Registration Dossier

Administrative data

Description of key information

Skin irritation: Non irritating to skin in vitro (Episkin method)
Eye irritation: Not corrosive to eyes or a severe irritant to eyes in vitro (BCOP); Not a mild nor a moderate irritant to rabbits eye based on read across to structural analogue, Cyclohexyl salicylate

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
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
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Date of inspection: 10-07-2012 Date of Signature: 13-02-2013
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.
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
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
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
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
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

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%

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 EPISKINTM 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).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was performed on 11 December 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Date of Inspection : 10 July 2012 Date of Signature : 05 February 2013
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.

Species:
cattle
Strain:
other: bovine cornea from slaughterhouse
Details on test animals or tissues and environmental conditions:
Not applicable
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST ITEM

-Amounts(s) applied (volume or weight with unit):
0.75 mL of the test item was applied to triplicate corneas.

-Concentration (if solution):
The test item was used as supplied.

VEHICLE
No vehicle used
Duration of treatment / exposure:
The undiluted test item was applied for 10 minutes followed by an incubation period of 120 minutes.
Duration of post- treatment incubation (in vitro):
Post-treatment incubation period of 120 minutes.
Number of animals or in vitro replicates:
triplicates for test item, positive and negative control.
Details on study design:
TEST SITE
-Area of exposure
0.75 mL of the test item was applied to triplicate corneas.

-% coverage:
The test item was topically applied to the cornea. The holders were gently tilted back and forth to ensure a uniform application of the item over the entire cornea.

-Type of wrap used:
None used

REMOVAL OF TEST ITEM
-Washing (if done):
At the end of the exposure period the test item was removed from the anterior chamber and each cornea was rinsed three times with fresh complete MEM containing phenol red before a final rinse with complete MEM without phenaol red.

-Time after start of exposure:
10 minutes post exposure

EVALUATION OF RESULTS
Results from the two test method endpoints, opacity and permeability, were combined in an empirically derived formula to generate an In Vitro Irritancy Score.

Opacity Measurement-
The change in opacity for each cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final opacity reading. These values were then corrected by subtracting from each the average change in opacity observed for the negative control corneas. The mean opacity value of each treatment group was then calculated by averaging the corrected opacity values of each cornea for that treatment group.

Permeability Measurement
The corrected OD492 was calculated by subtracting the mean OD492 of the negative control corneas from the OD492 value of each treated cornea. The OD492 value of each treatment group was calculated by averaging the corrected OD492 values of the treated corneas for the treatment group.

In Vitro Irritancy Score
The following formula was used to determine the in vitro score:
In Vitro Irritancy Score = mean opacity value + (15 x mean OD492 value)
Additionally, the opacity and permeability values were evaluated independently to determine whether the test item induced a response through only one of the two endpoints.

Visual Observation
The condition of the cornea was visually assessed immediately after rinsing and at the final opacity measurement.

DATA INTERPRETATION
A test item that induces an in vitro irritancy score greater than or equal to 55.1 is defined as an ocular corrosive or severe irritant.



Irritation parameter:
in vitro irritation score
Run / experiment:
test item
Value:
2.7
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
The test item induced an in vitro irritancy score of 2.7.
The corneas treated with the test item were clear post treatment and post incubation.

RESULTS

Corneal Opacity and Permeability Measurement

Individual and mean corneal opacity measurements and individual and mean corneal permeability measurements are given in Table 1.

Corneal Epithelium Condition

The condition of the cornea immediately after rinsing and at the final opacity measurement is given in Table 2.

The corneas treated with the test item were clear post treatment and post incubation. The corneas treated with the negative control item were clear post treatment and post incubation. The corneas treated with the positive control item were cloudy post treatment and post incubation.

In Vitro Irritancy Score

The results are summarised as follows:

Treatment

In Vitro Irritancy Score

Test Item

2.7

Negative Control

2.7

Positive Control

36.1

Criteria for an Acceptable Test

The positive control In Vitro irritancy Score was within the range of 30.9 to 67.7. The positive control acceptance criterion was therefore satisfied.

