[No public or meaningful name is available]

Endpoint summary

Description of key information

Skin irritation/corrosion:

Skin Corrosion Test (SCT, OECD 431): not skin corrosive

Skin Irritation Test (SIT, OECD 439): irritating to the skin

Based on the results observed and by applying the evaluation criteria it was concluded that Emuldur 3643 shows a skin irritation potential in the EpiDerm™ in vitro skin irritation and corrosion test strategy under the test conditions chosen.

Eye irritation/corrosion:

BCOP (OECD 437): no serious eye damage

EpiCocular (OECD 492): irritating to the eye

Based on the results of the BCOP and EpiOcular Tests and by applying the evaluation criteria it was concluded that Emuldur 3643 shows an eye irritation potential in the in vitro eye irritation test strategy under the test conditions chosen.

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: human-derived epidermal keratinocytes

Source strain:

not specified

Details on animal used as source of test system:

N/A: in vitro

Vehicle:

unchanged (no vehicle)

Details on test system:

The objective was to assess the skin irritation and corrosion potential of the test material. Using
the methods currently available, a single in vitro assay is not sufficient to cover the full range
of skin irritation/corrosion potential. Therefore, two in vitro assays were part of this in vitro skin
irritation and corrosion test strategy: The Skin Corrosion Test (SCT) and Skin Irritation Test
(SIT).
The present test is based on the experience that skin corrosive and irritant chemicals produce
cytotoxicity in human reconstructed epidermis after a short-term topical exposure. The test is
designed to predict a skin corrosion or irritation potential of a chemical by using the threedimensional
human epidermis model EpiDermTM. After application of the test material to the
stratum corneum surface of the EpiDermTM tissue, the induced cytotoxicity (= loss of viability)
is measured by a colorimetric assay. Cytotoxicity is expressed as the reduction of mitochondrial
dehydrogenase activity. The mitochondrial dehydrogenase reduces the yellow-colored watersoluble
3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) to the insoluble
blue-colored formazan. After isopropanol extraction of the formazan from the tissues, the
optical density of the extract is determined spectrophotometrically. The optical density of the
extracts of tissues treated with the test substance is compared to negative control values from
tissues and expressed as relative tissue viability.

MATERIALS AND TECHNICAL EQUIPMENT
Laminar flow bench: HERAsafe KS 18 (Thermo Electron Corporation)
CO2 incubator: Heraeus BBD 6220
Incubation conditions: 37°C ± 1°C, 5% ± 1% CO2, 90% ± 10% relative
humidity
Spectrophotometer: SunriseTM Absorbance Reader
For the determination of the optical density of colored extracts.
Measurement using a filter wavelength 570 nm without reference
filter
EpiDerm™ 200 kit: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia
containing:
24 EPI-200 tissues (reconstructed epidermis): surface 0.6 cm²
cultured in Millicells®  1 cm
Tissue for MTT
reduction control:
EPI-200 tissue that is killed by freezing at –20°C
Assay medium: Skin corrosion test: EPI-100-ASY assay medium
Skin irritation test: EPI-100-NMM assay medium
MTT diluent: Dulbecco's modified eagle's medium (DMEM)-based
medium used for diluting MTT (MatTek In Vitro Life Science
Laboratories, Bratislava, Slovakia / Sigma, Germany)
Wash buffer: Dulbecco's phosphate-buffered saline (PBS), w/o Ca2+, Mg2+
Detection agent: 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT)
(MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia /
Sigma, Germany), 1.0 mg / mL MTT diluent
Extracting agent: Isopropanol p.a.

CONTROLS
Negative control (NC): Deionized water (skin corrosion test)
PBS, sterile (skin irritation test)
Positive control (PC): 8 N potassium hydroxide solution (skin corrosion test)
5% (w/v) sodium dodecyl sulfate (SDS) in water (skin irritation test)
MTT reduction control
(KC):
Deionized water or test substance (skin corrosion test)
PBS, sterile, or test substance (skin irritation test)

TEST SYSTEM
Three-dimensional human epidermis model
The EpiDermTM model 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 multilayered stratum corneum
containing intercellular lamellar lipid layers arranged in patterns analogous to that found in vivo.
The EpiDermTM tissues (surface 0.6 cm²) are cultured on cell culture inserts (MILLICELLs,
10 mm ) specially prepared and available commercially as kits (EpiDerm™ 200) containing
24 tissues on shipping agarose.

Tissue model: EPI-200
Tissue Lot Number: 34145 (SCT, 1st test run, SIT) and 34161 (SCT, 2nd test run)
(Certificates of Analysis see Appendix)
Origin: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia

Control samples:

yes, concurrent negative control

Amount/concentration applied:

see "details on study design below"

Duration of treatment / exposure:

see "details on study design below"

Duration of post-treatment incubation (if applicable):

see "details on study design below"

Number of replicates:

see "details on study design below"

Details on study design:

EXPERIMENTAL PROCEDURE
Pretest for mesh compatibility
For liquid test substances, a nylon mesh can be used as spreading aid. In order to exclude a
reaction of the test substance with the mesh, the compatibility of the test substance with the
nylon mesh was checked in a pretest (experimental conduct in accordance with GLP, but
without a GLP status).
The test substance and the mesh were brought together on a slide, and the reaction was
observed after a 60-minute exposure.
An interaction between test substance and mesh was not noticed. However, it was judged that
the use of a mesh was not necessary for the test substance.

Pretest for direct MTT reduction
The direct reduction of MTT by a test substance interferes with the color density produced by
metabolic capacity of the tissue and would falsify the test results.
In order to assess the ability of the test material to reduce MTT directly, a pretest (experimental
conduct in accordance with GLP, but without a GLP status) was performed as described below.
The test substance was added to 0.9 mL MTT solution. The mixture was incubated in the dark
at about 37°C for 3 hours. A negative control (deionized water) was tested concurrently.
If the color of the MTT solution or (in case of water-insoluble test substances) the border to the
water phase turned blue / purple, the test substance was presumed to reduce MTT directly.
In case of direct MTT reduction, two freeze-killed control tissues (KC) per exposure time (skin
corrosion test) or three freeze-killed control tissues (KC) (skin irritation test) were treated
additionally with each the test article and the negative control in the same way as described in
section below.
Based on the result of the pretest, it was judged that application of killed control tissues is
necessary.

Basic procedure
Several test substances were tested in parallel within the present tests (test nos. 131 and 134)
by using the same control tissues per test run (NC and PC).

