o-chlorobenzenethiol

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From January 30th to March 4th, 2016.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

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

Reference substance name:

o-chlorobenzenethiol

EC Number:

228-667-5

EC Name:

o-chlorobenzenethiol

Cas Number:

6320-03-2

Molecular formula:

C6H5ClS

IUPAC Name:

2-chlorobenzene-1-thiol

Test material form:

liquid

In vitro test system

Test system:

isolated skin discs

Source species:

rat

Cell source:

other: The test was conducted with skin discs collected from three Wistar (outbred) female rats.

Source strain:

Wistar

Details on animal used as source of test system:

SOURCE ANIMAL
- Source: Conventional husbandry of laboratory animals of the Centre for Experimental Medicine at the Medical University in Katowice.
- Sex: females
- Age at study initiation: 21 days old.
- Housing: Rats were kept in a plastic cage covered with a wire bar lid. The dimensions of the cage were 58 x 37 x 21 cm.UV-sterilized wood shavings were used as bedding.
- Diet: Murigran standard granulated laboratory fodder ad libitum
- Water: Tap water ad libitum
- Acclimation period: 4 days of quarantine for observation.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 -22 ºC
- Humidity (%): 35 - 47%
- Air changes (per hr): 16 times/hour
- Photoperiod: 12 hours light/12 hours dark

Vehicle:

unchanged (no vehicle)

Details on test system:

SKIN DISC PREPARATION
- Procedure used: After the quarantine, the dorsal and flank hair from young, 25-day-old, female rats was carefully removed with a shaver. Then, the shaven area was washed with an antibiotic solution containing streptomycin (10 mg/mL), penicillin (10 μg/mL), and amphotericin (0.25 mg/mL) to inhibit bacterial growth. The animals were washed with antibiotics again on the third day after the first wash and used in the study within one day of the second wash. The animals were euthanized by intraperitoneal administration of morbital at a dose of 200 mg/kg bw within one day of the second wash.
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.
- Quality control for skin discs: Electrical resistance obtained with two of the isolated skin discs was > 10 kΩ.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 21 -22 ºC
- Temperature of post-treatment incubation (if applicable):

REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: the test item and the control items were removed by washing with a jet of tap water at up to 30°C.
- Observable damage in the tissue due to washing: no.
- Modifications to validated SOP: no.

DYE BINDING METHOD
- Dye used in the dye-binding assay: none / Sulforhodamine B
- Spectrophotometer:
- Wavelength:
- Filter:
- Filter bandwidth:

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

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if [complete, e.g. if the mean TER value is less than or equal to 5 kΩ and the skin disk is obviously damaged, or if the mean TER value is less than or equal to 5 kΩ, and the skin disc is showing no obvious damage, but the mean disc dye content is greater than or equal to the mean disc dye content of the 10M HCl positive control obtained concurrently.]
- The test substance is considered to be non-corrosive to skin if [complete, e.g. 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, and the mean disc dye content is well below the mean disc dye content of the 10M HCl positive control obtained concurrently.]
- Justification for the selection of the cut-off point(s) if different than recommended in TG 430:

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 150 µL (undiluted).

NEGATIVE CONTROL
- Amount(s) applied (volume or weight):
- Concentration (if solution):

POSITIVE CONTROL
- Amount(s) applied (volume or weight):
- Concentration (if solution):

Duration of treatment / exposure:

24 h

Test system

Type of coverage:

open

Preparation of test site:

shaved

Vehicle:

unchanged (no vehicle)

Controls:

other: Skin discs were used as control.

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 150 µL.

Duration of treatment / exposure:

24 hours

Observation period:

Not applicable.

Number of animals:

Discs were collected from 3 animals.

Details on study design:

TEST SITE
- Area of exposure: 20 mm each skin disk.

REMOVAL OF TEST SUBSTANCE
- Washing: Jet of tap water at up to 30ºC
- Time after start of exposure: 24 h.

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

transcutaneous electrical resistance (in kΩ)

Run / experiment:

1

Value:

4.63

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

Basis: mean Skin discs from animal number 1. Time point: 24 hours. Max. score: 4.89. Reversibility: Not applicable.. Remarks: 4.63 ± 0.32. (migrated information)

Results 2

Irritation / corrosion parameter:

transcutaneous electrical resistance (in kΩ)

Run / experiment:

2

Value:

4.87

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

Basis: mean Skin discs from animal number 2. Time point: 24 hours. Max. score: 5.01. Reversibility: other: Not applicable. Remarks: 4.87 ± 0.12. (migrated information)

Results 3

Irritation / corrosion parameter:

transcutaneous electrical resistance (in kΩ)

Run / experiment:

3

Value:

4.89

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

Basis: mean Skin discs from animal number 3. Time point: 24 hours. Max. score: 5.01. Reversibility: other: Not applicable.. Remarks: 4.89 ± 0.13. (migrated information)

Results 4

Irritation / corrosion parameter:

dye content (µg/disc)

Run / experiment:

1

Value:

67.07

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

Basis: mean : Test item, skin discs from animal number 1. Time point: Approximately 43 hours. Max. score: 68.75. Reversibility: other: Not applicable.. Remarks: 67.07 ± 1.51. (migrated information)

Results 5

Irritation / corrosion parameter:

dye content (µg/disc)

Run / experiment:

