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Description of key information

An in vitro skin corrosion (equivalent to OECD 431) and skin irritation assay (OECD 439) are available for the substance. For the eye irritation endpoint an in vitro study is available (OECD 437) alongside considerations for waiving further work. No studies are available to assess respiratory irritation.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Report date: 17 November 2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP compliance
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)
GLP compliance:
yes (incl. QA statement)
Remarks:
(13 February 2003)
Test system:
human skin model
Source species:
human
Cell type:
other: epithelial and formed into a stratified, cornified epithelium
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm Skin Model
- Tissue batch number(s): 6616 Kit D
- Date received: 3 August 2005
- Supplier: MatTek Corporation, Ashland, MA, USA

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37°C

ASSESSMENT OF DIRECT TEST MATERIAL REDUCTION OF MTT
A test substance may directly reduce MTT, thus mimicking the dehydrogenase activity of the cellular mitochondria. This property of the test substance is only a problem if, at the time of the MTT test (after the chemical has been rinsed off), there are still sufficient amounts of the test substance present on (or in) the tissues. To identify this possible interference, the test material was checked for its ability to reduce MTT directly.
Using a positive displacement pipette 100 μL of the test material was added to 900 μL of a 1.0 mg/mL MTT solution freshly prepared in assay medium and incubated at 37°C for 60 minutes. A negative control, 100 μL of sterile distilled water, was tested concurrently. If the MTT solution colour turned blue/purple (darkening) relative to the negative control, the test material was presumed to have reduced the MTT. Water insoluble test materials may show direct reduction only at the interface between the test material and the medium.

PREPARATION OF EPIDERM
The assay medium was pre-warmed to 37°C. Using sterile techniques, 900 μL of this assay medium was pipetted into the appropriate wells of pre-labelled 6-well plates for both the 3-minute and 60 minute exposures. Each well was labelled with details of the material to be applied to the tissue, and the exposure time to be used. EpiDerm tissues were transferred into the 6-well plates containing the pre-warmed assay medium 1 hour before dosing. This transfer was done under aseptic conditions in the biological safety cabinet, using sterile forceps. Care was taken to remove all maintenance agarose sticking to the outside of the cell culture inserts containing the EpiDerm tissues. The 6-well plates containing the EpiDerm samples were placed in an incubator at 37°C, 5% C02 for approximately 1 hour before dosing.

MAIN TEST
Before pre-incubation was complete, a 24-well plate was prepared for use as a "holding plate" for both the 3 minute and 60 minute exposures. This plate was used to maintain the viability of the tissue inserts between rinsing following chemical exposure and MTT loading. Another 24-well plate was prepared for the MTT loading. 300 μL of either pre-warmed assay medium (holding plate) or 300 μL of a 1.0 mg/ml MTT solution (MTT loading plate) was dispensed into each well using a pipette. The two plates were placed in the incubator in a humidified atmosphere (37°C, 5% C02) until required.

After pre-incubation was complete, the medium was aspirated and replaced with 0.9 mL of fresh assay medium. For the 60-minute exposure, 50 μL of sterile distilled water (negative control) was added to the first two tissues and the timer started. For the test material and the positive control material (8.0 N Potassium Hydroxide) 50 μL was also applied to the corresponding tissues.

The plate was placed back in the incubator in a humidified atmosphere (37°C, 5% C02) until the 60-minute exposure was complete.

When dosing for the 60-minute exposure was complete, the same procedure was repeated for the 3-minute exposure. Because the exposure time was so short, the tissues were dosed at regular intervals to allow for the time taken to rinse each insert following exposure and to ensure that each tissue received an equal exposure time. Rinsing was achieved by filling and emptying each tissue insert approximately 20 times using a constant soft stream of PBS to gently remove any residual test material. Excess PBS was removed by blotting the bottom of the insert with tissue paper. Each insert was placed in the prepared holding plate until all inserts were rinsed. They were then blotted and transferred to the 24-well plate prepared for MTT loading. The plate was incubated for 3 hours (37°C, 5% C02). The same rinsing and MTT loading procedure was repeated for the 60-minute plate when the exposure was complete.

After the 3-hour incubation was complete, the inserts were blotted and transferred to pre-labelled 24-well plates for MTT extraction. 2 mL of MTT extractant solution (isopropanol) was used to completely immerse each insert and the plate was covered with Nescofilm ™ to prevent isopropanol evaporation. The plates were placed in a refrigerator overnight to allow extraction to proceed.

After the extraction period was complete, the inserts were removed using forceps, and their contents were decanted back into the well that the insert came from. The contents of each well were mixed thoroughly using a pipette and for each tissue 3 x 200 μL aliquots of the blue formazan solution were transferred to the appropriate wells of a pre-labelled 96-well plate. 200 μL of isopropanol alone was added to the two wells designated as negative controls. The absorbency at 540 nm (OD540) of each well was measured using the Anthos 2001 microplate reader (Anthos Labtec Instruments, Salzburg, Austria).