Skin corrosion test:
From the day of arrival in the laboratory, tissues were kept in the refrigerator. At least 1 hour,
but not more than 1.5 hours before test substance application, tissues were transferred to
6-well plates with 0.9 mL assay medium and preconditioned in the incubator at 37°C. The
pre-incubation medium was replaced with fresh medium immediately before application.
Two tissues per exposure time (3 minutes at room temperature or 1 hour in the incubator as a
rule) and test group (test material, negative control and positive control; 12 tissues per test)
were used. In addition, two killed control tissues per exposure time were treated with the test
substance and the NC, respectively, to detect direct MTT reduction.
Fifty microliters (50 μL) undiluted liquid test substance were applied using a pipette.
Control tissues were treated concurrently with 50 μL deionized water (NC, NC KC) or with
50 μL 8 N potassium hydroxide solution (PC) or test substance (KC). A nylon mesh was placed
carefully onto the tissue surface of the NC and NC KC (test no. 131) afterwards.
The tissues were washed with PBS to remove residual test material 3 minutes or 1 hour after
start of the application treatment.
Rinsed tissues were kept in 24-well plates (holding plates) at room temperature on assay
medium until all tissues per application time were dosed and rinsed. The assay medium was
then replaced with MTT solution, and the tissues were incubated for 3 hours.
After incubation, the tissues were washed with PBS to stop the MTT incubation.
The formazan that was metabolically produced by the tissues was extracted by incubation of
the tissues in isopropanol. The optical density at a wavelength of 570 nm (OD570) of the extracts
was determined spectrophotometrically. Blank values were established of 4 microtiter wells
filled with isopropanol for each microtiter plate.
Skin irritation test:
On the day of arrival in the laboratory, the tissues were transferred to sterile 6-well plates with
0.9 mL assay medium and preconditioned in the incubator at 37°C. After 1 hour, the
pre-incubation medium was replaced with fresh medium and preconditioning continued for
18 ± 3 hours.
Three tissues were treated with the test substance, the PC and the NC, respectively.
In addition, three killed control tissues were used for the test substance and the NC,
respectively, to detect direct MTT reduction.
Thirty microliters (30 μL) undiluted liquid test substance were applied using a pipette.
Control tissues were treated concurrently with 30 μL sterile PBS (NC, NC KC) or with 30 μL
5% SDS (PC) or test substance (KC). A nylon mesh was placed carefully onto the tissue
surface of the NC, NC KC and PC afterwards.
The tissues were kept under the laminar flow hood at room temperature for 25 minutes overall
and for 35 minutes in the incubator.
The tissues were washed with sterile PBS to remove residual test material 1 hour after start of
application. Rinsed tissues were blotted on sterile absorbent paper and transferred into new
6-well plates pre-filled with 0.9 mL fresh medium. When all tissues were rinsed, the surface of
each tissue was dried carefully with a sterile cotton swab.
Subsequently, the tissues were placed into the incubator at 37°C for 24 ± 2 hours.
After 24 ± 2 hours, the tissues were transferred into new 6-well plates pre-filled with
0.9 mL fresh medium and placed into the incubator for an additional 18 ± 2-hour
post-incubation period.
After the post-incubation period, the assay medium was replaced with 0.3 mL MTT solution,
and the tissues were incubated in the incubator for 3 hours.
After incubation, the tissues were washed with PBS to stop the MTT incubation. The formazan
that was metabolically produced by the tissues was extracted by incubation of the tissues in
isopropanol. The optical density at a wavelength of 570 nm (OD570) of the extracts was
spectrophotometrically determined. Blank values were established of 4 microtiter wells filled
with isopropanol for each microtiter plate.

Data evaluation
Table(s) and/or figure(s) of measured parameters presented in the report were produced using
a PC-based tabular calculation software. The mean and individual data were not always
rounded, but the significant digits were produced by changing the display format.
Consequently, calculation of mean values by using the individual data presented in the report
will in some instances yield minor variations in value.
Principle The OD570 values determined for the various tissues are
measures of their viability. The quotient of the OD570 of tissues
treated with the test material and the mean OD570 values of the
NC (percent of control) is used for evaluating whether a test
material is skin corrosive or irritant.
Calculation of individual and
mean optical densities
The individual tissue OD570 is calculated by subtracting the
mean blank value of the respective microtiter plate from the
respective individual tissue OD570 value. The mean OD570 for a
test group of two tissues (skin corrosion test) or three tissues
(skin irritation test) treated in the same way is calculated.
Application of
measurements using killed
control tissues
In case of direct MTT reduction by the test substance, the OD570
values measured in the freeze-killed control tissues (KC) will be
used to correct the mean OD570 of the tissues treated with the
test substance (mean corrected OD570 KC). Since killed tissues
might still have a residual enzyme activity that is able to produce
some formazan net, OD570 KC is calculated by subtracting the
OD570 KC of the NC from the OD570 KC of the test substance.
In case the net OD570 KC is greater than zero, it is subtracted
from the respective mean OD570 to result in the mean corrected
OD570 KC. The mean corrected OD570 KC represents the
formazan production linked to the tissue viability and therefore
indicates the cytotoxic potency of the test substance.
Tissue viability The quantification of tissue viability is presented as the quotient
of the mean OD570 (or mean corrected OD570 KC if applicable)
divided by the respective OD570 NC value in percent for each
exposure time.

