Fatty acids, lanolin, lithium salts

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EC method B.40 tris: In vitro Skin Corrosion: Human Skin Test Method

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Fatty acids, lanolin, lithium salts

EC Number:

268-919-1

EC Name:

Fatty acids, lanolin, lithium salts

Cas Number:

68154-72-3

IUPAC Name:

Fatty acids, lanolin, lithium salts

Test material form:

solid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: SOAP 012-2
- Expiration date of the lot/batch: November 01, 2022
- Purity test date: 13 December 2017


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At +10 to +25°C, kept containers tightly closed in a cool, well-ventilated place, kept away from heat, sparks and open flame.


FORM AS APPLIED IN THE TEST (if different from that of starting material) 25 mg of test item were applied to the skin model with a surface area of 0.63 cm2 to uniformly cover the skin surface. The test item is a fine powder.

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

The test item was applied topically to a three-dimensional human skin model, comprising at least a reconstructed epidermis with a functional stratum corneum. Corrosive materials are identified by their ability to produce a decrease in cell viability (as determined, for example, by using the MTT reduction assay) below defined threshold levels at specified exposure periods. The principle of the human skin model assay is based on the hypothesis that corrosive chemicals are able to penetrate the stratum corneum by diffusion or erosion, and are cytotoxic to the underlying cell layers.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ (EPI-200-SCT)
- Tissue batch number(s): Lot no. 25878

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: At the end of the exposure period, the test item was carefully washed from the skin surface with Dulbecco's phosphate buffered saline (D-PBS)

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg MMT /mL
- Incubation time: 3 h
- Spectrophotometer: Tecan Sunrise Magellan Version 7.2
- Wavelength: 540 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: 1.968 ± 0.147 (acceptance criteria: 1.0-3.0)
- Barrier function: 6.8 h (acceptance criteria:4.77-8.72)
- Contamination: No contamination

NUMBER OF REPLICATE TISSUES: Two replicate tissues for each treatment (exposure periods) were employed

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
The frozen tissues were stored in the freezer (-20 ± 5°C).
The test item was applied to two freeze-killed tissues. In addition, two freeze-killed tissues were left untreated. The entire assay protocol was performed on the frozen tissues in parallel to the assay performed with the live EpiDerm tissues.

- Fresh tissues / killed tissues: Freeze-killed tissues. Due to interacting of the test item with the MTT measurement (reduction of MTT) an additional test with freeze-killed tissues had to be performed.
- N. of replicates : 2
- Method of calculation used:

For the MTT reducing test item, calculations were corrected by values of correspondent additional controls.
True viability = Viability of treated tissue – Interference from test chemical = OD tvt – OD kt
where OD kt = (mean OD tkt – mean OD ukt)
tvt = treated viable tissue kt = killed tissues
tkt = treated killed tissue ukt = untreated killed tissue (NC treated tissue)
If the interference by the test substance is greater than 30% of the negative control value, additional steps must be taken into account or the test substance may be considered incompatible with this test.

PREDICTION MODEL / DECISION CRITERIA
-The criteria of corrosivity associated with the EpiDermTM model are as follows:
- the test item is considered to be corrosive to skin and classified as category 1 (or optional category 1A ), if the viability after 3 minutes exposure is less than 50%;
- the test item is considered to be corrosive to skin and classified as sub-category 1B-and-1C, if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is less than 15%;
- the test item is considered to be non-corrosive to skin, if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg

NEGATIVE CONTROL : sterile deionised water
- Amount(s) applied (volume or weight): 50 µL


POSITIVE CONTROL : 8 N KOH
- Amount(s) applied (volume or weight): 50 µL

Duration of treatment / exposure:

1st period: 3 min; 2nd period: 60 min

Number of replicates:

Two replicate tissues for each treatment (exposure periods) were employed.

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minute ecposure

Value:

98.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1 hour exposure

Value:

95.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean optical density (OD) of the negative control of 2 tissues was 1.456 (3 minute exposure) or 1.552 (1-hour exposure) and therefore well within the acceptable range of ≥ 0.8 to ≤ 2.8
- Acceptance criteria met for positive control: The viability of cells treated with the positive reference item 8 N KOH was 11.2% or 4.4% (3-minute or 1-hour exposure, respectively) of the negative control and, hence well below the 15% cut-off value at the 1-hour exposure
- Acceptance criteria met for variability between replicate measurements: The difference of viability between the two tissue replicates (at 20 - 100% viability) was below the limit of acceptance of 30%.
All acceptance criteria were fulfilled.

Any other information on results incl. tables

Historical data of negative and positive controls
(Most recent background data from GLP studies of the years 2015 - 2017 (n = 14).

Material	Average OD (mean% difference ±SD)	Average viability [%] (mean% difference ±SD)	Range		No. of unqualified experi-ments
			Viability [%]	% difference
Short time incubation – 3‑min
Negative control (Non-Corrosive)	1.624 (2.5±2.4)	100 (2.5±2.4)	93.7–106.3	0.14–8.6	0^#1
8 N KOH (Corrosive)	0.126 (8.8±7.4)	7.6 (8.8±7.4)	2.0–15.6	<0.01–23.7	0
Long time incubation – 60‑min
Negative control (Non-Corrosive)	1.650 (4.0±5.0)	100 (4.0±5.0)	85.6–114.4	0.13–18.3	0^#1
8 N KOH (Corrosive)	0.090 (5.7±9.8)	5.9 (5.7±9.8)	2.0–12.6	0.30–38.4	0^#2

OD: Optical density. Viability for negative control is set = 100%

SD: Standard deviation

CV: Coefficient of variation

^#1     Unqualified results =       if the mean OD of the NC tissues is < 0.8 or > 2.8

                                            if difference in viability for duplicate tissues > 30%

^#2     Unqualified results =       8 N KOH: viability > 15% (1-hour exposure)

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the present test conditions Fatty acids, lanolin, lithium salts tested at two exposure periods of 3 minutes or 1 hour was non-cytotoxic and, hence, predicted to be non-corrosive to skin in an experiment employing an artificial three-dimensional model of human skin.

Executive summary:

The purpose of this study was to assess the corrosive properties of Fatty acids, lanolin, lithium salts to human skin,in an experiment with an artificial three-dimensional model of human skin. TheEpiDerm™model was employed.

The test item Fatty acids, lanolin, lithium salts, the negative control sterile deionised water or the positive reference item 8 N KOH were applied to the skin model surface at two exposure periods of 3 minutes or 1 hour.

Two tissues were used for each treatment and concurrent control groups. The optical density (OD) was determined byusing the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue) reduction assayand expressed as relative percentage of viability of the negative control-treated tissues.

In comparison to the negative controls, the mean viability of cells exposed to the test item was 98.1% after a 3-minute exposure period and 95.7% after a 1-hour exposure (corrected viability calculated for MTT reducing test items using freeze-killed control tissues) and hence ,the 3-minute and the1-hour exposure values were above the cut-off percentage cell viability values distinguishing corrosive from non-corrosive test items of ≥50% and ≥15%, respectively.Therefore, the test item was non-corrosive in this skin model and is predicted to be non-corrosive to human skin.

Conducted according to OECD Test Guideline 431 and GLP, the study is considered to be reliable without restriction (Klimisch 1).