Nicotine dihydrogen ditartrate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-10-12 to 2017-04-05

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study was performed in accordance with standard test protocols (OECD 429 resp. EU B.42) 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 429 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

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

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: AnLab Prague, Czech Republic
- Females: nulliparous and non-pregnant
- Age at study initiation: 8-10 weeks
- Weight at study initiation: 20-23 g
- Housing: Polypropylene cages, 5 animals per cage, supsended on stainless steel racks
- Diet (e.g. ad libitum): laboratory food ssniff (ssniff Spezialdiäten GmbH, Germany), served ad libitum, each day approximately at the same time
- Water (e.g. ad libitum): tap water for human consumption. Supply of drinking water was unlimited. Quality of drinking water was periodically monitored (including microbiological control) and recorded
- Acclimation period: 6 days prior to the start of treatment. Acclimation was according to standard operation procedures.
- Indication of any skin lesions: none

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 + - 3°C
- Humidity (%): 50 - 60%
- Air changes (per hr):central air conditioning
- Photoperiod (hrs dark / hrs light): 12-hour light/ 12-hour dark cycle

Study design: in vivo (LLNA)

Vehicle:

dimethyl sulphoxide

Concentration:

Pre-screen test: 100% (w/v)
Main study: 25%, 50%, 100% (w/v)
Main study (part 2): 5%, 10%, 25% (w/v)

Test item suspension was prepared by mixing of 1g of test item up to 1mL of vehicle.

No. of animals per dose:

Number of animals:
4 females - pre-screen test
Main Study
5 females – negative control (vehicle)
5 females – positive control
15 females – test item
Main Study (part 2)
5 females – negative control (vehicle)
5 females – positive control
15 females – test item

Details on study design:

PRE-SCREEN TESTS:
The pre-screen test was conducted under the same conditions as the main LLNA study, except there was no assessment of lymph node proliferation.

- Compound solubility: A solubility experiment was performed according to the recommendations given by OECD 429. The test item was insoluble in acetone:olive oil, 4:1 v/v, propylene glycol and dimethyl sulfoxide, but the most homogenous suspension was prepared by mixing of the test item with DMSO. Based on this DMSO was selected for preparation of suspension for application to an ear of the mice.
- Test procedure: All mice (2 mice/group) were observed daily for any clinical signs of toxicity or local irritation at the application site. Body weights were recorded pre-test and prior to the termination (day 6). Both ears of each mouse were assessed for any sign of irritation. Ear thickness was measured by a calliper on Day 1 (pre-dose), Day 3 and Day 6. Excessive local skin irritation is indicated by an erythema score ≥ 3 and/or an increase in ear thickness of ≥25%.

MAIN STUDY

Day 1: Each animal was identified and the body weight was recorded. To the dorsum of each ear 25μL of the appropriate dilution of the test item, or the vehicle alone was applied.
Days 2 and 3: The application procedure carried out on day 1 was repeated.
Days 4 and 5: No treatment.
Day 6: The body weight of each animal was recorded. 250μL of phosphate-buffered saline (PBS) containing 2 μCi (7.4 x 104 Bq) of 125I-iododeoxyuridine and 10-5M fluorodeoxyuridine was injected into all test and control mice via the tail vein. Five hours later, the animals were sacrificed. The draining auricular lymph nodes from each ear were excised and pooled in PBS for each experimental group, (pooled treatment group approach) and weighted.

Preparation of solutions:
- 25% a-Hexylcinnamaldehyde: 0.5 mL of -Hexylcinnamaldehyde at a concentration of 85% was diluted in 1.2 mL vehicle (Acetone/Olive Oil, 4:1, v/v). Stock and diluted solution of a-Hexylcinnamaldehyde were kept at laboratory temperature.
- 5% Trichloracetic acid: 2.5 g TCA was dissolved in 50 mL of deionized water. The solution was kept in the refrigerator.
- Phosphate buffered saline: One tablet of PBS was dissolved in 100 ml deionized water. The solution was kept in the refrigerator.
- 125I-iododeoxyuridine: 125I-iododeoxyuridine (1 mCi/mL) was diluted for final radioactivity of  2 μCi (7.4 x 104 Bq) in 250μL of PBS buffer. Diluted solution of 125I-iododeoxyuridine was prepared maximally 3 days before its application to animals. Stock and diluted solution of 125I-iododeoxyuridine were kept in refrigerator.
- Dose Preparation: The required amount of the test item (according to the concentration) was mixed in vehicle shortly before the administration. The test item in the amount of 1000 mg was mixed in vehicle up to volume of 1 ml (1000 mg/ml) and this was the highest achievable concentration (100% w/v). The lower concentrations were prepared by mixing of 500 mg 250 mg, 100 mg 50 mg in vehicle up to 1 ml – giving 50, 25, 10 and 5% w/v concentration. The preparations were made freshly before each dosing occasion.
- Dose Administration: The test item was administrated in volume of 25 L to the dorsum of each ear. The a-Hexylcinnamaldehyde (25%) as positive control and vehicle as a negative control were administrated in the same volume.

