Registration Dossier

Administrative data

Description of key information

This is good quality skin irritation data which indicates moderate skin irritation potential for 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).  We have a Klimisch 1 in-vitro data which has been interpreted as indicating eye irritation potential.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20 April 2010 and 04 May 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.
Qualifier:
according to
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)
Deviations:
no
GLP compliance:
yes
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
TEST ANIMAL
- Source:
One New Zealand White rabbit supplied by an accredited supplier.

- Age at study initiation:
Twelve to twenty weeks old.

- Weight at study initiation:
At the start of the study the animal weighed 2.44 kg.

- Housing:
The animal wasindividually housed in a suspended cage.

- Diet (e.g. ad libitum):
Certified Rabbit Diet. ad libitum.

- Water (e.g. ad libitum):
ad libitum

- Acclimation period:
At least Five days.


ENVIRONMENTAL CONDITIONS
- Temperature (°C):
17 to 23°C

- Humidity (%):
30 to 70%
- Air changes (per hr):
The rate of air exchange was at least fifteen changes per hour.

- Photoperiod (hrs dark / hrs light):
The lighting was controlled by a time switch to give twelve hours continuous light (06:00 to 18:00) and twelve hours darkness.
Type of coverage:
semiocclusive
Preparation of test site:
shaved
Vehicle:
other: Moistened sufficiently with 0.5 ml of distilled water
Controls:
no
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit):
0.5 g

- Concentration (if solution):
Not applicable.

VEHICLE
Not applicable.

- Concentration (if solution):
Not applicable.

- Lot/batch no. (if required):
Not given in study report.
Duration of treatment / exposure:
3-minutes, 1-hour and 4 hours
Observation period:
14 days
Number of animals:
1
Details on study design:
TEST SITE
- Area of exposure:
On the day of the test a suitable test site was selected on the back of the rabbit.

- % coverage:
Not given in study report.

- Type of wrap if used:
Test material was introduced under a 2.5 cm x 2.5 cm cotton gauze patch and placed in position on the shorn skin.


REMOVAL OF TEST SUBSTANCE
- Washing (if done):
Any residual test material removed by gentle swabbing with cotton wool soaked in distilled water.

- Time after start of exposure:
3-minute, 1-hour and 4 hours.


SCORING SYSTEM:
Approximately one hour following the removal of the patches, and 24, 48 and 72 hours later, the test sites were examined for evidence of primary irritation and scored according to the following scale (table below).
Additional observations were made on Days 7 and 14 to assess the reversibility of skin reactions.
Irritation parameter:
erythema score
Remarks:
Erythema/Eschar Formation
Basis:
animal: 69116 male
Time point:
other: Mean score at 24, 48 and 72 hours
Score:
2
Max. score:
2
Reversibility:
fully reversible
Remarks:
within 14 days
Remarks on result:
other: Brown discolouration of the epidermis, loss of skin elasticty and loss of skin flexibility noted at the 24, 48 and 72-Hour observations. A hardened light brown coloured scab was noted at the 7-Day observation.
Irritation parameter:
edema score
Basis:
mean
Time point:
other: 24, 48 & 72-hour mean score
Score:
2
Max. score:
2
Reversibility:
fully reversible
Remarks:
Fully reversible in 14-days
Irritant / corrosive response data:
RESULTS

Skin Reactions

3-Minute Exposure Period

The scores for erythema/eschar and oedema are given in Table 1.
Very slight erythema was noted at the treated skin site at the 24, 48 and 72 Hour observations.
The treated skin site appeared normal at the 7-Day observation.

1-Hour Exposure Period

The scores for erythema/eschar and oedema are given in Table 1.
Well-defined erythema and very slight oedema were noted at the treated skin site at the 24, 48 and 72 Hour observations. Crust formation, preventing accurate evaluation of erythema and oedema, was noted at the treated skin site at the 7 Day observation.
The treated skin site appeared normal at the 14 Day observation.