ACCEPTANCE CRITERIA
In case one of the acceptance criteria below was not met, repetition of the test was considered
(see section 4).
Barrier function and Quality
control (QC)
The supplier demonstrates that each batch of the model used
meets the defined production release criteria. MatTek
determines the ET50 value following exposure to Triton X-100
(1%) for each EpiDermTM batch. The ET50 must fall within an
established range based on a historical database of results.
The following acceptability range (upper and lower limit) for the
ET50 is established by the supplier as described in the cited
OECD guidelines.
Lower acceptance limit: ET50 = 4.0 hours
Upper acceptance limit: ET50 = 8.7 hours
EpiDerm QC (EPI-200) batch release see Appendix
Acceptance criteria for the
negative control (NC)
The absolute OD570 of the negative control tissues in the MTT
test is an indicator of tissue viability obtained in the testing
laboratory after the shipping and storing procedure and under
specific conditions of the assay. Tissue viability is acceptable if
the mean OD570 of the NC is  0.8. The mean OD570 of the NC
should not exceed 2.8.
Acceptance criteria for the
positive control (PC)
Skin corrosion test: 8 N potassium hydroxide solution is used
as positive reference. A tissue viability of <= 30% is acceptable
for the 3-minute exposure. Mean viability of the tissues exposed
for 1 hour should be <15%.
Skin irritation test: 5% SDS is used as PC and reflects the
sensitivity of the tissues used in the test conditions. A viability
of <= 20% is acceptable.
Acceptance criteria for the
variability of the tissues
Skin corrosion test: For every treatment, two tissues are treated
in parallel. In the range of 20% and 100% viability, variability
between the tissues is considered to be acceptable if the
coefficient of variation (CV) of % viability is <= 30%.
Skin irritation test: For every treatment, three tissues are treated
in parallel. The inter-tissue variability is considered to be
acceptable if the SD of % viability is <= 18%.
Acceptance criteria for the
killed controls (KC)
The OD570 of the tissues for the KC of the NC should be
<= 0.35. The OD570 value for direct MTT reduction of a test
substance should be <= 30% of the OD570 of the NC.

EVALUATION OF RESULTS
The evaluation of the in vitro skin irritation potential of the test substance is based on the results
of the Skin Corrosion Test (SCT) and the Skin Irritation Test (SIT).
If a test substance is not tested in both assays or an inconclusive result is obtained in one of
the studies, the test strategy may still lead to an overall evaluation when the result of a single
study gives a clear prediction. However, if both studies are inconclusive or contradictory results
are obtained, a test evaluation may not be possible.

Evaluation of results Skin Corrosion Test (SCT)
Skin corrosive potential of the test materials is predicted from the mean relative tissue viabilities
obtained after a 3-minute treatment compared to the negative control tissues treated
concurrently with deionized water. A chemical is considered as skin corrosive if the mean
relative tissue viability after the 3-minute treatment with a test material is decreased below
50%. In addition, materials with a viability of  50% after the 3-minute treatment are considered
as skin corrosive if the mean relative tissue viability after a 1-hour treatment with test material
is decreased below 15%.
A single test composed of at least two tissue replicates should be sufficient for a test chemical
when the result is unequivocal. However, in case of borderline results such as non-concordant
replicate measurements and/or mean percent tissue viability equal to
± 5% of the cutoff values cited above, a second test should be considered as well as a third
one in case of discordant results between the first two tests.

The following decision criteria apply:
Step 1: Identification of corrosives
Mean tissue viability (% of negative control)
< 50% after 3 min exposure => corrosive
≥ 50% after 3 min exposure and < 15% after 1 h exposure => corrosive
≥ 50% after 3 min exposure and ≥ 15% after 1 h exposure => non-corrosive
Step 2: Optional UN GHS subcategorization of corrosives identified in step 1
< 25% after 3 min exposure => UN GHS Cat 1A
≥ 25% after 3 min exposure => UN GHS Cat 1B or 1C

A “borderline“ range (50 ± 5%, 25 ± 5% and 15 ± 5%) was determined statistically using historic
BASF data and hence considers the variance of the test method. This evaluation is an
amendment to the evaluation provided in OECD Guideline 431.

Evaluation of results Skin Irritation Test (SIT)
The test chemical is identified as requiring classification and labelling according to UN GHS
(Category 2 or Category 1) if the mean percent tissue viability after exposure and posttreatment
incubation is less than or equal (≤) to 50%.
A single test composed of at least three tissue replicates should be sufficient for a test chemical
when the result is unequivocal. However, in case of borderline results such as non-concordant
replicate measurements and/or mean percent tissue viability equal to ± 5% of the cutoff value,
a second test should be considered as well as a third one in case of discordant results between
the first two tests.
The following decision criteria apply:
Mean tissue viability (% of negative control)
≤ 50 => No prediction can be made (UN GHS Category 2 or Category 1)
> 50 => Non-irritant (No UN GHS Category)
A “borderline“ range (50 ± 5%) was determined statistically using historic BASF data and hence
considers the variance of the test method. This evaluation is confirming the borderline range
provided in OECD Guideline 439.

Irritation / corrosion parameter:

% tissue viability

Remarks:

Skin Corrosion Test (3-minutes exposure)

Run / experiment:

mean of two tissues, final relative mean viability of tissues after KC correction [% of NC]:

Value:

90

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Remarks:

Skin Corrosion Test (1-hour exposure), 1st run

Run / experiment:

mean of two tissues, Final relative mean viability of tissues after KC correction [% of NC]:

Value:

58.6

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable because of methodological limitations

Irritation / corrosion parameter:

% tissue viability

Remarks:

Skin Corrosion Test (1-hour exposure), 2nd run

Run / experiment:

mean of two tissues, Final relative mean viability of tissues after KC correction [% of NC]:

Value:

70.2

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Remarks:

Skin Irritation Test (SIT)

Run / experiment:

mean of three tissues, Final relative mean viability of tissues after KC correction [% of NC]:

Value:

2.1

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

The following results were obtained in the EpiDerm™ skin corrosion/irritation test:

The test substance is able to reduce MTT directly. Therefore, an additional MTT reduction

control KC (freeze-killed control tissues) was introduced.

Results of the Skin Corrosion Test (SCT)

Two test runs of the 1-hour exposure period of the Skin Corrosion Test (SCT) were performed.

1st test run

The final relative mean viability of the tissues treated with the test substance determined after

an exposure period of 3 minutes was 90% (values for single tissues: 101.1% and 78.8%), and

it was 58.6% (values for single tissues: 44.4 and 72.8%) after an exposure period of 1 hour.

The acceptance criteria for the variability of the viable tissues treated with the test substance

was not met after the exposure period of 1 hour (CV% of viabilities: 34.3%).

Tissues treated with the positive control 8 N KOH showed a relative mean viability of 11.7%

(3-minute exposure) and 3.7% (1-hour exposure), reflecting the expected sensitivity of the

tissues.

The mean OD570 of the negative control (deionized water) fulfills the acceptance criteria (see

section 3.7. and Table 7) and demonstrates the validity of the assay.

As the acceptance criteria for the variability of the test substance treated tissues was not met

at the 1-hour exposure period, a 2nd test run (only 1-hour exposure) was performed to verify

the result.

2nd test run

The final relative mean viability of the tissues treated with the test substance determined after

an exposure period of 1 hour was 70.2% (values for single tissues: 67.8% and 72.7%). The

variability between the results of the tissues is within the acceptance range.

