Reaction mass of propylidynetrimethanol and 2-ethylpropane-1,3-diol and 5-ethyl-1,3-dioxane-5-methanol

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2009-09-28 to 2010-01-21

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

Draft from 20 March 2009 (Version 6), second circulation

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: ECVAM Skin Irritation Validation Study. Validation of the EpiSkin® Irritation Test –42 Hours Assay for the Prediction of Acute Skin Irritation of Chemicals, L’Oreal Recherche, January 2005.

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: ECVAM Skin Irritation Validation Study (SIVS). Performance Standards for Applying Human Skin Models to In Vitro Skin Irritation Testing (2007). ECVAM Statement on the Validity of in vitro Tests for Skin Irritation (2007).

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Dimethylolpropane Tech

IUPAC Name:

Dimethylolpropane Tech

Details on test material:

Dimethylolpropane Tech (DMP Tech), batch no. 3877810, a colourless to slightly yellow liquid, was received from Perstorp Holding AB on 04 September 2009 and was stored desiccated at ambient room temperature. The test item was supplied with an expiry date of 03 September 2010. A Certificate of Analysis was supplied with the test item.
A read across is proposed based on structural similarities between the substances.

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

skin obtained from plastic surgery from multiple donors

Justification for test system used:

Dermal irritation is defined as the production of reversible damage to the skin following the application of a test item. According to Annex VII of Regulation (EC) No. 1907/2006, there is a requirement for in vitro tests for skin irritation and corrosion. The Reconstructed human epidermis test method according to OECD TG 439 was validated by EURL ECVAM and gained regulatory acceptance to cover the skin irritation/corrosion endpoint.
The SkinEthic EpiSkin® irritation assay assesses the irritation potential of a test item by examining its cytotoxic effects after a defined exposure period and recovery time. The endpoint of the assay is the estimation of cell viability by assaying the reduction of MTT to its formazan metabolite by mitochondrial reductase. Irritant materials are identified by their ability to reduce cell viability below a threshold of 50% of the negative control value.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: SkinEthic EpiSkin®
EpiSkin® kits containing tissue culture units and assay medium/maintenance medium were supplied by Episkin SNC, Lyon, France. They are produced by culturing adult human keratinocytes on a collagen base in conditions which permit their terminal differentiation and the reconstruction of an epidermis with a functional horny layer (stratum corneum). The human keratinocytes come from mammary/abdominal samples obtained from healthy consenting donors during plastic surgery. HIV 1 & 2, HEP B and HEP C tests are carried out on the donors as well as verification of the bacteriological and fungal sterility of the cells. After a stage of immersed culture of the keratinocytes on the collagen support (3 days), during emersion (10 days) the epidermis differentiates and a horny layer is formed. The production of EpiSkin® is in accordance with the quality reference norms ISO 9001, ensuring traceability and reproducibility of the epidermal tissue as well as that of the quality control tests carried out on each batch of epidermis. The reproducibility of each batch is checked by histological analysis taking into account the general organisation, the stratification of the epidermis, the nucleation of the basal layer and the size of the intercellular spaces, adhesion of the basal layer to the support and the quantity of granular cells and the thickness of the horny layer. Each criterion is scored out of 4. The maximum score is 28. The minimum score for the criterion of acceptability of the model is 19.5. The reproducibility of the response of each EpiSkin® batch is tested against a reference irritant: SDS. The IC50 of the surfactant is measured by the MTT assay after 18 h of contact. The minimum score for acceptability of the model is 1.5 mg/mL.

After arrival at Charles River, the EpiSkin® kits were inspected for quality. The pH and temperature indicators were both acceptable. Each individual unit was then transferred to a single well of a 12 well microtiter plate containing maintenance medium (2 mL). The maintenance medium had been warmed to ca 37°C in a waterbath. Prior to dosing, all units were then incubated for ca 24 h at ca 37°C in a humidified atmosphere with 5% CO2.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37° C
- Temperature of post-treatment incubation (if applicable): 37° C

REMOVAL OF TEST MATERIAL AND CONTROLS
- After exposure to the test item or positive and negative controls for ca 15 min, the EpiSkin® units were washed by rinsing with Dulbecco’s phosphate buffered saline (25 mL). After washing, all skin units were blotted dry with tissue paper, transferred to fresh maintenance medium and incubated for ca 42 h at ca 37°C in a humidified atmosphere with 5% CO2.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- After the recovery period, all EpiSkin® units were transferred to a new 12 well microtiter plate containing MTT solution in assay medium (ca 0.3 mg/mL, 2 mL per well). Each unit was tapped gently on tissue paper to remove residual moisture before transferring to the MTT solution. The skin units were then incubated for ca 3 h at ca 37°C in a humidified atmosphere with 5% CO2.
After incubation, each skin unit was removed from the MTT solution and gently tapped dry on tissue paper to remove any excess moisture. The exposed skin was then completely removed from the unit using a specially designed biopsy punch. The epidermis was separated from the collagen matrix and both layers added to an appropriately labelled microfuge tube containing acidic isopropanol (500 μL). Samples were then stored at ca 4°C protected from light.
After ca 72 h storage, the samples were removed from the refrigerator and mixed by vortexing to ensure a homogenous mixture. Two aliquots (200 μL) were then added to a 96 well flat bottom microtiter plate for each sample, ensuring that no solid material was removed from the sample tube. Plates were analysed using an MRX plate reader using wavelength measurement at 550 nm. Absorbance values were calculated against the background acidified isopropanol sample contained on the plate.

