Fatty acids, C16-18, calcium salts

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was performed between 23 February 2010 and 26 February 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

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: OECD GUIDELINE FOR THE TESTING OF CHEMICALS 431

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Fatty acids C18-(unsaturated) lithium salts

IUPAC Name:

Fatty acids C18-(unsaturated) lithium salts

Details on test material:

Sponsor's identification : Fatty acids C18-(unsaturated) lithium salts
Description : off white solid
Batch number : SF 173
Date received : 21 January 2010
Expiry date : 21 January 2012
Storage conditions : room temperature in the dark

In vitro test system

Test system:

other: reconstituted human epidermis model

Source species:

other: reconstituted human epidermis model

Cell type:

other: reconstituted human epidermis model

Cell source:

other: reconstituted human epidermis model

Source strain:

other: reconstituted human epidermis model

Details on animal used as source of test system:

Reconstituted human epidermis model

Vehicle:

unchanged (no vehicle)

Details on test system:

TEST SITE
- Area of exposure:
20 mg of of the test material was applied to the epidermis surface. 100 µl of 0.9% w/v sodium chloride solution was added for wetting of the test material.

- % 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

Control samples:

no

Amount/concentration applied:

TEST MATERIAL

- The test Material was applied neat.

- Amount(s) applied (volume or weight with unit):
20 mg of of the test material was applied to the epidermis surface. 100 µl of 0.9% w/v sodium chloride solution was added for wetting of the test material.

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

Duration of post-treatment incubation (if applicable):

3, 60 & 240 minutes post exposure incubation

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

240 minutes

Value:

127.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Non-corrosive to skin

Remarks:

Reversibility: Not applicable.

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 minutes

Value:

128.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Non-corrosive to skin

Remarks:

Reversibility: Not applicable

Results 3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minutes

Value:

89.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Non-corrosive to skin

Remarks:

Reversibility: Not applicable

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. The test material was adequately rinsed from the tissues, however the results obtained showed that during rinsing 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 OD₅₄₀ values 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                      :          127.3%

60 minutes exposure                        :          128.9%

3 minutes exposure                          :          89.8%

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 7.0% 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	240 Minutes	0.128	100*
Positive Control	240 Minutes	0.009	7.0
Test Material	240 Minutes	0.163	127.3
	60 Minutes	0.165	128.9
	3 Minutes	0.115	89.8

*=     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:

other: Non-corrosive

Remarks:

Criteria used for interpretation of results: expert judgment

Conclusions:

The test material was considered to be Non-Corrosive to the skin and accredited the EU risk phrase of No label and a UN packing group Non-Corrosive.

Executive summary:

Introduction: The purpose of this test is to evaluate the corrosivity potential of the test material using the EPISKIN^TM in vitro Reconstituted Human Epidermis (RHE) Model after treatment periods of 3, 60 and 240 minutes. This method was designed to meet the requirements of the following:

OECD Guideline for the Testing of Chemicals No. 431 “In Vitro Skin Corrosion: Human Skin Model Test” (adopted 13 April 2004).

The EPISKIN^TM model is able to distinguish between corrosive and non-corrosive chemicals for all of the chemical types studied, and is also able to distinguish between known R35 (UN packing group I) and R34 (UN packing group II & III) chemicals.

Methods: Duplicate tissues were treated with the test material for exposure periods of 3, 60 and 240 minutes. At the end of the exposure period the test material was rinsed from each tissue before each tissue was taken for MTT loading. After MTT loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT-loaded tissues. 

At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 µl samples were transferred to the appropriate wells of a pre-labelled 96 -well plate. The optical density was measured at 540 nm.

Data are presented in the form of % viability (MTT reduction in the test material treated tissues relative to negative control tissues).

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

240 minutes exposure                     :          127.3%
60 minutes exposure                       :          128.9%
3 minutes exposure                         :          89.8%

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

Conclusion: The test material was considered to be Non-Corrosive to the skin and accredited the EU risk phrase of No label and a UN packing group Non-Corrosive.