2

Value:

80.51

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

Basis: mean : Test item, skin discs from animal number 2. Time point: Approximately 43 hours. Max. score: 81.27. Reversibility: other: Not applicable.. Remarks: 80.51 ± 0.82. (migrated information)

Results 6

Irritation / corrosion parameter:

dye content (µg/disc)

Run / experiment:

3

Value:

79.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

Basis: mean : Test item, skin discs from animal number 3. Time point: Approximately 43 hours. Max. score: 81.92. Reversibility: other: Not applicable.. Remarks: 79.7 ± 2.01. (migrated information)

Other effects / acceptance of results:

The test item was considered corrosive because the mean TER values obtained for the test item were less than or equal to (≤) 5 kΩ and the skin disks showed no obvious damage but the mean dye content was greater than or equal to (≥) the mean disc dye content of the 10M HCl positive control obtained concurrently.
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 skin changes.

Any other information on results incl. tables

Table 1. Results of the transcutaneous resistance test (TER).

Animal number	Tested substance	Skin disc number	TER value (kΩ)	Mean TER value ± SD (kΩ)
1	Positive control – 10M HCl	1	0.92	0.91 ± 0.01
		2	0.90
		3	0.90
	Negative control – distilled water	1	18.52	18.91 ± 0.36
		2	18.98
		3	19.23
	Test item	1	4.73	4.63 ± 0.32
		2	4.28
		3	4.89
2	Positive control – 10M HCl	1	0.83	0.84 ± 0.01
		2	0.85
		3	0.85
	Negative control – distilled water	1	17.03	17.34 ± 0.48
		2	17.89
		3	17.10
	Test item	1	5.01	4.87 ± 0.12
		2	4.78
		3	4.83
3	Positive control – 10M HCl	1	0.86	0.87 ± 0.01
		2	0.87
		3	0.88
	Negative control – distilled water	1	14.75	15.05 ± 0.32
		2	15.03
		3	15.38
	Test item	1	4.92	4.89 ± 0.13
		2	5.01
		3	4.75

 

Table 2. Gross changes on the surface of the treated skin discs.

Animal number	Tested substance	Skin disc number	Gross changes
1	Positive control – 10M 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 – 10M 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
3	Positive control – 10M 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

Table 3. Results of the treated disc Sulforhodamine B dye content determination.

Animal number	Tested substance	Skin disc number	Optical density at 565 nm	Dye c (µg/disc) oncentration	Mean ± SD (µg/disc) dye concentration
1	Positive control – 10M HCl	1	0.495	80.46	79.05 ± 1.38
		2	0.478	77.69
		3	0.486	78.99
	Negative control – distilled water	1	0.109	17.72	17.55 ± 0.74
		2	0.112	18.20
		3	0.103	16.74
	Test item	1	0.423	68.75	67.07 ± 1.51
		2	0.405	65.83
		3	0.410	66.64
2	Positive control – 10M HCl	1	0.503	81.76	79.75 ± 2.22
		2	0.493	80.13
		3	0.476	77.37
	Negative control – distilled water	1	0.101	16.42	16.74 ± 0.86
		2	0.099	16.09
		3	0.109	17.72
	Test item	1	0.500	81.27	80.51 ± 0.82
		2	0.496	80.62
		3	0.490	79.64
3	Positive control – 10M HCl	1	0.473	76.88	78.51 ± 1.44
		2	0.486	78.99
		3	0.490	79.64
	Negative control – distilled water	1	0.101	16.42	16.58 ± 0.59
		2	0.106	17.23
		3	0.099	16.09
	Test item	1	0.504	81.92	79.70 ± 2.01
		2	0.487	79.16
		3	0.480	78.02

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test item was corrosive to the skin according to the UN GHS classification system for the rat skin transcutaneous electrical resistance test.

Executive summary:

An in vitro skin corrosion study was conducted according to the Transcutaneous Electrical Resistance (TER) test method and OECD guideline 430 under GLP conditions. Skin discs were collected at a first stage from two female rats and at a second stage from one additional female rat, this was deemed necessary due to the disconcordant results between the first two testing runs. The skin discs were obtained from 29 day old animals, each of the three runs consisted of three skin replicates for the test item as well as three replicates for the positive (10M hydrochloric acid) and negative (distilled water) controls respectively. Before the test started, the transcutaneous electrical resistance of two skin discs were measured as quality control procedure, in all the cases the resistance values were superior than 10kΩ thus the other discs were appropriate for the testing. 150 µL of the test item and the positive and negative controls were applied for 24 hours and kept at 21 -22ºC, the TER was measured as recommended by the above mentioned guideline. Because the mean TER values were lower or equal to 5 kΩ and in absence of visual damage, a dye binding assay was included in the study. Following the TER assesment the magnesium sulphate was discharged from the tube and 150 µL of a 10 % solution of sulforhodamine B in distilled water were applied to the epidermal surface of each disc skin for 2 hours, the following procedures were conducted as recomended by the OECD guideline 430 and finally, the content of the dye was determined using a spectrophotometer at 565 nm. The test item was considered corrosive because the mean TER values obtained for the test item were less than or equal to (≤) 5 kΩ and the skin disks showed no obvious damage but the mean dye content was greater than or equal to (≥) the mean disc dye content of the 10M HCl positive control obtained concurrently.