NUMBER OF REPLICATE TISSUES: 2

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is 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 less than 15%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
yes, concurrent MTT non-specific colour control
Amount/concentration applied:
50 µL
Duration of treatment / exposure:
3-minute or 60-minute exposure
Number of replicates:
2 tissues
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3-minute exposure
Value:
83.6
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
60-minute exposure
Value:
66.1
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
QUALITY CRITERIA
The mean OD540 of the two negative control tissues were 1.895 for the 3-minute exposure and 1.824 for the 60-minute exposure. These both met test acceptance criteria (i.e. mean OD540 ≥ 0.8).
Potassium Hydroxide as an 8.0 N solution was used as a positive reference. A 3-minute application of 8.0 N Potassium Hydroxide revealed a mean relative tissue viability of 13.6%. The assay met the acceptance criteria (i.e. mean relative tissue viability of the 3 minute positive control was ≤ 30% ).
In addition to the positive and negative control quality criteria, a difference of > 30% between two tissues treated identically is regarded as a rejection criterion and re-testing of the chemical is recommended if the resulting viability is near to a classification eut-off. For all duplicate tissues in this study, the difference in viabilities between tissues was ≤ 30% in all cases.

Table 1: OD540 Values and % viabilities for the negative control, positive control and test material

Material

Exposure Time

OD5401

Mean OD5402

% Viability3

Mean % Viability4

Negative Control

3 Minutes

1.824

1.895 ± 0.100

N/A

100*

1.965

60 Minutes

1.773

1.824 ± 0.071

100*

1.874

Positive Control

3 Minutes

0.297

0.258 ± 0.056

15.7

13.6 ± 3.0

0.218

11.5

60 Minutes

0.116

0.118 ± 0.002

6.4

6.5 ± 0.1

0.119

6.5

Test Material

3 Minutes

1.601

1.583 ± 0.025

84.5

83.6 ± 1.3

1.565

82.6

60 Minutes

1.143

1.205 ± 0.087

62.7

66.1 ± 4.7

1.266

69.4

1 = OD540 representing each EpiDerm tissue

2 = Mean OD540 of duplicate EpiDerm tissues ± standard deviation

3 = Viability representing each EpiDerm tissue expressed as a percentage of the mean OD540 of either the 3 minute or 60 minute negative control tissue

4 = Mean viability of duplicate treated EpiDerm tissues ± standard deviation

* = Mean viability of negative control tissues set at 100%

Interpretation of results:
other: non-corrosive
Conclusions:
The relative mean viability of the test material-treated tissues was ≥ 50% after the 3-minute exposure and ≥ 15% after the 60-minute exposure.
Monofluoroethylene carbonate was considered to be non-corrosive.
Executive summary:

The study was performed to assess the corrosivity potential of the test material using the EpiDerm Skin Model (MatTek, Ashland, MA, USA). The test is based on the hypothesis that corrosivity potential is related to toxicity to the EpiDerm tissue. The study was validated by the inclusion of a positive control material, 8.0 N Potassium Hydroxide.

The experimental design of the study consists of a test for Direct Reduction of MTT by the test material, followed by the main test.

For the main test, duplicate EpiDerm tissues were treated with 50 μL of Monofluoroethylene carbonate and exposed for 3 minutes and 60 minutes. The tissues were incubated at 37°C in a humidified atmosphere of 5% C02 in air for the appropriate exposure times.

Duplicate negative and positive control tissues, treated with 50 μL sterile distilled water or 50 μL 8.0 N Potassium Hydroxide respectively, were also exposed for 3 minutes and 60 minutes.

At the end of the exposure period each EpiDerm tissue was rinsed using Dulbecco' s phosphate buffered saline (DPBS) and placed into a 'holding plate', until all of the tissues had been treated and rinsed. They were then transferred to an MTT 'loading plate', and incubated at 37°C for 3 hours in a humidified atmosphere of 5% C02 in air. At the end of this time, each EpiDerm tissue was blotted dry and placed into an MTT 'extraction plate' in order to extract all of the reduced MTT from the tissues.

At the end of the extraction period, the extracted MTT solution was mixed for each EpiDerm tissue and 3 x 200 μL samples for each tissue were transferred to the appropriate wells of a 96 well plate. The absorbency at 540nm (OD540) of each well was measured with the Anthos 2001 microplate reader. Data are presented in the form of % viability (MTT conversion relative to negative controls) for each of the two exposure times.

The relative mean viability of Monofluoroethylene carbonate treated tissues was 83 .6% after 3 minutes exposure and 66.1 % after 60 minutes exposure.

