3,5-dichlorobenzoyl chloride

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02 October 2015 to 30 October 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 431 (In Vitro Skin Corrosion: Human Skin Model Test)

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

Test material

Test material information

Constituent 1

Reference substance name:

3,5-dichlorobenzoyl chloride

EC Number:

220-813-6

EC Name:

3,5-dichlorobenzoyl chloride

Cas Number:

2905-62-6

Molecular formula:

C7H3Cl3O

IUPAC Name:

3,5-dichlorobenzoyl chloride

Test material form:

other: solid (unspecified)

Details on test material:

- Appearance: White solid
- Storage conditions of test material: At room temperature

Test animals

Species:

other: EpiDerm Skin Model

Strain:

other: not applicable

Details on test animals or test system and environmental conditions:

TEST SYSTEM
- EpiDerm Skin Model (EPI-200, Lot no.: 23220 kit Q; supplied by MatTek Corporation, Ashland, MA, USA)
The model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multi-layered, highly differentiated model of the human epidermis. It consists of organised basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDerm tissues (surface 0.6 cm²) were cultured on polycarbonate membranes of 10 mm cell culture inserts. The skin tissues were kept in the refrigerator the day they were received.

CELL CULTURE
- Tissues
On the day of receipt the tissues were kept on agarose and stored in the refrigerator. On the next day, at least one hour before starting the assay the tissues were transferred to 6-well plates with 0.9 mL DMEM medium (Dulbecco’s Modified Eagle’s Medium, supplemented DMEM medium, serum-free)
- MTT medium
MTT concentrate (5 mg/mL) diluted (1:5) with MTT diluent (supplemented DMEM).
- Environmental conditions
All incubations, with the exception of the test material incubation of 3 minutes at room temperature, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100 % (actual range 77 - 88 %), containing 5.0 ± 0.5 % CO₂ in air in the dark at 37.0 ± 1.0 °C (actual range 36.5 - 36.7 °C).

Test system

Type of coverage:

open

Preparation of test site:

other: not applicable

Vehicle:

unchanged (no vehicle)

Controls:

other: Negative control: Milli-Q water; Positive control: Potassium hydroxide (8.0 normal solution)

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 μL
- Test Material Preparation: The test material was heated to approximately 30 °C to obtain a homogeneous liquid sample. The liquid test material was applied undiluted (50 μL) directly on top of the tissue.

Duration of treatment / exposure:

3 minutes of exposure and 1 hour of exposure

Observation period:

Following test material exposure, tissues were incubated for 3 hours and extracted overnight

Number of animals:

Two tissues were used per exposure time and one per control

Details on study design:

TEST FOR THE INTERFERENCE OF THE TEST MATERIAL WITH THE MTT ENDPOINT
A test material may interfere with the MTT endpoint if it is coloured and/or it is able to directly reduce MTT. The cell viability measurement is affected only if the test material is present on the tissues when the MTT viability test is performed.

TEST FOR COLOUR INTERFERENCE BY THE TEST MATERIAL
The test material was checked for possible colour interference before the study was started. Some non-coloured test materials may change into coloured materials in aqueous conditions and thus stain the skin tissues during the 1-hour exposure. To assess the colour interference, approximately 120 mg of the test material or 50 μL Milli-Q water as a negative control were added to 0.3 mL Milli-Q water. The mixture was incubated for approximately 1 hour at 37.0 ± 1.0 °C in the dark. At the end of the exposure time the mixture was shaken and it was checked if a blue / purple colour change was observed.
In case the test material induces colour interference in aqueous conditions, in addition to the normal procedure, two tissues must be treated with test material for 3 minutes and two tissues for 1-hour. Instead of MTT solution these tissues will be incubated with DMEM medium.

TEST FOR REDUCTION OF MTT BY THE TEST MATERIAL
The test material was checked for possible direct MTT reduction before the study was started. To assess the ability of the test material to reduce MTT, approximately 80 mg of the test material was added to 3 mL MTT solution (1 mg/mL) in phosphate buffered saline. The mixture was incubated for approximately 1 hour at 37.0 ± 1.0 °C. A negative control, sterile Milli-Q water was tested concurrently. At the end of the exposure time it was checked if a blue / purple colour change was observed.
In case the test material reacts with the MTT medium in addition to the normal 1-hour procedure, two freeze-killed tissues treated with test material and two freeze-killed non treated tissues must be used for the cytotoxicity evaluation with MTT.

APPLICATION/TREATMENT OF THE TEST MATERIAL
The day after tissue receipt, at least 1 hour before the assay was started the tissues were transferred to 6-well plates containing 0.9 mL DMEM medium per well. The level of the DMEM medium was just beneath the tissue. The plates were incubated for approximately 2 hours at 37.0 ± 1.0 °C. The medium was replaced with fresh DMEM medium just before the test material was applied. The test was performed on a total of 4 tissues per test material together with a negative control and positive control. Two tissues were used for a 3-minute exposure to the test material and two for a 1-hour exposure.
50 μL of the undiluted test material was added into the 6-well plates on top of the skin tissues. The remaining tissues were treated with 50 μL Milli-Q water (negative control) and with 50 μL 8 N KOH (positive control), respectively.
After the exposure period, the tissues were washed with warmed phosphate buffered saline to remove residual test material; however since the test material stuck to the tissues it was difficult to remove. Rinsed tissues were kept in 24 well plates on 300 μL DMEM medium until 6 tissues (= one application time) were dosed and rinsed.

