7-methyl-2H-benzo-1,5-dioxepin-3(4H)-one

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05 - 07 October 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted according to OECD test Guideline No. 431 without any deviation.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

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:

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

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

UK GLP Compliance Programme (inspected on 05 July 2016 / signed on 28 October 2016)

Test material

Test material information

Constituent 1

Reference substance name:

7-methyl-2H-benzo-1,5-dioxepin-3(4H)-one

EC Number:

249-320-4

EC Name:

7-methyl-2H-benzo-1,5-dioxepin-3(4H)-one

Cas Number:

28940-11-6

Molecular formula:

C10H10O3

IUPAC Name:

7-methyl-3,4-dihydro-2H-1,5-benzodioxepin-3-one

Test material form:

solid: particulate/powder

Details on test material:

- Physical state: Powder, white solid
- Storage condition of test material: Ambient temperatures (10-30°C) and humidity, in darkness (may be used in day/artificial light), under air atmosphere.

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

Following the REACH bottom-up strategy, the EpiDerm™ Human Skin Model method was used to assess skin corrosion as recommended in the OECD test guideline No. 431.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ Reconstructed Human Epidermis Model Kit, MatTek, Bratislava, Slovakia.
- Tissue batch number(s): 23361
- Production Date: 03 October 2016
- Shipping date: 03 October 2016
- Delivery date: 04 October 2016
- Date received: 04 October 2016
- Date of initiation of testing: 06 October 2016

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 3 and 60 minutes at 37 °C in a humidified atmosphere of 5% CO2
- Temperature of post-treatment incubation: 3 h at 37 °C in a humidified atmosphere of 5% CO2

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: Rinsing was performed using a wash bottle and was achieved by filling and emptying each tissue approximately 20 times under a constant soft stream of DPBS for approximately 40 seconds to gently remove any residual test item.
- Observable damage in the tissue due to washing: no noted damage to the test item tissue culture surfaces following the rinsing step.
- Modifications to validated SOP: none reported

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1.0 mg/mL MTT solution
- Incubation time: 3 hours at 37 °C, 5% CO2 in air
- Spectrophotometer: Anthos 2001 microplate reader
- Wavelength: 562 nm
- Filter: not reported
- Filter bandwidth: filter band pass ± 15
- Linear OD range of spectrophotometer: not reported

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
In order to confirm the integrity of the test system a comparison was made with historical data for negative and positive controls obtained by the laboratory in the previous six months. All values for negative and positive controls were within the historical range and this was taken to indicate the adequate functioning of the test system.
- Viability: OD (540-570 nm) = 1.739 ± 0.134 (acceptance criteria: between 1.0-3.0)
- Barrier function: ET-50 = 6.71 hours (acceptance criteria: between 4.77-8.72 hours)
- Morphology: Tissue viability and the barrier function test are within acceptable ranges and indicate appropriate formation of the epidermal barrier, the presence of a functional stratum corneum, a viable basal cell layer, and intermediate spinous and granular layers.
- Contamination: sterile (Bacteria, yeast and other fungi not detected)
- Reproducibility: For the previous 39 experiments conducted between April 2016 and September 2016 using this test method, the mean OD of the positive control was 0.087 ± 0.022 after 3 minutes of exposure and 0.078 ± 0.023 after 60 minutes of exposure. The mean percentage viability was 4.7 ± 0.9 after 3 minutes of exposure and 4.4 ± 0.8 after 60 minutes of exposure (The assay establishes the acceptance criterion for an acceptable test if the relative mean tissue viability for the positive control treated tissues was ≤15% relative to the negative control treated tissues after 1 hour exposure). In this same period the mean OD of the negative control was 1.922 ± 0.239 after 3 minutes of exposure and 1.866 ± 0.238 after 60 minutes of exposure (The assay establishes the acceptance criterion for an acceptable test if the mean OD for the negative control treated tissues was ≥0.8 and ≤2.8 for every exposure time).

NUMBER OF REPLICATE TISSUES: Duplicate tissues for test item, negative and positive controls

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Killed tissues
- Procedure used to prepare the killed tissues : Freeze-killed tissues were prepared prior to the study by placing untreated EPIDERMTM tissues in an empty 12 well plate and storing in a freezer (-14 to -30 °C) for a minimum of 24 hours. Before use each tissue was thawed by placing in 0.9 mL of assay medium for approximately 1 hour at room temperature.
- N. of replicates : 2
- Method of calculation used: True viability = mean OD (treated viable tissues) - [OD (treated killed tissues)-OD (untreated killed tissues)]

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 50%, or 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 substance 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 of the unformulated test item (undiluted) wetted with 25 μL of sterile water to increase tissue surface contact.

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL of sterile distilled water was used as supplied

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL of 8.0 N Potassium Hydroxide was used as supplied

Duration of treatment / exposure:

3 and 60 minutes.

