Methyl 2-[[3-[4-(1,1-dimethylethyl)phenyl]-2-methylpropylidene]amino]benzoate

Endpoint summary

Description of key information

Skin corrosion: Corrosive properties are not anticipated due to absence of acids and base functional groups in its chemical structure and absence of skin and eye irritation.

Skin irritation (OECD TG 439): Not skin irritating

Eye irritation (OECD TG 438): Not eye irritating

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

This study was conducted between 05 January 2017 and 28 April 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study is considered to be reliability 1 as it has been conducted according to OECD Test Guideline 439 using the EPISKIN™ Reconstructed Human Epidermis Model and in compliance with GLP.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

28 July 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Identification: Lilial ME Anthranilate
Purity: 85.62%*
Appearance: Yellow viscous liquid (paste-like**)
Expiry Date: 10 May 2019
Storage Conditions: At room temperature, under N2-atmosphere
Stability in Solvent: Not indicated by the Sponsor
* dose calculation will not be adjusted to purity
** determined by Envigo CRS GmbH laboratory staff

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: The EpiSkin™ tissue consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis.

Source strain:

not specified

Details on test system:

EpiSkin™ Kit Components Needed for the Assay
EpiSkin™ Kit Lot No.: 17-EKIN-006 and 17-EKIN-017
1 Sealed 12-well plate Contains 12 inserts with EpiSkin™ tissues on agarose
1 12-well plate For MTT viability assay
1 bottle Assay Medium Basic medium for use in MTT assays
1 bottle EpiSkin™ Maintenance Medium Basic medium for incubations

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

Test Item Preparation

The test item was heated in a water-bath to 65 ± 2 °C for melting purpose. Afterwards, the melted test item was cooled to room temperature. Each 10 µL of the test item were applied to triplicate EpiSkin™ tissues.

Duration of treatment / exposure:

15 Minutes

Duration of post-treatment incubation (if applicable):

42 hours post-exposure incubation

Number of replicates:

3. Triplicate tissues were treated with: test substance, positive control or negative control.

Details on study design:

Study Design
MTT-Solution
MTT Formazan salt was dissolved in DMEM or assay medium to reach a final concentration of 0.3 mg/mL.

Cell Culture
EpiSkin™ kits are purchased from SkinEthic Laboratories (69007 Lyon, France). The EpiSkin™ tissue consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiSkin™ tissues (surface 0.38 cm²) are cultured on specially prepared cell culture inserts.
EpiSkin™ tissues were shipped at ambient temperature on medium-supplemented agarose gels in a 12-well plate and reached Envigo CRS GmbH on 07 February 2017 and on 25 April 2017. On the day of the experiments the pre-incubation phase of the EpiSkin™ tissues started.

est for Direct MTT Reduction and Colour Interference
Prior to the start of the test, the test item’s colour interference potential had to be evaluated. For this purpose the test item (approximately 10 µL) was mixed with 90 µL of deionised water in a pre-experiment. The test item/water mixture was gently shaken for 15 minutes at room temperature. Deionised water was used as control.
The test item did not dye water during the incubation period.
For correct interpretation of results it is necessary to assess the ability of the test item to directly reduce MTT. To test for this ability approximately 10 µL of the test item was added to 2 mL of MTT solution (0.3 mg/mL) and the mixture was incubated in the dark at
37 ± 1.5 °C (5 ± 0.5% CO2) for 3 hours. MTT solution was used as control.
Since the colour did not turn blue/purple, the test item was not considered to be a MTT reducer.

EXPERIMENTAL PERFORMANCE
Due to a borderline result in the 1st main experiment, a 2nd main experiment was performed according to the recommendation in the OECD TG 439 and to the Sponsor’s request.

Pre-warming of EpiSkin™ Tissues
Under sterile conditions using sterile forceps, the inserts were transferred into 12-well plates containing the pre-warmed (37 ± 1.5 °C) maintenance medium. The EpiSkin™ tissues were incubated for 23 to 24 hours in both main experiments.

Treatment
Both main experiments:
The negative control, positive control and the test item were added into the insert atop the concerning EpiSkin™ triplicate tissues. The 12-well plates were treated for 15 minutes.
After the end of the treatment interval the inserts were immediately removed from the 12-well plate. The tissues were gently rinsed with PBS to remove any residual test material. The test item could not be rinsed from the tissues satisfactorily. Excess PBS was removed by gently shaking the inserts and blotting the bottom with blotting paper. The inserts were placed in the plates with 2 mL maintenance medium. The tissues were incubated for 42 hours at 37 ± 1.5 °C, 5 ± 0.5% CO2.

