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Administrative data

Description of key information

Skin Irritation

The relative mean viability of the test item treated tissues was 4.7% after the 15 -minute exposure period.

Eye Irritation in vitro

Under the conditions of this study, the test material induced serious eye damage.

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:
6 June 2012 - 11 June 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
Recommended in international guidelines
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN™ Reconstructed Human Epidermis Model Kit, supplied by SkinEthic Laboratories, Lyon, France.
- Delivery date: 5 June 2012

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: Room temperature
- Temperature of post-treatment incubation: 37°C

TEST FOR DIRECT MTT REDUCTION
A test material may interfere with the MTT endpoint, if it is able to directly reduce MTT and at the same time is present on or in the tissues when the MTT viability test is performed. To identify this possible interference, the test material is checked for the ability to directly reduce MTT according to the following procedure: 10 µL of the test material was added to 2 mL of a 0.3 mg/mL MTT solution freshly prepared in assay medium. The solution was incubated in the dark at 37°C, 5% CO2 in air for 3 hours. Untreated MTT solution was used as a control. If the MTT solution containing the test material turns blue or purple, the test material is presumed to have reduced the MTT and the determination of skin irritation potential would be performed in parallel on viable and water killed tissues for quantitative correction of the results.


MAIN TEST

PRE-INCUBATION: DAY 0
2 mL of maintenance medium, warmed to approximately 37°C, was pipetted into the first column of 3 wells of a pre labelled 12 well plate. Each epidermis unit was transferred into the maintenance medium filled wells (3 units per plate). A different 12-well plate was used for the test material and each control material. The tissues were incubated at 37°C, 5 % CO2 in air overnight.

APPLICATION OF TEST MATERIAL: DAY 1
2 mL of maintenance medium, warmed to approximately 37°C, was pipetted into the second column of 3 wells of the 12 well plate. Triplicate tissues were treated with the test material for an exposure period of 15 minutes. The test material was applied topically to the corresponding tissues ensuring uniform covering. 10 µL of the test material was then applied to the epidermal surface. Triplicate tissues treated with 10 µL of DPBS served as the negative controls and triplicate tissues treated with 10 µL of SDS 5% w/v served as the positive controls. To ensure satisfactory contact with the positive control material the SDS solution was spread over the entire surface of the epidermis using a pipette tip (taking particular care to cover the center). After a 7 minute contact time the SDS solution was re spread with a pipette tip to maintain the distribution of the SDS for the remainder of the contact period (re-spreading is not required for the negative control or test material). The plates were kept in the biological safety cabinet at room temperature for 15 minutes.

REMOVAL OF TEST MATERIAL AND CONTROLS
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 Ca²+ and Mg²+. 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 material. The rinsed tissues were transferred to the second column of 3 wells containing 2 mL of maintenance medium in each well. The rinsed tissues were incubated at 37°C, 5% CO2 in air for 42 hours.

MTT LOADING/ FORMAZAN EXTRACTION: DAY 3
Following the 42 hour post-exposure incubation period each 12-well plate was placed onto a plate shaker for 15 minutes to homogenise the released mediators in the maintenance medium. 1.6 mL of the maintenance medium from beneath each tissue was transferred to pre labelled micro tubes and stored in a freezer at 14 to 30°C for possible inflammatory mediator determination.
2 mL of a 0.3 mg/mL MTT solution, freshly prepared in assay medium, was pipetted into the third column of 3 wells of the 12-well plates. The tissues were transferred to the MTT filled wells, being careful to remove any excess maintenance medium from the bottom of the tissue insert by blotting on absorbent paper. The tissues were incubated for 3 hours at 37°C, 5% CO2 in air. At the end of the 3 hour incubation period each tissue was placed onto absorbent paper to dry. A total biopsy of the epidermis was made using the EPISKIN™ biopsy punch. The epidermis was carefully separated from the collagen matrix using forceps and both parts (epidermis and collagen matrix) placed into labelled 1.5 mL micro tubes containing 500 µL of acidified isopropanol, ensuring that both the epidermis and collagen matrix were fully immersed. Each tube was plugged to prevent evaporation and mixed thoroughly on a vortex mixer. The tubes were refrigerated at 1 to 10°C until day 6 of the experiment, allowing the extraction of formazan crystals out of the MTT-loaded tissues.

