Pyrolysis Reaction Product of Pinane

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

20 September 2018 to 31 October 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

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)

Version / remarks:

29 July 2016

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EU method B.40 Bis: In Vitro Skin Corrosion: Human Skin Model Test

Version / remarks:

30 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Pyrolysis Reaction Product of Pinane

EC Number:

947-964-7

Molecular formula:

n. a. for UVCB

IUPAC Name:

Pyrolysis Reaction Product of Pinane

In vitro test system

Test system:

human skin model

Remarks:

The EPISKIN-model is a 3-dimensional human epidermis model obtained after a 13-days culture period of adult human-derived epidermal keratinocytes seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen.

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: reconstructed human epidermis units are contained in the EpiSkin(TM)SM kits, supplier: SKINETHIC Laboratories; 4, rue Alexander Fleming, 69366 – LYON Cedex 07, France

Justification for test system used:

The EPISKIN model has been validated for corrosivity testing in an international trial; it is considered to be suitable for this study (STATEMENT ON THE SCIENTIFIC VALIDITY OF THE EPISKINTM TEST (AN IN VITRO TEST FOR SKIN CORROSIVITY); ECVAM JRC Environment Institute, European Commission; Ispra; 03 April 1998).

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN model
- Tissue batch number: 18-EKIN-038

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature

REMOVAL OF TEST MATERIAL AND CONTROLS
The skin units were rinsed thoroughly with approximately 25 mL PBS 1 x solution to remove all of the test material from the epidermal surface. The rest of the PBS was removed from the epidermal surface with a suitable pipette tip linked to a vacuum source (care was taken to avoid damaging the epidermis).

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 3 h
- Linearity range of spectrophotometer: 0.2136 – 3.1752

NUMBER OF REPLICATE TISSUES: 2

PREDICTION MODEL / DECISION CRITERIA
- The test substance is not considered to be corrosive to skin if the mean tissue viability is ≥ 35 % after 4 hours exposure.
- The test substance is considered to be corrosive to skin (Cat 1 A) if the mean tissue viability is < 35 % after 3 min exposure.
- The test substance is considered to be corrosive to skin (Cat 1 B or C) if the mean tissue viability is ≥ 35 % after 3 min exposure and < 35 % after 1 hour exposure OR ≥ 35 % after 1 hour exposure and < 35 % after 4 hours exposure

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 50 µL
NEGATIVE CONTROL
- Amount applied: 50 µL
POSITIVE CONTROL
- Amount applied: 50 µL

Duration of treatment / exposure:

4 h

Number of replicates:

2

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

103

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
The mean OD value of the two negative control tissues was 0.763.
The positive control result showed 7 % viability.
The difference of viability between the two tissue replicates was between 2.7 % to 9.3 %.
All validity criteria were within acceptable limits. Therefore, the study can be considered as valid.

Any other information on results incl. tables

Table 1: OD values and viability percentages of the controls: Controls Optical Density (OD) Viability (%) Δ% Negative Control: NaCl (9 g/L saline) 1 0.799 105 9.3 2 0.728 95 mean 0.763 100   Positive Control: Glacial acetic acid 1 0.063 8 2.7 2 0.043 6 mean 0.053 7

 

Table 2: OD values and viability percentages of the test item (including corrected values):

	Optical Density (OD)		Viability (%)	Δ%
Test substance	1	0.758	99	7.1
	2	0.812	106
	mean	0.785	103

 

Applicant's summary and conclusion

Interpretation of results:

other: non-corrosive, further information necessary to decide on irritation potential and final classification

Conclusions:

The results obtained from this in vitro skin corrosion test, using the EPISKIN model, indicated that the test item reveals no skin corrosion potential under the utilised testing conditions.

Executive summary:

The study was conducted to determine the skin corrosion potential of the test item. Disks of EPISKIN (two units) were treated with the test item and incubated for 4 hours (±10 min) at room temperature. Exposure of the test material was terminated by rinsing with PBS 1x solution. The viability of each disk was assessed by incubating the tissues for 3 hours (±15 min) with 5±1 % CO2 in a ≥ 95% humidified atmosphere and protected from light. The formazan precipitated was then extracted using acidified isopropanol and quantified spectrophotometrically. NaCl (9 g/L saline) and glacial acetic acid treated epidermis were used as negative and positive controls, respectively. The test item did not show significantly reduced cell viability in comparison to the negative control after four hours of exposure. Both individual tissue viabilities were above 35 % of the mean negative control value. The average test item treated tissue viability was 103 %. Positive and negative controls showed the expected cell viability values within acceptable limits. The experiment was considered to be valid. The results obtained from this in vitro skin corrosion test, using the EPISKIN model, indicated that the test item reveals no skin corrosion potential. This result can be used together with further information on the irritative nature of the substance to decide on its final classification.