IL-1 α Immunoassay
Samples of all treatment groups were taken from the wells in both experiments. The plates were shaken for approximately 15 minutes to homogenise the released mediators in the medium before sampling. At least 1.6 mL medium from each well was taken and was stored in the freezer at –20 °C. Although, the results derived from the MTT assay (see chapter 4.4 and 8.1) were borderline in the 1st main experiment and contrarily to the 1st main experiment in the 2nd experiment, the IL-1 α concentration in the medium was not determined according to the Sponsor’s request, and the taken samples were discarded after report finalization.

MTT Assay
A 12-well plate was filled with 2 mL assay medium containing 0.3 mg/mL MTT per well.
After the treatment procedure was completed for all tissues the cell culture inserts were transferred from the holding plates to the MTT-plates. After a 3 hour incubation period (37 ± 1.5 °C, 5 ± 0.5% CO2) MTT solution was aspirated from the wells and the wells were rinsed three times with PBS. Tissue samples were cut out of the inserts with a biopsy punch and transferred into plastic vials. The tissue samples were immersed into extractant solution by gently pipetting 0.5 mL extractant solution (isopropanol containing 0.04 N HCl) into each vial. The tissue samples were completely covered by isopropanol. The vials were sealed to inhibit isopropanol evaporation. The formazan salt was extracted for nearly 3 hours (1st main experiment) or about 2.5 hours (2nd main experiment), respectively, while shaking at room temperature.
Per tissue sample 2  200 µL aliquots of the formazan blue solution were transferred into a 96-well flat bottom microtiter plate. OD was read in a microplate reader (Versamax® Molecular Devices, 85737 Ismaning, Germany, version 4.7.1) with 570 nm filter. Mean values were calculated from the 2 wells per tissue sample.

Data Recording
The data generated were recorded in the laboratory protocol. The results were presented in tabular form, including experimental groups with the test item, negative and positive controls.
DATA EVALUATION
The mean OD of the three negative control tissues was calculated after blank correction. This value corresponds to 100% tissue viability in the current test. For each individual tissue treated with the test item or the positive control the individual relative tissue viability is calculated according to the following formula:

Relative viability (%) = (Mean OD test item or positive control)/ (mean OD negative vontrol) x 100

For the test item and the positive control the mean relative viability ± rel. standard deviation of the three individual tissues will be calculated and used for classification according to the following prediction model:
in vitro result in vivo prediction
mean tissue viability < 50% irritant (I)
mean tissue viability > 50% non-irritant (NI)

Acceptability of the Assay
The absolute OD 570 nm of the negative control tissues in the MTT test is an indicator of tissue viability obtained after the shipping and storing procedure and under specific conditions of the assay. Tissue viability is meeting the acceptance criterion if the mean OD of the three tissues is > 0.6 till ≤ 1.5.
The rel. standard deviations in between tissues of the same treatment group should be ≤ 18%.
An assay is meeting the acceptance criterion if mean relative tissue viability of the positive control is ≤ 40%.
The data of the quality control (determined by SkinEthic Laboratories, 69007 Lyon, France) of the respective EpiSkin™ lot is mentioned in the present report (the acceptance limit of the IC50 should be between 1.5 and 3.0 mg/mL after 18 hours treatment with SLS

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

15 Minute exposure/42h observation

Value:

66.1

Negative controls validity:

valid

Remarks:

Set to 100%

Positive controls validity:

valid

Remarks:

8.3

Remarks on result:

no indication of irritation

Results after treatment with Lilial ME Anthranilate and controls

1^stMain Experiment
Dose Group	Absor-bance 570 nm Tissue 1*	Absor-bance 570 nm Tissue 2*	Absor-bance 570 nm Tissue 3*	Mean Absor-bance of 3 Tissues	Relative Absor-bance [%] Tissue 1, 2 + 3**	Relative Standard Deviation [%]	Rel. Absor-bance [% of Negative Control]***
Negative Control	0.896	1.007	0.760	0.888	101.0 113.4 85.6	13.9	100.0
Positive Control	0.075	0.085	0.098	0.086	8.4 9.6 11.1	13.8	9.7
Test Item	0.440	0.434	0.446	0.440	49.5 48.9 50.3	1.4	49.6
2^ndMain Experiment
Negative Control	0.611	0.595	0.607	0.604	101.1 98.4 100.4	1.4	100.0
Positive Control	0.045	0.058	0.048	0.050	7.4 9.6 8.0	13.4	8.3
Test Item	0.396	0.402	0.400	0.399	65.5 66.6 66.2	0.8	66.1

