Reaction mass of (2Z)-5-[(1R,3R,6S)-2,3-dimethyltricyclo[2.2.1.02,6]hept-3-yl]-2-methyl-2-penten-1-ol and (2Z)-2-methyl-5-[(1S,2R,4R)-2-methyl-3-methylenebicyclo[2.2.1]hept-2-yl]-2-penten-1-ol

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11-13 December 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted according to OECD test Guideline No. 431 without any deviation.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

18 June 2019

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

UK GLP Compliance Monitoring Programme (inspected on 21/08/2018 / signed on 19/11/2018)

Test material

Test material information

Constituent 1

Reference substance name:

5-(2,3-dimethyltricyclo[2.2.1.02,6]hept-3-yl)-2-methylpent-2-en-1-ol, stereoisomer

EC Number:

204-102-8

EC Name:

5-(2,3-dimethyltricyclo[2.2.1.02,6]hept-3-yl)-2-methylpent-2-en-1-ol, stereoisomer

Cas Number:

115-71-9

Molecular formula:

C15H24O

IUPAC Name:

5-(2,3-dimethyltricyclo[2.2.1.0~2,6~]hept-3-yl)-2-methylpent-2-en-1-ol

Constituent 2

Reference substance name:

[1S-[1α,2α(Z),4α]]-2-methyl-5-(2-methyl-3-methylenebicyclo[2.2.1]hept-2-yl)-2-penten-1-ol

EC Number:

201-027-2

EC Name:

[1S-[1α,2α(Z),4α]]-2-methyl-5-(2-methyl-3-methylenebicyclo[2.2.1]hept-2-yl)-2-penten-1-ol

Cas Number:

77-42-9

Molecular formula:

C15H24O

IUPAC Name:

2-methyl-5-(2-methyl-3-methylenebicyclo[2.2.1]hept-2-yl)pent-2-en-1-ol

Test material form:

liquid: viscous

Details on test material:

- Appearance: Colourless viscous liquid
- Storage conditions: Dry area, protected from light in a refrigerator (temp 2-8°C) stored under nitrogen in a closed container after every opening

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: cells obtained from a single or multiple donors

Details on animal used as source of test system:

Not applicable

Justification for test system used:

Following the REACH top-down strategy, the EPIDERM™ Reconstructed Human Epidermis Model method was used to assess skin corrosion.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ Reconstructed Human Epidermis Model Kit, supplied by MatTek
- Tissue batch number: 30893
- Delivery date: 11 December 2019
- Date of initiation of testing: 12 December 2019

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

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: Rinsing was achieved by filling and emptying each tissue under a constant soft stream of Dulbecco’s Phosphate Buffered Saline (DPBS) (without Ca++ Mg++) for approximately 40 seconds, to gently remove any residual test item. Excess DPBS was removed by blotting the bottom of the tissue insert with tissue paper
- Observable damage in the tissue due to washing: none
- Modifications to validated SOP: none

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: Labtech LT 4500 microplate reader
- Wavelength: 570 nm
- Filter:Yes
- Filter bandwidth: 10 nm
- Linear OD range of spectrophotometer: upper limit 2.6

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: 1.895 +/- 0.072 (Acceptance criteria: 1.0-3.0) <=> Pass
- Barrier function: 4.85 hrs (Acceptance criteria: 4.77-8.72 hrs) <=> Pass
- Morphology: Appropiate formation of the epidermal barrier, presence of functional stratum corneum, viable basal cell layer, and intermediate spinous and granular layers
- Contamination: Sterile (Acceptance criteria: No contamination) <=> Pass
- Reproducibility: Coefficient of Variation between replicates was < 30%

NUMBER OF REPLICATE TISSUES: 2

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

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 50%, or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15%.
- The test substance is considered to be non-corrosive to skin if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 μL

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL of sterile distilled water was used as supplied

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL of 8.0 N Potassium Hydroxide was used as supplied

Duration of treatment / exposure:

Tissues were treated with the test item for exposure periods of 3 and 60 minutes.