The tissues treated with the positive control 8 N KOH showed a relative mean viability of 4.7%

(1-hour exposure), reflecting the expected sensitivity of the tissues.

The mean OD570 of the negative control (deionized water) fulfills the acceptance criteria (see

section 3.7. and Table 7) and demonstrates the validity of the assay.

Overall, the obtained results of the Skin Corrosion Test (SCT) did not indicate a corrosive

potential of the test substance.

Results of the Skin Irritation Test (SIT)

The final relative mean viability of the tissues treated with the test substance determined after

an exposure period of 1 hour with an about 42-hour post-incubation was 2.1%. The variability

between the results of the tissues is within the acceptance range.

The tissues treated with the positive control 5% SDS showed a relative mean viability of 2.8%,

reflecting the expected sensitivity of the tissues.

The mean OD570 of the negative control (PBS) fulfills the acceptance criteria (see section 3.7.

and Table 8) and demonstrates the validity of the assay.

In the Skin Irritation Test (SIT) the test substance was assessed to have a skin irritation

potential.

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

Based on the results observed and by applying the evaluation criteria it was concluded that Emuldur 3643 shows a skin irritation potential in the EpiDerm™ in vitro skin irritation and corrosion test strategy under the test conditions chosen.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

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 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Deviations:

no

GLP compliance:

yes

Species:

human

Details on test animals or tissues and environmental conditions:

TEST SYSTEM
Three-dimensional human cornea model
The EpiOcularTM model (OCL-200) is a three-dimensional, non-keratinized tissue construct
composed of normal human-derived epidermal keratinocytes used to model the human corneal
epithelium (compare Figure 1). The EpiOcularTM tissues (surface 0.6 cm²) are cultured on cell
culture inserts (MILLICELLs, 10 mm diameter) and are available commercially as kits
(EpiOcular™ 200) containing 24 tissues on shipping agarose.
Tissue model: OCL-200
Tissue Lot Number: 34910 (Certificate of Analysis see Appendix)
Supplier: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia

ANALYSES
No analysis of the test-substance preparation was performed because the test substance was
applied undiluted.

Vehicle:

unchanged (no vehicle)

Controls:

yes

Amount / concentration applied:

see details on study design

Duration of treatment / exposure:

see details on study design

Duration of post- treatment incubation (in vitro):

see details on study design

Number of animals or in vitro replicates:

2

Details on study design:

EXPERIMENTAL PROCEDURE
Pretest for direct MTT reduction
The direct reduction of MTT by a test substance interferes with the color density produced by
metabolic capacity of the tissue and would falsify the test results.
In order to assess the ability of the test material to reduce MTT directly, a pretest (experimental
conduct in accordance with GLP, but without a GLP status) was performed as described below.
The test substance was added to 0.9 mL MTT solution. The mixture was incubated in the dark
at about 37°C for 3 hours. A negative control (deionized water) was tested concurrently.
If the color of the MTT solution or (in case of water-insoluble test substances) the border to the
water phase turned blue / purple, the test substance was presumed to reduce MTT directly.
In case of direct MTT reduction, two freeze-killed control tissues (KC) were treated additionally
with each the test article and the negative control in the same way as described in section 3.6.
Based on the result of the pretest, it was judged that application of killed control tissues is
necessary.

Basic procedure
Several test substances were tested in parallel within the present test (test no. 129) using the
same control tissues (NC and PC).
Two tissues were treated with each the test substance, the PC and the NC.
In addition, two killed tissues were used for each the test substance and the NC to detect direct
MTT reduction.
There are two separate protocols for liquids and solids differing in exposure time and postincubation
period. Due to the physical condition of the test substance, the protocol for liquids
was applied.

Pre-incubation of the tissues
On the day of arrival in the laboratory, the tissues were transferred to sterile 6-well plates with
1 mL assay medium and preconditioned in the incubator at 37°C. After 1 hour, the
pre-incubation medium was replaced with fresh medium, and preconditioning continued in the
incubator at standard culture conditions for 16 – 24 hours.

Pretreatment of the tissues
After pre-incubation, the tissues were pretreated with 20 μL PBS in order to wet the tissue
surface. The tissues were incubated at standard culture conditions for 30 minutes.

Application of the test substance
Using a pipette, fifty microliters (50 μL) undiluted liquid test substance were applied covering
the whole tissue surface.
Control tissues were applied concurrently with 50 μL sterile deionized water (NC, NC KC) or
with 50 μL methyl acetate (PC) or test substance (KC).
After application, the tissues were placed into the incubator until the total exposure time of
30 minutes was completed.

Removal of the test substance and postincubation period
In order to remove the test substance, the tissues were washed with sterile PBS. For this
purpose, the tissues were immersed and swiveled three times in each of three beakers filled
with PBS. Washed tissues were immersed immediately into 12-well plates pre-filled with
5 mL/well pre-warmed medium (post-soak immersion) to remove residual test substance.
After 12 minutes of post-soak immersion, each tissue was dried on absorbent paper and
transferred to fresh 6-well plates filled with 1 mL/well pre-warmed medium.
Subsequently, the tissues were incubated at standard culture conditions for 2 hours (postincubation
period).

MTT incubation
After the post-incubation period, the assay medium was replaced with 0.3 mL MTT solution,
and the tissues were incubated in the incubator for 3 hours.
After incubation, the tissues were washed with PBS to stop the MTT incubation.
The formazan that was metabolically produced by the tissues was extracted by incubation of
the tissues in isopropanol. The optical density at a wavelength of 570 nm (OD570) of the extracts
was spectrophotometrically determined. Blank values were established of 4 microtiter wells
filled with isopropanol for each microtiter plate.

Data evaluation
Table(s) of measured parameters that are presented in the report were produced using a
computer-based tabular calculation software. The mean and individual data were not always
rounded, but the significant digits were produced by changing the display format. As a
consequence, calculation of mean values by using the individual data presented in the report
will in some instances yield minor variations in value.
Principle
The OD570 values determined for the various tissues are a
measure of their viability. The ratio of the OD570 of tissues
treated with the test material and the mean OD570 values of the
NC (percent of control) is used for evaluating whether a test
material was an irritant.

Calculation of individual and mean optical densities
The corrected measured OD570 value for each individual tissue
was calculated by subtracting the mean blank value of the
respective microtiter plate from the respective individual tissue
OD570 value. The mean OD570 for a test group of two tissues
treated in the same way was calculated.