CELL VIABILITY MEASUREMENT
The DMEM medium was replaced by 300 μL MTT-medium and tissues were incubated for 3 hours at 37 °C in air containing 5 % CO₂. After incubation the tissues were washed with PBS and formazan was extracted with 2 mL isopropanol over night at room temperature. The amount of extracted formazan was determined spectrophotometrically at 570 nm in triplicate with the TECAN Infinite® M200 Pro Plate Reader.
Cell viability was calculated for each tissue as percentage of the mean of the negative control tissues. Skin corrosion potential of the test material was classified according to remaining cell viability following exposure of the test material with either of the two exposure times.

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

other: other: OD570

Value:

0.624

Remarks on result:

other:

Remarks:

Basis: mean. Time point: 3 minute application. Remarks: ± 0.165 (SD). (migrated information)

Results 2

Irritation / corrosion parameter:

other: other: OD570

Value:

0.634

Remarks on result:

other:

Remarks:

Basis: mean. Time point: 1 hour application. Remarks: ± 0.379 (SD). (migrated information)

In vivo

Irritant / corrosive response data:

The test material was checked for colour interference in aqueous conditions and possible direct MTT reduction by adding the test material to MTT medium. The solutions did not turn blue / purple and a blue / purple precipitate was not observed, therefore it was concluded that the test material did not interfere with the MTT endpoint.
Skin corrosion is expressed as the remaining cell viability after exposure to the test material. The relative mean tissue viability obtained after the 3-minute and 1-hour treatments with the test material compared to the negative control tissues was 29 and 32 % respectively. As the mean relative tissue viability for the test material was below 50 % after the 3-minute treatment it is considered to be corrosive.
The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range at the 1 hour treatment and just above the laboratory historical control data range at the 3 minute treatment. The mean relative tissue viability following 3-minute exposure to the positive control was 8 %.
The maximum inter-tissue variability in viability between two tissues treated identically was less than 24 % and the maximum difference in percentage between the mean viability of two tissues and one of the two tissues was less than 14 % for the positive and negative control. It was therefore concluded that the test system functioned properly. For the test material, the maximum inter-tissue variability in viability between two tissues treated identically was less than 60 % and the maximum difference in percentage between the mean viability of two tissues and one of the two tissues was less than 43 %. However since the test material showed a clear positive response after the 3-minute treatment, this did not influence the study outcome.

ACCEPTABILITY OF THE ASSAY
The inter-tissue variability in viability between two tissues and the maximum difference in percentage between the mean viability of two tissues and one of the two tissues treated with the test material were above the acceptability criteria (up to 32 and 19 % at the 3-minute treatment and up to 59 and 42 % at the 1 hour treatment period, respectively).
Since at the 3-minute treatment the inter-tissue variability in viability between two tissues and the maximum difference in percentage between the mean viability of two tissues and one of the two tissues treated with the test material were just above the acceptability criteria and both viabilities were clearly below 50 % (34 and 23 %) this test material induces a clearly positive response.

Any other information on results incl. tables

Table 1: Mean absorption in the in vitro skin corrosion test

	3 minute application (OD570)				1 hour application (OD570)
	A	B	Mean	SD	A	B	Mean	SD
Negative control	2.087	2.273	2.180	± 0.131	2.234	1.743	1.989	± 0.347
Test material	0.741	0.508	0.624	± 0.165	0.901	0.366	0.634	± 0.379
Positive control	0.153	0.201	0.177	± 0.034	0.149	0.115	0.132	± 0.024

Duplicate exposures are indicated by A and B.

Values are corrected for background absorption (0.0427). Isopropanol was used to measure the background absorption.

Table 2: Mean tissue viability in the in vitro skin corrosion test

	3 minute application viability (percentage of control)	1 hour application viability (percentage of control)
Negative control	100	100
Test material	29	32
Positive control	8	7

Applicant's summary and conclusion

Interpretation of results:

Category 1A (corrosive) based on GHS criteria

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

Under the conditions of this study, the test material is corrosive in the in vitro skin corrosion test.

Executive summary:

The potential of the test material to cause skin corrosion was investigated using the in vitro skin corrosion test with a human skin model in accordance with the standardised guidelines OECD 431 and EU Method B.40 bis under GLP conditions.

The assay investigates the ability of the test material to induce skin corrosion on a human three dimensional epidermal model (EpiDerm (EPI-200)). The possible corrosive potential of the test material was tested through topical application for 3 minutes and 1 hour and assessing the viability of the tissue. The test material was applied undiluted (50 μL) directly on top of the skin tissue. Milli-Q water and 8 N potassium hydroxide served as the negative and positive controls, respectively.

The positive control had a mean relative tissue viability of 8 % after 3 minutes exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range at the 1 hour treatment and just above the laboratory historical control data range at the 3 minute treatment. The acceptability criteria for the maximum inter-tissue variability in viability between two tissues treated identically and the maximum difference in percentage between the mean viability of two tissues and one of the two tissues were met for the negative and positive control, indicating that the test system functioned properly.

Skin corrosion is expressed as the remaining cell viability after exposure to the test material. The relative mean tissue viability obtained after 3-minute and 1-hour treatments with the test material compared to the negative control tissues was 29 and 32 %, respectively. As the mean relative tissue viability for the test material was below 50 % after the 3-minute treatment, the test material is considered to be corrosive.

Under the conditions of this study, the test material is corrosive in the in vitro skin corrosion test.