Duration of post-treatment incubation (if applicable):

not applicable

Number of replicates:

Duplicate tissues for test item, negative and positive controls

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minutes exposure

Value:

103.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100%

Positive controls validity:

valid

Remarks:

5.5%

Remarks on result:

other: No indication of corrosion

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 minutes exposure

Value:

8.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100%

Positive controls validity:

valid

Remarks:

5.5%

Remarks on result:

other: Positive indication of corrosion

Other effects / acceptance of results:

TISSUE VIABILITY:
The relative mean viability of the test item treated tissues was 103.7 and 8.2% after 3 and 60 minutes exposure, respectively.
The relative mean viability of the negative control treated tissues was 100 % after 3 and 60 minutes exposure.
The relative mean viability of the positive control treated tissues was 5.5 after 3 and 60 minutes exposure.

- OTHER EFFECTS:
- Visible damage on test system: none reported
- Direct-MTT reduction: The MTT solution containing the test item turned blue which indicated that the test item directly reduced MTT. Direct reduction was <30% relative to the negative control and therefore acceptable.
- Colour interference with MTT: No

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD562 for the negative control treated tissues was 1.709 for the 3 Minute exposure period and 1.742 for the 60 Minute exposure period. The negative control acceptance criteria were therefore satisfied.
- Acceptance criteria met for positive control: The relative mean tissue viability for the positive control treated tissues was 5.5% relative to the negative control following the 60 Minute exposure period. The positive control acceptance criterion was therefore satisfied.
- Acceptance criteria met for variability between replicate measurements: In the range 20 to 100% viability the Coefficient of Variation between the two tissue replicates of each treatment group did not exceed 30%. The acceptance criterion was therefore satisfied.
- Historical data: In order to confirm the integrity of the test system a comparison was made with historical data for negative and positive controls obtained by the laboratory in the previous six months. All values for negative and positive controls were within the historical range and this was taken to indicate the adequate functioning of the test system.

Any other information on results incl. tables

Table 7.3.1/1: Mean OD562 Values and Viabilities for the Negative Control Item, Positive Control Item and Test Item

Tissue	Exposure Period	Mean OD562 of individual tissues	Mean OD562of duplicate tissues (tvt)	Corrected OD562 of tissues (tvt-[tkt-ukt])	Standard Deviation	Coefficient of Variation (%)	Relative Mean Viability (%)
Negative Control	3 Minutes	1.625	1.709	-	0.119	7.0	100*
		1.793
	60 Minutes	1.774	1.742	-	0.046	2.6
		1.709
Positive Control	3 Minutes	0.099	0.094	-	0.008	N/A	5.5
		0.088
	60 Minutes	0.099	0.096	-	0.005	N/A	5.5
		0.092
Test Item	3 Minutes	1.733	1.776	1.772	0.060	3.4	103.7
		1.818
	60 Minutes	0.254	0.269	0.144	0.021	7.6	8.2
		0.283

tvt = treated viable tissues

tkt = treated killed tissues

ukt = untreated killed tissues

3 minute exposure corrected mean OD₅₆₂:

x̅ (0.157 + 0.156) = 0.157 (tkt) – x̅ (0.159 + 0.147) = 0.153 (ukt) = 0.004

60 minute exposure corrected mean OD562:

x̅ (0.319 + 0.255) = 0.287(tkt) – x̅ (0.174 + 0.149) = 0.162 (ukt) = 0.125

3 minute exposure direct MTT reduction relative to the negative control = 0.2%
60 minute exposure direct MTT reduction relative to the negative control = 7.2%

* = The mean % viability of the negative control tissue is set at 100%

Applicant's summary and conclusion

Interpretation of results:

Category 1B (corrosive) based on GHS criteria

Remarks:

combination of category 1B-and-1C

Conclusions:

Under the experimental conditions of this study, the test substance is classified as H314 “Causes severe skin burns and eye damage”, combination of category 1B-and-1C, according to EU CLP Regulation (EC) No 1272/2008 and to the GHS.

Executive summary:

An in vitro skin irritation study was performed according to the OECD Guideline 431 and in compliance with GLP, using the EpiDerm™ Human Skin Model.

 

Duplicate tissues were treated with 25 mg of the unformulated test item (undiluted) wetted with 25 μL of sterile water to increase tissue surface contact for exposure periods of 3 and 60 minutes. The test item was found to directly reduce MTT. Direct reduction was <30% relative to the negative control and therefore acceptable. Thus additional non-viable tissues were incorporated into the testing for correction purposes. Negative and positive control groups were treated for each exposure period. At the end of the exposure period the test item was rinsed from each tissue before each tissue was taken for MTT‑loading. After MTT loading each tissue was placed in 2 mL Isopropanol for MTT extraction. At the end of the formazan extraction period each well was mixed thoroughly and triplicate 200 mL samples were transferred to the appropriate wells of a pre-labeled 96‑well plate. The optical density (OD) was measured at 562 nm (OD₅₆₂). Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

The relative mean viability of the test item treated tissues was 103.7 and 8.2% after 3 and 60 minutes exposure, respectively. The relative mean viability of the positive control treated tissues was 5.5% after 3 and 60 minutes exposure. The quality criteria required for acceptance of results in the test were satisfied.

Under the experimental conditions of this study, the test substance is classified as H314 “Causes severe skin burns and eye damage”, combination of category 1B-and-1C, according to EU CLP Regulation (EC) No 1272/2008 and to the GHS.

This study is considered as acceptable and satisfies the requirement for skin icorrosion endpoint.