Registration Dossier

Diss Factsheets

Administrative data

Description of key information

The results of the skin irritation test according to OECD 439 together with the results of the skin corrosion test according to OECD 431 with the test item 2 -hexyldecanoic acid show that the test item has to be classified in Category 2 “Irritating to skin” but does not have to be classified in Category 1 “Corrosive”.


The result obtained in the in vitro eye irritation study according to OECD 438 in the Isolated Chicken Eye test method lead to the category "no prediction can be made". Therefore it was required to conduct an in vivo eye irritation test according to OECD 405. This experiment showed that the test substance 2 -hexyldecanoic acid is not irritating to the eyes.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19. - 21.April 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP compliant
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:
Certificate of Compliance with Good Laboratory Practices according to Directives 2004/9/CE and 2004/10/CE, Groupe Interministeriel des Produits Chimiques, Republique Francaise, Certificat n°: 2017/33, dated 27 April 2017
Specific details on test material used for the study:
- Lot/batch No.of test material: 05297/MA
- Expiration date of the lot/batch: Retest date October 2021
- Storage condition of test material: room temperature
Test system:
human skin model
Source species:
human
Cell type:
other: reconstructed epidermis of normal human keratinocytes
Cell source:
foreskin from a single donor
Vehicle:
unchanged (no vehicle)
Details on test system:
Human skin model
The 0.50 cm² reconstructed epidermises (Episkin SA, RHE/S/17 Batch No. 19-RHE-041) were received on 19 March 2019. The same day, the insert (filter + epidermis) was gently removed from the agarose while avoiding leaving agarose on the polycarbonate filter. The inserts were placed in 6 wells culture plate which had been previously filled with 300 µL of growth medium (Episkin SA, batch No. 19 SGM 031) during 2 hours and 20 minutes. Then just before treatment, the inserts were placed in 24 wells culture plate which had been previously filled with 300 µL of maintenance medium (Episkin SA, batch No. 19 SMM 011).

Technical Data, safety sheet and certificate of analysis:
SkinEthic RHE/Human Epidermis (RHE/S/17): 0.5 cm² reconstructed epidermis for normal human keratinocytes. Cells are grown on inert polycarbonate filters in chemically defined medium, for 17 days.
Origin: Foreskin
The product was prepared and packaged using aseptic techniques. Store in an incubator at 37 °C, 5 % CO2 with saturated humidity.
Histology: HES stained paraffin section: Multi-layered, highly differentiated epidermis consisting of organized basal, spinous and granular layers, and a multilayered stratum corneum.
6.5 cell layers
cell viability: 570 nm optical density, MTT test OD > 1.5 (CV = 1.3 %)
barrier function integrity test: exposure time inducing 50 % viability using Triton X-100 1 %: 4 <= ET50 <= 10 h: 6.6 h
biological safety: on blood of the donors was verified the absence of HIV1 and 2 antibodies, hepatitis C antibodies and hepatitis B antigen HBs. On cells from the donors were verified the absence of bacteria, fungia and mycoplasma.
suggested expiration date: March 25, 2019
certified and released by Michel Bataillon, Quality Control Manager
Manufactured in accordance to the ISO9001 quality system of Episkin
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied: 16 μL
- Concentration: undiluted, used as supplied

NEGATIVE CONTROL
- Amount(s) applied: 16 µL

POSITIVE CONTROL
- Amount(s) applied: 16 µL
- Concentration (if solution): 5% w/v
Duration of treatment / exposure:
42 minutes
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
3
Details on study design:
Treatment
The test item was applied as supplied, at the dose of 16 µL to the epidermal surface of 3 living human skin models during 42 minutes at room temperature. To ensure a good contact with the epidermises, during all the treatment period, the test item was recovered with a nylon mesh provided by Episkin SA.

In the same experimental conditions, a positive control (16 µL of 5% sodium dodecyl sulfate - SDS) and a negative control (16 µL of distilled water – ADL Prochilab - Batch No. 180517) were carried out. The 5% SDS solution was prepared by weighing 0.5 g of SDS (SIGMA Batch No. STBG6142V) in a 10 mL volumetric flask q.s. 10 mL of distilled water. Then, the preparation was magnetically stirred, just before the treatment. To ensure a good contact with the epidermises, during all the treatment period, the control items were recovered with a nylon mesh provided by Episkin SA.

Grading of reactions
42 minutes after the test item application, the nylon mesh was removed and the human epidermises were washed with 25 x 1 mL of DPBS (Dutscher - Batch No. 8041018). The rinsed tissues were checked for any coloration and noted to be whitish, comparable coloration to that of the negative control tissues. They were incubated for a 42 hours post-treatment incubation period in fresh medium at 37°C, 5% CO2. Then, the epidermises were put in contact with the MTT solution.

The cell viability was quantified by measurement of the cell succinate dehydrogenase activity. This enzyme is responsible for the MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue; CAS No. 298-93-1)] reduction into blue formazan crystal that is quantitatively measured by Optical Density (OD) after extraction from tissues. The measured OD are proportional to the number of living cells. The skin samples were placed in 300 µL of a MTT solution at 1.0 mg/mL for 3 hours and 02 minutes at 37°C, 5% CO2. The precipitated blue formazan product was then extracted using isopropanol during 2 hours and 01 minute under gentle agitation in the dark, and the concentration of formazan was determined by measuring the OD at 570 nm, just after dilution of the extracts 1:2 in isopropanol. The OD was measured in triplicate of MTT extract.