Application of measurements using killed control tissues
In case of direct MTT reduction by the test substance, the OD570
values measured in the freeze-killed control tissues (KC) will be
used to correct the mean OD570 of the tissues treated with the
test substance (mean corrected OD570 KC). Since a killed tissue
might still have a residual enzyme activity that is able to produce
some formazan net, OD570 KC is calculated by subtracting the
OD570 KC of the NC from the OD570 KC of the test substance.
In case the net OD570 KC is greater than zero, it is subtracted
from the respective mean OD570 to result in the mean corrected
OD570 KC. The mean corrected OD570 KC represents the
formazan production linked to the tissue viability and therefore
indicates the cytotoxic potency of the test substance.

Tissue viability
The quantification of tissue viability is presented as ratio of the
mean OD570 (or mean corrected OD570 KC if applicable) divided
by the respective OD570 NC value in percent.

ACCEPTANCE CRITERIA
In case one of the acceptance criteria below was not met, repetition of the test was considered.
Barrier function and Quality control (QC)
The supplier demonstrates that each batch of the model used
meets the defined production release criteria. MatTek
determines the ET50 (min) value following exposure to 100 μL
of 0.3% Triton X-100 for each EpiOcular™ EIT (OCL-200)
batch. The ET50 must fall within an established range based on
a historical database of results.
The following acceptability range (upper and lower limit) for the
ET50 is established by the supplier as described in the cited
OECD Guideline.
Lower acceptance limit: ET50 = 12.2 min
Upper acceptance limit: ET50 = 37.5 min
EpiOcular QC (OCL-200) batch release see Appendix

Acceptance criteria for the NC
The absolute OD570 of the NC tissues in the MTT test is an
indicator of tissue viability obtained in the testing laboratory
after the shipping and storing procedure and under specific
conditions of the assay. Tissue viability is acceptable if the
mean OD570 of the NC is  0.8. The mean OD570 of the NC
should not exceed 2.8.

Acceptance criteria for the PC
Methyl acetate used as PC usually leads to a tissue viability of
approx. 25%. A viability of < 50% is acceptable.

Acceptance criteria for tissue variability
Two tissues were treated under the same conditions.
A variability between the tissues is considered to be acceptable
if the relative difference of the viability is < 20%.

Acceptance criteria for the KC
The OD570 of the killed control tissues treated as negative
control should be <= 0.35. The value for direct MTT reduction of
a test substance should be <= 30% of the NC.

EVALUATION OF RESULTS
The eye irritation potential of the test material is predicted from the mean relative tissue
viabilities compared to the negative control tissues treated concurrently with sterile water. A
chemical is considered as "non-irritant” (no UN GHS Category) if the mean relative tissue
viability with a test material is greater than 60%.
A single test composed of at least two tissue replicates should be sufficient for a test chemical
when the result is unequivocal. However, in case of borderline results such as non-concordant
replicate measurements and/or mean percent tissue viability equal to ± 5% of the cut-off value,
a second test should be considered as well as a third one in case of discordant results between
the first two tests.
The following decision criteria apply:
Mean tissue viability (% of negative control)
<= 60 % No prediction can be made
> 60 % No UN GHS Category

A “borderline“ evaluation (60 ± 5%) was determined statistically using historic BASF data and
hence considers the variance of the test method. This evaluation is confirming the borderline
range provided in OECD Guideline 492.

HISTORIC CONTROL DATA
Historic control values of negative and positive controls collected over an appropriate period
are presented in section 4.2. These data demonstrate the reproducibility of results and
robustness of the procedures. They are used to derive suitable acceptance criteria (see
section 3.7.) for the test system.

Irritation parameter:

mean percent tissue viability 

Run / experiment:

negative control viable tissue

Value:

100

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

mean percent tissue viability 

Run / experiment:

negative control freeze-killed control tissues

Value:

1.7

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Irritation parameter:

mean percent tissue viability 

Run / experiment:

Test substance viable tissue

Value:

13.8

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Irritation parameter:

mean percent tissue viability 

Run / experiment:

Test substance freeze-killed tissue

Value:

3

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Irritation parameter:

mean percent tissue viability 

Run / experiment:

Final relative mean viability of tissues after KC correction [% of NC]:

Value:

10.7

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Irritation parameter:

mean percent tissue viability 

Run / experiment:

positive control viable tissue

Value:

35.2

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

Due to the ability of the test substance to reduce MTT directly, KC tissues were applied in
parallel. The results of the KC tissues indicate an increased MTT reduction (relative mean
viability 3.0% of NC). Thus, for the test substance the final relative mean viability is given after
KC correction.

Interpretation of results:

Category 2 (irritating to eyes) based on GHS criteria

Conclusions:

Based on the results observed and by applying the evaluation criteria described in chapter 3.8., it was concluded that Emuldur 3643 shows an eye irritation potential in the EpiOcular™ in vitro eye irritation test under the test conditions chosen.
The test method does not allow for the evaluation of serious eye damage. The result does not exclude a serious eye irritation potential of the test substance. For final assignment of a risk phrase at present, results from another study would be needed.

Reference 2

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

Details on test animals or tissues and environmental conditions:

Study Controls
Concurrent controls were used for several ICCR-Roßdorf GmbH studies performed simultaneously.

Positive Control
Positive Control 1: 2-Ethoxyethanol (purity: 99%)
Positive Control 2: Dimethylformamide (DMF, purity 100%)

Negative Control
Negative Control 1: Saline (0.9% NaCl in deionised water)
Negative Control 2: deionised water

Test Item Preparation
The test item was tested undiluted (liquid, non-surfactant).

Test System and Supporting Information
Test System
Test System: Freshly isolated bovine cornea (14 – 24 month old donor cattle)
Rationale: OECD 437
Source: AB Schlachthof GmbH & Co. KG, 63739 Aschaffenburg, Germany

Incubation Medium
The incubation medium consists of MEM, supplemented with 1.1 g sodium bicarbonate, 5 mL L-glutamine, 5 mL penicillin/streptomycin per 500 mL medium (final concentration of 100 units penicillin per mL medium, and 100 μg streptomycin per mL medium). Immediately before starting the test, MEM was supplemented with 1% fetal calf serum (FCS). MEM containing all supplements is called cMEM.
The OECD guideline 437 recommends the use of EMEM which is in composition and osmolarity equivalent to the MEM, thus MEM can be used without restriction.