*       Mean of two replicate wells after blank correction

**       relative absorbance per tissue [rounded values]: 100 x Absorbance tissue / mean absorbance negative control

***       relative absorbance per treatment group [rounded values]: 100 x mean absorbance test item / mean absorbance negative control

The optical pre-experiment (colour interference pre-experiment) to investigate the test item’s colour change potential in water did not lead to a change in colour.

Optical evaluation of the MTT-reducing capacity of the test item after 3 hour incubation with MTT-reagent did not show blue colour.

The mean relative absorbance value of the test item, corresponding to the cell viability, was reduced to 49.6% (threshold for irritancy: ≤ 50%) in the 1^stmain experiment, consequently the test item was irritant to skin.

In the 2^ndmain experiment the cell viability decreased to 66.1%. This value is above the threshold for irritancy. According this result, the test item has to be classified as non-irritant to skin.

1.1                  Discussion

Thisin vitrostudy was performed to assess the irritation potential of Lilial ME Anthranilate by means of the Human Skin Model Test.

Due to a borderline result in the 1st main experiment, a 2nd main experiment was performed according to the recommendation in the OECD TG 439 and to the Sponsor’s request.

The test item passed the MTT and colour interference pre-tests. Consequently, additional tests with freeze-killed or viable tissues were not necessary.

In both experiments each three tissues of the human skin model EpiSkin™ were treated with the test item, the negative or the positive control for 15 minutes.

The test item and the positive and negative controls were washed off the skin tissues after 15 minutes treatment, and after further incubation for approximately 42 hours (recovery period) the tissues were treated with the MTT solution for 3 hours following 2.5 to 3 hours extraction of the colorant from the cells. The amount of extracted colorant was determined photometrically at 570 nm.

After treatment with the negative control the absorbance values were well within the required acceptability criterion of mean OD³0.6 till ≤ 1.5 for the 15 minutes treatment interval (values between 0.633 and 1.044), thus showing the quality of the tissues.

Treatment with the positive control induced a decrease in the relative absorbance as compared to the negative control to 9.7% and to 8.3%, respectively, thus ensuring the validity of the test system.

The rel. standard deviations between the % variability values of the test item, the positive and negative controls were below 14% (threshold of the "OECD TG 439 Guideline for the Testing of Chemicals 439:In vitroSkin Irritation: Reconstructed Human Epidermis Test Method”: 18%), thus ensuring the validity of the study.

After treatment with the test item the mean relative absorbance value decreased to 49.6% in the 1^stmain experiment (threshold for irritancy: > 50%). Due to this borderline result, a 2^ndmain experiment was performed according to the Sponsor’s request and to the recommendation of the OECD TG 439. The determined tissue viability in the 2^ndmain experiment was 66.1%. This value is above the irritancy threshold. The Sponsor did not request performance of a 3^rdexperiment according to the recommendation in the OEDC TG 439 in case of discordant results between the first two runs. Therefore, prediction of the skin irritating potential of the test item cannot be made.

Possibly, the fact, that the test item could not be sufficiently removed from the tissues after exposure due to its sticky consistency, is responsible for the two different outcomes of the main experiments. Perhaps, the tissue surface was pasted by the test item in the 1^stmain experiment stronger than in the 2^ndmain experiment, therefore, the MTT assay resulted in a weaker reaction than in the 2^ndmain experiment. Additionally, it is imaginable, that the remaining test item prolonged the exposure period during 42 hours recovery time. In that case, in the 1^stmain experiment possibly a higher amount of the test item stick on the tissues than in the 2^ndmain experiment, leading to a higher level of irritating potential.

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, given that result of the 1st run is just slightly below the cut-off value and result of the 2nd run is clearly negative, it can be stated that the overall result is negative.

Executive summary:

Thisin vitrostudy was performed to assess the irritation potential of Lilial ME Anthranilate by means of the Human Skin Model Test according to OECD TG 439.