The measurement of OD at 570 nm of formazan extracts was performed in triplicate samples using the ELx800 absorbance microplate reader (controlled every year and calibrated if necessary) supplied by BioTek and the validated software Gen5 ELISA V1.05.11 supplied by BioTek.

DATA EVALUATION
Viability (%): (mean OD test item / OD negative control) x100
The OD values obtained for each test item were used to calculate a percentage of viability relative to the negative control, which was arbitrarely set to 100%.

ACCEPTABILITY
- SD (standard deviation) <= 18%
- Negative control: OD value of the 3 replicates in the range >= 0.8 and <= 3.0, the optical density was measured after a 1:2 dilution of the formazan extracts in isopropanol; the acceptability criteria should be in the range >=0.4 and <= 1.5 for the negative control.
Irritation / corrosion parameter:
% tissue viability
Value:
3.3
Negative controls validity:
valid
Positive controls validity:
valid
Remarks:
1.3 %
Remarks on result:
positive indication of irritation
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: none, the rinsed tissues were checked for any coloration and noted to be whitish, comparable coloration to that of the negative control tissues
- Direct-MTT reduction:
The direct interaction of MTT with the test item was checked by adding 16 µl of the test item to 300 µl of the solution of MTT at 1 mg/ml (same condition as in the main test). A yellow solution was observed after 3 hours of incubation at approx. 37°C. Therefore, there is no direct interaction between the test item and MTT.
- Colour interference with MTT:
The coloration potential of the test item in water was checked by adding 16 µl of the test item to 300 µl of distilled water. A colourless solution was obtained after 3 hours of incubation at approx. 37°C. The coloration potential of the test item in isopropanol was checked by adding 16 µl of the test item to 1.5 ml of isopropanol. A colourless solution was obtained after 2 hours of incubation at room temperature. Therefore the test item will not interfere with the MTT assay and there is no need to add non-specific coloration controls to the study.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, the mean viability was 100 %, the mean OD for the negative control treated tissues was 0.909 and the standard deviation value of the viability was 12.6.
- Acceptance criteria met for positive control: yes, relative mean tissue viability for the positive control treated tissues was 1.3%, the mean OD was 0.012 and the standard deviation value of the viability was 0.1.
- Acceptance criteria met for variability between replicate measurements: yes, standard deviation calculated from individual tissue viabilities of test item treated tissues was 0.2.

Table 1: Individual and average values of OD after 42 minutes exposure

   Well ID  OD  Mean OD/ disc *  Mean OD/product  Viability (%)

Mean viability (%)**

 SD viability   Conclusion
Negative control  SPL1  0.992 / 1.031 / 1.022 1.015   0.909 111.6   100.0 12.6   -
  SPL 2  0.907 / 0.934 / 0.938 0.926  

 101.8

 

 

 

 

SPL 3 

0.783 / 0.785 / 0.793 

0.787

 

 86.5

 

 

 

Positive control 

SPL 4 

0.011 / 0.012 / 0.012 

 0.011

 0.012

 1.2

1.3

 0.1

 irritant

 

SPL 5

 0.014 / 0.012 / 0.012

 0.012

 

 1.3

 

 

 

 

SPL 6

0.013 / 0.014 / 0.014

 0.013

 

 1.4

 

 

 

Test item 

SPL 10

0.030/ 0.029 / 0.028

 0.029

 0.030

3.2

 3.3

 0.2.3 Irritant or corrosive
 

 SPL 11

 0.028 / 0.029/ 0.027

 0.028

 

3.1 

 

 

 

 

SPL 12

 0.033 / 0.032 / 0.032

 0.032

 

3.5

 

 

 

 * = mean of 3 values (triplicate of the same extract)

OD: optical density

SPL: sample

SD: standard deviation

Interpretation of results:
other: additional in vitro corrosive test required to interprete the results
Conclusions:
The mean percent viablity of the treated tissues was 3.3 %, versus 1.3% of the positive control (5% Sodium Dodecyl Sulfate). The test item has to be classified in Category 2 “Irritating to skin” or in Category 1 “Corrosive”.
Executive summary:

The aim was to evaluate the possible irritating effects of test item 2-hexyldecanoic acid after topical application on in vitro human reconstructed epidermis (SkinEthic RHE® model).

The test item 2 -hexyldecanoic acid was applied as supplied, at the dose of 16 µL, to 3 living Reconstructed Human epidermis (SkinEthic RHE® model) during 42 minutes. The application was followed by a rinse with 25 mL of DPBS and a 42 hours and 00 minutes postincubation period at 37°C, 5% CO2. Cell viability was then measured by enzymatic conversion of the vital dye MTT into a blue formazan salt that was quantitatively measured after extraction from tissues.  The experimental protocol was established in accordance with O.E.C.D. Test Guideline No. 439 adopted 28 July 2015 and the Test method B.46 of Council regulation No. 761/2009 dated 23 July 2009 (EU Journal L220) - ATP Council regulation No. 440/2008 of 30 May 2008 (E.U. Journal L142).

The mean percent viability of the treated tissues was 3.3, versus 1.3 % in the positive control (5% Sodium Dodecyl Sulfate).  

In accordance with the Regulation EC No. 1272/2008 and in absence of information on skin corrosion, the test item 2 -hexyldecanoic acid has to be classified in Category 2 “Irritating to skin” or in Category 1 “Corrosive”.