Preparation of cell suspensions: Cell suspension of lymph node cells from pooled treatment groups were prepared by gentle
mechanical disaggregation by glass homogenizer. Lymph node cells were centrifuged (SOPA-00156-BIO) by 600g at 4°C for 10 min. Suspension of cells were precipitated with 5% trichloroacetic acid (TCA) at 4°C for 18-20h. Pellets were centrifuged by 2000g at 4°C for 5 min., re-suspended in 1 ml TCA and transferred to gamma counting tubes for 125I-counting.

Determination of cellular proliferation (incorporated radioactivity): Incorporation of 125I-iododeoxyuridine was measured by 125I-counting on Automatic Gamma Counter Wizard2 (Perkin Elmer) as counts per minute (CPM). The incorporation is expressed as disintegrations per minute (DPM)/treatment group. DPM were calculated according to formula:
DPM= CPM/ absolute detector efficiency
(Absolute detector efficiency for Gamma Counter Wizard2 2470 = 66.73%)

Clinical observation: Animals were carefully observed once daily for any clinical symptoms, either of local irritation at the application site or of the systemic toxicity. All observations were systematically recorded with individual records being maintained for each animal.

Body Weight: The animal body weights were determined at the start of the test and at the scheduled day of euthanasia of the animals.

Calculation of results:
Results are expressed as the Stimulation Index (SI). The SI was obtained by dividing the pooled radioactive incorporation for each treatment group by the radioactive incorporation of the pooled vehicle control group; this yields a mean SI. A substance is regarded as sensitizer in the LLNA test when SI≥3.
EC3 value, which induce stimulation indices, is determined by linear interpolation of points on dose-response curve, immediately above and below of SI value, according to the equation: EC3=c+[(3-d)/(b-d)]x(a-c)
a – higher concentration, b – SI of higher concentration, c – lower concentration d – SI of lower concentration
If all points are below the stimulation index of three, no EC3 value can be stated.

Main study (part 2):

The experimental part finished on 02.11.2016 did not allow to finalise the evaluation of the sensitisation potential of the test item due to systemic toxic effects in the main study in two dose groups. For this reason additional animals groups (positive control, negative control and 5, 10 and 25% concentrations of test item) were added.

Clinical Observations:
Animals were carefully observed for any clinical symptoms, either of local irritation at the application site or systemic toxicity.

Body Weights:
The animal body weights were measured prior to the first treatment and at the scheduled sacrifice.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

The mean values and standard deviations were calculated.

Results and discussion

Positive control results:

The sensitivity and reliability of the experimental technique employed was assessed by use of α-hexyl cinnamaldehyde dissolved in acetone/olive oil (4+1 v/v) (compound listed in OECD 429 Guideline) which is known to have skin sensitisation properties in mice. The periodic positive control experiment was performed in April 2014 using CBA/CaOlaHsd mice. The S.I. results were stated as follows (see also table in "Any other information on materials and methods incl. tables"):
Main study:
SI (positive control)= 12.79

Main study (part 2):
SI (positive control)= 6.08

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

1.49

Test group / Remarks:

5% Nicotine bitartrate dihydrate

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: See "Any other information on results incl. tables"

Results 3

Key result

Parameter:

SI

Value:

2

Test group / Remarks:

10% Nicotine bitartrate dihydrate

Results 4

Key result

Parameter:

SI

Value:

2.06

Test group / Remarks:

25% Nicotine bitartrate dihydrate

Results 5

Key result

Parameter:

EC3

Remarks on result:

not determinable

Cellular proliferation data / Observations:

see "Any other information on results incl. tables"

Any other information on results incl. tables

Pre-screen test

After the test item application, no signs of local irritation at the application site (erythema, redness,

oedema, scab formation) or systemic toxicity (pilo-erection, change in grooming activity, behaviour

activity, tremor, apathy, respiratory problems, ataxia, convulsions, gasping, aggressiveness) were

observed.