Due to borderline results in the 1^stmain experiment, the main experiment was performed twice according to the recommendation of the OECD TG 439.

The test item did not reduce MTT (pre-test for direct MTT reduction), and it did not dye deionised water when mixed with it (pre-test for colour interference). Consequently, additional tests with freeze-killed or viable tissuesto determine correction factors for calculating the true viability in the main experiment were not necessary.

In each main experiment three tissues of the human skin model EpiSkin™ were treated with the test item, the negative control (deionised water), or the positive control (5% sodium lauryl sulfate) for 15 minutes on triplicate tissues each.

After treatment with the negative control the absorbance values were well within the required acceptability criterion of mean OD³0.6 till ≤ 1.5 for the 15 minutes treatment interval in both main experiments, thus showing the quality of the tissues.

In both main experiments treatment with the positive control induced a sufficient decrease in the relative absorbance as compared to the negative control for the 15 minutes treatment interval thus ensuring the validity of the test system.

Main experiment 1:

After treatment with the test item the mean relative absorbance value decreased to 49.6%. This value is below the threshold for irritancy of ≤ 50%. The test item is considered to possess an irritant potential. Due to this borderline result, a 2^ndmain experiment was performed.

Main experiment 2:

After treatment with the test item the mean relative absorbance value decreased to 66.1%. This value is above the threshold for irritancy of ≤ 50%. The test item is not considered to possess an irritant potential. According to the recommendation of the OECD TG 439 a 3^rdmain experiment should be performed in case of inconsistent results for confirmation purpose. On request of the Sponsor, a 3^rdmain experiment was not performed.

In conclusion, given that result of the 1st run is just slightly below the cut-off value and result of the 2nd run is clearly negative, it can be stated that the overall result is negative.

Reference 2

Endpoint:

skin corrosion: in vitro / ex vivo

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

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:

12-12-2016 to 08-02-2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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)

GLP compliance:

yes (incl. QA statement)

Remarks:

Triskelion B.V., Utrechtseweg 48, 3700 AV, Zeist

Species:

other: Eyes of male or female chickens (ROSS, spring chickens)

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Slaughterhouse v.d. Bor, Nijkerkerveen, The Netherlands
- Characteristics of donor animals: Approximately 7 weeks old, male or female chickens, body weight range approximately 1.5-2.5 kg, were used as eye donors.
- Storage, temperature and transport conditions of ocular tissue: Heads of the animals were cut off immediately after sedation of the animals by electric shock and incision of the neck for bleeding, and before they reached the next station on the process line. The heads were placed in small plastic boxes on a bedding of paper tissues moistened with isotonic saline. Next, they were transported to the testing facility. During transportation, the heads were kept at ambient temperature.
- Time interval prior to initiating testing: Within 2 hours after kill, eyes were carefully dissected and placed in a superfusion apparatus.
- Indication of any existing defects or lesions in ocular tissue samples: No
- Indication of any antibiotics used: No

Vehicle:

unchanged (no vehicle)

Amount / concentration applied:

TEST MATERIAL
Because of its high viscosity the test substance was warmed to about 60 °C to facilitate collection by means of the syringe.

Details on study design:

SELECTION AND PREPARATION OF ISOLATED EYES
Within 2 hours after kill, eyes were carefully dissected and placed in a superfusion apparatus using the following procedure: First the eye-lids were carefully removed without damaging the cornea and a small drop of Fluorescein sodium 2.0% w/v was applied to the corneal surface for a few seconds and subsequently rinsed off with isotonic saline at ambient temperature. Next, the head with the fluorescein-treated cornea was examined with a slit-lamp microscope (Slit-lamp 900 BP, Haag-Streit AG, Liebefeld-Bern, Switzerland) to ensure that the cornea was not damaged. If undamaged (e.g., fluorescein retention and corneal opacity scores of ≤ 0.5), the eye was further dissected from the head without damaging the eye or cornea. Care was taken to remove the eye-ball from the orbit without cutting off the optical nerve too short. The enucleated eye was placed in a stainless steel clamp with the cornea positioned vertically and transferred to a chamber of the superfusion apparatus. The clamp holding the eye was positioned in such a way that the entire cornea was supplied with isotonic saline from a bent, stainless steel tube, at a target rate of 0.10-0.15 mL/min. The chambers of the superfusion apparatus as well as the saline were temperature controlled at approximately 32 °C (water pump set at 36.4 °C). After placing in the superfusion apparatus, the eyes were examined again with the slit-lamp microscope to ensure that they were not damaged. An accurate measurement was taken at the corneal apex of each eye. Eyes with a corneal thickness deviating more than 10% of the average corneal thickness of the eyes, eyes showing opacity (score higher than 0.5), or were unacceptably stained with fluorescein (score higher than 0.5) indicating the cornea to be permeable, or eyes that showed any other signs of damage, were rejected as test eyes and replaced.

