Rectified Hydrocarbons by-products from synthetic process of Turpentine and acid, alcohols fraction

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 October 2018 - 26 October 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

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)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Rectified Hydrocarbons by-products from synthetic process of Turpentine and acid, alcohols fraction

EC Number:

949-141-8

Molecular formula:

Not available since an UVBC substance.

IUPAC Name:

Rectified Hydrocarbons by-products from synthetic process of Turpentine and acid, alcohols fraction

Test material form:

liquid

In vitro test system

Test system:

human skin model

Remarks:

SkinEthic RHE® model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: foreskin (number of donors not specified)

Source strain:

not specified

Justification for test system used:

The SkinEthic RHE® model has been validated for irritation testing (Validation study based on the original ECVAM Performance Standards (21) in 2008) and its use is recommended by the relevant OECD guideline for irritation testing (OECD No. 439); therefore, it was considered to be suitable for this study.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: SkinEthic RHE® model
- Tissue batch number(s): 18-RHE-123
- Delivery date: 23/10/2018
- Expiration date: 29/10/2018
- Date of initiation of testing: 23/10/2018

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

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 25 x 1 mL of DPBS
- Observable damage in the tissue due to washing: no
- Modifications to validated SOP: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 300 µL of a MTT solution at 1.0 mg/mL
- Incubation time: 3 hours at 37°C, 5% CO2
- Spectrophotometer: ELx800 absorbance microplate reader (BioTek)
- Wavelength: 570 nm
- Linear OD range of spectrophotometer: not specified.

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: OD=1.4 (CV = 7.1%) specification OD > 0.7. Historical negative control mean OD range = 0.653-1.194 (measured after a 1:2 dilution of the extracts in isopropanol).
- Barrier function: 4.5 h (Specification 4.0h < ET50< 10.0h)
- Morphology: 5.5 Cell layers, absence of significant histological abnormalities, well differentiated epidermis, specification > 4
- Contamination: no

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE: no interference.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to skin (or corrosive) if the viability after 42 minutes exposure and 42 hours of post-treatment incubation is less than or equal to 50%.
- The test substance is considered to be non-irritant to skin if the viability after 42 minutes exposure and 42 hours of post-treatment incubation is greater than 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 16 μL (32 μL/cm2)

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 16 µL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 16 µL
- Concentration (if solution): 5% SDS

Duration of treatment / exposure:

42 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean

Value:

4.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

(distilled water)

Positive controls validity:

valid

Remarks:

2.0% viability (5% SDS)

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

The mean percent viability of the treated tissues was 4.3% versus 2.0% in the positive control (5% Sodium Dodecyl Sulfate). Therefore, the test item must be considered as skin irritant (cat 2) or skin corrosive (Cat 1).

- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: no
- Colour interference with MTT: no

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes. A full demonstration of proficiency was performed for the EpiSkin-SM model, plus a reduced validation with the SkinEthic RHE model. Adequate results were obtained for the evaluated chemicals.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, SD of the negative control group was 2.9% (acceptablility criteria, SD ≤ 18%) and OD mean was 0.613 (measured after a 1:2 dilution of the formazan extracts in isopropanol; the acceptability criteria
should be in the range ≥ 0.4 and ≤1.5).
- Acceptance criteria met for positive control: yes, SD of the positive control group was 0.1% (acceptablility criteria, SD ≤ 18%) and mean viability was 2.0% which is much lower than 50%.
- Acceptance criteria met for variability between replicate measurements: yes. SD of test item was 1.6% (acceptablility criteria, SD ≤ 18%).

Any other information on results incl. tables

Table 1. Summary of results.

	Skin	OD	Mean OD /disc (#)	Mean OD / product	Viability %	Mean viability %	SD Viability	Conclusion
Negative Control	1	0.633	0.635	0.613	102.7	100.0	2.9
		0.633
		0.638
	2	0.573	0.599		96.9
		0.621
		0.603
	3	0.615	0.621		100.4
		0.621
		0.626
Positive Control	4	0.011	0.012	0.012	1.9	2.0	0.1	Irritant
		0.012
		0.012
	5	0.012	0.012		1.9
		0.012
		0.013
	6	0.011	0.013		2.1
		0.013
		0.014
Test item	19	0.014	0.015	0.026	2.4	4.3	1.6	Irritant or corrosive
		0.015
		0.016
	20	0.032	0.032		5.2
		0.031
		0.033
	21	0.032	0.032		5.2
		0.032
		0.031

# mean of 3 values (triplicate of the same extract)

OD: optical density

Acceptability criteria:

SD≤18%.

Negative control: OD value of the 3 replicates in the range ≥ 0.8 and ≤ 3.0. The optical density was measured after a 1:2 dilution of the formazan extracts in isopropanol; the acceptability criteria should be in the range ≥ 0.4 and ≤1.5 for the negative control.

Applicant's summary and conclusion

Interpretation of results:

other: classified as corrosive (Cat 1) or irritant (Cat 2) (CLP Regulation EC no. 1272/2008)

Conclusions:

In the in vitro skin irritation RHE method, the mean percent viability of the treated tissues was 4.3% versus 2.0% in the positive control (5% Sodium Dodecyl Sulfate). Therefore, the test item must be considered as skin irritant (cat 2) or skin corrosive (Cat 1).

Executive summary:

An in vitro skin irritation test of the test item was performed in a reconstructed human SkinEthic RHE® model, according to OECD TG 439 (GLP study). Three epidermis units were treated with 16 μL test item for 42 minutes at room temperature. Exposure of the test item was terminated by rinsing with 25 x 1 mL of DPBS. The epidermis units were then incubated at 37°C for 42 hours in an incubator with 5% CO2. The viability of each disk was assessed by incubating the tissues with MTT, extracting the precipitated formazan crystals using isopropanol during 2 hours under gentle agitation in the dark, and measuring the concentration of formazan by determining the OD at 570 nm, just after dilution of the extracts 1:2 in isopropanol. Under test conditions, the mean percent viability of the treated tissues was 4.3%, versus 2.0% in the positive control (5% Sodium Dodecyl Sulfate). Therefore, the test item must be considered as skin irritant (cat 2) or skin corrosive (Cat 1).