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP compliant
Qualifier:
according to guideline
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Certificate of Compliance with Good Laboratory Practices according to Directives 2004/9/CE and 2004/10/CE, Groupe Interministeriel des Produits Chimiques, Republique Francaise, Certificat n°: 2017/33, dated 27 April 2017
Specific details on test material used for the study:
- Lot/batch No.of test material: 05297/MA
- Expiration date of the lot/batch: Retest date October 2021
- Storage condition of test material: room temperature
Test system:
human skin model
Source species:
human
Cell type:
other: reconstituted epidermis
Source strain:
not specified
Details on test system:
Human skin model
The 0.6 cm² reconstituted epidermis (epiCS, Cell Systems – Batch No.100-AI0836-1) were received on 15 May 2019. The insert (filter + epidermis) was gently removed from the agarose while avoiding leaving agarose on the polycarbonate filter. The insert was placed in a 6 wells culture plate which had been previously filled with 1 mL of culture medium (Cell Systems Batch No. 305-AI1018). The culture dishes were incubated at 37±2°C, 5% CO2 during 21 hours and 55 minutes before treatment. Just before the treatment, the culture medium was replaced by a new culture medium (Cell Systems, Batch No. 305-AI1018).

Certificate of Analysis epiCS Reconstructed Human Epidermis (SkinInvitro GmbH, Troisdorf, 14.5.2019)
Production desription: epiCS is a 3-D, fully differentiated human epidermis equivalent, reconstructed from normal human primary epidermal keratinocates
Product number: CS-1001
Lot number: 100-AI0836-1
Date of manufacture: 13.05.2019
Insert size: 0.6 cm²

Biological contaminants of primary cells
HIV1, HBV, HCV: negative
bacteria, yeast, mycoplasma: negative

histology:
No cornified layers: 5
No vital cell layers: 4

Barrier function
Target Result
ET-50 < 2-5h > 4 h 06 minutes
Viability 2h Triton X-100: 79.39%
Viability 3.5 h Triton X-100: 59.93%
Viability 5h Triton X-100: 32.89%

Evaluation of direct interaction with MTT: The direct interaction of MTT with the test item was checked by adding 50 µL of the test item to 1 mL of the solution of MTT at 1 mg/mL (same conditions as in the main test). A yellow solution with test item deposit at the bottom of the well was observed after 3 hours of incubation between 37.1°C and 37.4°C, 5% CO2. > Therefore, there is no direct interaction between the test item and MTT.

Coloration potential and spectral analysis of the test item: In water: The coloration potential of the test item in water was checked by adding 50 µL of the test item to 300 µL of distilled water. A colourless solution was obtained after 1 hour and 40 minutes of incubation between 37.1°C and 37.4°C, 5% CO2.

In isopropanol: The coloration potential of the test item in isopropanol was checked by adding 50 µL of the test item to 2 mL of isopropanol. A colourless solution was obtained after 2 hours of incubation at room temperature. > No significant coloration appeared. Therefore the test item will not interfere with the MTT assay.



Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
test item was applied as supplied at the dose of 50 µL
Duration of treatment / exposure:
3 minutes and 1 h
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 minutes application
Value:
103.8
Positive controls validity:
valid
Remarks:
19.17 % viability
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1 hour application
Value:
83.4
Positive controls validity:
valid
Remarks:
0.41 % viability
Other effects / acceptance of results:
Acceptability criteria:  The difference of viability between the 2 tissues treated with the negative control during 3 minutes was higher than 30% (40.9%). Considering the results obtained (both epidermises have a viability much higher than 50% and are clearly classified as “Non-corrosive”), this deviation is considered as without impact on the conclusion of the study.

Table 1: Assessment of the skin corrosion individual and average values after 3 minutes of exposure

Application: 50 µl of the test item as supplied on 0.6 cm² human skin model

   Well ID  OD  Mean OD/disc (#)  Mean OD/product Viability %   Mean viability %  Viability difference between replicates %
Negative control  SPL 1

0.716

0.704

0.719

0.713   0.592  120.44 100.00   40.9
  SPL 2

0.456

0.461

0.494

 0.471    79.56  
Positive control SPL 3

0.100

0.116

0.131 

0.116  0.114   19.59  19.17  0.8
  SPL 4

0.106

0.110

0.115

0.111     18.75    
Test item SPL 5

0.544

0.647

0.651

 0.614  0.615  103.72  103.80  0.2
  SPL 6

0.524

0.681

0.640

 0.615    103.89    

Note #: mean of 3 values OD: optical density SPL: sample

Acceptability criteria:  - Negative control: mean OD of the tissue replicates should be ≥ 0.8 and ≤ 2.8 for epiCS® model, for every exposure time. As the extract was diluted at 1:3 just before the OD measure, the acceptability criteria should be in the range ≥ 0.3 and ≤ 0.9 for the negative control.

- Positive control: mean viability of the tissue replicates exposed for 1 hour, expressed as % of the negative control, should be < 20% for epiCS model. - Test item: in the range 20-100% viability, and for ODs ≥ 0.3, difference of viability between the two tissue replicates should not exceed 30%.