Experimental Design and Study Conduct
Collection of Bovine Eyes
Freshly isolated bovine eyes of donor cattle were collected from the abattoir. Excess tissue was removed from the excised eyes. The isolated eyes were stored in HBSS containing 1% (v/v) penicillin/streptomycin (100 units/mL penicillin and 100 μg/mL streptomycin) in the cooled slaughterhouse and during transportation on the same morning to the laboratory using a Styrofoam box. The corneas were isolated on the same day after delivery of the eyes.

Preparation of Corneas
All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity and scratches were discarded. The cornea was carefully removed from the eye using scalpel and rounded scissors. A rim of about 2 mm of tissue (sclera) was left for stability and handling of the isolated cornea. The corneas were directly used in the BCOP test on the same day.
Each isolated cornea was mounted in a specially designed cornea holder according to the description given in OECD guideline 437, which consists of anterior and posterior compartments, which interface with the epithelial and the endothelial sides of the cornea, respectively. The endothelial side of the cornea was positioned against the sealing ring (O-ring) of the posterior part of the holder. The cornea was gently flattened over the O-ring but stretching was avoided. The anterior part of the holder was positioned on top of the cornea and fixed in place with screws. Both compartments of the holder were filled with incubation medium. The posterior compartment was filled first to return the cornea to its natural convex position. Care was taken to assure that no air bubbles were present within the compartments.
For equilibration, the corneas in the holder were incubated in a vertical position for about one hour at 32 ± 1 °C in a water-bath. At the end of the incubation period, the medium was changed before basal opacity measurement (t0).
Only corneas with a value of the basal opacity < 7 were used. Sets of three corneas were used for treatment with the test item and for the negative and positive controls.

Vehicle:

unchanged (no vehicle)

Controls:

yes

Amount / concentration applied:

see details on study design

Duration of treatment / exposure:

see details on study design

Observation period (in vivo):

see details on study design

Duration of post- treatment incubation (in vitro):

see details on study design

Number of animals or in vitro replicates:

3

Details on study design:

Exposure of the Corneas to the Test Groups
The corneas were distributed as follows:
Negative Control Saline: 3 corneas
Negative Control Deionised Water: 3 corneas
Positive Control 2-Ethoxyehtanol: 3 corneas
Positive Control Dimethylformamide: 3 corneas
Test Item: 3 corneas

The anterior compartment received the test item or the negative or positive controls at a volume of 0.75 mL on the surface of the corneas, via open chamber method, respectively. The corneas were incubated in a horizontal position at 32 ± 1 °C in the water-bath.
The incubation time lasted ten minutes (± 30 seconds).
After exposure, the test item or control items, respectively, were rinsed off from the application side with EMEM containing phenol red for at least three times or more until phenol red was still discoloured (yellow or purple), or the test item was still visible. Since the test item proved difficult to remove by the rinsing method, the front cover of the holder was opened and the cornea was carefully washed using a gentle stream of incubation medium. Once the medium is free of test item the corneas are given a final rinse with cMEM without phenol red. Fresh cMEM was added into the anterior compartment. Then the corneas were incubated at 32 ± 1 °C for further two hours in a vertical position, followed by a second opacity reading (t130). The opacity measurement is described in chapter 3.6.
In the second step of the assay, permeability of the corneas was determined. The permeability measurement is described in chapter 3.7.

Opacity Measurement
The opacitometer determines changes in the light transmission passing through the corneas, and displays a numerical opacity value. This value was recorded in a table. The opacitometer OP_KiT opacitometer (Electro Design, 63-Riom France) was calibrated as described in the manual and the opacity of each of the corneas was determined by reading each holder placed in the photoreceptor compartment for treated cornea.

Permeability Determination
Following the opacity readings, the permeability endpoint was measured as an indication of the integrity of the epithelial cell sheets. After the final opacity measurement was performed, the incubation medium was removed from both chambers. The posterior chamber was filled with fresh cMEM first. Then the anterior compartment was filled with 1 mL of a 0.4% (w/v) sodium fluorescein solution in HBSS. Corneas were incubated again in a horizontal position for 90 ± 5 minutes in a water-bath at 32 ± 1 °C. Complete medium from the posterior compartment was removed, well mixed and transferred into a 96 well plate and the optical density at 490 nm (OD490) was measured with a microplate reader (Versamax® Molecular Devices). The absorbance values were determined using the software SoftMax Pro Enterprise (version 4.7.1).

Data Recording
The data generated were recorded in the raw data file. The results are presented in tabular form, including experimental groups with the test item, negative, and positive controls.

Data Evaluation
Opacity
The change of opacity value of each treated cornea or positive and negative control corneas is calculated by subtracting the initial basal opacity from the post treatment opacity reading (t130 – t0), for each individual cornea.
The average change in opacity of the negative control corneas is calculated and this value is subtracted from the change in opacity of each treated cornea or positive control to obtain a corrected opacity.

Permeability
The corrected OD490 value of each cornea treated with positive control or test item is calculated by subtracting the average negative control cornea value from the original permeability value for each cornea.

In vitro Irritancy Score (IVIS) Calculation
The following formula is used to determine the IVIS of the negative control:
IVIS = opacity value + (15 x OD490 value)
The following formula is used to determine the corrected IVIS of the positive control and the test item:
IVIScorr = (opacity value – mean of opacity of negative control) + 15 x (permeability value – mean permeability of the negative control)
The mean IVIS value of each treated group was calculated from the individual IVIS values.
Depending on the score obtained, the test item is classified into the following category according to OECD guideline 437:
IVIS ≤ 3 => no category
IVIS > 3; ≤ 55 => no prediction can be made
IVIS > 55 => Category 1

Acceptability of the Assay
The Acceptability Criteria are only valid for Saline and 2-Ethoxyethanol since for Aqua deion. and Dimethylformamide a historical control data base is not sufficient yet.
The test was acceptable if
• the positive control gives an IVIS that falls within two standard deviations of the current historical mean (updated every three months and annexed to this report), and if
• the negative control responses result in opacity and permeability values that are less than the established upper limits for background opacity and permeability values for bovine corneas treated with the respective negative control.
A single testing run composed of at least three corneas should be sufficient for a test chemical when the resulting classification is unequivocal. In cases of borderline results in the first testing run, a second testing run will be considered, as well as a third one in case of discordant mean IVIS results between the first two testing runs. A result in the first testing run is considered borderline if the predictions from the 3 corneas are non-concordant, such that:
• 2 of the 3 corneas give discordant predictions from the mean of all corneas, or,
• 1 of the 3 corneas gives a discordant prediction from the mean of all 3 corneas, and the discordant result is >10 IVIS units from the cut-off threshold of 55.
• If the repeat testing run corroborates the prediction of the initial testing run (based upon the mean IVIS value), then a final decision can be taken without further testing. If the repeat testing run results in a non-concordant prediction from the initial testing run (based upon the mean IVIS value), then a third and final testing run should be conducted to resolve equivocal predictions, and to classify the test chemical. It may be permissible to waive further testing for classification and labelling in the event any testing run results in a GHS Category 1 prediction.

