2,2,2',2'-tetrakis(hydroxymethyl)-3,3'-oxydipropan-1-ol

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28.09.2009 to 22.01.2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Proprietary GLP study conducted according to current guidelines

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: Draft OECD Guideline. In Vitro Skin Irritation: Reconstructed Human Epidermis (RHe) Test Method. 20 March 2009 (Version 6) 2nd Circulation.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

2,2,2',2'-tetrakis(hydroxymethyl)-3,3'-oxydipropan-1-ol

EC Number:

204-794-1

EC Name:

2,2,2',2'-tetrakis(hydroxymethyl)-3,3'-oxydipropan-1-ol

Cas Number:

126-58-9

Molecular formula:

C10H22O7

IUPAC Name:

2,2'-[oxybis(methylene)]bis[2-(hydroxymethyl)propane-1,3-diol]

Specific details on test material used for the study:

Di-Penta 93, batch no. 3810066489, a white crystalline powder, was received from Perstorp Holding AB, and was stored protected from light at ambient room temperature. The Sponsor supplied Test Item Data Sheet stated that the batch was produced on 15 December 2008 and “expects not to expire within at least five years after production. The shelf-life is two years from dispatch.”

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: human keratinocytes come from mammary/abdominal samples obtained from healthy consenting donors during plastic surgery.

Vehicle:

water

Details on test system:

The EpiSkin® reconstituted human epidermis models were obtained from 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 surfactantmn mn, 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.

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 (ca 10 mg) 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

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

10 mg ± 2mg was applied onto the exposed surface of the EpiSkin. The surface of the EpiSkin was moistened with sterile, ultra-pure water (5 µL) prior to dosing. The surface area of the EpiSkin was ca 0.38 cm² and therefore the mean application rate was ca 23.6 mg/cm².

Duration of treatment / exposure:

Exposure: 15 minutes
Observation: 42 hours

Number of replicates:

Three viable EpiSkin units

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Remarks:

Percentage Viability of EpiSkin

Run / experiment:

Time point: 42 hours.

Value:

90.29

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Remarks:

Remarks: SD 5.19%.

Other effects / acceptance of results:

The negative control results were within the acceptance criteria defined in the ECVAM validation SOP.
The positive control results were within the acceptance criteria defined in the ECVAM validation SOP.
Exposure to Di-Penta 93 resulted in an EpiSkin® viability of 90.29% ± 5.19% of the negative control value. Percentage viabilities are shown in Table 1.

Any other information on results incl. tables

MTT Direct Reduction Test:

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 Di-Penta 93 did not reduce MTT to formazan after ca. 3 h incubation.

Table 1: Viability of EpiSkin Cultures

Test item	Mean Viability (%) after 42 hours	SD (%)	CV (%)
Di-Penta 93	90.29	5.19	5.75
5% SDS Solution (positive control)	10.40	4.10	39.42
PBS Solution (negative control)	100.00	7.82	7.82

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Di-Penta 93 is non-irritant when tested within the EpiSkin® in vitro irritation assay.

Executive summary:

Di-Penta 93 was tested for dermal irritation in vitro, using the SkinEthic EpiSkin® in vitro irritation assay. The endpoint of the assay was the estimation of cell viability by assaying the reduction of methylthiazoldiphenyl-tetrazolium bromide (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. A preliminary test was conducted to assess the ability of Di-Penta 93 to directly reduce MTT to formazan. Di-Penta 93 did not interact with the MTT solution. The irritation potential was assessed by applying Di-Penta 93 (ca 10 mg) directly onto the surface of three viable EpiSkin® reconstructed human epidermis units for ca 15 min. Di-Penta 93 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 added to acidic isopropanol. The 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 Di-Penta 93 resulted in an EpiSkin® viability of 90.29% ± 5.19% 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, Di-Penta 93 is non-irritant (“no category” in accordance with GHS classification) when tested within the EpiSkin® in vitro irritation assay.