Table 2: Assessment of the skin corrosion individual and average values after 1 hour of exposure

Application: 50 µl of the test item as supplied on 0.6 cm² human skin model

   Well ID  OD  Mean OD/disc (#)  Mean OD/product Viability %   Mean viability %  Viability difference between replicates %
Negative control  SPL 11

0.667

0.832

0.807

0.769   0.726  105.92 100.00   11.8
  SPL 12

0.653

0.664

0.730

 0.683    94.08  
Positive control SPL 13

0.002

0.000

0.001

0.001  0.003   0.14  0.41  0.6
  SPL 14

0.005

0.005

0.005

0.005     0.69    
Test item SPL 15

0.545

0.544

0.579

 0.556  0.606  76.58  83.40  13.6
  SPL 16

0.624

0.642

0.698

 0.655    90.22    

Note #: mean of 3 values OD: optical density SPL: sample

Acceptability criteria:  - Negative control: mean OD of the tissue replicates should be ≥ 0.8 and ≤ 2.8 for epiCS® model, for every exposure time. As the extract was diluted at 1:3 just before the OD measure, the acceptability criteria should be in the range ≥ 0.3 and ≤ 0.9 for the negative control.

- Positive control: mean viability of the tissue replicates exposed for 1 hour, expressed as % of the negative control, should be < 20% for epiCS model. - Test item: in the range 20-100% viability, and for ODs ≥ 0.3, difference of viability between the two tissue replicates should not exceed 30%.

Table 3: Assessment of the skin corrosion after 3 minutes and after 1 hour exposure

         Mean viability (%)
   3 min exposure 1 hour exposure  conclusion 
 Positve control  19.17  0.41  corrosive sub-category 1B/C

 Test item

 103.80

 83.40

 Non-corrosive

Note

The item is to be classified as non-corrosive:

➢ If the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15 %

The item is to be classified as corrosive:

➢ If  the viability after 3 minutes exposure is strictly less than 50%, or

➢ If the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is strictly less than 15 %

Interpretation of results:
Category 2 (irritant) based on GHS criteria
Remarks:
not corrosive; together with the result of the OECD 439
Conclusions:
In accordance with the Regulation (EC) No. 1272/2008 and the results of skin irritation (HSMI-PH19/0104) according to OECD 439, the results obtained under these experimental conditions enable to conclude that the test item 2-hexyldecanoic acid does not have to be classified in Category 1 “Corrosive” but has to be classified in Category 2 “Irritating to skin”.
Executive summary:

The aim of the study was to evaluate the possible corrosive effects of the test item 2 -hexyldecanoic acid after topical administration on in vitro human reconstituted epidermis (epiCS®, supplied by CellSystems®).

The test item 2 -hexyldecanoic acid was applied as supplied, at the dose of 50 µL, to 2 living Human skin model surfaces (epiCS®, supplied by CellSystems®) during 3 minutes and 1 hour. The application was followed by a rinse with 20 mL of DPBS. Cell viability was then measured by enzymatic conversion of the vital dye MTT into a blue formazan salt that was quantitatively measured after extraction from tissues.  The experimental protocol was established in accordance with the O.E.C.D. Test Guideline No. 431 dated 29 July 2016 and the method B.40 bis of the Council regulation No. 440/2008 of 30 May 2008.  

3 minutes and 1 hour after the test item application, the mean percent viability of the epidermis skins treated with the test item were 103.80% (considered as 100%) and 83.40%, versus 19.17% and 0.41%, respectively, with the positive control item (potassium hydroxide 8N).

In accordance with the Regulation (EC) No. 1272/2008 and the results of skin irritation (HSMI-PH19/0104) according to OECD 439, the results obtained under these experimental conditions enable to conclude that the test item 2 -Hexyldecanoic acid does not have to be classified in Category 1 “Corrosive” but has to be classified in Category 2 “Irritating to skin”.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Referenceopen allclose all

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
May 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP compliant
Qualifier:
according to guideline
Guideline:
OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
Version / remarks:
adopted 25 June 2018
Deviations:
yes
Remarks:
Deviation is considered as without impact on the results of the study.
GLP compliance:
yes (incl. QA statement)
Remarks:
Certificate of Compliance with Good Laboratory Practices according to Directives 2004/9/CE and 2004/10/CE, Groupe Interministeriel des Produits Chimiques, Republique Francaise, Certificat n°: 2017/33, dated 27 April 2017
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: supplied by the sponsor, Batch no.: 05297/MA
- Expiration date of the lot/batch: October 2021
- Purity test date: 27.10.2017
- Storage condition of test material: room temperature
Species:
other: isolated chicken eye
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: slaughterhouse Etablissement Brun, 33820 Etauliers, France, eyes collected from chicken killed for human consumption
- Number of animals: not mentioned
- Characteristics of donor animals: approx. 7 weeks old, 1.5 - 2.5 kg, sex not mentioned
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Because eyes were dissected in the laboratory, the intact heads were transported from the slaughterhouse at ambient temperature in plastic boxes humidified with towels moistened with physiological saline. Eyes were enucleated at test laboratory and transferred to a chamber of the superfusion apparatus such that the entire cornea was supplied with the physiological saline drip in the range of 0.1 to 0.15 ml/min. at temperatures between 32.3 and 32.4 °C.
- Indication of any existing defects or lesions in ocular tissue samples: none
- Indication of any antibiotics used: not mentioned
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied: 30 µl


