Reaction mass of 2-methylbutyl salicylate and pentyl salicylate

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was performed between 04 December 2012 and 10 December 2012

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:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection: 10-07-2012 Date of Signature: 13-02-2013

Test material

Test material information

Constituent 1

Reference substance name:

Pentyl salicylate

EC Number:

218-080-2

EC Name:

Pentyl salicylate

Cas Number:

2050-08-0

Molecular formula:

C12H16O3

IUPAC Name:

Pentyl 2-hydroxybenzoate

Constituent 2

Reference substance name:

2-methylbutyl salicylate

EC Number:

256-972-3

EC Name:

2-methylbutyl salicylate

Cas Number:

51115-63-0

Molecular formula:

C12H16O3

IUPAC Name:

2-methylbutyl 2-hydroxybenzoate

Test material form:

liquid

Specific details on test material used for the study:

Sponsor's identification : Amyl Salicylate
CAS number : 2050-08-0
Description : clear colourless liquid
Batch number : PE00022143
Purity : 99.99%
Date received : 03 October 2011
Expiry date : 19 August 2012
Storage conditions : room temperature in the dark

This study was scheduled to be conducted after the test item expiry date of 19 August 2012. The sponsor was therefore contacted prior to commencement of the study. The sponsor tested the test item in order to re-validate the quality of the batch with the conclusion that the quality of the batch is comparable to the specification given in the test item Technical Data Sheet originally supplied with the test item. This study therefore proceeded as scheduled.

In vitro test system

Test system:

human skin model

Remarks:

EPISKIN model

Source species:

human

Cell type:

other: EPISKINTM reconstructed human epidermis model

Cell source:

other: EPISKINTM reconstructed human epidermis model

Source strain:

other: EPISKINTM reconstructed human epidermis model

Justification for test system used:

Recommended model for OECD 439 studies.

Vehicle:

unchanged (no vehicle)

Details on test system:

TEST SITE
Area of exposure:
10 µl of the test item was applied to the epidermis surface.

PERCENTAGE COVERAGE:
The test item was applied topically to the corresponding tissues ensuring uniform covering.

EXPOSURE TIME:
15 Minutes post exposure.

TEST ITEM REMOVAL:
At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing DPBS with Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of DPBS to gently remove any residual test item.


SCORING SYSTEM
Classification of irritation potential is based upon relative tissue viability following the 15 minute exposure period followed by the 42 hour post-exposure incubation period according to the following:

Mean tissue viability is ≤50% : Irritant

Mean tissue viability is >50% : Non-Irritant

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

10 µl of the test item was applied to the epidermis surface. 

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

42 hour post exposure incubation

Number of replicates:

triplicates

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean triplicates Test item

Value:

82

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

8,1% viability

Remarks on result:

no indication of irritation

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Treatment with test item: 1st replicate

Value:

92.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Remarks:

OD540: 0.778

Results 3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Treatment with test item: 2nd replicate

Value:

89.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Remarks:

OD540: 0.753

Results 4

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Treatment with test item: 1st replicate

Value:

64.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Remarks:

OD540: 0.544

Any other information on results incl. tables

RESULTS

Direct MTT Reduction

The MTT solution containing the test item did not turn blue which indicated that the test item did not directly reduce MTT.

Test Item, Positive Control Item and Negative Control Item

The individual and mean OD₅₄₀ values, standard deviations and tissue viabilities for the test item, negative control item and positive control item are given in Table 1. The mean viabilities and standard deviations of the test item and positive control, relative to the negative control are also given in Table 1.The relative mean viability of the test item treated tissues was 82.0% after a 15-Minute exposure period.

Quality Criteria

The relative mean tissue viability for the positive control treated tissues was 8.1% relative to the negative control treated tissues and the standard deviation value of the percentage viability was 1.2%. The positive control acceptance criterion was therefore satisfied.

The mean OD₅₄₀ for the negative control treated tissues was 0.844 and the standard deviation value of the percentage viability was 13.9%. The negative control acceptance criterion was therefore satisfied.

The standard deviation calculated from individual percentage tissue viabilities of the three identically treated tissues was 15.2%. The test item acceptance criterion was therefore satisfied.

Table1 : Mean OD₅₄₀ Values and Percentage Viabilities for the Negative Control Item, Positive Control Item and Test Item

Item	OD540 of tissues	Mean OD540 of triplicate tissues	±SDof OD540	Relative individual tissue viability (%)	Relative mean viability (%)	± SD of Relative mean viability (%)
Negative Control Item	0.861	0.844	0.117	102.0	100*	13.9
	0.719			85.2
	0.951			112.7
Positive Control Item	0.062	0.069	0.010	7.3	8.1	1.2
	0.080			9.5
	0.064			7.6
Test Item	0.778	0.692	0.128	92.2	82.0	15.2
	0.753			89.2
	0.544			64.5

SD=   Standard deviation

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

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test item was considered to be Non-Irritant (NI).

Executive summary:

Introduction: The purpose of this test was to evaluate the skin irritation potential of the test item using the EPISKIN^TM reconstructed human epidermis model after a treatment period of 15 minutes followed by a post-exposure incubation period of 42 hours. The principle of the assay was based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test item by means of the colourimetric MTT reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan salt (within the mitochondria of viable cells) in the test item treated tissues relative to the negative controls. The concentration of the inflammatory mediator IL-1α in the culture medium retained following the 42-Hour post-exposure incubation period is also determined for test items which are found to be borderline non-irritant based upon the MTT reduction endpoint. This complimentary end‑point will be used to either confirm a non-irritant result or will be used to override the non-irritant result. This method was designed to be compatible with the following:

OECD Guidelines for the Testing of Chemicals No. 439 “In Vitro Skin Irritation” (adopted 22 July 2010)

Method B.46 of Commission Regulation (EC) No. 440/2008/EC

Method: Triplicate tissues were treated with the test item for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 hours. At the end of the post-exposure incubation period each tissue was taken for MTT-loading. The maintenance medium from beneath each tissue was transferred to pre-labelled micro tubes and stored in a freezer for possible inflammatory mediator determination. After MTT loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT-loaded tissues. 

At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 µl samples were transferred to the appropriate wells of a pre-labelled 96-well plate. The optical density was measured at 540 nm.

Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

Results: The relative mean viability of the test item treated tissues was 82.0% after the 15-Minute exposure period.

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

Conclusion: The test item was considered to be Non-Irritant (NI).