Irritation parameter:

in vitro irritation score

Run / experiment:

Emuldur 3643 mean IVIS

Value:

0.06

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

in vitro irritation score

Run / experiment:

negative control (saline) mean IVIS

Value:

1.26

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

in vitro irritation score

Run / experiment:

negative control (deionised water) mean IVIS

Value:

1.47

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

in vitro irritation score

Run / experiment:

positive control 2-Ethoxyethanol mean IVIS

Value:

93.3

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Irritation parameter:

in vitro irritation score

Run / experiment:

positive control Dimethylformamide mean IVIS

Value:

96.04

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

DISCUSSION

This in vitro study was performed to assess the corneal damage potential of Emuldur 3643 by means of the BCOP assay using fresh bovine corneas.

After a first opacity measurement of the fresh bovine corneas (t0), the neat test item Emuldur 3643, the positive, and the negative controls were applied to the different corneas and incubated for 10 minutes at 32 ± 1 °C. After the incubation phase, the test item as well as the positive and the negative controls were each rinsed from the corneas. Further, the corneas were incubated for another 120 minutes at 32 ± 1 °C in incubation medium, and opacity was measured for a second time (t130).

After the opacity measurements, permeability of the corneas was determined by measuring spectrophotometrically the transfer of sodium fluorescein after incubation in a horizontal position for 90 minutes at 32 ± 1 °C.

With the negative control (saline and deion. water), neither an increase of opacity nor permeability of the corneas could be observed (saline mean IVIS = 1.26 and deion.water mean IVIS = 1.47).

The positive controls (2-Ethoxyethanol; Dimethylformamide) showed clear opacity and distinctive permeability of the corneas (mean IVIS 2-Ethoxyethanol = 93.30; Dimethylformamide = 96.04) corresponding to a classification as serious eye damaging (CLP/EPA/GHS (Cat 1)).

The test item Emuldur 3643 was tested undiluted. Relative to the negative control saline, the test item Emuldur 3643 caused an increase of the corneal opacity and permeability. The calculated mean IVIS was 0.06.

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, according to the current study and under the experimental conditions reported, Emuldur 3643 does not show serious eye damage.
The test method according to the regulatory accepted protocol at the time of reporting does not allow for the evaluation of eye irritation. The result does not exclude an irritation potential of the test substance. For final assignment of a risk phrase at present, results from another study would be needed.

Reference 3

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Deviations:

no

GLP compliance:

yes

Species:

other: summary report for BCOP and EIT studies

Details on test animals or tissues and environmental conditions:

summary report for BCOP and EIT studies

Vehicle:

not specified

Amount / concentration applied:

summary report for BCOP and EIT studies

Duration of treatment / exposure:

summary report for BCOP and EIT studies

Observation period (in vivo):

summary report for BCOP and EIT studies

Duration of post- treatment incubation (in vitro):

summary report for BCOP and EIT studies

Number of animals or in vitro replicates:

summary report for BCOP and EIT studies

Details on study design:

summary report for BCOP and EIT studies

Irritation parameter:

in vitro irritation score

Remarks:

BCOP

Run / experiment:

Test substance

Value:

0.06

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

mean percent tissue viability 

Remarks:

EIT

Run / experiment:

Test substance

Value:

10.7

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Interpretation of results:

Category 2 (irritating to eyes) based on GHS criteria

Conclusions:

Based on the results of the BCOP and EpiOcular Tests and by applying the evaluation criteria it was concluded that Emuldur 3643 shows an eye irritation potential in the in vitro eye irritation test strategy under the test conditions chosen.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation/corrosion

The objective of the in vitro studies according to OECD 431 and 439 guidelines and GLP was to assess the skin irritation and corrosion potential of Emuldur 3643. Using the methods currently available, a single in vitro assay is not sufficient to cover the full range of skin irritating/corrosion potential.

Therefore, two in vitro assays were part of this in vitro skin irritation and corrosion test strategy:

The Skin Corrosion Test (SCT) and Skin Irritation Test (SIT), which were conducted in parallel.

The potential of the test substance to cause dermal corrosion/irritation was assessed by a single topical application of 50 μL (skin corrosion test) or 30 μL (skin irritation test) undiluted test substance to a reconstructed three-dimensional human epidermis model (EpiDerm™).

For the skin corrosion test, two EpiDerm™ tissues were incubated with the test substance for 3 minutes and 1 hour, each. In addition to the test substance, 50 μL per tissue of a negative control (deionized water) and of a positive control (8 N KOH) were applied to two tissues each per exposure period.

The skin irritation test was performed with three EpiDerm™ tissues, which were incubated with the test substance for 1 hour followed by a 42-hour post-incubation period. In addition to the test substance, 30 μL per tissue of a negative control (PBS) and of a positive control (5% SDS) were applied to three tissues each.

Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure/post-incubation by using a colorimetric test. The reduction of mitochondrial dehydrogenase activity measured by reduced formazan production after incubation with a tetrazolium salt (MTT) was chosen as endpoint. The formazan production of the epidermal tissues treated with the test substance is compared to that of negative control tissues. The quotient of the values indicates the relative tissue viability.

The following results were obtained in the EpiDerm™ skin corrosion/irritation test:

The test substance is able to reduce MTT directly. Therefore, an additional MTT reduction control KC (freeze-killed control tissues) was introduced.

Results of the Skin Corrosion Test (SCT)

Two test runs of the 1-hour exposure period of the Skin Corrosion Test (SCT) were performed.

1st test run

The final relative mean viability of the tissues treated with the test substance determined after an exposure period of 3 minutes was 90% (values for single tissues: 101.1% and 78.8%), and it was 58.6% (values for single tissues: 44.4 and 72.8%) after an exposure period of 1 hour.

