3-methyl-1,5-pentanediyl diacrylate

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 April 2013 - 29 April 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

see below

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 439 (In vitro dermal irritation)

Deviations:

no

Principles of method if other than guideline:

The study was performed in accordance with study plan with the following deviations from the agreed study plan:
. § Test for direct MTT reduction with the test item: due to a typing error in the study plan, the volume of MTT solution used for the negative control in the preliminary test for direct MTT reduction which is specified in the study plan is incorrect. Indeed, the volume of MTT solution used in this study was 2 mL (as described in CiToxLAB’s SOP) instead of 2.2 mL. This deviation was considered not to have compromised the validity or integrity of the study,

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

3-methyl-1,5-pentanediyl diacrylate

EC Number:

264-727-7

EC Name:

3-methyl-1,5-pentanediyl diacrylate

Cas Number:

64194-22-5

Molecular formula:

C12H18O4

IUPAC Name:

3-methyl-5-(prop-2-enoyloxy)pentyl prop-2-enoate

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

other: reconstructed human epidermis

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
Dates of experimental phase: from 23 April 2013 to 29 April 2013

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. Excess D-PBS was removed by blotting the bottom of the tissue culture insert with absorbent paper.
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 (± 1) hours.

POSITIVE CONTROL
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 no treatment

yes, concurrent positive control

Amount/concentration applied:

Amount applied per tissue: 10 ± 2 µL

Duration of treatment / exposure:

Exposure period of 15 minutes, followed by a rinsing

Duration of post-treatment incubation (if applicable):

MTT-loading after a 42h-incubation period following rinsing. Observation of MTT-> formazan transformation by viable cells, after a 3-hour MTT incubation

Number of replicates:

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

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

15 minute exposure (treated)

Value:

6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Preliminary tests
In the preliminary tests, the test item was found not to have direct MTT reducing properties or a 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 a 42-hour recovery period, the relative mean viability of the tissues treated with the test item was 6% with a standard deviation of 1%. As the mean viability was < 50% after the MTT reduction, the results met the criteria for an in vitro classification as irritant (R38) to skin.

Applicant's summary and conclusion

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

Under the experimental conditions of this study, the test item is considered to be irritant to skin.

Executive summary:

The objective of this study was to evaluate the skin irritation potential of the test item using the Episkin^TMreconstructed 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’s 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 topically applied on triplicate tissues and incubated at room temperature for 15 (± 1) minutes. At the end of the treatment period, each tissue was rinsed with D-PBS and incubated for 42 (± 1) hours at, 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).

 

Results

 

Preliminary tests

In the preliminary tests, the test item was found not to have direct MTT reducing properties or a 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 a 42-hour recovery period, the relative mean viability of the tissues treated with the test item was 6% with a standard deviation of 1%. As the mean viability was < 50% after the MTT reduction, the results met the criteria for an in vitro classification as irritant (R38) to skin.

 

Conclusion

 

Under the experimental conditions of this study, the test item is considered to be irritant to skin.

 

According to the results of this study, the classification of the test item should be:

- irritant (R38) (Directive 67/548/EEC) and category 2 (H315) (Regulation (EC) No. 1272/2008).