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Toxicological information

Skin irritation / corrosion

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Administrative data

skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-10-10 to 2016-10-14
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
The study was performed in accordance with standard test protocols (OECD 439 resp. EU B.46) in a quality controlled laboratory. The study is valid according to criteria mentioned in the test protocols.

Data source

Reference Type:
study report
Report Date:

Materials and methods

Test guideline
according to
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
GLP compliance:
yes (incl. certificate)

Test material

Test material form:
solid: particulate/powder
Details on test material:
white or almost white powder
Specific details on test material used for the study:
- Homogeneity: homogeneous
- Appearance: white powder

- Storage condition of test material: Room Temperature (20 ± 5°C)

In vitro test system

Test system:
human skin model
Cell type:
non-transformed keratinocytes
Cell source:
other: human-derived epidermal keratinocytes, cultured to form a multi-layered, highly differentiated model of the human epidermis
unchanged (no vehicle)
Details on test system:
The test system is a commercially available EpiDermTM-Kit, procured by MatTek.
The EpiDermTM tissue consists of human-derived epidermal keratinocytes which have
been cultured to form a multi-layered, highly differentiated model of the human epidermis.
It consists of organized basal, spinous and granular layers, and a multi-layered stratum
corneum containing intercellular lamellar lipid layers representing main lipid classes analogous
to those found in vivo. The EpiDermTM tissues are cultured on specially prepared cell
culture inserts.

EpiDermTM tissues were procured from MatTek In Vitro Life Science Laboratories, Bratislava.
Designation of the kit: EPI-200-SIT
Day of delivery: 11. Oct. 2016
Batch no.: 23362
Amount/concentration applied:
Amounts of Test Item

Tissue 1: 25.7 mg
Tissue 2: 25.2 mg
Tissue 3: 25.1 mg
Duration of treatment / exposure:
Tissues were dosed in 1-minute-intervals. After dosing the last tissue, all plates were
transferred into the incubator for 35 minutes at 37 ± 1°C and 5.0 ± 0.5% CO2.
1 hour after the first application, the inserts were removed from the plates using sterile forceps
and rinsed immediately in 1-minute-intervals.
After rinsing, each tissue was blotted with sterile cellulose tissue and then transferred into
a new 6-well-plate with fresh assay medium (0.9 mL).
Then, the tissues were set in the incubator for 23 hours and 27 minutes at 37 ± 1°C and
5.0 ± 0.5% CO2.
Duration of post-treatment incubation (if applicable):
After post-incubation, 0.9 mL assay medium were filled in the lower row of the 6-well-plate.
The tissues were removed from the incubator and shaken for 5 minutes (120 rpm). Then
the inserts were transferred into the lower row of the 6-well-plate and set into the incubator
for 19 hours and 20 minutes for post-incubation at 37 ± 1°C and 5.0 ± 0.5% CO2.
Number of replicates:
3 Tissues

Test system

Details on study design:
MTT Assay
After a total incubation time of 42 hours and 47 minutes, a 24-well-plate was prepared with
300 μL freshly prepared MTT-medium in each well. The tissues were blotted on the bottom
and then transferred into the 24-well-plate. Then the 24-well-plate was set into the incubator
for 3 hours at 37 ± 1°C and 5.0 ± 0.5% CO2.
After this time, the MTT-medium was aspirated and replaced by DPBS buffer. This was
then aspirated, too, and replaced several times.
At last, each insert was thoroughly dried and set into the empty, pre-warmed 24-well-plate.
Into each well, 2 mL isopropanol were pipetted, taking care to reach the upper rim of the
insert. The plate was then shaken (120 rpm) for 2 hours at room temperature.
After 2 hours, the inserts were pierced with an injection needle, taking care that all colour
was extracted. The inserts were then discarded and the content of each well was thoroughly
mixed in order to achieve homogenisation.
From each well, two replicates with 200 μL solution (each) were pipetted into a 96-wellplate
which was read in a plate spectrophotometer at 570 nm.

The photometric absorbance of the negative controls is considered as 100%. For each
replicate of test item and positive control, tissue viability is calculated as % photometric
absorbance compared with the mean of the negative controls

Results and discussion

In vitro

Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Vehicle controls validity:
not applicable
Negative controls validity:
Positive controls validity:
Remarks on result:
no indication of irritation

Any other information on results incl. tables

Absorbance Values negative control, test item and positive control (OD 570nm)

 Designation  Measurement  Negative Control  Nicotine bitartrate dihydrate  Positive Control
 Tissue 1     1  2.021  1.934  0.097
 2  2.033  1.898  0.097
 Tissue 2     1  2.050  2.005  0.098
 2  2.026  2.010  0.097
 Tissue 3     1  1.994  2.094  0.096
 2  1.954  2.078  0.096

Mean absorbance values

 Designation  Negative Control  Nicotine bitartrate dihydrate  Positive Control
 Mean - blank (tissue 1)  1.987  1.876  0.057
 Mean - blank (tissue 2)  1.998  1.968  0.058
 Mean - blank (tissue 3)  1.934  2.046  0.056
 Mean of the three tissues  1.973  1.963  0.057

% Viability

 Designation  Nicotine bitartrate dihydrate  Positive Control
 % viability (tissue 1)  95.1%  2.9%
 % viability (tissue 2)  99.7%  2.9%
 % viability (tissue 3)  103.7%  2.8%
 % viability (mean)  99.5%  2.9%
 +/- SD of mean viability (%)  4.3%  0.1%

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
The test item is considered as non-irritant to skin.
After the treatment, the relative absorbance values were reduced to 99.5%. This value is
above the threshold for skin irritation (50%).
The optical density of the negative control was well within the required acceptability criterion
of 0.8 ≤ mean OD ≤ 2.8.
The positive control has met the acceptance criterion too, for thus ensuring the validity of
the test system.
Variation within replicates was within the accepted range for negative control, positive control
and test item (required: ≤ 18%).
For these reasons, the result of the test is considered valid.
Executive summary:

Three tissues of the human skin model EpiDermTM were treated with Nicotine bitartrate

dihydrate for 60 minutes.

The test item was applied directly to each tissue and spread to match the tissue size

(0.63 cm2; as indicated by the supplier).

DPBS-buffer was used as negative control, 5% SDS solution was used as positive control.

After treatment with the negative control, the absorbance values were within the required

acceptability criterion of 0.8 ≤ mean OD ≤ 2.8, OD was 2.0. The positive control showed

clear irritating effects. Relative absorbance was reduced to 2.9 % (required: < 20%).

Variation within the tissue replicates was acceptable (required: ≤ 18%).

After the treatment with the test item, the relative absorbance values were reduced to

99.5 %. This value is above the threshold for skin irritation potential (50%).

Therefore, Nicotine bitartrate dihydrate is considered as non-irritant to skin in the

Reconstructed Human Epidermis (RHE) Test Method.