4-Hour Exposure Period

The scores for erythema/eschar and oedema are given in Table 2.
Very slight erythema was noted at the treated skin site one hour after patch removal. Well-defined erythema, slight oedema, light brown discolouration and loss of skin elasticity and flexibility were noted at the treated skin site at the 24, 48 and 72-Hour observations. A hardened, light brown coloured scab, preventing accurate evaluation of erythema and oedema, was noted at the treated skin site at the 7 Day observation.
The treated skin site appeared normal at the 14 Day observation.

The individual scores for erythema/eschar and oedema are given in the folowing tables.

IndividualSkin Reactions - 3 -Minute and 1 -Hour exposures


Skin Reaction

Observation Time
(following patch removal)

Individual Scores - Rabbit Number and Sex

69116Male

3-Minute Exposure

1-Hour Exposure

Erythema/Eschar Formation

Immediately

0

0

1 Hour

0

0

24 Hours

1

2

48 Hours

1

2

72 Hours

1

2

7 Days

0

?eCf

14 Days

0

0

Oedema Formation

Immediately

0

0

1 Hour

0

0

24 Hours

0

1

48 Hours

0

1

72 Hours

0

1

7 Days

0

?od

14 Days

0

0


Cf=     Crust formation

?e =    Adverse skin reactions prevent accurate evaluation of erythema

?od =  Adverse skin reactions prevent accurate evaluation of oedema


Table2              Skin ReactionsFollowing 4-Hour Exposure

Skin Reaction

Observation Time
(following patch removal)

Individual Scores – Rabbit Number and Sex

69116Male

Erythema/Eschar Formation

Immediately

0

1 Hour

1

24 Hours

2BrLeLf

48 Hours

2BrLeLf

72 Hours

2BrLeLf

7 Days

?eSp

14 Days

0

Oedema Formation

Immediately

0

1 Hour

0

24 Hours

2

48 Hours

2

72 Hours

2

7 Days

?od

14 Days

0

Sum of 24 and 72-hour Readings (S)       :          8

Primary Irritation Index (S/2)                   :          8/2 = 4.0

Classification                                        :          MODERATE IRRITANT


Br=     Light brown discolouration of the epidermis

Le =    Loss of skin elasticity

Lf =     Loss of skin flexibility

Sp =   Hardened light dark brown coloured scab

?e =    Adverse skin reactions prevent accurate evaluation of erythema

?od =  Adverse skin reactions prevent accurate evaluation of oedema

The test material produced a primary irritation index of 4.0 and was classified as a MODERATE IRRITANT to rabbit skin according to the Draize classification scheme. No corrosive effects were noted.

 Interpretation of Results

Calculation of Primary Irritation Index and Grading of Irritancy Potential Using the Draize Scheme

The scores for erythema and oedema at the 24 and 72-hour readings were totalled for the three test rabbits (12 values) and this total was divided by six to give the primary irritation index of the test material. The test material was classified according to the following scheme devised by Draize J H (1959) "Dermal Toxicity" In: Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics. Association of Food and Drug Officials of the,,, p.47:

Primary Irritation Index

Classification of Irritancy

     0

Non-irritant

> 0 to 2

Mild irritant

> 2 to 5

Moderate irritant

> 5 to 8

Severe irritant

If irreversible alteration of the dermal tissue is noted in any rabbit, as judged by the Study Director, which include ulceration and clear necrosis or signs of scar tissue, the test material is classified as corrosive to rabbit skin. Classification according to Draize may, therefore, not be applicable.