ABSORBANCE/ OPTICAL DENSITY MEASUREMENTS: DAY 6
At the end of the formazan extraction period each tube was mixed thoroughly on a vortex mixer to produce a homogenous coloured solution. For each tissue, duplicate 200 µL samples were transferred to the appropriate wells of a pre labelled 96 well plate. 200 µL of acidified isopropanol alone was added to the two wells designated as ‘blanks’. The optical density was measured (quantitative viability analysis) at 540 nm (without a reference filter) using the Anthos 2001 microplate reader.

NUMBER OF REPLICATE TISSUES: 3

DATA EVALUATION
Quantitative MTT Assessment (Percentage Tissue Viability)
For the test material the relative mean tissue viabilities obtained after the 15 minute exposure period followed by the 42 hour post exposure incubation period were compared to the mean of the negative control treated tissues (n=3). The relative mean viabilities were calculated in the following way:
Relative mean viability (%) = (mean OD562 of test material / mean OD562 of negative control) x100
Classification criteria for in vitro interpretation:
Relative mean tissue viability is ≤ 50%: Irritant,
Relative mean tissue viability is > 50%: Non-irritant.

QUALITY CRITERIA
- The assay establishes the acceptance criterion for an acceptable test if the relative mean tissue viability for the positive control treated tissues is ≤ 40% relative to the negative control treated tissues, and the standard deviation (SD) value of the percentage viability is ≤ 18 %.
- The assay establishes the acceptance criterion for an acceptable test if the mean OD562 for the negative control treated tissues is ≥ 0.6 and ≤ 1.5, and the SD value of the percentage viability is ≤ 18%.
- The assay establishes the acceptance criterion for an acceptable test if the standard deviation calculated from individual percentage tissue viabilities of the three identically treated tissues is ≤ 18%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount applied: 10 µL

NEGATIVE CONTROL
- Amount(s) applied: 10 µL

POSITIVE CONTROL
- Amount applied: 10 µL
- Concentration: 5 % w/v
Duration of treatment / exposure:
15 minutes
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean
Value:
4.7
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid

Table 1 Mean OD540 Value and Percentage Viabilities for the Negative Control Item, Positive Control Item and the Test Item

Item

OD540 of Tissues

Mean OD540 of Triplicate Tissues

± SD of OD540

Relative Individual Tissue Viability (%)

Relative Mean Viability (%)

± SD of mean Relative Viability (%)

Negative Control

0.846

 

0.791

 

0.056

107.0

 

100*

 

7.1

0.793

100.3

0.734

92.8

Positive Control

0.043

 

0.036

 

0.006

5.4

 

4.5

 

0.8

0.031

3.9

0.034

4.3

Test Material

0.041

 

0.037

 

0.004

5.2

 

4.7

 

0.5

0.033

4.2

0.036

4.6

SD = Standard Deviation

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

Interpretation of results:
Category 2 (irritant) based on GHS criteria
Conclusions:
Under the conditions of the study the test material requires classification as a skin irritant (category 2) in accordance with EU criteria.
Executive summary:

The skin irritation potential of the test material was determined in accordance with the standardised guidelines OECD 439 and EU Method B.46. under GLP conditions using a human skin model.

The purpose of this test was to evaluate the skin irritation potential of the test material using the EPISKINreconstructed human epidermis model after a treatment period of 15 minutes followed by a postexposure incubation period of 42 hours. 

During the study, triplicate tissues were treated with the test material for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 hours. The test material was found to have the potential to cause colour interference with the MTT endpoint therefore additional tissues were incorporated into the testing to correct for this. At the end of the postexposure incubation period each tissue was taken for MTT-loading. The maintenance medium from beneath each tissue was transferred to prelabelled 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 MTTloaded 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 prelabelled 96well plate. The optical density was measured at 540 nm.

The relative mean viability of the test material treated tissues was 4.7% after the 15‑minute exposure period and 42‑hours postexposure incubation period. The quality criteria required for acceptance of results in the test were satisfied.