The animal body weights were measured prior to the first treatment and at the scheduled sacrifice.

No marked changes of mean body weight were observed during the test. The individual and mean

values of animal body weights of the treated group are shown in the Table below.

Pre-screen test: Initial and terminal body weights (g)

Mouse no.	Initial body weight	Terminal body weight
1	22.10	21.94
2	21.13	20.81
Mean	21.62	21.38
S.D.	0.686	0.799

No erythema was observed in both mice after test item administration. On day 1 (pre dose), day 3 and day 6 the ear thickness was measured.

The erythema score and the increase in ear thickness did not meet the criteria which are considered as signs for excessive local skin irritation (erythema score ≥ 3; ear thickness increase ≥25%).

For calculation of mean and S.D. values of body weights and ear thickness MS Excel was used.

Main Study (part 1)

Based on the observations recorded in the preliminary test, the concentration of 100, 50 and 25% was selected for the main test.

- Clinical Observations: The daily clinical observation of the animals did not show visible clinical signs.

- Body Weights: The animal body weights were measured prior to the first treatment and at the scheduled sacrifice. The decrease of body weight was observed in all treated groups and at concentrations of 50% and 100%, the decrease was statistically significant. The individual and mean values of animal body weights of the control group, positive control group and the three test item treated groups are shown in the Table below.

For calculation of mean, S.D. and t-test values of body weights MS Excel was used.

Mean body weights - Main study (part 1)

	Initial body weight (g) [S.D]	Terminal body weight (g) [S.D]
Control (vehincle)	21.20 [0.787]	21.45 [1.017]
Positive control	21.46 [1.459]	21.96 [1.595]
Nicotine bitartrate dihydrate (100%)	21.10 [1.946]	17.18* [1.614]
Nicotine bitartrate dihydrate (50%)	19.97 [0.784]	18.05* [0.738]
Nicotine bitartrate dihydrate (25%)	21.71 [0.477]	21.26 [1.021]

Lymph Node Proliferation

In comparison with the control group, an increase of the pooled lymph node weight was recorded at all used concentrations. The pooled lymph node weights of treated groups were 0.0278g for 25% concentration, 0.0282g for 50% concentration and 0.0414g for 100% concentration of tested item. The lymph node weight of control group and positive control group were 0.0324g and 0.0838g, respectively. The DPM values for the three treated groups were 1021 (25%), 1143 (50%) and 1754 (100%), respectively. The SI values for the three treated groups were 1.86 (25%), 2.08 (50%) and 3.19 (100%), respectively. The lymph node weights, DPM and SI values are shown in the table below.

Lymph node, DPM, SI values - Main study (part 1)

	Lymph node weight (g)	Number of lymph nodes	DPM	SI
Control	0.0324	10	550	1.0
Positive Control	0.0838	10	7037	12.79
Nicotine bitartrate dihydrate (25%)	0.0278	10	1021	1.86
Nicotine bitartrate dihydrate (50%)	0.0282	10	1143	2.08*
Nicotine bitartrate dihydrate (100%)	0.0414	10	1754	3.19*

* values determined for these doses were excluded from evaluation because of a significant decrease of body weight >10%.

Main study (part 2)

The experimental part finished on 02.11.2016 did not allow to finalise the evaluation of the sensitisation potential of the test item due to systemic toxic effects in the main study in two dose groups. For this reason additional animals groups (positive control, negative control and 5, 10 and 25% concentrations of test item) were added.

Clinical Observations

Animals were carefully observed for any clinical symptoms, either of local irritation at the application site or systemic toxicity. The daily clinical observation of the animals did not show visible clinical signs.

Body Weights

The animal body weights were measured prior to the first treatment and at the scheduled sacrifice. The individual and mean values of animal body weights of the control group, positive control group and the three test item treated groups are shown in Table 7. For calculation of mean, S.D. and t-test values of body weights MS Excel was used.

