Registration Dossier

Administrative data

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21 September 2014 - 26 September 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 430 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test Method (TER))
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Name of test material (as cited in study report): 4-amino-5-(ethylsulphonyl)-o-anisic acid
- Physical state: Solid
- Analytical purity: 99.81%
- Lot/batch No.: MP1032.31

In vitro test system

Test system:
isolated skin discs
Source species:
rat
Source strain:
Wistar
Details on animal used as source of test system:
SOURCE OF THE BIOLOGICAL MATERIAL:
TEST ANIMALS:
- Source: Centre for Experimental Medicine at the Medical University in Katowice
- Age at study initiation: 21 days old (hair follicles in dormant phase)
- 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): 21-23 ºC
- Humidity (%): 55-70%
- Air changes (per hr): about 16 times/hour
- Photoperiod (hrs dark / hrs light): 12 hours light/12 hours dark
Vehicle:
water
Details on test system:
SKIN DISC PREPARATION
- Procedure used:The animals were euthanized by intraperitoneal administration of morbital at a dose of 200 mg/kg b.w.
After euthanasia, the dorso-lateral skin of each animal was removed and stripped of excess subcutaneous fat by carefully peeling it away from the skin using a paper towel. The skin discs were cut out using a scalpel. Each skin disc was placed over one of the ends of a PTFE (polytetrafluoroethylene) tube, ensuring that the epidermal surface was in contact with the tube. A rubber ‘O’ ring was press-fitted over the end of the tube to hold the skin in place and excess tissue is trimmed away. The rubber ‘O’ ring was then carefully sealed to the end of the PTFE tube with petroleum jelly. The tube was supported by a spring clip inside a receptor chamber containing MgSO4 solution (154 mM). The skin disc should be fully submerged in the MgSO4 solution. As many as 11 skin discs with a diameter of 20-mm each were obtained from a single rat skin. Two of them were used to control the quality of the procedure, whereas the remaining nine were used for the purpose of the experiment.
The age of the animals was particularly important. The age of 29 days ensures that the hair follicles are in the dormant phase before adult hair growth begins [SOP/T/57].

- Quality control for skin discs:
Before the test, the electrical resistance of two skin discs obtained from each rat was measured as a quality control procedure for each animal skin. Both discs should give resistance values greater than 10 kΩ for the remainder of the discs to be used for the test.

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

REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: 1 time washing with jet of tap water at up to 30ºC.
- Observable damage in the tissue due to washing:No

DYE BINDING METHOD
- Dye used in the dye-binding assay: [none / MTT / Sulforhodamine B / other:] : Not necessary,since all TER values for the test item were higher than 5 kΩ and there were not any visible changes on the skin discs.

NUMBER OF INDEPENDENT TESTING RUNS: 3 replicates

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be corrosive to skin if the mean TER value obtained for the test item is less than or equal to 5 kΩ and the skin disc is obviously damaged.
- The test substance is considered to be non-corrosive to skin if the mean TER value obtained for the test item is greater than 5 kΩ, or the mean TER value is less than or equal to 5 kΩ, and the skin disc shows no obvious damage.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
VEHICLE
- Amount(s) applied (volume or weight with unit):test item moistened with 150 μL of destiled water.

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

POSITIVE CONTROL
- Amount(s) applied (volume or weight):150 μL.
Duration of treatment / exposure:
24 hours
Number of replicates:
3

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
transcutaneous electrical resistance (in kΩ)
Run / experiment:
MEAN TER value, TEST 1
Value:
ca. 15.75
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Remarks:
SD+-041
Irritation / corrosion parameter:
transcutaneous electrical resistance (in kΩ)
Run / experiment:
MEAN TER value TEST 2
Value:
ca. 19.57
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Remarks:
SD+- 0.43
Irritation / corrosion parameter:
other: Morphologics effects
Remarks on result:
not measured/tested
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system:No

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: YES - the mean TER values were higher than 5 kΩ
- Acceptance criteria met for positive control: YES - the mean TER value is less than 5 kΩ.

The mean TER values for the test item were higher than 5 kΩ and there were not any visible changes on the skin discs.

Any other information on results incl. tables

On the grounds of the study, the test item belongs to a group of substances which 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.

Table No.1: Results of the control transcutaneous electrical resistance test (TER):

Animal number

Skin disc number

TER value (kΩ)

1

1

17.14

2

16.57

2

1

14.72

2

13.98

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.

Table No.2: 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

0.90

0.90 ± 0.01

2

0.89

3

0.90

Negative control – distilled water

1

18.75

18.63 ± 0.46

 

2

18.13

3

19.02

Test item

1

15.28

15.75 ± 0.41

 

2

16.03

3

15.95

2

Positive control –

36% HCl

1

0.88

0.89 ± 0.01

2

0.88

3

0.90

Negative control – distilled water

1

19.78

19.29 ± 1.11

 

2

19.01

3

19.09

Test item

1

19.74

19.57 ± 0.52

 

2

19.08

3

19.88

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 15.75 kΩ (animal no. 1) and 19.57 kΩ (animal no. 2).

Table No.3: 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:
non-corrosive/severe irritant.
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. 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 15.75 kΩ (animal no. 1) and 19.57 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.