Under the conditions of the study the test material requires classification as a skin irritant (category 2) in accordance with EU criteria.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (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:
19 January 2017 - 02 February 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Compliant to GLP and testing guidelines; adequate consistence between data, comments and conclusions.
Qualifier:
according to
Guideline:
other: EU method B.47 (In vitro eye irritation)
Qualifier:
according to
Guideline:
other: OECD guideline 437 (In vitro eye irritation)
GLP compliance:
yes (incl. certificate)
Species:
other: bovine cornea
Strain:
other: not applicable
Details on test animals or tissues and environmental conditions:
Species: bovine cattle.
Origin: bovine eyes were obtained from freshly slaughtered cattle at the abattoir EVA, Saint Pierre sur Dives, France.
Age: as French Authorities avoid the use of any organs from the head of bovines aged more than 12 months, bovine cattle were up to 12 months old (typically, 5 to 8 months old).
Reason for choice: bovine corneas are recommended by Regulatory Authorities for this type of study. They are adapted for the evaluation of potential ocular irritants since they are part of the target organ.
Transport from Supplier to CiToxLAB France: the eyes were transported to CiToxLAB France at ambient temperature, immerged in buffered Hanks medium containing an antibiotic [Hank’s Balanced Salts Solution (HBSS) plus penicillin/streptomycin (100 units/100 µg/mL final)]. A container with smooth internal surfaces was used for the transport to avoid damage to the corneas.
Upon arrival at CiToxLAB France, the selection and preparation of corneas was performed as soon as possible. At each step of the preparation procedure, care was taken to avoid touching the corneas in order not to damage them.

Selection: a careful macroscopic examination was performed on all eyes to detect the presence of any defects (opacity, scratches, pigmentation, etc). Any eyes with defects were discarded. The examination was performed under a lamp, using HBSS in order to keep the eyes moistened and shiny. Particular attention was paid to the corneas and the eyes were swivelled in order to observe the fringe areas and any scratches directly under the light.
Preparation of the selected corneas: the tissues surrounding the eyeball were carefully pulled away and the cornea, surrounded by approximately 2 to 3 mm of sclera, was dissected out. The isolated corneas were stored in HBSS until all corneas had been prepared.
Washing of the corneas: the corneas were washed for 15 minutes, three times, in HBSS plus penicillin/streptomycin (100 units/100 µg/mL final) at room temperature. The corneas were used within a maximum of 24 hours.
Storage of the corneas: as the corneas were not used immediately, they were stored after washing. Each cornea was stored individually in 12 mL of M199 medium containing 5% dextran, plus penicillin/streptomycin, at +4°C, for a maximum of 24 hours before use.

(Pre)Incubation T°C: 32°C
Dates of experimental phase: from 30 January 2017 to 02 February 2017.
Vehicle:
unchanged (no vehicle)
Controls:
other: in vitro negative and positive controls
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 µL
- Concentration (if solution): undiluted.

VEHICLE
- Amount(s) applied (volume or weight with unit): not applicable
- Concentration (if solution): not applicable
- Lot/batch no. (if required): not applicable
- Purity: not applicable.
Duration of treatment / exposure:
Exposure period of 10 minutes (+/- 30 seconds), followed by rinsing.
Observation period (in vivo):
Opacity measurement:
- before treatment
- after 2-hour incubation in water

Permeability measurement:
- after 90-min incubation in water (and other procedures), following the 2nd opacity measurement
Number of animals or in vitro replicates:
Not applicable
Triplicate corneas for each timepoint and tested substance (test item, negative control, positive control)
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Rinsing: the anterior part of the eye was emptied and then rinsed 7 times with pre-warmed cMEM containing phenol red. Despite the rinsing performed, residual test item was noted over each treated cornea.
NEGATIVE CONTROL:
As the test item was tested undiluted (i.e. in its original form), 0.9% Sodium Chloride (0.9% NaCl) was used as negative control.
Since several test items were assayed concurrently, the negative control was shared.

POSITIVE CONTROL:
As the test item was tested using a 10-minute treatment, the positive control was Absolute Ethanol. It was used neat and sampled on the day of use.
Since several test items were assayed concurrently, the positive control was shared.

SCORING SYSTEM/TOOL
- Opacity:
Using an opacitometer
The average change in opacity during exposure is determined. It is corrected by subtracting the average negative control value from values in positive control and test item.
- Permeability:
Using a spectrophotometer: optical density (OD) at 490 nm wavelength
The optical density is corrected by subtracting the average negative control value from values in positive control and test item.
- Scoring:
In vitro irritancy score (IVIS) = Corrected Opacity + (15 x Corrected OD)

Interpretation: see below
Irritation parameter:
in vitro irritation score
Value:
77
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Interpretation of results:
Category 1 (irreversible effects on the eye) based on GHS criteria
Conclusions:
The test item was identified as a test chemical inducing serious eye damage (UN GHS Category 1).