Duration of treatment / exposure:
10 seconds, then rinsed twice from the eye with 10 ml of physiological saline at ambient temperature
Duration of post- treatment incubation (in vitro):
Treated corneas were evaluated at 30, 75, 120, 180, and 240 minutes after post-treatment rinse
Number of animals or in vitro replicates:
3 replicates for the test item, 3 replicates for the positive control, 1 replicate for the negative control
Details on study design:
SELECTION AND PREPARATION OF ISOLATED EYES
The eyelids were carefully excised, taking care not to damage the cornea. Then, the eye was further dissected from the skull, taking care not to damage the cornea. The eyeball was pulled from the orbit by holding the nictitating membrane firmly with surgical forceps, and the eye muscles were cut with a bent, blunt-tipped scissor. When the eye was removed from the orbit, a visible portion of the optic nerve should be left attached. Once removed from the orbit, the eye was placed on an absorbent pad and the nictitating membrane and other connective tissue were cut away. The enucleated eye was mounted in a stainless steel clamp with the cornea positioned vertically. The clamp was then transferred to a chamber of the superfusion apparatus. The clamps were postioned in the superfusion apparatus such that the entire cornea was supplied with the physiological saline drip (in the range 0.1 to 0.15 mL/min). The chambers of the superfusion apparatus was temperature controlled between 32.3 and 32.4°C. After being placed in the superfusion apparatus, the eyes were examined with a slit-lamp microscope to ensure that they have not been damaged during the dissection procedur. Corneal thickness was also measured at this time at the corneal apex using the depth measuring device on the slit-lamp microscope. Eyes with (i) a fluorescein retention score of <0.5, (ii) corneal opacity >0.5, or, (iii) any additional signs of damage were replaced. For eyes that are not rejected based on any of these criteria, individual eyes with a corneal thickness deviating more the 10% from the mean value for all eyes were also rejected and replaced.

EQUILIBRATION AND BASELINE RECORDINGS
All examined and approved eyes were incubated between 45 and 64 minutes to equilibrate them to the test system prior to dosing. Following the equilibration period, a zero reference measurement was recorded for corneal thickness and opacity to serve as baseline. The fluorescein score determined at dissection was used as the baseline measurement for that endpoint.

NUMBER OF REPLICATES
3 replicates for the test item, 3 replicates for the posisitve control, 1 replicate for the negative control

NEGATIVE CONTROL USED
yes, physiological saline

POSITIVE CONTROL USED
yes, Benzalkonium chloride, 5% in physiological saline

APPLICATION DOSE AND EXPOSURE TIME
Immediately following the zero reference measurements, the eye (in its holder) was removed from the superfusion apparatus, placed in a horizontal position, and 30 µL of the test item was applied, as supplied, to the cornea such that the entire surface of the cornea was evenly covered with the test item. The test item was applied for 10 seconds and then rinsed from the eye with 20 mL of physiological saline at ambient temperature. The eye (in its holder) was subsequently returned to the superfusion apparatus in the original upright position.

OBSERVATION PERIOD
Treated corneas were evaluated pretreatment and starting at 30, 75, 120, 180, and 240 minutes (± 5 minutes) after the post-treament rinse.

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: rinsed twice from the eye with 10 mL of physiological saline at ambient temperature
- Indicate any deviation from test procedure in the Guideline : The eyes were incubated between 45 and 64 minutes instead of between 45 and 60 minutes, as initially scheduled. As the results obtained with the eyes treated with the negative control which has the longest duration of acclimation were conformed to what expected, this deviation is considered as without impact on the results of the study.

METHODS FOR MEASURED ENDPOINTS:
All observations of the cornea and measurement of corneal thickness were performed using a Haag-Streit BP900 slit-lamp microscope with depth-measuring device no. I. For the measurement of corneal thickness the slit-width was set at 9 1/2, equally 0.095 mm. Corneal opacity was calculated by using the area of the cornea that was most densely opacified for scoring. The mean corneal opacity value for all test eyes was calculated for all observation time points. Based on the highest mean score for corneal opacity as observed at any time point, an overall category score was then given for each test or control item. The endpoints evaluated were corneal opacity, swelling, fluorescein retention, and morphological effects (e.g. pitting or loosening of the epithelium). All of the endpoints, with the exception of fluorescein retention were determined at each of the above time points. Corneal swelling was determined from corneal thickness measurements made with an optical pachymeter on a slit-lamp microscope and was expressed as a percentage. The mean percentage of corneal swelling for all test eyes was calculated for all observation time points. Based on the highest mean score for corneal swelling, as observed at any time point, an overall category score was then given for each test item. The mean fluorescein retention value for all test eyes was calculated for the 30-minute observation time point only, which was used for the overall category score given for each test or control item. Morphological effects include "pitting" of corneal epithelial cells, "loosening" of epithelium, "roughening" of the corneal surface and "sticking" of the test item to the cornea. The classification of these findings is subjective to the interpretation of the investigator.

SCORING SYSTEM:
- Mean corneal swelling (%) : according to guideline
- Mean maximum opacity score : according to guideline
- Mean fluorescein retention score at 30 minutes post-treatment ; according to guideline

DECISION CRITERIA: decision criteria as indicated in the TG was used.
Irritation parameter:
cornea opacity score
Remarks:
maximal mean score
Value:
3
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: corresponding to ICE class IV
Irritation parameter:
fluorescein retention score
Remarks:
mean score
Value:
0.8
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: corresponding to ICE class II
Irritation parameter:
percent corneal swelling
Remarks:
maximal mean swelling
Value:
8
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: corresponding to ICE class II
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: At all examination time points no morphological effects were noted in the negative control and the test item treated eyes. The eyes treated with the positive control showed blisters on the cornea from 30 minutes post-dose in all 3 tested eyes.

