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Toxicological information

Skin irritation / corrosion

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Administrative data

skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to the O.E.C.D. test guideline 439 with GLP compliance.

Data source

Reference Type:
study report
Report Date:

Materials and methods

Principles of method if other than guideline:
The study protocol met the requirements of the OECD test guideline, “In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method” 439.
GLP compliance:

Test material

Details on test material:
As per IUCLID Sections 1.1. 1.2. and 4.1.

Test animals

other: Study was in vitro.
Details on test animals and environmental conditions:
Study was in vitro.

Test system

unchanged (no vehicle)
Amount / concentration applied:
Neat 100%
Duration of treatment / exposure:
One hr.
Observation period:
42 +/- 2 hr.
Number of animals:
None, study was in vitro.
Details on study design:
EpiDermTM Skin Kit (MatTek Corporation), was used for the assessment. The EpiDerrntm’ tissues were stored at 2-8°C until use. On the day prior to testing, EpiDermTM Maintenance Medium was set to room temperature prior to use. Nine-tenths mL of Maintenance Medium were aliquotted into the appropriate wells of 6-well plates. The EpiDermTM tissues were transferred aseptically into the 6-well plates. The EpiDermTM tissues were then incubated at 37±1°C in a humidified atmosphere of 5±1% CO2 in air (standard culture conditions) for 60±5 minutes. After 60 minutes, the EpiDermTM tissues were transferred to appropriate wells containing 0.9 mL of fresh warmed (to 37°C) Maintenance Medium. The plates were returned to the incubator for 18 ±3 hours to acclimate the tissues.

The EpiDermTM tissues were treated in triplicate with the test substance or positive control for 60±1 minutes. All of plates were transferred to the incubator for 35 +/- 1 minutes at standard culture conditions. After 35 minutes, all of the plates were removed from the incubator, placed into the laminar flow hood and kept at room temperature until the exposure period was completed. After 60 ± 1 minutes of test or control substance exposure, the tissues were rinsed with sterile, CMF-DPBS by filling and emptying the tissue insert 15 times. The bottoms of the tissue inserts were blotted on sterile paper towels and the inserts were transferred to new 6-well plates containing 0.9 mL of flesh warmed (to 37°C) Maintenance Medium. Residual test substance was observed, therefore sterile cotton-tipped applicators pre-moistened with CMFDPBS were used to attempt to remove any residual test substance from the tissue surface. The tissues were then placed into the incubator at standard culture conditions for a post-treatment expression incubation of 42 ± 2 hours.

In order to access cell viability a lOX stock of MTT (3-[4,5 - dimethylthiazol-2-ylj - 2,5 - diphenyltetrazoliuni bromide) prepared in PBS (filtered at time of batch preparation) was thawed and diluted in warm MTT Addition Medium to produce a 1.0 mg/mL solution no more than two hours before use. Three hundred microliters of the MIT solution were added to each designated well of a pre-labeled 24-well plate. After the total 42 ± 2 hours post-exposure expression incubation, the 6-well plates were removed from the incubator. Each tissue was blotted on a sterile paper towel and transferred to an appropriate well containing 0.3 mL of MTT’ solution. The 24-well MTT plates were incubated at standard culture conditions for 3 ± 0.1 hours.

After the 3 ± 0.1 hour incubation, the EpiDermTM tissues were submerged, gently swirled, and rinse media decanted in a beaker containing approximately 150 mL of CMF-DPBS three times. The tissue was then blotted on absorbent paper, cleared of excess liquid, and transferred to a prelabelled 24-well plate containing 2.0 mL of isopropanol in each designated well. The plate was covered with parafilm and shaken for at least 2 hours at room temperature to extract the MTT. isopropanol were added to the wells designated as blanks. The absorbance at 570 nm (0D570) of each well was measured with a Molecular Devices Vmax plate reader with the AUTOMIX function selected.

Mean corrected 0D570 values were calculated for each individual test substance and control tissue from the duplicate aliquots. The group mean of the corrected 0D570 values for the negative controls were calculated. The % of Control viability calculations were made for each individual tissue to access viability.

Results and discussion

In vitro

Irritation / corrosion parameter:
other: other: EpiDerm in vitro cell viability.
Vehicle controls validity:
Negative controls validity:
Positive controls validity:
Remarks on result:
other: see remark
Basis: mean EpiDerm tissue cultures.. Time point: 42 +/- 2 hr.. Reversibility: no data Measurment was cell viability.. Remarks: Relative % viability of the test substance treated cultures was 30.1 +/- 28.3.. (migrated information)

Any other information on results incl. tables

The test substance was attempted to be removed from the exposed EpiDermTM tissues using cotton-tipped applicators pre-moistened with DPBS. A dissecting scope was used to check for residual test substance before and after use of the pre-wetted cotton swabs. The residual test substance could not be completely removed from the EpiDermml tissues. The residual test substance prolonged the exposure to the tissues, which may have influenced the toxic/irritant effect. Furthermore, mechanical damage may have also reduced cell viability.

Applicant's summary and conclusion

Due to issues of removal of the test substance, an epoxy resin material from the EpiDerm cell cultures the findings from this in vitro study, are based on Expert Judgement, not be valid. Therefore, this in vitro data cannot be used to access the skin irritating potential of the test substance.
Executive summary:

The test substance, 4,4'-Isopropylidenedicyclohexanol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, was evaluated for its potential in cause skin irritation in an O.E.C.D. 439 test guideline GLP, SKIN IRRITATION TEST (SIT)

USING THE EPIDERM’ SKIN MODEL. Due to issues of removal of the test substance, an epoxy resin material from the EpiDerm cell cultures the findings from this in vitro study, are based on Expert Judgement, not be valid. Therefore, this in vitro data cannot be used to access the skin irritating potential of the test substance.