Executive summary:

The objective of this study was to evaluate the potential irritant and corrosive properties of the test item, COOLACT 10, to the eye. The Bovine Corneal Opacity and Permeability (BCOP) test method can identify chemicals inducing serious eye damage and chemicals not requiring classification for eye irritation or serious eye damage.

The design of this study was based on the guideline OECD Guideline 437 and the study was performedin compliance with CiToxLAB France standard operating procedures and with the OECD Principles of Good Laboratory Practice.

 

Method

Corneas obtained from freshly slaughtered calves were mounted in corneal holders. Both chambers of the corneal holder were filled with complemented MEM culture media (cMEM) and pre-incubated for 1 hour and 5 minutes (± 5 minutes) at +32°C.

A single experiment was performed using three corneas for each treated series (test item, positive and negative controls).

Before the treatment, a first opacity measurement was performed on each cornea using an opacitometer.

The test item was applied undiluted, in a single experiment using a treatment time of 10 minutes and using the open-chamber method. Negative and positive controls were applied using the same treatment time and using the closed-chamber method. At the completion of the treatment period, all items were removed from the front opening of the anterior chamber and the epithelia were rinsed.

The corneas were then incubated for 2 hours (± 10 minutes) at +32°C before a second opacity measurement was performed.

After the second opacity measurement, the medium of the anterior chamber was removed and filled with a fluorescein solution. The holders were then incubated vertically for 90 minutes (± 5 minutes) at +32°C.

At the end of the incubation period, the optical density of the solution from the posterior chamber of each holder was measured in order to determine the permeability of the cornea. Each cornea was then observed for opaque spots and other irregularities.

 

Results

Macroscopic examination

Opacity and fluorescein fixation were observed on the corneas treated with the test item.

 

In Vitro Irritancy Score

All acceptance criteria were fulfilled. The study was therefore considered as valid.

The mean In Vitro Irritancy Score (IVIS) of the test item-treated corneas was: 77.

 

As the mean IVIS was > 55, the test item was considered asa testchemicalinducing serious eye damage (UN GHS Category 1).

Conclusion

Under the experimental conditions of this study, the test item was identified asinducing serious eye damage (UN GHS Category 1).

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

SKIN IRRITATION

The key study was carried out to evaluate the skin irritation potential of the substance using the EPISKIN reconstructed human epidermis model in accordance with OECD Guidelines for the Testing of Chemicals No. 439 “In Vitro Skin Irritation” (adopted 22 July 2010) and Method B.46 of Commission Regulation (EC) No. 440/2008/EC.

 

The test consisted of 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.

The relative mean viability of the test item treated tissues was 4.7 % after the 15-Minute exposure period.

 

Supporting information is provided in the form of two studies conducted on formulations containing the substance and a prediction calculated by the OECD QSAR toolbox.

In the first supporting study, the primary skin irritation of the test material was assessed in Dunkin-Hartley guinea pigs. The guinea-pigs were exposed to a 5, 10, 30 and 50 % concentration in acetone at four sites on the flank. Readings of dermal reactions were taken at 24 and 48 hours. Under the conditions of the test, the solutions of the test material tested were found to be non-irritating. Minor reactions were noted in two guinea pigs at 24 hours at a 50 % concentration, however, these were very light. The study did not follow a guideline and due to being performed with a preparation was assigned a reliability score of 3 in accordance with the criteria set forth by Klimisch (1997).

 

In the second supporting study, the cumulative skin irritation of the test material was assessed over 21 days, at 0, 1, 2.5, 5 and 10 % in ethanol in Albino rabbits. The test was performed in accordance with the method of Marzulli et al (1975) Fd. Cosmet. Toxicol. 13: 533-540. The test material was applied to five open sites on the shaved back on each of six rabbits. Any reactions were scored, and the total cumulative irritation of all the rabbits at each concentration was calculated. Under the conditions of the test, no reactions were noted with the test material dosed for 21 days up to 2.5 %. The mean cumulative scores for the 10 and 5 % concentrations (the concentrations at which the only reactions were observed) were 7.7 and 1.7, respectively out of a maximum of 84. The study did not follow a guideline and due to being performed with a preparation was assigned a reliability score of 3 in accordance with the criteria set forth by Klimisch (1997).