EQUILIBRATION AND BASELINE RECORDINGS
Each eye provided its own baseline values for corneal swelling, corneal opacity and fluorescein retention. For that purpose, after an equilibration period of 45-60 minutes, the corneal thickness of the eyes was measured again to determine the zero reference value for corneal swelling calculations.

NUMBER OF REPLICATES
Negative control: 1
Positive control: 3
Test group: 3

NEGATIVE CONTROL USED
Physiological saline

POSITIVE CONTROL USED
Benzalkonium Chloride 5%

APPLICATION DOSE AND EXPOSURE TIME
30 μL for 10 seconds

OBSERVATION PERIOD
240 minutes

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: 20 mL saline. After rinsing, each eye in the holder was returned to its chamber.
- Indicate any deviation from test procedure in the Guideline: none

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Slit-lamp microscope examination
- Damage to epithelium based on fluorescein retention: Slit-lamp microscope examination
- Swelling: measured with optical pachymeter on a slit-lamp microscope; slit-width setting: set at 0.095 mm
- Others: After the final examination, the test substance treated eyes, the negative and positive control eyes were preserved in a neutral aqueous phosphate-buffered 4% solution of formaldehyde. The corneas were embedded in paraffin wax, sectioned at ca 4 μm and stained with PAS (Periodic Acid-Schiff). The microscopic slides were subjected to histopathological examination.

SCORING SYSTEM:
Defined scoring scales were used for each parameter to define the severity of effects into four categories (I-IV).
- Mean corneal swelling (%): According to OECD 438 guideline. Examination of the eyes after 0, 30, 75, 120, 180, and 240 minutes
- Mean maximum opacity score: According to OECD 438 guideline. Examination of the eyes after 0, 30, 75, 120, 180, and 240 minutes
- Mean fluorescein retention score at 30 minutes post-treatment: According to OECD 438 guideline.

DECISION CRITERIA: According to OECD 438 guideline

Irritation parameter:

percent corneal swelling

Run / experiment:

slit-lamp examination

Value:

4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: maximum mean values

Irritation parameter:

cornea opacity score

Run / experiment:

slit-lamp examination

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: maximum mean values

Irritation parameter:

fluorescein retention score

Run / experiment:

slit-lamp examination

Value:

0.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Slit-lamp examination: The test substance caused very slight corneal swelling (mean of 4%), no opacity and no or very slight fluorescein retention (mean of 0.3). The negative control eye did not show any corneal effect and demonstrated that the general conditions during the tests were adequate. The positive control BAC 5% caused severe corneal effects and demonstrated the ICE test valid to detect severe eye irritants.

Microscopic examination of the corneas treated with the test substance revealed very slight erosion of the epithelium in one cornea. Microscopic examination of the cornea treated with the negative control (saline) did not reveal any abnormalities. Microscopic examination of the corneas treated with the positive control BAC 5% revealed slight or severe erosion and very slight or slight vacuolation of the epithelium and endothelial necrosis (two corneas).

Interpretation of results:

other: Classification is not warranted according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.

Conclusions:

Under the test conditions (OECD 438 and GLP) the test substance is not considered to be an eye irritant in accordance with the criteria outlined in Annex I of the CLP regulations (1272/2008/EC).

Executive summary:

In accordance to OECD guideline 438 and GLP, the test substance was examined for its in vitro eye irritating potential using the Isolated Chicken Eye (ICE) Test. In the ICE test, 3 eyes were exposed to 30 µL test substance for 10 seconds. In addition, one negative control eye (30 µL saline) and three positive control eyes (30 µL Benzalkonium Chloride (BAC)) were tested. After the exposure the eyes were rinsed with 20 mL saline and were examined at approximately 0, 30, 75, 120, 180, and 240 minutes after treatment. The test substance caused very slight corneal swelling (mean of 4%), no opacity and no or very slight fluorescein retention (mean of 0.3). The negative control eye did not show any corneal effect and demonstrated that the general conditions during the tests were adequate. The positive control BAC 5% caused severe corneal effects and demonstrated the ICE test valid to detect severe eye irritants. Microscopic examination of the corneas treated with the test substance revealed very slight erosion of the epithelium in one cornea. Microscopic examination of the cornea treated with the negative control (saline) did not reveal any abnormalities. Microscopic examination of the corneas treated with the positive control BAC 5% revealed slight or severe erosion and very slight or slight vacuolation of the epithelium and endothelial necrosis (two corneas). Based on these results, the test substance is considered to be not eye irritating.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Additional information

