P-1401

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 June 2015 - 25 July 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 430 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test Method (TER))

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Test material form:

solid: particulate/powder

In vitro test system

Test system:

isolated skin discs

Source species:

rat

Cell type:

non-transformed keratinocytes

Cell source:

foreskin from multiple donors

Source strain:

Wistar

Details on animal used as source of test system:

SOURCE ANIMAL
- Source:Centre for Experimental Medicine at the Medical University in Katowice
- Sex: Female
- Age at study initiation (in days):At the beginning of the experiment, the animals were 21 days old. The skin discs used in the experiment were obtained from two 29-day-old female rats.
- Housing: Plastic cage covered with a wire bar lid. Dimensions: 58 x 37 x 21 cm. Bedding: UV-sterilized wood shavings.
- Diet (e.g. ad libitum): ad libitum, "Murigran” standard granulated laboratory fodder
- Water (e.g. ad libitum): ad libitum, tap water
- Acclimation period: 3 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20ºC
- Humidity (%): 49-56%
- Air changes (per hr): about 16 times/hour
- Photoperiod (hrs dark / hrs light): 12 hours light/12 hours dark

Justification for test system used:

The Transcutaneous Electrical Resistance (TER) test method is a validated and accepted method for the classification of substances as Cat. 1 or non-corrosive.

Vehicle:

unchanged (no vehicle)

Remarks:

Moistened with 150 µL distilled water.

Details on test system:

SKIN DISC PREPARATION
- Procedure used:
- Quality control for skin discs: Electrical resistance obtained with two of the isolated skin discs was greater than 10 kΩ.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 21-22 ºC

REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: One.
- Observable damage in the tissue due to washing: Not rpeorted.
- Modifications to validated SOP: Not reported.


NUMBER OF INDEPENDENT TESTING RUNS / EXPERIMENTS TO DERIVE FINAL PREDICTION: Two.

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be corrosive to skin if the mean TER value is less than or equal to 5 kΩ and the skin disk is obviously damaged.
- The test substance is considered to be non-corrosive to skin if the mean TER value obtained for the test substance is greater than 5 kΩ, or if the mean TER value is less than or equal to 5 kΩ, and the skin disc is showing no obvious damage.

The dye binding procedure was not necessary in this case since all TER values for the test item were higher than 5 kΩ and there were not any visible changes on the skin discs.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): A sufficient amount of the solid was applied evenly to the disc to ensure that the whole surface of the epidermis was covered. Then it was moistoned with 150 µL distilled water.

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 150 µL distilled water

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 150 µL 36 % hydrochloric acid.
- Concentration (if solution): 36 %

Duration of treatment / exposure:

24 hours

Number of replicates:

Three skin discs obtained per animal per run.
Two runs.

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

transcutaneous electrical resistance (in kΩ)

Run / experiment:

1 (mean of three skin discs)

Value:

10.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

transcutaneous electrical resistance (in kΩ)

Run / experiment:

2 (mean of three skin discs)

Value:

12.64

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: The gross examination showed that the positive control skin discs exhibited skin perforation, whereas the negative control skin discs and the ones treated with the test item did not reveal any changes.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes

Any other information on results incl. tables

Results of the control transcutaneous electrical resistance test (TER):

Animal number	Skin disc number	TER value (kΩ)
1	1	14.41
	2	17.28
2	1	17.21
	2	16.98

The skin discs gave the resistance values greater than 10 kΩ; therefore, the remainder of the skin discs of the animals could have been used in the experiment.

Results of the transcutaneous electrical resistance test (TER):

Animal number	Tested substance	Skin disc number	TER value (kΩ)	Mean TER value ± SD (kΩ)
1	Positive control – 36% HCl	1	0.81	0.81 ± 0.01
		2	0.82
		3	0.80
	Negative control – distilled water	1	19.72	19.19 ± 0.46
		2	18.95
		3	18.90
	Test item	1	10.11	10.30 ± 0.35
		2	10.70
		3	10.08
2	Positive control – 36% HCl	1	0.87	0.87 ± 0.01
		2	0.86
		3	0.87
	Negative control – distilled water	1	11.93	12.13 ± 0.20
		2	12.13
		3	12.32
	Test item	1	12.35	12.64 ± 0.39
		2	12.49
		3	13.09

The concurrent mean values for the positive and negative controls were within the acceptable ranges for the method:

Positive control: 0.5-1.0 kΩ

Negative control: 10 -25 kΩ

The mean TER results for the skin discs treated with the test item were equal to 10.30 kΩ (animal no. 1) and 12.64 kΩ (animal no. 2).

Gross changes on the surface of the treated skin discs:

Animal number	Tested substance	Skin disc number	Gross changes
1	Positive control – 36% HCl	1	perforation
		2	perforation
		3	perforation
	Negative control – distilled water	1	no changes
		2	no changes
		3	no changes
	Test item	1	no changes
		2	no changes
		3	no changes
2	Positive control – 36% HCl	1	perforation
		2	perforation
		3	perforation
	Negative control – distilled water	1	no changes
		2	no changes
		3	no changes
	Test item	1	no changes
		2	no changes
		3	no changes

The gross examination showed that the positive control skin discs exhibited skin perforation, whereas the negative control skin discs and the ones treated with the test item did not reveal any changes.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Remarks:

CLP Implementation.

Conclusions:

The substance do not lead to skin corrosion/severe irritation. The mean TER values for the test item were higher than 5 kΩ and there were not any visible changes on the skin discs.

Executive summary:

The in vitro skin corrosion: transcutaneous electrical resistance test (TER) was performed according to OECD Guideline 430 Guideline and EU Method B.40. (GLP study). Skin discs used in the experiment were obtained from two 29-day-old rats. The test item (ground to a powder) was uniformly applied to the epidermal surface of the skin disc placed inside a tube. Positive (36% hydrochloric acid) and negative (distilled water) controls were conducted concurrently. Three skin discs obtained from each animal were used for the test item and three for each control item. The test item and the control items were evenly applied to the discs for 24 hours and kept at 21-22°C. Then, they were removed by washing with a jet of tap water and the surface tension of the skin was reduced by adding 70% ethanol. After removing the ethanol the tissue was hydrated by the addition of 3 mL of a solution of MgSO4 (154 mM). A LCR 6401 low-voltage, alternating current databridge was used to measure the electrical resistance of the skin in kΩ by placing the databridge electrodes on either side of the skin disc. The skin discs were subjected to a gross examination. The mean TER results were equal to 10.30 kΩ (animal no. 1) and 12.64 kΩ (animal no. 2). The concurrent positive and negative control values fell within the acceptable ranges for the method. Gross examinations of the skin discs did not reveal any pathological changes. On the grounds of the study, it may be stated that the test item do not lead to skin corrosion/severe irritation. The mean TER values for the test item were higher than 5 kΩ and there were not any visible changes on the skin discs.