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

Monofluoroethylene carbonate was considered to be non-corrosive in vivo.

Endpoint:
skin irritation: 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
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
GLP compliance:
yes
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
foreskin from a single donor
Source strain:
not specified
Vehicle:
water
Details on test system:
TEST SYSTEM:
LabCyte EPI-MODEL24
3D human epidermis model
Model was stored at rooom temperature until culture.

PREPARATION OF LabCyte EPI-MODEL 24 (Day -1):
- Assay medium warmed for ca. 30 minutes to 37°C
- assay medium (0.5mL/well) add to the first row of 24-well assay plate
- epidermis tissues were transferred into assay medium filled wells of the 1st row
- plate included overnight in CO2 incubator

APPLICATION OF TEST SUBSTANCES AND RINSING:
- 25 microlitre of the test substance and control were applied to the surface of the epidermis tissues using a micropipette
- Assay plate tilted to allow liquid to spread over surface of tissues and allowed to stand for 15 minutes at room temperature
- After 15 minutes, the tissues were removed and rinsed with wash buffer
- Rinsing was repeated 15 times so all residual test substance or controls was removed from epidermis surface

POST-INCUBATION (Day 0 - 2)
- Tissues were transferred to assay plate with fresh assay medium (3rd row of plate)
- Allowed to stand in CO2 incubator for 42 hours

MTT ASSAY (Day 2)
- MTT medium warmed for ca. 30 minutes to 37°C
- Assay plates removed from incubator
- MTT medium added to assay plate (4th row) and tissues transferred into MTT medium
- Allowed to stand in CO2 incubator for 3 hours

FORMAZAN EXTRACTION AND MEASUREMENT (Day 2 - 3)
- Tissues removed from MTT medium and transferred into 1.5mL microtube
- Isopropanol (300 microlitre) was added to each microtube to soal the entire tissue
- Microtubes incubated in the refigerator overnight in order to extract pigments

OPTICAL DENSITY MEASUREMENTS OF EXTRACTS
- Microtubes were shaken to mix the solution, 200 microlitres transferred to 96-well plate
- Optical density measured using a microplate reader (wavelength: 570nm and 650 nm)
- measured OD was determined by substracting 650 nm OD from 570 nm OD

CALCULATION OF TISSUE VIABILITY
Tissue viability (%) = mean measured OD/ Mean measured OD (negative contol) x 100

DECISION CRITERIA
Tissue viability classifiaction:
- Tissue viability is - Tissue viability is > 50%; non-irritant
Control samples:
yes, concurrent vehicle
yes, concurrent positive control
Amount/concentration applied:
25 microlitres
Duration of treatment / exposure:
15 minutes
Duration of post-treatment incubation (if applicable):
42 hours after rinsing
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Remarks:
mean
Value:
17.7
Vehicle controls validity:
valid
Positive controls validity:
valid
Remarks:
2.7% tissue viability
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: no
- Colour interference with MTT: no

DEMONSTRATION OF TECHNICAL PROFICIENCY:
The standard deviation value of three tissue viabilities for the positive control and negative control was less than 3%, indicating that the system functioned properly

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

Table 1: Individual OD values mean OD and individual tissue viability

 Treatment

 Individual OD

(570/650 nm)

Mean OD

(570/650 nm) 

 Individual tissue viability

(% of control)

 Standard deviation

(%)

Negative control 
0.8954  0.9267        96.6  3.0       
 0.9344  100.8
 0.9503  102.5

 Test substance

 0.1449

 

 

0.1636 

 

 

 15.6

1.8 

 0.1721

18.6 

 0.1739

 18.8

 Positive control

 0.0212

 

 

 0.0247

 

 

 2.3

 

 

 

0.3 

 

 0.0255

 2.8

 0.274

 3.0

Interpretation of results:
Category 2 (irritant) based on GHS criteria
Conclusions:
It was concluded that the test substance is irritant in the in vitro skin irritation test under the experimental conditions of the test.
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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
Deviations:
no
GLP compliance:
yes
Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Slaughterhouse (Kumamoto Chuou Meat Centre Co., Ltd, 548, Subayashi, Toyono-machi, Uki-shi, Kumamoto 861-4307, Japan
- Number of animals: Fifteen eyes excised from animals
- Characteristics of donor animals (e.g. age, sex, weight): 23 to 25 months old.
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Transported in a cooler with Hanks' balanced salt solution.
- Time interval prior to initiating testing: Used on day of receipt
- indication of any existing defects or lesions in ocular tissue samples: There were no eyes exhibiting defects.
- Indication of any antibiotics used: None
Vehicle:
physiological saline
Remarks:
Isotonic sodium chloride solution
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 µL (test substance heated so tested in liquid form)
- Concentration (if solution): NA used as supplied

