubation period was 50.2%.

Conclusion.  According to the protocol followed the test material was considered to be a Non-Irritant (NI).