Nicotine dihydrogen ditartrate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-11-28 to 2016-12-01

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study was performed in accordance with standard test protocols (OECD 492) in a quality controlled laboratory. The study is valid according to criteria mentioned in the test protocols.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

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 (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

nicotine bitartrate dihydrate

IUPAC Name:

nicotine bitartrate dihydrate

Constituent 2

Reference substance name:

6019-06-3

Cas Number:

6019-06-3

IUPAC Name:

6019-06-3

Constituent 3

Reference substance name:

Nicotine dihydrogen ditartrate

EC Number:

200-607-2

EC Name:

Nicotine dihydrogen ditartrate

Cas Number:

65-31-6

Molecular formula:

C10-H14-N2.2C4-H6-O6

IUPAC Name:

nicotine dihydrogen ditartrate

Test material form:

solid: particulate/powder

Remarks:

powder

Details on test material:

white or almost white powder

Specific details on test material used for the study:

- Homogeneity: homogeneous
- Appearance: white powder


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room Temperature (20 ± 5°C)

Test animals / tissue source

Species:

other: see "Details on test animals and environmental conditions"

Strain:

other: see "Details on test animals and environmental conditions"

Details on test animals or tissues and environmental conditions:

Commercially available EpiOcularTM kit.
The EpiOcularTM tissue consists of primary human-derived epidermal keratinocytes which
have been cultured to form a stratified squamous epithelium similar to that found in the
human cornea. It consists of highly organized basal cells. These cells are not transformed
or transfected with genes to induce an extended life span. The EpiOcularTM tissues are
cultured in specially prepared cell culture inserts with a porous membrane through which
nutrients can pass to the cells. The tissue surface is 0.6 cm2.

EpiOcularTM tissues were procured from MatTek In Vitro Life Science Laboratories,
Mylnské Nivy 73, 82105 Bratislava, Slovakia.
Designation of the kit: OCL-212-EIT
Day of delivery: 29. Nov. 2016
Batch no.: 23754

Test system

Vehicle:

unchanged (no vehicle)

Amount / concentration applied:

A MTT stock solution of 5 mg/mL in DPBS buffer was prepared and stored in aliquots of
2 mL at – 20 ± 5 °C. 2 mL of the stock solution were thawed and diluted with 8 mL of assay
medium (resulting in 1 mg/mL). This MTT-solution with the concentration of 1 mg/mL
was used in the test.
For the pre-test (testing the ability of direct formazan reduction), the concentrate was
thawed and diluted with serum-free MEM directly before use. For the main test, the concentrate
was thawed and diluted with assay medium directly before use.

Duration of treatment / exposure:

50 μL of the controls and a defined amount of the test item (see table
7.2-a) were applied in duplicate in 1- minute- intervals. At the beginning of each experiment
(application of negative controls), a stop watch was started. After dosing the last tissue,
all plates were transferred into the incubator for 6 hours and 3 minutes at 37 ± 1 °C,
5 ± 1 % CO2 and 80 – 100 % relative humidity. At the end of exposure time, the inserts
were removed from the plates in 1-minute-intervals using sterile forceps and rinsed immediately.

Duration of post- treatment incubation (in vitro):

The tissues were immediately transferred into 5 mL of assay medium in pre-labelled 12-well plate for 25 minutes post
soak at room temperature.
After that, each insert was blotted on absorbent material and transferred into a pre-labelled
6-well plate, containing 1 mL assay medium. For post-treatment incubation, the tissues
were incubated for 18 hours at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity.
After the post-treatment incubation, the MTT Assay was performed.

Number of animals or in vitro replicates:

Test item, negative control and positive control were applied in duplicate.