	Initial body weight (g) [S.D]	Terminal body weight (g) [S.D]
Control (vehincle)	19.27 [0.653]	19.68 [0.797]
Positive control	18.89 [0.369]	19.63 [0.280]
Nicotine bitartrate dihydrate (5%)	19.62 [0.407]	21.07 [0.957]
Nicotine bitartrate dihydrate (10%)	18.29 [0.426]	19.05 [0.273]
Nicotine bitartrate dihydrate (25%)	17.74 [0.405]	18.01 [0.279]

Lymph Node Proliferation

In comparison with the control group, an increase of the pooled lymph node weight was recorded at concentrations 10 and 25%. The pooled lymph node weights of treated groups were 0.0396g for 5% concentration, 0.0426g for 10% concentration and 0.0443g for 25% concentration of tested item. The lymph node weight of control group and positive control group were 0.0391g and 0.0621g, respectively. The DPM values for the three treated groups were 2000 (5%), 2676 (10%) and 2758 (25%), respectively. The SI values for the three treated groups were 1.49 (5%), 1.99 (10%) and 2.06 (25%), respectively. The lymph node weights, DPM and SI values are shown in table below.

Lymph node weight, DPM, SI values - Main study (part 2)

	Lymph node weight (g)	Number of lymph nodes	DPM	SI
Control	0.0391	10	1340	1.0
Positive Control	0.0621	10	8141	6.08
Nicotine bitartrate dihydrate (25%)	0.0396	10	2000	1.49
Nicotine bitartrate dihydrate (50%)	0.0426	10	2676	2.00
Nicotine bitartrate dihydrate (100%)	0.0443	10	2758	2.06

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Remarks:

The EC3 value could not be determined.

Conclusions:

The skin sensitizing potential of Nicotine bitartrate dihydrate was assessed using the murine local lymph node assay (LLNA), according to OECD Guideline No. 429. In this study the test item was tested at concentrations of 5, 10, 25, 50, and 100% in dimethyl sulfoxide. At concentration of 100% and 50% a significant decrease of body weight >10% without any other signs of toxicity within the observation period were determined, which is likely based on a systemic toxic effect of the test item. Because of that, the SI values determined for these doses were excluded from evaluation. Based on the Stimulation Indices (SI) below of three determined at concentration of 5, 10, and 25% the Nicotine bitartrate dihydrate is not considered to be a skin sensitizer under the conditions of this study. The EC3 value could not be determined.

Executive summary:

The sensitization potential of Nicotine bitartrate dihydrate was evaluated using the Local Lymph Node Assay (LLNA). The LLNA has been developed to determine the allergic contact sensitization potential of chemicals.

Based on the recommendations of the OECD Guideline 429 and solubility evaluation, the test item was dissolved in Dimethyl sulfoxide (DMSO). As a positive control 25% a-Hexylcinnamicaldehyde was used. The Pre-screen test was performed using a dose of 100 % (w/v). Based on the observations recorded in the preliminary test, the concentration of 100 % was selected as top dose for the main test. Five female mice (CBA/Ca) per group were topically exposed (dorsum of both ears) to the test item at concentrations of 25%, 50% and 100%, to the positive control and to the vehicle only. Lymphocyte proliferation was measured using incorporation of radioactive 125I-iododeoxyuridine and 10-5M fluorodeoxyuridine in the draining lymph nodes. The radioactive incorporation was expressed as disintegrations per minute (DPM)/pooled treatment group and compared with DPM value from the vehicle control group and expressed as the Stimulation Index (SI). After application of the test item at three concentrations (25%, 50% and 100% w/v) the animals did not show visible clinical symptoms of local irritation. At concentrations of 50% and 100% the decrease of body weight was observed. The decrease of body weight was > 10% and statistically significant.

Due to the systemic toxic effect (body weight decrease) in the main study in two dose groups (100 and 50%) the additional testing was needed (see section 8.3. Main study part 2). In the repeated Main study the additional groups (positive control, negative control and test item concentrations of 5, 10 and 25%) were tested under the same test conditions. Stimulation Indices (SI) of 1.49, 2.00 and 2.06 were determined with the test item at concentration of 5, 10, and 25% in dimethyl sulfoxide, respectively. The EC3 value could not be calculated, as all measured points were below the Stimulation Index of three. The test item Nicotine bitartrate dihydrate is not considered a skin sensitizer under the test conditions of this study.