Interpretation of results:
moderately irritating
Remarks:
Migrated information EU - R38 "Irritating to skin". Criteria used for interpretation of results: other: EU and Draize
Conclusions:
The test material produced a primary irritation index of 4.0 and was classified as a MODERATE IRRITANT to rabbit skin according to the Draize classification scheme (based on one rabbit only). No corrosive effects were noted.
The test material produced positive criteria and was considered to be an irritant according to EU labelling regulations Commission Directive 2001/59/EC. The symbol “Xi”, the indication of danger “Irritant” and the risk phrase R 38 “IRRITATING TO SKIN” are therefore required. This corresponds to a CLP/GHS classification of Category 2 for skin irritation.

Executive summary:

Introduction. 

The study was performed to assess the irritancy potential of the test material to the skin of the New Zealand White rabbit. The method was designed to meet the requirements of the following:

§        OECD Guidelines for the Testing of Chemicals No. 404 “Acute Dermal Irritation/Corrosion” (adopted 24 April 2002)

§        Method B4 Acute Toxicity (Skin Irritation) of Commission Regulation (EC) No. 440/2008

Results. 

3-Minute and 1-Hour semi-occluded applications of the test material to the intact skin of one rabbit produced no corrosive effects.

A single 4-Hour, semi-occluded application of the test material to the intact skin of one rabbit produced well-defined erythema and slight oedema. Other skin reactions noted were light brown discolouration of the epidermis, loss of skin elasticity and flexibility and hardened, light brown coloured scab (preventing accurate evaluation of erythema and oedema). The treated skin site appeared normal at the 14‑Day observation.

Conclusion. 

The test material produced a primary irritation index of 4.0 and was classified as a MODERATE IRRITANT to rabbit skin according to the Draize classification scheme (based on one rabbit only). No corrosive effects were noted.

The test material produced positive criteria and was considered to be an irritant according to EU labelling regulations Commission Directive 2001/59/EC. The symbol “Xi”, the indication of danger “Irritant” and the risk phrase R 38 “IRRITATING TO SKIN” are therefore required.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation
Remarks:
other: in-vitro
Type of information:
experimental study
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.
Qualifier:
no guideline available
Deviations:
not applicable
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
Species:
other: Reconstructed Human Corneal Model
Strain:
other: Reconstructed Human Corneal Model
Details on test animals or tissues and environmental conditions:
Not applicable
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
Irritation parameter:
other: viability of cells
Basis:
other: mean viability of cells
Time point:
other: Day 2
Reversibility:
other: Not applicable
Remarks on result:
other: Viability after 60 minute exposure and 16 hour post exposure incubation 50.2%
Irritant / corrosive response data:
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%.

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)

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).
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

This is good quality skin irritation data which indicates moderate skin irritation potential for 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). We have in-vitro data which has been interpreted as indicating eye irritation potential, based on the knowledge that for this chemical class, in-vitro studies tend to underestimate irritation potential. It is not felt to be scientifically justified on animal welfare grounds to do an in-vivo study. The available information is sufficient for classification and labeling purposes.


Justification for selection of skin irritation / corrosion endpoint:
This study is a Klimisch 1 GLP study to current guidelines, the result is consistent with an earlier less compliant study (Guest 1991) where the test substance was not so clearly specified.

Justification for selection of eye irritation endpoint:
The available study is an in-vitro study which is interpreted as indicating eye irritation potential, supported by the skin irritation data, where the in-vitro test was shown to significantly underestimate the skin irritation potential. It is not scientifically justified to carry out an animal study to confirm the in-vitro data given the weight of evidence of irritation data for this substance.

Effects on skin irritation/corrosion: moderately irritating

Effects on eye irritation: moderately irritating

Justification for classification or non-classification

This is good quality skin irritation data which indicates moderate to severe skin irritation potential for 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). This results in an EU CLP (GHS) classification as a Category 2 Skin irritant with hazard phrase H315. The in-vitro skin irritation study, was interpreted as positive as the viablility of 50.2 was so close to the 50% threshold and we have good evidence that our fatty amine chemistry is less active in-vitro than in the rabbit. Based on this it is justified to also classify as Category 2 for Eye irritation with Hazard statement H319.