DEMONSTRATION OF TECHNICAL PROFICIENCY:
The combination of the three endpoints for the test ISOCARB 16 (2-Hexyldecanoic acid) was 2 x II, 1 x IV, therefore the outcome of the study was "no prediction can be made".
The combination of the three endpoint for the negative control (physiological saline) wsa 3 x I, therefore the negative control is classified as "No category", as expected.
The combination of the three endpoints for the positive control (5% Benzalkonium chloride) was 3 x IV, therefore the positive control is classified as "Corrosive/Severe Irritant", as expected.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Range of historical values if different from the ones specified in the test guideline: not different

Table #1: Values for Evaluation of Corneal Lesions after Treatment with Test Item

 Endpoint measured  Eye No. Time (min)                

 

 

 0

 30

 75

 120

 180

 240

 Corneal opacity

 13

 0

 0

 1

3

 3

 3

 

 14

 0

 0

 1

3

 3

 3

 

 15

 0

 1

 3

 3

 3

31

 Mean

 

 0.0

 0.3

 1.7

 3.0

 3.0

 3.0

 ICE class

 

IV 

 Fluoreescein retention

13

 0.5

 1

 -

 -

 -

 -

 

14

 0.5

 0.5

 -

 -

 -

 -

 

15

 0.5

 1

 -

 -

 -

 -

 Mean

 

 0.5

 0.8

 -

 -

 -

 -

 ICE class

 

 II

 -

 -

 -

-

 Corneal thickness

13

 0.50

 0.50

 0.50

 0.52

 0.52

 0.52

 

14

 0.48

 0.48

 0.50

 0.52

 0.53

 0.55

 

15

 0.53

 0.53

 0.53

 0.56

 0.56

 0.56

 Corneal swelling (%)

13

 -

 0

 0

 4

 4

 4

 

14

 -

 0

4

 8

 10

 15

 

15

 -

 0

 0

6

 6

 5

 Mean

 

 -

 0

1

 6

7

 8

 ICE class

 

II

 Combination of 3 endpoints

 

 2 x II, 1 x IV

 Classification

 

 no prediction can be made 

Table #2: Values for Evaluation of Corneal Lesions after Treatment with Positive Control (5% (w/v) Benzalkonium chloride

 Endpoint measured  Eye No. Time (min)           

 

 

 0

 30

 75

 120

 180

 240

 Corneal opacity

 1

 0

 3

 3

 3

 3

 3

 

 2

 0

 3

 3

 3

 3

 3

 

 3

 0

 3

 3

 3

 3

 3

 Mean

 

 0.0

 3.0

 3.0

 3.0

 3.0

 3.0

 ICE class

 

IV 

 Fluoreescein retention

 1

 0.5

 3

 -

 -

 -

 -

 

 2

 0.5

 3

 -

 -

 -

 -

 

 3

 0.5

 3

 -

 -

 -

 -

 Mean

 

 0.5

 3.0

 -

 -

 -

 -

 ICE class

 

 IV

 -

 -

 -

-

 Corneal thickness

 1

 0.46

 0.52

 0.62

 0.65

 0.70

 0.72

 

 2

 0.48

 0.54

 0.61

 0.62

 0.64

 0.64

 

 3

 0.52

 0.53

 0.59

 0.61

 0.72

 0.78

 Corneal swelling (%)

 1

 -

 13

 35

 41

 52

 57

 

 2

 -

 13

 27

 29

 33

 33

 

 3

 -

 2

 13

 17

328

50

 Mean

 

 -

 10

 18

 22

 30

 31

 ICE class

 

IV

 Combination of 3 endpoints

 

 3 x IVI

 Classification

 

Category 1: Corrosive / Severe irritant 

Table #3: Values for Evaluation of Corneal Lesions after Treatment with Negative Control (Physiological Saline)

 Endpoint measured  Eye No. Time (min)           

 

 

 0

 30

 75

 120

 180

 240

 Corneal opacity

 16

 0

 0

 0

 0

 0

 0

 ICE class

 

 Fluoreescein retention

 16

 0.5

 0.5

 -

 -

 -

 -

 ICE class

 

 I

 -

 -

 -

-

 Corneal thickness

 16

 0.48

 0.48

 0.48

 0.48

 0.48

 0.48

 Corneal swelling (%)

 16

 -

 0

 0

 0

 0

 0

 ICE class

 

I

 Combination of 3 endpoints

 

 3 x I

 Classification

 

 No category 

Interpretation of results:
study cannot be used for classification
Conclusions:
The results obtained under these experimental conditions lead to the category "no prediction can be made", as defined in the guideline OECD No. 438, therefore the test item is not predicted as causing serious eye damage (Category I) or as not classified for eye irritation/serious eye damage (No Category) with the Isolated Chicken Eye test method.
Executive summary:

The study was performed according with the OECD Test Guideline No. 438 in compliance with GLP. The aim of the study was to evaluate the possible ocular corrosive or severe irritating effects of the test item after administration on enucleated chicken eyes. The test item was applied, as supplied, at the dose of 30 µl, to 3 enucleated chicken eyes, during 10 seconds, then the eyes were rinsed twice with 10 ml of physiological saline. Three eyes were treated in the same manner with a positive control and one eye with a negative control. Damages by the test item were assessed by determination of corneal swelling, opacity, and fluorescein retention at 30, 75, 120, 180 and 240 minutes post-dose. The ocular reactions observed in eyes treated with the test item were: max. mean score of corneal opacity: 3.0, corresponding to ICE class IV; mean score of fluorescein retention: 0.8, corresponding to ICE class II; max. mean corneal swelling: 8%, corresponding to ICE class II. The combination of the three endpoints for the positive control, 5% Benzalkonium chloride, was 3 x IVI. Therefore, the positive control is classified as "Corrosive/Severe Irritant", as expected. The combination of the three endpoints for the negative control, physiological saline, was 3 x I. Therefore, the negative control is classified as "No Category", as expected.