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- The following calculations were made:
Optical Density (OD) = OD raw - OD blank mean
Individual Viabilities (%) = [OD test / Mean OD negative control] x 100
Relative Viabilities (5%) = % Viability = [OD test / OD negative control] x 100

The assay was deemed acceptable if the following criteria were met: The mean OD value of the 3 negative control tissues was ≥0.6 and the Standard Deviation value (SD) of the % viability was ≤18; The mean % viability of the 3 positive control tissues was ≥30% and the SD was ≤18; The mean % viability SD of the 3 treated tissues was ≤18.

NUMBER OF REPLICATE TISSUES: Three

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Prior to conducting the irritation assay, it was first necessary to determine if the test item was itself capable of reducing methylthiazoldiphenyl-tetrazolium bromide (MTT) to its formazan metabolite. Since the assay depends on the ability of viable skin cells to reduce MTT to formazan, any reduction by the test item would interfere with the integrity of the results. The test item (10 μL) was added to MTT (2 mL, ca 0.3 mg/mL) in PBS in a glass universal vial (3 replicates) and incubated at ca 37°C in a humidified atmosphere with 5% CO2 for ca 3 h. Formazan production was assessed by visual inspection. Three replicates of the positive control (eugenol, 10 μL) and the negative control (sterile, ultra-pure water, 10 μL) were tested in parallel to demonstrate the efficacy of the MTT solution.

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

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to skin in accordance with GHS category 2 if the viability after 15 minutes exposure and post-treatment incubation is less than 50%.
- The test substance is considered to be non-corrosive to skin if the viability after 15 minutes exposure is greater than or equal to 50%. Depending on country/regional regulatory requirements, the test chemical may be considered as “no category” if the tissue viability after exposure and post-treatment incubation is more than (>) 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: DMP Tech was applied onto the exposed surface of three viable EpiSkin® reconstructed human epidermis units using a Gilson positive displacement pipette set to deliver 10 μL. The test item was gently spread over the entire exposed skin surface using the tip of the pipette, taking care to avoid damaging the epidermis. The surface area of the EpiSkin® is ca 0.38 cm² and therefore the application rate was ca 26.3 μL/cm².

VEHICLE
- Amount(s) applied: Test substance was applied unchanged (no vehicle)

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

After exposure to the test item or positive and negative controls for ca 15 min, the EpiSkin® units were washed by rinsing with Dulbecco’s phosphate buffered saline (25 mL). After washing, all skin units were blotted dry with tissue paper, transferred to fresh maintenance medium and incubated for ca 42 h at ca 37°C in a humidified atmosphere with 5% CO2.

Number of replicates:

Three EpiSkin® units

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean of three replicates after 15 minutes

Value:

87.91

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

The results of the assay were similar for the three viable EpiSkin® units dosed with DMP Tech. Exposure to DMP Tech resulted in a mean EpiSkin® viability of 87.91% ± 11.33% of the negative control value.

OTHER EFFECTS: No other effects reported.
- Visible damage on test system: After arrival at Charles River, the EpiSkin® kits were inspected for quality. The pH and temperature indicators were both acceptable.
- Direct-MTT reduction: The test was scored by visual assessment of the formation of the purple-coloured formazan. The positive control (eugenol) reduced the MTT solution to formazan almost immediately, generating a dark purple colour before incubation. The negative control (sterile, ultra-pure water) and DMP Tech did not reduce MTT to formazan after ca 3 h incubation.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control results were within the acceptance criteria defined in the ECVAM validation SOP.
- Acceptance criteria met for positive control: The positive control results were within the acceptance criteria defined in the ECVAM validation SOP.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, DMP Tech is non-irritant (“no category” in accordance with CLP classification) when tested within the EpiSkin® in vitro skin irritation assay.

Executive summary:

In an in vitro skin iritation study performed according to OECD Guideline 439 (Reconstructed Human Epidermis Test Method), Dimethylolpropane Tech (DMP Tech) was tested in the EpiSkin® model for dermal irritation. Cell viability served as a parameter for irritation potential and was determined by the specific reduction of methylthiazoldiphenyl-tetrazolium bromide (MTT) to its formazan metabolite, which was measured spectrophotometrically. Irritant materials are identified by their ability to reduce cell viability below a threshold of 50% of the negative control value.

A volume of 10 µL DMP Tech was applied unchanged onto the exposed surface (ca. 0.38 cm²) of three viable EpiSkin® reconstructed human epidermis units derived from mammary/abdominal samples of healthy at an application rate was ca 26.3 μL/cm² for 15 minutes. DMP Tech was then washed from the surface of the EpiSkin® and the units returned to the incubator for a recovery period of ca 42 h.

After the recovery period, the skin units were transferred to assay medium containing MTT (0.3 mg/mL) and returned to the incubator for ca 3 h. Biopsies of the EpiSkin® membranes were then removed and extracted with acidic isopropanol.

Formazan production was assessed by measuring the optical density of the extract at 550 nm and the viability of each individual tissue was calculated as a percentage of the mean negative control viability. Exposure to DMP Tech resultedin an EpiSkin® viability of 87.91% ± 11.33% of the negative control value. The positive and negative controls, conducted in parallel, were within the defined acceptance criteria and demonstrated the efficacy of the test system.

In conclusion, DMP Tech is non-irritant (“no category” in accordance with GHS classification) when tested within the EpiSkin® in vitro irritation assay.