The acceptance criteria for the variability of the viable tissues treated with the test substance was not met after the exposure period of 1 hour (CV% of viabilities: 34.3%).

Tissues treated with the positive control 8 N KOH showed a relative mean viability of 11.7% (3-minute exposure) and 3.7% (1-hour exposure), reflecting the expected sensitivity of the tissues.

The mean OD570 of the negative control (deionized water) fulfills the acceptance criteria (see section 3.7. and Table 7) and demonstrates the validity of the assay.

As the acceptance criteria for the variability of the test substance treated tissues was not met at the 1-hour exposure period, a 2nd test run (only 1-hour exposure) was performed to verify

the result.

2nd test run

The final relative mean viability of the tissues treated with the test substance determined after an exposure period of 1 hour was 70.2% (values for single tissues: 67.8% and 72.7%). The variability between the results of the tissues is within the acceptance range.

The tissues treated with the positive control 8 N KOH showed a relative mean viability of 4.7% (1-hour exposure), reflecting the expected sensitivity of the tissues.

The mean OD570 of the negative control (deionized water) fulfills the acceptance criteria (see section 3.7. and Table 7) and demonstrates the validity of the assay.

Overall, the obtained results of the Skin Corrosion Test (SCT) did not indicate a corrosive potential of the test substance.

Results of the Skin Irritation Test (SIT)

The final relative mean viability of the tissues treated with the test substance determined after an exposure period of 1 hour with an about 42-hour post-incubation was 2.1%. The variability between the results of the tissues is within the acceptance range.

The tissues treated with the positive control 5% SDS showed a relative mean viability of 2.8%, reflecting the expected sensitivity of the tissues.

The mean OD570 of the negative control (PBS) fulfills the acceptance criteria (see section 3.7. and Table 8) and demonstrates the validity of the assay.

In the Skin Irritation Test (SIT) the test substance was assessed to have a skin irritation potential.

Based on the results observed and by applying the evaluation criteria described in chapter 3.8., it was concluded that Emuldur 3643 shows a skin irritation potential in the EpiDerm™ in

vitro skin irritation and corrosion test strategy under the test conditions chosen.

Eye irritation/corrosion

BCOP

This in vitro study was performed to assess the corneal damage potential of Emuldur 3643 by means of the BCOP assay using fresh bovine corneas (OECD 437 guideline and GLP).

After a first opacity measurement of the fresh bovine corneas (t0), the neat test item, the positive, and the negative controls were applied to corneas fixed in an incubation chamber in horizontal position for 10 minutes at 32 ± 1 °C. The posterior chamber contained incubation medium. After the incubation phase the test item, the positive, and the negative controls were each rinsed from the corneas. Further, the corneas were incubated for another 120 minutes at 32 ± 1 °C in a vertical position, while the anterior chamber contained incubation medium as well. Afterwards, opacity was measured a second time (t130).

After the opacity measurements, permeability of the corneas was determined by measuring spectrophotometrically the transfer of sodium fluorescein after incubation in a horizontal position for 90 minutes at 32 ± 1 °C.

With the negative controls (saline and deion. water), neither an increase of opacity nor permeability of the corneas could be observed (saline mean IVIS = 1.26 and deion.water mean IVIS = 1.47)

The positive controls (2-Ethoxyethanol; DMF) showed clear opacity and distinctive permeability of the corneas corresponding to a classification as serious eye damaging (EU CLP/EPA/GHS (Cat 1)).

Relative to the negative control, the test item Emuldur 3643 caused an increase of the corneal opacity and permeability. The calculated mean in vitro irritancy score was 0.06.

According to OECD 437 (see table in chapter 3.9.3) Emuldur 3643 does not show serious eye damage.

The test method according to the regulatory accepted protocol at the time of reporting does not allow for the evaluation of eye irritation. The result does not exclude an irritation potential of the test substance. For final assignment of a risk phrase at present, results from another study would be needed.

EIT

The potential of Emuldur 3643 to cause ocular irritation was assessed by a single topical application of 50 μL undiluted test substance to a reconstructed three-dimensional human cornea model (EpiOcular™).

Two EpiOcular™ tissues were incubated with the test substance for 30 minutes followed by a 2-hour post-incubation period.

In addition to the test substance, 50 μL per tissue of a negative control (deionized water) and a positive control (methyl acetate) were applied to two tissues each.

Tissue destruction was determined by measuring the metabolic activity of the tissues after exposure/post-incubation by using a colorimetric test. The reduction of mitochondrial dehydrogenase activity measured by reduced formazan production after incubation with a tetrazolium salt (MTT) was chosen as endpoint. The formazan production of the epidermal tissues treated with the test substance is compared to that of negative control tissues. The ratio of the values indicates the relative tissue viability.

The following results were obtained in the EpiOcular™ eye irritation test:

The test substance is able to reduce MTT directly. Therefore, an additional MTT reduction control KC (freeze-killed control tissues) was introduced.

The final relative mean viability of the tissues treated with the test substance was 10.7%. The variability between the results of the tissues is within the acceptance range.

The tissues treated with the positive control methyl acetate showed a relative mean viability of 35.2%, reflecting the expected sensitivity of the tissues.

The mean OD570 of the negative control (deionized water) fulfills the acceptance criteria (see section 3.7. and 4.2.) and demonstrates the validity of the assay.

Based on the results observed and by applying the evaluation criteria described in chapter 3.8., it was concluded that Emuldur 3643 shows an eye irritation potential in the EpiOcular™ in vitro eye irritation test under the test conditions chosen.

The test method does not allow for the evaluation of serious eye damage. The result does not exclude a serious eye irritation potential of the test substance. For final assignment of a risk phrase at present, results from another study would be needed.

Summary of BCOP and EpiOcular (EIT):

Based on the results of the BCOP and EpiOcular Tests and by applying the evaluation criteria it was concluded that Emuldur 3643 shows an eye irritation potential in the in vitro eye irritation test strategy under the test conditions chosen.

Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008.

Based on the results of the SCT and SIT, the test material is irritating to the skin. As a result the substance is classified as skin irritant Cat. 2 under Regulation (EC) No 1272/2008. 

Based on the results of the BCOP and EpiOcular test, the test material is irritating to the eye. As a result the substance is classified as eye irritant Cat. 2 under Regulation (EC) No 1272/2008.