[No public or meaningful name is available]

Skin irritation / corrosion

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Reaction Product of 1,1,1-Trimethylol propane triacrylate with aziridine and 1-cyclooctyl-1,4-diazocane and 4-hydroxy-4-methylpentan-2-one

Molecular formula:

C6H12N2 - C47H77O16N4

IUPAC Name:

Reaction Product of 1,1,1-Trimethylol propane triacrylate with aziridine and 1-cyclooctyl-1,4-diazocane and 4-hydroxy-4-methylpentan-2-one

Test material form:

liquid

Details on test material:

Name of test item: Emuldur 3643
Test item No.: 19/0071-2
Batch identification: 27771924U0
Purity: 100 % (UVCB*, for details see analytical characterization No.20L00150) *Substances of unknown or variable composition, complex reaction products.
Homogeneity: The test item was homogeneous by visual inspection.
Storage stability: The stability of the test item under storage conditions over the study period was guaranteed by the sponsor, and the sponsor holds this responsibility.
Expiry date: July 15, 2021
Storage conditions: Ambient (RT)
Physical state / color: Liquid / colorless to yellowish, clear

In vitro test system

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"

Test system

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.

Results and discussion

In vitro

Results

Results 1

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

Results 2

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

Results 3

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

Results 4

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

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.