 

In the final piece of supporting information, the severe skin irritation potential of the substance was evaluated using the Severe Skin Irritation Model from the Danish EPA pre-programmed into the OECD QSAR Toolbox version 3.0.0.995. The substance was within the prediction domain of the model.

From the available data, it can be concluded that the substance is not anticipated to be a severe skin irritant; it is therefore anticipated that the substance is irritating to rabbit skin, but not corrosive.

Justification for selection of skin irritation / corrosion endpoint:

The key study was conducted in accordance with the standardised guidelines OECD 439 and EU Method B.46 under GLP conditions. In accordance with the criteria of Klimisch (1997) it was awarded a reliability score of 1. As such, it is considered an accurate assessment of the irritancy potential of the substance.

Effects on skin irritation/corrosion: irritating

EYE IRRITATION

Eye Irritation (in vitro)

The potential of the test material to cause eye damage was determined in accordance with the standardised guideline OECD 437, under GLP conditions. The irritat and corrosive properties of the test material to the eye were evaluated using the Bovine Corneal Opacity and Permeability (BCOP) test. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Corneas obtained from freshly slaughtered calves were mounted in corneal holders. Both chambers of the corneal holder were filled with complemented MEM culture media (cMEM) and pre-incubated for 1 hour and 5 minutes ± 5 minutes at 32 °C. A single experiment was performed using three corneas for each treated series (test material, positive and negative controls). Before the treatment, a first opacity measurement was performed on each cornea using an opacitometer. The test material was applied undiluted, in a single experiment using a treatment time of 10 minutes and using the open-chamber method. Negative and positive controls were applied using the same treatment time and using the closed-chamber method. At the completion of the treatment period, all items were removed from the front opening of the anterior chamber and the epithelia were rinsed. The corneas were then incubated for 2 hours ± 10 minutes at 32 °C before a second opacity measurement was performed. After the second opacity measurement, the medium of the anterior chamber was removed and filled with a fluorescein solution. The holders were then incubated vertically for 90 ± 5 minutes at 32 °C. At the end of the incubation period, the optical density of the solution from the posterior chamber of each holder was measured in order to determine the permeability of the cornea. Each cornea was then observed for opaque spots and other irregularities.

Opacity and fluorescein fixation were observed on the corneas treated with the test material. The mean In Vitro Irritancy Score (IVIS) of the test material-treated corneas was: 77. As the mean IVIS was > 55, the test item was considered as a test chemical inducing serious eye damage.

All acceptance criteria were fulfilled and the study was therefore considered as valid.

Under the conditions of this study, the test material induced serious eye damage.

Supporting information is provided in the form of two studies conducted on formulations containing the substance. Both supporting studies were in vivo studies conducted in accordance with the standardised guideline OECD 405.

In the first supporting study, the irritation potential of the test material as a 1.0 % formulation in olive oil to the rabbit eye was assessed in a study conducted in accordance with the standardised guideline OECD 405. Under the conditions of the test, no irritation was observed in any animal at any of the observation time points.

In the second supporting study, the irritation potential of the test substance as a 10 % formulation in olive oil to the rabbit eye was assessed in a study conducted in accordance with the standardised OECD guideline at the time. Under the conditions of the test, no irritation was observed in any animal.

Although the supporting studies were conducted in accordance with standardised guidelines they were conducted on formulations containing the substance at 1.0% and 10%, respectively. As such it is not possible to ascertain the effects of the neat substance and therefore the studies cannot be regarded as being reliable.

Justification for selection of eye irritation endpoint:

The key study was conducted in accordance with the standardised guidelines OECD 437 under GLP conditions. In accordance with the criteria of Klimisch (1997) it was awarded a reliability score of 1. As such, it is considered an accurate assessment of the irritancy potential of the substance.

Effects on eye irritation: serious eye damage (category 1)

Justification for classification or non-classification

Skin Irritation

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance requires classification with respect to skin irritation as Category 2 (H315: Causes skin irritation).

Eye Irritation

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance requires classification with respect to eye irritation as Category 1 (H318: Causes serious eye damage).