In accordance with the Regulation (EC) No. 1272/2008, the result obtained under these experimental conditions lead to the category "no prediction can be made", as defined by the OECD guideline No. 438. Therefore the test item is not predicted as causing serious eye damage (Catefory I)  or as not classified for eye irritation/serious eye damage (No category) with the Isolated Chicken Eye test method.

Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
July 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP compliant
Qualifier:
according to guideline
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Version / remarks:
October 9th, 2017
Deviations:
yes
Remarks:
Due to animal welfare reasons analgesia was used, for details see section "Any other information on materials and methods incl. tables" below
GLP compliance:
yes (incl. QA statement)
Remarks:
Certificate of Compliance with Good Laboratory Practices according to Directives 2004/9/CE and 2004/10/CE, Groupe Interministeriel des Produits Chimiques, Republique Francaise, Certificat n°: 2017/33, dated 27 April 2017
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: supplied by sponsor, Batch no.: 05297/MA
- Expiration date of the lot/batch: Retest date October 2021
- Purity test date: 27 October 2017
- Storage condition of test material: room temperature
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: Granja San Bernardo (Tulebras, Navarra - Spain)
- Age at study initiation: 9 or 13 weeks old
- Housing: single housing, animals were provided with environmental enrichment (Aspen brick)
- Diet: ENVIGO - 2030 C, ad libitum
- Water: Tap water from public distribution system ad libitum
- Acclimation period: minimum 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 to 23
- Humidity (%): 30 to 70
- Air changes (per hr): at least 10 per hour
- Photoperiod (hrs dark / hrs light): 12/12
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent no treatment
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied: 0.1 ml
Duration of treatment / exposure:
single administration, in each animal the test article was introduced into the conjunctival sac of one eye after gently pulling the lower lid away from the eyeball, afterwards the eyelid was closed for 1 second, unlimited exposure (not rinsed).
Observation period (in vivo):
1 hour, 24, 48 and 72 hours following treatment.
Number of animals or in vitro replicates:
3 male
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing: no

SCORING SYSTEM: acc. to guideline
Irritation parameter:
iris score
Remarks:
animal #1
Basis:
mean
Time point:
24/48/72 h
Score:
0
Max. score:
0
Irritation parameter:
cornea opacity score
Remarks:
animal #1
Basis:
mean
Time point:
24/48/72 h
Score:
0
Max. score:
0
Irritation parameter:
chemosis score
Remarks:
animal #1
Basis:
mean
Time point:
24/48/72 h
Score:
0
Max. score:
0
Irritation parameter:
conjunctivae score
Remarks:
animal #1
Basis:
mean
Time point:
24/48/72 h
Score:
0.3
Max. score:
1
Reversibility:
fully reversible within: 48 h
Irritation parameter:
iris score
Remarks:
animal #2
Basis:
mean
Time point:
24/48/72 h
Score:
0
Max. score:
0
Irritation parameter:
cornea opacity score
Remarks:
animal #2
Basis:
mean
Time point:
24/48/72 h
Score:
0.3
Max. score:
1
Reversibility:
fully reversible within: 48 h
Irritation parameter:
chemosis score
Remarks:
animal #2
Basis:
mean
Time point:
24/48/72 h
Score:
0.3
Max. score:
1
Reversibility:
fully reversible within: 48 h
Irritation parameter:
conjunctivae score
Remarks:
animal #2
Basis:
mean
Time point:
24/48/72 h
Score:
0.7
Max. score:
2
Reversibility:
fully reversible within: 48 h
Irritation parameter:
iris score
Remarks:
animal #3
Basis:
mean
Time point:
24/48/72 h
Score:
0
Irritation parameter:
cornea opacity score
Remarks:
animal #3
Basis:
mean
Time point:
24/48/72 h
Score:
0.3
Max. score:
1
Reversibility:
fully reversible within: 48 h
Irritation parameter:
chemosis score
Remarks:
animal #3
Basis:
mean
Time point:
24/48/72 h
Score:
0.3
Max. score:
1
Reversibility:
fully reversible within: 48 h
Irritation parameter:
conjunctivae score
Remarks:
animal #3
Basis:
mean
Time point:
24/48/72 h
Score:
1
Max. score:
2
Reversibility:
fully reversible within: 72 h
Irritant / corrosive response data:
The ocular reactions observed during the study have been slight to moderate and totally reversible: - at the conjunctivae level: a slight to moderate redness noted 1 hour after the test item instillation in all animals and totally reversible between Days 2 and 3, associated with a slight chemosis noted 1 hour after the test item instillation in all animals and totally reversible between Days 1 and 2. - at the corneal level: a slight opacity noted 24 hours after the test item instillation in two animals and totally reversible on Day 2.
Other effects:
none

Table 1: Irritant/corrosive response data at each observation time:

 Score at time point/Reversibility Cornea  Iris  Conjuntivae Redness  Conjunctiva Chemosis
 24 h  0/1/1  0/0/0 1/1/2 0/1/1
 48 h  0/0/0  0/0/0 0/1/1 0/0/0
 72 h  0/0/0  0/0/0 0/0/0 0/0/0
 Mean 0.22 0 0.67 0.22
 Reversibility yes yes yes yes
Interpretation of results:
GHS criteria not met
Conclusions:
Based on the result of this study, the substance 2-Hexyldecanoic acid is not irritating to the eyes.
Executive summary:

The test item 2-Hexyldecanoic acid was instilled, as supplied, into the eye of three New Zeland rabbits at the dose of 0.1 ml. The experimental protocol was established on the basis of the official method as defined in the OECD Test Guideline No. 405 dated October 9th, 2017 and performed in accordance with the Principles of GLP. The ocular reaction observed during the study have been slight to moderate and totally reversible within 72 hours.

