3,3,5-trimethylcyclohexyl acrylate

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 November 2015 -- 22 December 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: EU Method B.46 (In vitro dermal irritation)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 439 (In vitro dermal irritation)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

3,3,5-trimethylcyclohexyl acrylate

EC Number:

289-200-9

EC Name:

3,3,5-trimethylcyclohexyl acrylate

Cas Number:

86178-38-3

Molecular formula:

C12H20O2

IUPAC Name:

3,3,5-trimethylcyclohexyl prop-2-enoate

Test material form:

liquid

In vitro test system

Test system:

human skin model

Source species:

human

Details on animal used as source of test system:

EpiskinTM Model Kit (0.38 cm2 tissues) supplied by SkinEthic Laboratories, Lyon, France.
Medium and Incubation T°C: 37°C

Vehicle:

unchanged (no vehicle)

Details on test system:

REMOVAL OF TEST SUBSTANCE
- Rinsing: at the end of the treatment period, each tissue was removed from the well of the treatment plate, and rinsed with D-PBS. Rinsing was achieved by gently filling and emptying several times each tissue with D PBS to gently remove any residual test or control items. The rinsed tissues were transferred to the second column of 3 wells containing 2 mL of maintenance medium in each well and the plates were incubated at +37°C, 5% CO2 in a humidified incubator for 42 hours.

POSITIVE CONTROL
Name: Sodium Dodecyl Sulphate (SDS) at a 5% (w/v) aqueous solution.

NEGATIVE CONTROL
Name: Dulbecco’s Phosphate-Buffered Saline (D-PBS).

SCORING SYSTEM:
- Optical density (OD) was measured at 570 nm:
Relative mean viability (%) = 100 x mean cOD(test item) / mean cOD(negative control)
where:
- mean cOD Negative Control = mean ODNC – mean ODblank
- mean cOD Test Item = mean ODTI – mean ODblank

Interpretation: see below

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

Amount per tissue: 10 µL

Duration of treatment / exposure:

Exposure period of 15 minutes, followed by rinsing
MTT-loading after a 42h-incubation period. Observation of MTT-> formazan transformation by viable cells.

Number of replicates:

Triplicate tissues for each tested substance (test item, negative control, positive control).

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

treated

Value:

86

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

In the preliminary tests, the test item was found not to have direct MTT reducing properties or colouring potential.
All acceptance criteria for the negative and positive controls were fulfilled. The study was therefore considered to be valid.
Following a 15 minutes exposure and 42 hours of recovery period, the relative mean viability of the tissues treated with the test item was 86% with a Standard Deviation of 22%. Despite a Standard Deviation higher than 18% was noted, all viability data of test item-treated tissues were above the cut-off of 50%, and the
determination of IL-1a concentrations was used to consolidate the trend for a non-irritant response.
Therefore, the IL-1a concentrations in culture media samples retained from the three negative controls and the three test item-treated tissues were analyzed by ELISA. The IL-1a concentration value of one tissue was found below the Limit Of Quantification (< 5.00 pg/mL). Consequently, the mean IL-1a concentration
from the three test item-treated tissues was not calculated. The IL-1a concentration values of the two other test item-treated tissues were found < 60 pg/mL (39.1 and 6.30 pg/mL).
Therefore, the results met the criteria for an in vitro classification as non-irritant to skin.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test item is considered to be non-irritant to skin.

Executive summary:

The objective of this study was to evaluate the skin irritation potential of the test item, using the EpiskinTM reconstructed human epidermis model.

The study design was based upon international guidelines (OECD Guideline No. 439 and Commission Regulation (EC) No. 761/2009, B.46). The study was conducted in compliance with CiToxLAB France standard operating procedures and the principles of Good Laboratory Practice.

 

Methods

 

Preliminary tests were performed to detect the ability of the test item to directly reduce MTT as well as its colouring potential.

Following the preliminary tests, the skin irritation potential of the test item was tested in the main test. The test item and both the negative and positive controls were applied topically on triplicate tissues and incubated at room temperature for 15 minutes. At the end of the treatment period, each tissue was rinsed with D-PBS and incubated for 42 hours at +37°C, 5% CO₂ in a humidified incubator. The cell viability was then assessed by means of the colourimetric MTT reduction assay.

Relative viability values were calculated for each tissue and expressed as a percentage of the mean viability of the negative control tissues which was set at 100% (reference viability).

In addition, the concentration of the inflammatory mediator IL-1a was evaluated in the culture medium retained following the 42-hour recovery period. This quantification, based on an ELISA assay, was performed since the mean relative viability of the test item-treated tissues was > 50% following the MTT reduction assay.

 

Results

 

Preliminary tests

In the preliminary tests, the test item was found not to have direct MTT reducing properties or colouring potential.

 

Main test

All acceptance criteria for the negative and positive controls were fulfilled. The study was therefore considered to be valid.

Following a 15 minutes exposure and 42 hours of recovery period, the relative mean viability of the tissues treated with the test item was 86% with a Standard Deviation of 22%. Despite a Standard Deviation higher than 18% was noted, all viability data of test item-treated tissues were above the cut-off of 50%, and the determination of IL-1a concentrations was used to consolidate the trend for a non-irritant response.

Therefore, the IL-1a concentrations in culture media samples retained from the three negative controls and the three test item-treated tissues were analyzed by ELISA. The IL-1a concentration value of one tissue was found below the Limit Of Quantification (< 5.00 pg/mL). Consequently, the mean IL-1a concentration from the three test item-treated tissues was not calculated. The IL-1a concentration values of the two other test item-treated tissues were found < 60 pg/mL (39.1 and 6.30 pg/mL).

Therefore, the results met the criteria for an in vitro classification as non-irritant to skin.

Conclusion

The test item is considered to be non-irritant to skin.