Table 1          Individual and Mean Corneal Opacity and Permeability Measurements

Treatment

Cornea Number

Opacity

Permeability (OD)

In vitroIrritancy Score

Pre-Treatment

Post-Treatment

Post-Incubation

Post-Incubation-Pre‑Treatment

Corrected Value

 

Corrected Value

Negative Control

1

4

5

8

4

 

0.051

 

 

2

3

2

4

1

 

0.046

 

 

3

2

2

3

1

 

0.033

 

 

 

 

 

 

2.0*

 

0.043+

 

2.7

Positive Control

4

2

31

27

25

23.0

1.155

1.112

 

5

2

30

25

25

23.0

0.872

0.829

 

6

1

26

24

24

22.0

0.792

0.749

 

 

 

 

 

 

22.7·

 

0.896·

36.1

Test Item

7

2

5

7

5

3.0

0.019

0.0

 

8

1

4

6

5

3.0

0.020

0.0

 

9

2

5

6

4

2.0

0.034

0.0

 

 

 

 

 

 

2.7·

 

0.0·

2.7


OD = Optical density  * = Mean of the post incubation-pre‑treatment values  + = Mean permeability       · = Mean corrected value


Table 2          Corneal Epithelium Condition

Treatment

Cornea Number

Observation

Post Treatment

Post Incubation

Negative Control

1

clear

clear

2

clear

clear

3

clear

clear

Positive Control

4

cloudy

cloudy

5

cloudy

cloudy

6

cloudy

cloudy

Test Item

7

clear

clear

8

clear

clear

9

clear

clear


Interpretation of results:
GHS criteria not met
Conclusions:
The test item was considered not to be an ocular corrosive or severe irritant.
Executive summary:

Introduction. A study was performed to assess the ocular irritancy potential of the test item to the isolated bovine cornea. The method was designed to be compatible with the following:

OECD Guidelines for the Testing of Chemicals No. 437 (2009) “Bovine Corneal Opacity and Permeability Assay”

Method. The undiluted test item was applied for 10 minutes followed by an incubation period of 120 minutes. Negative and positive control items were tested concurrently. The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined in an empirically derived formula to generate an In Vitro Irritancy Score (IVIS). 

Results. The in vitro Irritancy scores are summarised as follows:

Treatment

In Vitro Irritancy Score

Test Item

2.7

Negative Control

2.7

Positive Control

36.1

Conclusion. The test item was considered not to be an ocular corrosive or severe irritant.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation:

OECD 439, GLP Study.

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).

The relative mean viability of the test item treated tissues was82.0% after the 15-Minute exposure period.

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

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

Eye irritation:

OECD 437, GLP study

The undiluted test item was applied for 10 minutes followed by an incubation period of 120 minutes. Negative and positive control items were tested concurrently. The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined in an empirically derived formula to generate an In Vitro Irritancy Score (IVIS).

The in vitro Irritancy score (IVIS) of the test item was found to be 2.7.

Conclusion: The test item was considered not to be an ocular corrosive or severe irritant.

Read across justification:

To address toxicological endpoints as part of the REACH registration of the REACH registration of Amyl Salicylate (Target Substance) it is proposed to read-across to Cyclohexyl Salicylate (Source Substance).

The use of read-across works within the spirit of REACH and the stated aim of the legislation to reduce animal testing where possible.

The Target Substance and Source Substance have been characterised using the categories and databases present in the OECD [Q]SAR Toolbox. From the profiling, it can be seen that the two substances share structural similarities and also ‘mechanistic action’ similarities which are both general and endpoint specific.

For classification purposes this demonstrates that read across is valid and Amyl salicylate is not classified in accordance with CLP based on the Source substance, Cyclohexyl salicylate also not being classified.

Justification for classification or non-classification

The test substance was assessed for skin and eye irritation. There was no potential for skin irritation (Episkin method) within the framework of Regulation (EC) No. 1272/2008 (CLP) or in accordance with Regulation 67/548/EEC.

Furthermore, the substance is not corrosive to eyes or a severe irritant to eyes in an in vitro (BCOP) where the IVIS score was below 3. As such, no classification in accordance with Regulation (EC) No. 1272/2008 (CLP) or in accordance with Regulation 67/548/EEC is required.