Skin corrosion:

Corrosive properties are not anticipated due to absence of acids and base functional groups in its chemical structure and absence of eye irritation.

In vitro skin irritation:

The skin irritation/corrosion potential of the test substance, Lilial ME Anthranilate was assessed according to OECD Test Guideline 439 using a Reconstructed Human EpiDermis (RHE).

Eeach three tissues of the human skin model EpiSkin™ were treated with the test item, the negative or the positive control for 15 minutes.

The test item and the positive and negative controls were washed off the skin tissues after 15 minutes treatment, and after further incubation for approximately 42 hours (recovery period) the tissues were treated with the MTT solution for 3 hours following 2.5 to 3 hours extraction of the colorant from the cells. The amount of extracted colorant was determined photometrically at 570 nm.

After treatment with the negative control the absorbance values were well within the required acceptability criterion of mean OD³0.6 till ≤ 1.5 for the 15 minutes treatment interval (values between 0.633 and 1.044), thus showing the quality of the tissues.

Treatment with the positive control induced a decrease in the relative absorbance as compared to the negative control to 9.7% and to 8.3%, respectively, thus ensuring the validity of the test system.

The rel. standard deviations between the % variability values of the test item, the positive and negative controls were below 14% (threshold of the "OECD TG 439 Guideline for the Testing of Chemicals 439:In vitroSkin Irritation: Reconstructed Human Epidermis Test Method”: 18%), thus ensuring the validity of the study.

After treatment with the test item the mean relative absorbance value decreased to 49.6% in the 1^stmain experiment (threshold for irritancy: > 50%). Due to this borderline result, a 2^ndmain experiment was performed. The determined tissue viability in the 2^ndmain experiment was 66.1%. This value is above the irritancy threshold.

In conclusion, given that result of the 1st run is just slightly below the cut-off value and result of the 2nd run is clearly negative, it can be stated that the overall result is negative.

In vitro eye irritation:

In accordance to OECD guideline 438 and GLP, the test substance was examined for its in vitro eye irritating potential using the Isolated Chicken Eye (ICE) Test. In the ICE test, 3 eyes were exposed to 30 µL test substance for 10 seconds. In addition, one negative control eye (30 µL saline) and three positive control eyes (30 µL Benzalkonium Chloride (BAC)) were tested. After the exposure the eyes were rinsed with 20 mL saline and were examined at approximately 0, 30, 75, 120, 180, and 240 minutes after treatment. The test substance caused very slight corneal swelling (mean of 4%), no opacity and no or very slight fluorescein retention (mean of 0.3). The negative control eye did not show any corneal effect and demonstrated that the general conditions during the tests were adequate. The positive control BAC 5% caused severe corneal effects and demonstrated the ICE test valid to detect severe eye irritants. Microscopic examination of the corneas treated with the test substance revealed very slight erosion of the epithelium in one cornea. Microscopic examination of the cornea treated with the negative control (saline) did not reveal any abnormalities. Microscopic examination of the corneas treated with the positive control BAC 5% revealed slight or severe erosion and very slight or slight vacuolation of the epithelium and endothelial necrosis (two corneas).Based on these results, the test substance is considered to be not eye irritating.

Justification for classification or non-classification

Skin corrosion:Corrosive properties are not anticipated due to absence of acids and base functional groups in its chemical structure and absence of eye irritation.

Skin irritation: Based on the results of the in vitro skin irritation study which shows first result to be slightly below the cut-off value and 2nd run clearly negative it is concluded that the substance is negative for skin irritation. The substance does not need to be classified for skin irritation according to EU Classification, Labeling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008 and its amendments.

Eye irritation:Based on the negative results in the eye irritation test the substance does not need to be classified for this endpoint according to EU Classification, Labeling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008 and its amendments.