Details on study design:

A 24-well-plate was prepared with 300 μL freshly prepared MTT-reagent in each well. The
tissue inserts were blotted on absorbent material and then transferred into the MTT solution.
The plate was incubated for 190 minutes at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 %
relative humidity.
At last, each insert was thoroughly dried and set into a pre-labelled 6-well-plate, containing
2 mL isopropanol, taking care that no isopropanol is flowing into the tissue insert. The
plate was firmly sealed to avoid evaporation of the solvent and then shaken for 2 hours at
room temperature.
The inserts were removed from the 6-well plate and discarded. 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.
Eight wells with 200 μL isopropanol were pipetted also. The plate was read in a plate
spectrophotometer at 570 nm.

-Positive control: Methyl acetate (C3H6O2, CAS No. 79-20-9)
-Negative control: Sterile demineralised water
-Storage of test item: The test item was stored in the test facility in a closed vessel at room temperature (20 ±
5°C).

Calculation:
 Calculation of mean OD of the blank isopropanol (ODBlk)
 Subtraction of mean ODBlk of each value of the same experiment (corrected values)
 Calculation of mean OD of the two replicates for each tissue
 Calculation of mean OD of the two relating tissues for controls and test item

Results and discussion

In vitro

Results

Irritation parameter:

other: % viability (formazan production)

Value:

2.3

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

Absorbance Values Negative Control, Positive Control and Test Item (OD at 570 nm)

Designation	Measurement	Negative control	Positive control	Nicotine bitartrate dihydrate
Tissue 1	1	1.733	0.585	0.077
	2	1.735	0.591	0.076
Tissue 2	1	1.923	0.618	0.080
	2	1.891	0.663	0.080

Mean Absorbance Negative Control, Positive Control and Test Item

Designation	Negative Control	Positive Control	Nicotine bitartrate dihydrate
Mean - blank (Tissue 1)	1.697	0.551	0.040
Mean - blank (Tissue 2)	1.870	0.604	0.043

%Viability Positive Control and Test Item

Designation	Positive control	Nicotine bitartrate dihydrate
%Viability (Tissue 1)	30.9%	2.2%
%Viability (Tissue 2)	33.8%	2.4%
%Viability Mean	32.4%	2.3%

Values for negative control and for positive control were within the range of historical data

of the test facility.

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Conclusions:

According to the OECD Guideline 492, the EpiOcularTM Eye Irritation Test does not allow
discrimination between eye irritation/reversible effects on the eye (Category 2) and serious
eye damage/irreversible effects on the eye (Category 1). For these purposes, further testing
with other suitable test methods is required.
Under the conditions of the test system, Nicotine bitartrate dihydrate is considered
as at least eye irritant in the EpiOcularTM Eye Irritation Test.
After treatment with the test item, the relative absorbance values were reduced to 2.3 %.
This value is below the threshold for eye irritation potential (≤ 60%).
All validity criteria were met. The criterion for optical density of the negative control was
fulfilled: The OD value was 1.8 (> 0.8 and < 2.5).
The positive control induced a decrease in the relative absorbance as compared to the
negative control to 32.4%.
Variation within the replicates was acceptable (≤20 %).
For these reasons, the result of the test is considered as valid.

Executive summary:

One valid experiment was performed.

The test item Nicotine bitartrate dihydrate was applied to a three-dimensional human cornea

tissue model in duplicate for an exposure time of 6 hours and 3 minutes.

The solid test item was applied to each tissue.

After treatment, the respective substance was rinsed from the tissue; then, cell viability of

the tissues was evaluated by addition of MTT, which can be reduced to formazan. The

formazan production was evaluated by measuring the optical density (OD) of the resulting

solution.

Demineralised water was used as negative control; Methyl acetate was used as positive

control.

The controls showed the following results: After treatment with the negative control, the

absorbance values were within the required acceptability criterion of 0.8 ≤ mean OD ≤ 2.5,

OD was 1.8. The positive control showed clear eye irritating effects, the relative absorbance

value was reduced to 32.4 % (< 50%).

Variation within tissue replicates was acceptable (≤ 20%).

After treatment with the test item, the relative absorbance values were reduced to 2.3 %.

This value is below the threshold for eye irritation potential (≤ 60%).