Reaction product of o-cresol and camphene, obtained by addition, hydrogenation and distillation

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 Jun - 04 Aug 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 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

adopted: 28 Jul 2015

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Version / remarks:

Commission Regulation (EC) 440/2008, 1st ATP 2009

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Hess. Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany

Test material

Test material information

Constituent 1

Reference substance name:

Reaction product of o-cresol and camphene, obtained by addition, hydrogenation and distillation

EC Number:

944-329-6

Molecular formula:

C17H28O

IUPAC Name:

Reaction product of o-cresol and camphene, obtained by addition, hydrogenation and distillation

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: EpiDerm™

Source strain:

not specified

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ (MatTek Corporation, Bratislava, Slovakia)
- Tissue batch number: 25834
- Delivery date: 01 Aug 2017
- Date of initiation of testing: 01 Aug 2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1.5 °C for 35 min in the incubator; thereafter at room temperature for 25 min in a sterile bench

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: Tissues were gently rinsed with DPBS at least 15 times in order to remove any residual test material. After the rinsing the inserts were submerged in DPBS at least 3 times. Afterwards the inserts were once again rinsed with DPBS from the inside and the outside.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h
- Spectrophotometer: microplate reader (Versamax, Molecular Devices, Softmax Pro v.4.7.1)
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: The quality of the EpiDerm™ tissue was assessed by undertaking an MTT cell viability test. The determined OD (540 - 570 nm) was 1.866 ± 0.143 (acceptance criteria: 1.0 - 3.0).
- Barrier function: The barrier function was assessed by determination of the exposure time required to reduce tissue viability by 50% (ET-50) upon application of 100 µL of 1% Triton X-100. The ET-50 value was determined to be 6.02 h (acceptance criteria: 4.77-8.72 h).
- Contamination: The cells used to produce the EpiDerm™ tissue were screened for the presence of viruses, bacteria, yeast and other fungi.

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
Since the test substance did not directly reduce MTT, an additional test with freeze-killed tissues was not performed.

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

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to skin if the viability after 1 hour exposure is less or equal than 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 30 µL

NEGATIVE CONTROL
- Amount applied: 30 µL

POSITIVE CONTROL
- Amount applied: 30 µL
- Concentration: 5% aqueous solultion

Duration of treatment / exposure:

60 min

Duration of post-treatment incubation (if applicable):

approximately 42 h

Number of replicates:

triplicates for each treatment and control group

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean value of 3 tissues

Run / experiment:

60 min

Value:

75.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Direct MTT reduction: The test substance was not considered to be a MTT reducer.
- Colour interference with MTT: The test substance did not change colour when mixed with deionised water. Also its intrinsic colour was not intensive.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control OD (1.436, 1.405 and 1.404) was in the range of the required acceptability criterion of mean OD ≥ 0.8 and ≤ 2.8 for the 60 min treatment interval thus showing the quality of the tissues.
- Acceptance criteria met for positive control: Exposure to the positive control induced a decrease in the relative absorbance as compared to the negative control to 4.2% thus confirming the validity of the test system.
- Acceptance criteria met for variability between replicate measurements: The relative standard deviations of the 3 identical replicates of the test substance and negative control in the main test were below 4.9% (threshold of OECD 439: <18%).

Any other information on results incl. tables

Table 2. Results after treatment with the test substance and controls

	Mean absorbance at 570 nm *			Mean absorbance of 3 tissues blank corrected	Rel. absorbance (%) **			Rel. SD (%)	Rel. absorbance (% of negative control)***
	Tissue 1	Tissue 2	Tissue 3		Tissue 1	Tissue 2	Tissue 3
Negative control	1.436	1.405	1.404	1.415	101.5	99.3	99.2	1.3	100.0
Positive control	0.056	0.061	0.060	0.059	3.9	4.3	4.3	4.9	4.2
Test substance	1.033	1.097	1.062	1.064	73.0	77.5	75.0	3.0	75.2

* Mean of 3 replicate wells after blank correction (blank values: well 1 and 2: 0.038; well 3: 0.037)

** Relative absorbance per tissue (rounded values): 100 × (absorbance tissue) / (mean absorbance negative control)

*** Relative absorbance per treatment group (rounded values): 100 × (mean absorbance test item/positive control) / (mean absorbance negative control)

Applicant's summary and conclusion

Interpretation of results:

other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No. 1272/2008

Conclusions:

Under the conditions of the conducted test, the test substance did not possess irritating properties towards reconstructed human epidermis tissue in the EpiDerm™ model.

Executive summary:

Under the conditions of the conducted test, the test substance did not possess irritating properties towards reconstructed human epidermis tissue in the EpiDerm™ model. The tissue viablilty was 75.2%.