Duration of post-treatment incubation (if applicable):

3 h with MTT

Number of replicates:

Duplicate tissues for test item, negative and positive controls

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minute exposure

Value:

114.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

4.9

Remarks on result:

no indication of irritation

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 minutes exposure

Value:

116.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

4.3

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: none
- Direct-MTT reduction: no
- Colour interference with MTT: no

DEMONSTRATION OF TECHNICAL PROFICIENCY:
All values for negative and positive controls were within the historical range (historical data for negative and positive controls obtained by the laboratory in the previous six months).

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD570 for the negative control treated tissues was 1.866 for the 3 Minute exposure period and 2.008 for the 60 Minute exposure period. The negative control acceptance criteria were therefore satisfied.
- Acceptance criteria met for positive control: The relative mean tissue viability for the positive control treated tissues was 4.3% relative to the negative control following the 60 Minute exposure period. The positive control acceptance criterion was therefore satisfied.
- Acceptance criteria met for variability between replicate measurements: In the range 20 to 100% viability the Coefficient of Variation between the two tissue replicates of each treatment group did not exceed 30%. The acceptance criterion was therefore satisfied .

Any other information on results incl. tables

Table 7.3.1/1: Mean OD570 Values and Viabilities for the Negative Control Item, Positive Control Item and Test Item

 

Tissue	Exposure Period	MeanOD570of individual tissues	Mean OD570of duplicate tissues	Standard Deviation	Coefficient of Variation (%)	Relative Mean Viability (%)
Negative Control	3 Minutes	1.908	1.866	0.059	3.2	100*
		1.824
	60 Minutes	1.946	2.008	0.087	4.3
		2.069
Positive Control	3 Minutes	0.102	0.091	0.016	na	4.9
		0.080
	60 Minutes	0.090	0.086	0.006	na	4.3
		0.082
Test Item	3 Minutes	2.254	2.135	0.169	7.9	114.4
		2.015
	60 Minutes	2.201	2.333	0.187	8.0	116.2
		2.465

OD=Optical density

*=         The mean percentage viability of the negative control tissue is set at 100%

na=        Not applicable

Relative mean viability (%) = (Mean OD570 of test item / Mean OD570 of negative control) x 100

Coefficient of Variation = (standard deviation / mean OD of duplicate tissues) x 100

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test item was considered to be non-corrosive to the skin, according to the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

Executive summary:

An in vitro skin corrosion study was performed according to the OECD Guideline 431 and in compliance with GLP, using the EPIDERM^TM reconstructed human epidermis model.

 

Duplicate tissues were treated with the test item for exposure periods of 3 and 60 minutes. Negative and positive control groups were treated for each exposure period. At the end of the exposure period the test item was rinsed from each tissue before each tissue was taken for MTT‑loading. After MTT-loading each tissue was placed in 2 mL of isopropanol for MTT extraction.

At the end of the formazan extraction period each well was mixed thoroughly and triplicate 200 mL samples were transferred to the appropriate wells of a pre-labeled 96‑well plate. The optical density (OD) was measured at 570 nm (OD₅₇₀).

 

The MTT solution containing the test item did not turn blue and indicate the test item did not reduce MTT. The solution containing the test item did not become colored and indicate the test item did not have the potential to cause color interference.

 

The relative mean viability of the test item treated tissues was 114.4 and 116.2% after 3 and 60 minutes exposure, respectively. The relative mean viability of the negative control treated tissues was set to 100 % after 3 and 60 minutes exposure. The relative mean viability of the positive control treated tissues was 4.9 and 4.3% after 3 and 60 minutes exposure, respectively.

 

The quality criteria required for acceptance of results in the test were satisfied.

 

The test item was considered to be non-corrosive to the skin, according to the Regulation (EC) No 1272/2008 (CLP) and to the GHS.