Diallyl hexahydrophthalate

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 December 2016 - 16 August 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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:

yes

Remarks:

The draft report was issued later than stated in the study plan, and Szabolcs Gáty became responsible for QA on Feb 15 2017, and no longer a member of Test Facility Management. These deviations had no adverse affect the results or study integrity.

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Commission Regulation (EC) No 440/2008, Annex Part B, B.40.Bis: “In Vitro Skin Corrosion: Human Skin Model Test”, Official Journal of the European Union No. L142 (31 May 2008)

Deviations:

yes

Remarks:

The draft report was issued later than stated in the study plan, and Szabolcs Gáty became responsible for QA on Feb 15 2017, and no longer a member of Test Facility Management. These deviations had no adverse affect the results or study integrity.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Diallyl hexahydrophthalate

EC Number:

237-580-1

EC Name:

Diallyl hexahydrophthalate

Cas Number:

13846-31-6

Molecular formula:

C14H20O4

IUPAC Name:

1,2-bis(prop-2-en-1-yl) cyclohexane-1,2-dicarboxylate

Test material form:

liquid

Remarks:

Colourless

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source of test material: Osaka Soda Co., Ltd
- Name: MDAC (Chemical name: 1,2-Cyclohexanedicarboxylic acid, di-2-propenyl ester)
- Appearence: Colourless liquid
- Batch No. of test material: 40201
- Expiration date of the batch: 26 January 2019
- Purity test date: 99.2% (CoA)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Controlled room temperature (15-25°C, ≤ 70% Relative Humidity), protected from light
- Stability under test conditions: Assumed stable for the duration of the study
- Solubility, stability of the test substance in the solvent/vehicle: The test item was applied as supplied, no formulation was required.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: None, the test item was applied as supplied, no formulation was required.

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: source not stated

Justification for test system used:

The EPISKINTM(SM) model has been validated for corrosivity testing in an ECVAM international trial (Fentem, 1998) and its use is recommended by the relevant OECD guideline for corrosivity testing (OECD No. 431). It was therfore considered as suitable for this study.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN TM(SM) (Manufacturer: SkinEthic, France, Batch No.: 16-EKIN-050, Expiry date: 19 December 2016) a three-dimensional human epidermis model.
- Tissue batch number(s): Batch No.: 16-EKIN-050,
- Date of initiation of testing: 15 December 2016
- Date of completion of testing:16 December 2016

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: Incubated for 4 hours (±10 min) at room temperature (22.2-24.3°C) covered with plate lids.

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: After incubation, all test item treated tissues and positive control tissues were removed and rinsed thoroughly with PBS solution to remove all residual test item or positive control material from the epidermal surface. Likewise, negative control tissues were processed accordingly. The remaining PBS was removed from the epidermal surface using a pipette (without touching the epidermis).
- Observable damage in the tissue due to washing: None

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: MTT solution (0.3 mg/mL). 2 mL of MTT working solution was added to each well below the skin units.
- Incubation time: At 37°C in an incubator with 5% CO2 for 3 hours, protected from light .
- Spectrophotometer: Optical Density (OD) measured using a plate reader (a blank sample containing 2 mL of acidified isopropanol was processed in parallel with MTT solution). The instrument was verified by measuring a verification plate (Manufacturer: Thermo Fisher Scientific, Catalogue Number: 240 72800, Serial Number: 0920-14, Date of calibration: 22 August 2016, calibration is valid until August 2018) at the required wavelength on each day before use.
- Wavelength: 570 nm.

- Contamination: No contamination during the study. EPISKINTM(SM) kits are manufactured according to defined quality assurance procedures (certified ISO 9001). All biological components of the epidermis and the kit culture medium have been tested for the presence of viruses, bacteria and mycoplasma. The quality of the final product was assessed by undertaking a MTT cell viability test and a cytotoxicity test with sodium dodecyl sulphate (SDS). These quality control experiments were conducted at SkinEthic laboratories (supplier of the EpiSkinTM(SM).

NUMBER OF REPLICATE TISSUES: Two replicates per test-item were used. Two negative controls and two positive controls were also run in each assay. As the test item had an MTT interacting potential, two additional test item-treated killed epidermis and two negative control treated killed epidermis were used.

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Killed tissues : As the test item had an MTT interacting potential, two additional test item-treated killed epidermis and two negative control treated killed epidermis were used.
- Procedure used to prepare the killed tissues (if applicable): For killed epidermis, living epidermis units (Manufacturer: SkinEthic, France, Batch No.: 16-EKIN-033, Expiry Date: 22 August 2016) were placed in a 12 well plate with 2 mL of distilled water, then incubated (37°C with 5% CO2, in a >95% humidified atmosphere for 48 hours (±1 hour). At the end of the incubation period the water was discarded and the dead epidermis units frozen on 19 August 2016, frozen units can be used up to 6 months). Before use, the killed tissues were thawed at room temperature (at least 30 minutes in 2 mL of Maintenance Medium). Further use of killed tissues was similar to that of living tissues.
- N. of replicates : Two additional controls on killed epidermis (two test item treated and two negative control treated skin units) were also used to determine the MTT interacting potential of the test item
- Method of calculation used: Additional control samples were used to determine the OD value derived from non-specific reduction of the MTT. The measured OD value was corrected by the result of the additional controls before calculation of viability% as follows:

Non-specific MTT reduction calculation (NSMTT):
NSMTT = [(ODKT- ODKNC) / ODNC] × 100
OD KNC: negative control treated killed tissues OD
OD KT: test item treated killed tissues OD
OD NC: negative control OD

If NSMTT is ≤ 50%, then true MTT metabolic conversion (TODTT) had to be undertaken as follows:
TODTT = [ODTT – (ODKT – ODKNC)]
ODTT: test item treated viable tissues

– The % relative viability (RV%) for each test item replicate was calculated relative to the mean negative control:
RV1 % = [TODTT1 / mean ODNC] × 100
RV2 % = [TODTT2 / mean ODNC] × 100
– The mean value of the 2 individual relative viability % for test item was calculated:
Mean Relative Viability % = (RV1 % + RV2 %) / 2

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

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin: if both disks have mean viability of <35% = Corrosive (at the corresponding incubation period), or if the mean value is <35% and the variability is less than 50%.

- The test substance is considered to be non-corrosive to skin: if both disks have mean viability of ≥35% = Non Corrosive, or If the mean value is ≥35% and the variability is less than 50% = Non Corrosive

Exceptions: If the classification is not made with these criteria, retest with 2 more disks. Take the mean of the 4 disks to classify as above or below 35%. Outlier values may be excluded where there are scientific reasons, such as where application or rinsing is difficult and that the Study Director considers that a result is not representative.

Classification Packing group Criteria for In Vitro interpretation

UN GHS / EU-CLP Sub Category 1A If corrosive after 3 min exposure
Sub Category 1B If not corrosive after 3 min exposure and corrosive after 1 hour exposure
Sub Category 1C If not corrosive after 1 hour exposure and corrosive after 4 hours exposure

Non corrosive If not corrosive after 4 hours exposure.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume with unit): 50 μL of test item was applied evenly to each of two test units and each additional control skin units.
- Concentration (if solution): The test item was applied as supplied, no formulation was required. No correction for purity of the test item was applied.

NEGATIVE CONTROL
- Amount(s) applied (volume):50 μL of physiological saline was added to each of the two negative control skin units and each of the two negative control treated killed epidermis units.
- Concentration (if solution): 0.9% (w/v)

POSITIVE CONTROL
- Amount(s) applied (volume): 50 μL of glacial acetic acid was added to each of the two positive control skin units.
- Concentration (if solution): not specifed

Duration of treatment / exposure:

The plates with the treated epidermis units were incubated for 4 hours (±10 min) at room temperature (22.2 - 24.3°C) covered with the plate lids. Following rinsing at the end of the incubation time, MTT solution (2 mL of 0.3 mg/mL MTT working solution) was added to each well below the skin units. The lid was replaced and the plate incubated at 37°C in an incubator with 5% CO2 for 3 hours, protected from light.

Number of replicates:

In this assay, two replicates per test item were used.Two negative controls and two positive controls were also run in each assay. As the test item had an MTT
interacting potential, two additional test item-treated killed epidermis and two negative control treated killed epidermis were used.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Test Item (MDAC)

Value:

127.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: The results indicate the test item (MDAC) is non-corrosive to the skin.

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: None
- Direct-MTT reduction: 50 μL of test item was added to 2 mL MTT working solution and mixed. The mixture was incubated at 37°C in an incubator with 5 % CO2, in a >95% humidified atmosphere for 3 hours and then any colour change was observed. After three hours incubation, purple colour in the mixture was detected; therefore additional controls were used in the experiment. Based on the results of the additional controls on killed epidermis, the calculated NSMTT was 0.9%. This was considered to be significant, thus correction with Non-Specific MTT (NSMTT) were made.

- Colour interference with MTT: 10 μL of the test item was added to 90 μL of deionised water and isopropanol. The mixture was shaken for about 15 minutes at room temperature and then colour was checked (unaided visual assessment). As no coloured solution was detected and the test item had no intrinsic colour, no additional colour controls were needed in the experiment to determine the Non Specific Colour (the test item showed no ability to stain the epidermis).
.
DEMONSTRATION OF TECHNICAL PROFICIENCY:
After receipt, the two indicators (pH and temperature) of the delivered kits were checked and based on the observed colours, the epidermis units were acceptable for study use.
The mean OD value of the two negative control tissues was in the recommended range (0.856).
The two positive control treated tissues showed 0.9% viability demonstrating the proper performance of the assay.
The difference of viability between the two test item-treated tissue samples in the MTT assay was 0.1%.
The difference of viability between the two negative control tissue samples in the MTT assay was 10.6%.
The mean OD value of the blank samples (acidified isopropanol) was 0.046.
All these parameters were within acceptable limits and therefore the study was considered to be valid.


ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for variability between replicate measurements: Yes

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Following exposure with MDAC, the mean cell viability was 127.7% compared to the negative control (after adjustment for non-specific MTT reduction). This is above the threshold of 35%, therefore the test item was considered non-corrosive. The experiment met the validity criteria, therefore the study was considered to be valid.

In conclusion, in this in vitro EPISKIN™(SM) model test with MDAC (Batch number: 40201), the results indicate that the test item is non-corrosive to the skin.