Reaction products of N-Phenyl-1-Naphthylamine with Nonene, branched

Endpoint summary

Description of key information

Skin irritation/corrosion:

Skin Irritation Test (SIT, OECD 439): not irritating to the skin

Eye irritation/corrosion:

EpiOcular (OECD 492): not irritating to the eye

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

Version / remarks:

18 June 2019

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).
However, in the current case, the results derived with SIT alone were sufficient for a final
assessment. Therefore, further testing in SCT was waived.
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 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): PBS, sterile
Positive control (PC): 5% (w/v) sodium dodecyl sulfate (SDS) in water
MTT reduction control (KC): PBS, sterile, or test substance

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: 30881 (Certificate 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:

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

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 3.6.3.
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. 125) by using
the same control tissues (NC and PC).
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.
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 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 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.

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 / corrosion parameter:

% tissue viability

Remarks:

test substance, viable tissues

Run / experiment:

mean

Value:

86.2

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:

negative control, viable tissues

Run / experiment:

mean

Value:

100

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:

positive control, viable tissues

Run / experiment:

mean

Value:

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:

Slight compound residues (brown-red colored) remained on all tissues treated with the test
substance after the washing procedure.
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 5.7 % of NC). Thus, for the test substance, the final relative mean viability is given after
KC correction.

Interpretation of results:

GHS criteria not met

Conclusions:

Based on the results observed and by applying the evaluation criteria it was concluded that XPDL 958 does not show a skin irritation potential in the EpiDerm™ in vitro skin irritation and corrosion test strategy under the test conditions chosen.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not 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)

Version / remarks:

18 June 2019

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 ∅) and are available commercially as kits
(EpiOcular™ 200) containing 24 tissues on shipping agarose.
Tissue model: OCL-200
Tissue Lot Number: 30669 (Certificate of Analysis see Appendix)
Supplier: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia

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 designsee details on study design

Duration of post- treatment incubation (in vitro):

see details on study design

Number of animals or in vitro replicates:

see details on study design

Details on study design:

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

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. 122) 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. The washing procedure was intensified by careful wiping with UltraClean™ LASIK
Expanded Eye Spears (Beaver Visitec International) to remove as much compound residues
as possible. Washed tissues were immersed immediately into 12-well plates pre-filled with
5 mL/well pre-warmed medium (post-soak immersion) to remove further 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 

Remarks:

test substance, viable tissues

Run / experiment:

mean

Value:

96.5

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:

test substance after KC correction

Run / experiment:

mean

Value:

94.9

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:

negative control, viable tissue

Run / experiment:

mean

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 

Remarks:

positive control, viable tissue

Run / experiment:

mean

Value:

18.9

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

Slight compound residues (brown-red colored) remained on the KC tissues treated with the
test substance after the washing procedure.
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 1.6 % of NC). Thus, for the test substance the final relative mean viability is given after
KC correction.

Interpretation of results:

GHS criteria not met

Conclusions:

Based on the results observed and by applying the evaluation criteria it was concluded that XPDL 958 does not show an eye irritation potential in the EpiOcular™ in vitro eye irritation test under the test conditions chosen.

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

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

Details on test animals or tissues and environmental conditions:

summary report

Vehicle:

not specified

Amount / concentration applied:

summary report

Duration of treatment / exposure:

summary report

Observation period (in vivo):

summary report

Duration of post- treatment incubation (in vitro):

summary report

Number of animals or in vitro replicates:

summary report

Details on study design:

summary report

Irritation parameter:

in vitro irritation score

Remarks:

BCOP

Run / experiment:

not conducted due to clear result in the EIT

Remarks on result:

not measured/tested

Irritation parameter:

mean percent tissue viability 

Remarks:

EIT

Run / experiment:

Test substance

Value:

94.9

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Interpretation of results:

GHS criteria not met

Conclusions:

Based on the results observed in the EpiOcular Test alone and by applying the evaluation criteria, it was concluded that XPIDL 958 does not show an eye irritation potential in the in vitro eye irritation test strategy under the test conditions chosen.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation/corrosion

The objective was to assess the skin irritation and corrosion potential of XPDL 958. 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, OECD 431) and Skin Irritation Test (SIT, OECD 439).

However, in the current case the results derived with SIT alone were sufficient for a final assessment. Therefore, further testing in SCT was waived.

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

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 irritation test:

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

Slight compound residues (brown-red colored) remained on all tissues treated with the test substance after the washing procedure. However, this did not influence the validity of the study,

since freeze-killed control tissues (KC) were used and the final relative mean viability of the test substance is given after correction with the KC value.

In order to avoid contamination of the extract solution with the compound residues, all tissues treated with the test substance were extracted only from below without piercing.

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 80.5%.

The results of the KC tissues indicate slightly increased MTT reduction (relative mean viability 5.7 % of NC).

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

Application of the positive control 5% SDS showed a relative mean viability of the tissues of 2.0% and reflects the expected sensitivity of the tissues.

The mean OD570 of the negative control (PBS) fulfills the acceptance criteria and demonstrates the validity of the assay.

Based on the results observed and by applying the evaluation criteria it was concluded that XPDL 958 does not show a skin irritation potential in the EpiDerm™ in vitro skin irritation and corrosion test strategy under the test conditions chosen.

Eye irritation/corrosion

The potential of XPDL 958 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™, OECD 492).

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.

Slight compound residues (brown-red colored) remained on the KC tissues treated with the test substance after the washing procedure. However, the results of the KC tissues indicate

only slightly increased MTT reduction. Thus, the final relative mean viability of the test substance is not substantially affected by the KC correction.

In order to avoid contamination of the extract solution with the compound residues, all tissues treated with the test substance were extracted only from below without piercing.

The final relative mean viability of the tissues treated with the test substance was 94.9%.

The results of the KC tissues indicate slightly increased MTT reduction (relative mean viability 1.6 % of NC).

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

Application of the positive control methyl acetate showed a relative mean viability of the tissues of 18.9% and reflects 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 XPDL 958 does not show an eye irritation potential in the EpiOcular™ in vitro eye irritation test 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 SIT, the test material is not irritating to the skin.

Based on the results of the EpiOcular test, the test material is not irritating to the eye.

As a result the substance is not classified under Regulation (EC) No 1272/2008 with regards to skin or eye irriation.