Reaction mass of 1,2,3,4,4a,5,6,7-octahydro-2,5,5-trimethyl-2-naphthol and 4-(2,6,6-trimethyl-1-cyclohexen-1-yl)butan-2-one and 4-(2,6,6-trimethyl-2-cyclohexen-1-yl)butan-2-one

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 September 2017 - 25 September 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:

28 July 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:

Amended by EC No. 640/2012 OJ No. L193, 20 July 2012.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

dd. 03 November 2015

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of 1,2,3,4,4a,5,6,7-octahydro-2,5,5-trimethyl-2-naphthol and 4-(2,6,6-trimethyl-1-cyclohexen-1-yl)butan-2-one and 4-(2,6,6-trimethyl-2-cyclohexen-1-yl)butan-2-one

EC Number:

915-318-3

Cas Number:

41199-19-3, 17283-81-7 and 31499-72-6

IUPAC Name:

Reaction mass of 1,2,3,4,4a,5,6,7-octahydro-2,5,5-trimethyl-2-naphthol and 4-(2,6,6-trimethyl-1-cyclohexen-1-yl)butan-2-one and 4-(2,6,6-trimethyl-2-cyclohexen-1-yl)butan-2-one

Test material form:

liquid

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

skin obtained from plastic surgery from multiple donors

Justification for test system used:

In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimise the need of in vivo testing. One of the validated in vitro skin irritation tests is the EPISKIN test, which is recommended in international guidelines (e.g. OECD and EC).

Vehicle:

unchanged (no vehicle)

Details on test system:

TEST SYSTEM
- EPISKIN Small Model (TM) (EPISKIN-SM (TM), 0.38 cm^2, Batch no.: 17-EKIN-038); a three-dimensional human epidermis model, which consists of adult human-derived epidermal keratinocytes which have been seeded in 12-well plates on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen and cultured for 13 days.

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Histology scoring: 21.0 ± 1.2 (CV=5.6%)
- IC50: 2.2 mg/mL
- Expiration date: 25 September 2017

SKIN DISC PREPARATION
- Procedure used: On the day of receipt the tissues were transferred to 12-well plates and preincubated with prewarmed Maintenance Medium for 23 hours at 37°C

ENVIRONMENTAL CONDITIONS (set to maintain)
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation: 37.0 ± 1.0°C (actual: 36.3-46.4°C)
- Humidity: 80 - 100% (actual: 34-94%)

REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: 1, with phosphate buffered saline
- Observable damage in the tissue due to washing: no

- Interference with the MTT endpoint was tested before the study by adding 10 μL of the test item to 90 μL of MTT solution (test for color interference) and by adding 25 μL of the test item to 2 mL MTT solution (0.3 mg/mL in PBS).

DYE BINDING METHOD
- Dye used in the dye-binding assay: MTT (0.3 mg/mL in PBS)
- Incubation time: 3 hours
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader.
- Wavelength: 570 nm

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to skin if the relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours of post incubation is ≤ 50% of the mean viability of the negative controls.
- The test substance is considered to be non-irritant to skin if the relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours of post incubation is > 50% of the mean viability of the negative controls.

SCORING SYSTEM:
- Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment. Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues. Skin irritation is expressed as the remaining cell viability after exposure to the test item.

ACCEPTABILITY CRITERIA:
a)  The absolute mean OD570 (optical density at 570 nm) of the three tissues of the negative control should reasonably be within the laboratory historical control data range and the Standard Deviation value (SD) of the % viability should be ≤18.
b) The mean relative tissue viability of the positive control should be ≤40% relative to the negative control and the Standard Deviation value (SD) of the % viability should be ≤18.
c) The SD calculated from individual % tissue viabilities of the three identically treated replicates should be ≤18.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST ITEM:
- Amount applied: 25 µL

NEGATIVE CONTOL:
- Amount applied: 25 µL Phosphate buffered saline

POSITIVE CONTROL
- Amount applied: 25 µl
- Concentration: 5% (aq) Sodium dodecyl sulphate
- Re-spread after 7 minutes contact time

Duration of treatment / exposure:

15 ±0.5 minutes

Duration of post-treatment incubation (if applicable):

42 hours and 3 hours with MTT

Number of replicates:

3

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Remarks:

Mean of 3 replicates

Value:

19

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

Mean tissue viability: 5.4%

Other effects / acceptance of results:

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

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, the absolute mean OD570 of the three tissues of the negative control were within the laboratory historical control data range (i.e. 1.201 ± 0.180) and the SD of the % viability was <18% (i.e. 7.2%)
- Acceptance criteria met for positive control: yes, the mean relative tissue viability of the positive control was <50% (i.e. 5.4%) and the SD of the % viability was <18% (i.e. 1.4%).
- Acceptance criteria met for variability between replicate measurements: yes, the SD calculated from individual % tissue viabilities of the three identically treated replicates was <18% (i.e. 15%).

- Since the mean relative tissue viability for the test item was below 50% the test item is considered to be irritant.

Any other information on results incl. tables

Table 2 Individual OD measurements (570 nm)

	A (OD570)	B (OD570)	C (OD570)
Negative control OD570measurement 1 OD570measurement 2	1.3365 1.3054	1.1564 1.1426	1.2816 1.2394
POLYAMBROL OD570measurement 1 OD570measurement 2	0.1277 0.1251	0.4796 0.4644	0.2158 0.2052
Positive control OD570measurement 1 OD570measurement 2	0.1289 0.1202	0.1018 0.1044	0.0947 0.0907

OD = Optical density

Triplicate exposures are indicated by A, B and C.

Table 3 Historical data for skin irritation studies

	Negative control (absorption; OD570)	Positive control (absorption; OD570)
Range	0.676 – 1.336	0.036 – 0.549
Mean	1.01	0.16
SD	0.16	0.10
n	155	154

SD = Standard deviation

n = Number of observations

The above mentioned historical control data range of the controls were obtained by collecting all data over the period of November 2013 to November 2016.

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Remarks:

Additional information on corrosion is needed for classification.

Conclusions:

In an in vitro skin irritation study, performed according to OECD guideline 439 and GLP principles, Polyambrol was found to be irritant to the skin (mean tissue viability of 19%).