In conclusion, the results obtained under the experimental conditions of this study show that the test substance is not irritating to the eyes. In accordance with the Regulation EC No. 1272/2008 on classification, labelling and packaging of substances and mixtures, no symbol and warning label are required.

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The test item 2 -hexyldecanoic acid was tested in an in vitro skin irritation study according to OECD 439. The test item was applied as supplied, at the dose of 16 µL, to 3 living Reconstructed Human epidermis (SkinEthic RHE® model) during 42 minutes. The application was followed by a rinse with 25 mL of DPBS and a 42 hours and 00 minutes postincubation period at 37°C, 5% CO2. Cell viability was then measured by enzymatic conversion of the vital dye MTT into a blue formazan salt that was quantitatively measured after extraction from tissues. The mean percent viability of the treated tissues was 3.3, versus 1.3 % in the positive control (5% Sodium Dodecyl Sulfate).  Accordance with the Regulation EC No. 1272/2008 2 -hexyldecanoic acid has to be classified in Category 2 “Irritating to skin” or in Category 1 “Corrosive”.

Therefore an in vitro skin corrosion test according to OECD 431 was additional conducted. 2 -hexyldecanoic acid was applied as supplied, at the dose of 50 µL, to 2 living Human skin model surfaces (epiCS®, supplied by CellSystems®) during 3 minutes and 1 hour. The application was followed by a rinse with 20 mL of DPBS. Cell viability was then measured by enzymatic conversion of the vital dye MTT into a blue formazan salt that was quantitatively measured after extraction from tissues. 3 minutes and 1 hour after the test item application, the mean percent viability of the epidermis skins treated with the test item were 103.80% (considered as 100%) and 83.40%, versus 19.17% and 0.41%, respectively, with the positive control item (potassium hydroxide 8N). In accordance with the Regulation (EC) No. 1272/2008 and the results of skin irritation (HSMI-PH19/0104) accorindg to OECD 439, the results obtained under these experimental conditions enable to conclude that the test item 2 -Hexyldecanoic acid does not have to be classified in Category 1 “Corrosive” but has to be classified in Category 2 “Irritating to skin”.

An in vitro eye irritation test according OECD 438 was conducted to evaluate the possible ocular corrosive or severe irritating effects of the test item after 2 -hexyldecanoic acid administration on enucleated chicken eyes. The test item was applied, as supplied, at the dose of 30 µl, to 3 enucleated chicken eyes, during 10 seconds, then the eyes were rinsed twice with 10 ml of physiological saline. Three eyes were treated in the same manner with a positive control and one eye with a negative control. Damages by the test item were assessed by determination of corneal swelling, opacity, and fluorescein retention at 30, 75, 120, 180 and 240 minutes post-dose. The ocular reactions observed in eyes treated with the test item were: max. mean score of corneal opacity: 3.0, corresponding to ICE class IV; mean score of fluorescein retention: 0.8, corresponding to ICE class II; max. mean corneal swelling: 8%, corresponding to ICE class II. The combination of the three endpoints for the positive control, 5% Benzalkonium chloride, was 3 x IVI. Therefore, the positive control is classified as "Corrosive/Severe Irritant", as expected. The combination of the three endpoints for the negative control, physiological saline, was 3 x I. Therefore, the negative control is classified as "No Category", as expected.

In accordance with the Regulation (EC) No. 1272/2008, the result obtained under these experimental conditions lead to the category "no prediction can be made", as defined by the OECD guideline No. 438. Therefore the test item 2 -hexyldecanoic acid is not predicted as causing serious eye damage (Catefory I)  or as not classified for eye irritation/serious eye damage (No category) with the Isolated Chicken Eye test method.

Therefore it was required to conduct an in vivo eye irritation test according to OECD 405 to determine the eye irritation properties of the test item. 2-Hexyldecanoic acid was instilled, as supplied, into the eye of three New Zeland rabbits at the dose of 0.1 ml. The ocular reaction observed during the study have been slight to moderate and totally reversible within 72 hours. In conclusion, the results obtained under the experimental conditions of this study show that the test substance 2 -hexyldecanoic acid is not irritating to the eyes. In accordance with the Regulation EC No. 1272/2008 on classification, labelling and packaging of substances and mixtures, no symbol and warning label are required.

Supporting are the results of the eye irritation study with the similar substance 2-butyloctanoic acid according to OECD 405. The Analogue has a slight irritant effect in the eye. The observed reaction was not sufficiently severe to require classification of the test item.

Justification for classification or non-classification

In accordance with the Regulation EC No. 1272/2008 the results of the skin irritation test according to OECD 439 together with the results of the skin corrosion test according to OECD 431 with the test item 2 -hexyldecanoic acid show that the test item has to be classified in Category 2 “Irritating to skin” but does not have to be classified in Category 1 “Corrosive”.

According to the classification criteria of regulation (EC) 1272/2008 the test item 2 -hexyldecanoic acid has not to be classified as eye irritant.