VEHICLE
- Amount(s) applied (volume or weight with unit): 750 µL
- Concentration (if solution): NA used as supplied
Duration of treatment / exposure:
Corneas were incubated for 10 minutes
Duration of post- treatment incubation (in vitro):
2 hours in a low temperature incubator
Number of animals or in vitro replicates:
Three corneas were selected for each group
Details on study design:
SELECTION AND PREPARATION OF CORNEAS : The corneal holder, MEM and cMEM were warmed in a water batch until use. Corneas were dissected with a 2 to 3 mm rim of sclera remaining. Isolated corneas were mounted in corneal holders with anterior and posterior compartments which interface with epithelial and endothelial slides of the cornea, respectively. Both compartments were filled with cMEM. Corneas were incubated for 1 hour in a low temperature incubator (set to 32°C). After the incubation period, the medium was removed from both compartments and replaced with fresh cMEM. Opacity determinations were performed on each of the corneas using an opacitometer. The opacity of each cornea was read against a cMEM filled chamber, and the initial opacity was calculated according to the equation in the test guideline. Corneas that had an initial opacity reading higher than 7 were not used. Nine corneas were chosen for the experiment excluding those with high opacity.

QUALITY CHECK OF THE ISOLATED CORNEAS: The eyes were checked for unacceptable defects such as opacity, scratches and neovascularization before use.

NUMBER OF REPLICATES : Three corneas were selected for each group

NEGATIVE CONTROL USED: Physiological saline

POSITIVE CONTROL USED : N,N-dimethylformamide

APPLICATION DOSE AND EXPOSURE TIME : 700 µL (10 minutes exposure time)

TREATMENT METHOD: closed chamber

POST-INCUBATION PERIOD: After the exposure incubation corneas were washed three times with MEM after which the corneas were rinsed a final time with cMEM. These were then incubated for a further 2 hours in a low temperature incubation (set at 32°C).

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: 3 washing steps

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Opacity determinations were performed using an opacitometer
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader (OD490)
- Others (e.g, pertinent visual observations, histopathology): Each cornea was visually observed (e.g. tissue peeling, residual test substance, non-uniform opacity patterns).

SCORING SYSTEM: The final opacity for each cornea was calculated by subtracting the initial opacity reading from the post-treatment reading. The mean of the final opacity value of each treatment group was calculated. To calculate the IVIS score, values obtained by subtracting the mean final opacity of the negative control from that of the test substance or positive control were used for the calculation. For permeability each value (OD490) was corrected for the background value (OD490 of cMEM only).

DECISION CRITERIA: Decision criteria as per the test guideline used.
Irritation parameter:
in vitro irritation score
Run / experiment:
2
Value:
>= 10.9 - <= 11.3
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
IVIS scores of 0.2 to 1.1
Positive controls validity:
valid
Remarks:
IVIS scores of 92.9 to 99.7
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: None noted

DEMONSTRATION OF TECHNICAL PROFICIENCY: Results as expected based on historical control data for BCOP studies

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: NA
Interpretation of results:
study cannot be used for classification
Conclusions:
Since the test substance induced an IVIS score > 3 and < 55, no prediction could be made for eye irritation or serious eye damage.
Endpoint:
eye irritation: in vitro / ex vivo
Data waiving:
other justification
Justification for data waiving:
the study does not need to be conducted because the substance is classified as skin irritation and the available information indicates that it should be classified as eye irritation (Category 2)
Reason / purpose for cross-reference:
data waiving: supporting information
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

For the skin irritation endpoint, the in vitro corrosion assay provided a negative result indicating that the test substance would not be considered corrosive. However, the in vitro skin irritation assay provided a positive result indicating the substance has skin irritation potential. As such the substance is regarded as a skin irritant category 2 (H315) in accordance with the CLP Regulation (EC No. 1272/2008, as amended).

With regards to eye irritation an in vitro BCOP assay is available which provided an inconclusive result and accordingly cannot be used for classification purposes. In accordance with Column 2 Annex VII of REACH: "the substance is classified as skin irritation and the available information indicates that it should be classified as eye irritation (Category 2)".

This substance was originally submitted and notified under the Dangerous Substances Directive (67/548/EEC) and was classified for skin and eye irritation under that Directive by the orginal data holder.  These classifications have since been upgraded to GHS as H315 (Causes skin irritation) and H319 (Causes serious eye irritation) and had been so notified to the ECHA C & L inventory.  The original data owners were unwilling to take on the duties of REACH Lead Registrant and we have therefore purchased access to their original data for use in this registration.  Since the data is the same we feel that it is important to maintain the same classifications agreed with the relevant National Competent Authorities for the original DSD notifications. We have therefore kept the classification as H315 and H319 and further testing on these endpoints is not warranted.