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Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was performed between 17 March 2010 and 19 March 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: OECD GUIDELINE FOR THE TESTING OF CHEMICALS 431
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Ethanol, 2,2'-iminobis-, N-C12-18-alkyl derivs.
EC Number:
276-014-8
EC Name:
Ethanol, 2,2'-iminobis-, N-C12-18-alkyl derivs.
Cas Number:
71786-60-2
Molecular formula:
Not applicable
IUPAC Name:
2,2'-(C12-18 evennumbered alkyl imino) diethanol
Details on test material:
Sponsor's identification : Bis (2-hydroxyethyl) coco alkylamine
Description : pale brown viscous liquid
Batch number : S-001016
Date received : 08 July 2009
Expiry date : 26 June 2017
Storage conditions : approximately 4°C in the dark under nitrogen

Test animals

Species:
other: reconstituted human epidermis model
Strain:
other: reconstituted human epidermis model
Details on test animals or test system and environmental conditions:
Not applicable

Test system

Type of coverage:
other: Topical
Preparation of test site:
other: Not applicable
Vehicle:
other: No vehicle used
Controls:
no
Amount / concentration applied:
TEST MATERIAL

- The test Material was applied neat.

- Amount(s) applied (volume or weight with unit):
50 µl of the test material was applied to the epidermis surface.

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

VEHICLE
No vehicle used
Duration of treatment / exposure:
3, 60 & 240 minutes post exposure incubation
Observation period:
Not applicable
Number of animals:
Not applicable
Details on study design:
TEST SITE
- Area of exposure:
50 µl of the test material was applied to the epidermis surface.
- % coverage:
The test material was applied topically to the corresponding tissues ensuring uniform covering.

- Type of wrap if used:
None used

REMOVAL OF TEST SUBSTANCE
- Washing (if done):
At the end of each exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing Phosphate Buffered Saline Dulbeccos (PBS) with Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of PBS to gently remove any residual test material.

- Time after start of exposure:
3, 60 or 240 minutes post exposure

SCORING SYSTEM:
Quantitative MTT Assessment (percentage tissue viability)
The corrosivity potential of the test material was predicted from the relative mean tissue viabilities obtained after the 3, 60 and 240-minute treatments, compared to the mean of the negative control tissues (n=2) treated with 0.9% w/v sodium chloride solution. The relative mean viabilities were calculated in the following way:

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

Classification of corrosivity potential was based on relative viabilities for each exposure time according to the following prediction model:

Treatment Time (minutes) Relative Mean Tissue viability Prediction
(% of negative control) EU Risk Phrase UN Packing Group
3 <35 Corrosive R35 I
3/60 ≥35 / <35 Corrosive R34 II
60/240 ≥35 / <35 Corrosive R34 III
240 ≥35 Non-Corrosive No label Non-Corrosive

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 min exposure
Value:
109
Vehicle controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
60 min exposure
Value:
114.8
Vehicle controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
240 min
Value:
94
Vehicle controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation

Any other information on results incl. tables

RESULTS

Direct MTT Reduction

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

Test Material, Positive Control Material and Negative Control Material

Mean OD540values and viabilities for the negative control, positive control material and test material are given in Table 1.

The relative mean viability of the test material treated tissues was as follows:

240 minutes exposure                      :          94.0%

60 minutes exposure                        :          114.8%

3 minutes exposure                          :          109.5%

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

Following the 3, 60 and 240 Minute exposure periods the test material treated tissues appeared blue which was considered to be indicative of viable tissue.

Quality Criteria

The relative mean tissue viability for the positive control treated tissues was 18.9% relative to the negative control treated tissues following the 240-minute exposure period. The positive control acceptance criterion was therefore satisfied.

Table 1 : Mean OD540 Values and Viabilities for the Negative Control, Positive Control Material and Test Material

Material

Exposure Period

Mean OD540 of duplicate tissues

Relative mean viability (%)

Negative Control Material

240 Minutes

0.169

100*

Positive Control Material

240 Minutes

0.032

18.9

Test Material

240 Minutes

0.159

94.0

60 Minutes

0.194

114.8

3 Minutes

0.185

109.5


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


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

Material

Exposure Period

Tissue 1

Tissue 2

Negative Control Material

240 Minutes

-

-

Positive Control Material

240 Minutes

++

++

Test Material

240 Minutes

-

-

60 Minutes

-

-

3 Minutes

-

-

MTT visual scoring scheme
-          =         blue tissue (viable)
+         =         blue/white tissue (semi-viable)
++       =         tissue is completely white (dead)

Applicant's summary and conclusion

Interpretation of results:
study cannot be used for classification
Conclusions:
The skin irritation property of the registration substance is assessed based on the read-across approach.
The read-across supporting substance CAS 71786 -60 -2 was non-corrosive in in-vitro test system (OECD 431). The obtained result is considered as of limited relevance to the true corrosive property of the test material or the registration substance.
Executive summary:

The skin corrosivity property of the registration substance is assessed based on the read-across approach.

The read-across supporting substance CAS 71786 -60 -2 was investigated for its skin irritation property according to the OECD Guideline 431.

The EPISKIN TM was treated with the test material for exposure periods of 3, 60 and 240 minutest and and viability of the tissue was investigated using MTT assay. No deviating values were obtained for treated and negative control substance treated tissues. The obtained result is indicative of non-corrosivity of the test material.

Comments on the results obtained in the in-vitro test systems: Generally, the fatty amine compounds are considered corrosive/irritating to skin based on the rabbit studies, in which the skin lesions became evident only after some days of observation period. Considering that there is actually no possibility to detect delayed tissue damage in the introduced in-vitro test system, the result obtained in this test system is likely to be of limited relevance.