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Administrative data

Description of key information

One study (LLNA) is available for skin sensitisation. This key study is GLP compliant and of high quality (Klimisch 1).

The Local Lymph Node Assay (LLNA) provides a rational basis for risk assessment to the sensitising potential of the test item in man. No further testing is needed for the assessment of this endpoint.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2014-09-10 to 2014-10-14
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: 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.
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: CBA/CaOlaHsd , Harlan Laboratories B.V., AD Horst / The Netherlands
- Age at study initiation: 9 - 10 weeks
- Weight at study initiation: 19.3-22.8 g
- Housing: group
- Cage Type: Makrolon Type II (pre-test) / III (main study), with wire mesh top
- Diet (e.g. ad libitum): 2018C Teklad Global 18% protein rodent diet (certified), ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: At least 5 days prior to the start of dosing under test conditions after health examination

ENVIRONMENTAL CONDITIONS
- Temperature (°C): temperature 22 ± 2°C
- Humidity (%): relative humidity approx. 45-65% (The relative humidity in the animal room was between approximately 45 - 100 % instead of 45 – 65% for few hours. This deviation does not affect the validity of the study)
- Photoperiod (hrs dark / hrs light): artificial light 6.00 a.m. - 6.00 p.m.

IN-LIFE DATES: daily
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
0.5%; 1% and 2% (which are approximately 12.2; 24.4 and 48.8 mg/kg body weight, calculated with the mean body weight of the animals before start of treatment and a density of nicotine of 1 g/mL)
No. of animals per dose:
4
Details on study design:
TREATMENT PREPARATION AND ADMINISTRATION:
RANGE FINDING TESTS:
- Compound solubility: A solubility experiment was performed according to the recommendations given by OECD 429. The highest test item concentration, which can be technically used, was 100% of the undiluted test item. Test item solution at different concentrations was prepared using acetone/olive oil (4+1, v/v) as vehicle.
- Irritation: The highest concentration tested was the highest concentration that could be achieved whilst avoiding systemic toxicity and excessive local skin irritation as confirmed by three pre-experiments (see "Any other information on materials and methods incl. tables").

MAIN STUDY
- Each test group of mice was treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 0.5, 1, and 2% (w/v) in acetone/olive oil (4+1, v/v).
- The application volume, 25 µL/ear/day, was spread over the entire dorsal surface ( diameter 8 mm) of each ear once daily for three consecutive days.
- A further group of mice (control animals) was treated with an equivalent volume of the relevant vehicle alone (control animals).
- Five days after the first topical application (day 6) 250 µL of phosphate-buffered saline containing 20.4 µCi of ³H-methyl thymidine (equivalent to 81.5 µCi/mL ³HTdR) were injected into each test and control mouse via the tail vein.
- Approximately five hours after treatment with ³HTdR all mice were euthanised by using CO2, which was, after harvesting of the lymph nodes, followed by cervical dislocation to ensure death.
- The draining lymph nodes were rapidly excised and pooled for each experimental group (8 nodes per group).
- Single cell suspensions (in phosphate buffered saline) of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 µm mesh size).
- After washing two times with phosphate buffered saline (approx. 10 mL) the lymph node cells were resuspended in 5% trichloroacetic acid (approx. 3 mL) and incubated at approximately +4 °C for at least 18 hours for precipitation of macromolecules.
- The precipitates were then resuspended in 5% trichloroacetic acid (1 mL) and transferred to scintillation vials with 10 mL of scintillation liquid and thoroughly mixed.
- The level of ³HTdR incorporation was then measured in a beta-scintillation counter.
- Similarly, background ³HTdR levels were also measured in two 1 mL-aliquots of 5% trichloroacetic acid. The beta-scintillation counter expresses ³HTdR incorporation as the number of radioactive disintegrations per minute.
- The proliferative response of the lymph node cells is expressed as the number of radioactive disintegrations per minute per lymph node (DPM/lymph node) and as the ratio of ³HTdR incorporated into lymph node cells of test animals relative to that recorded for lymph nodes of control animals (Stimulation Index; S.I.).
- Before DPM/lymph node values were determined, mean scintillation-background DPM was subtracted from test and control raw data.

A test item is regarded as a sensitiser in the LLNA if the following criteria are fulfilled:
• First, that exposure to at least one concentration of the test item resulted in an incorporation of ³HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the Stimulation Index.
• Second, that the data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.

In addition to the sensitising reactions the following observations and data were recorded during the test and observation period:
Mortality / Viability: At least once daily from experimental start to necropsy.

Body weights: In the pre-test: prior to the first application and prior to sacrifice. In the main experiment: prior to the first application and prior to treatment with ³HTdR.

Ear thickness: In the pre-test prior to the first application of the test item (day 1), on day 3 and before sacrifice (day 6).

Ear weights: In the pre-test after sacrifice; biopsy punches were taken from each ear.

Clinical signs (local / systemic): Clinical signs (local irritation at the application site or systemic toxicity) were recorded at least once daily. Especially the treatment sites were observed carefully.

Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
The mean values and standard deviations were calculated.
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"):
0%: 1.00
5%: 2.51
10%: 1.76
25%: 6.79
Calculation of EC3 value:
EC3: (10% - 25%) [(3 - 6.79) / (1.76 - 6.79)] + 25 = 13.7% with the co-ordinates of the two pairs of data lying immediately above and below the S.I. value of 3 on the LLNA dose response plot.

Key result
Parameter:
SI
Value:
1.06
Test group / Remarks:
0.5 % nicotine
Key result
Parameter:
SI
Value:
0.91
Test group / Remarks:
1 % nicotine
Key result
Parameter:
SI
Value:
0.92
Test group / Remarks:
2% nicotine
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: See "Any other information on results incl. tables"

Viability / Mortality

No deaths occurred during the study period.

Clinical Signs

From day 1 to 3, the animals treated with 2% test item concentration showed reduced spontaneous activity and on days 1 (here only animal no. 15), 4, and 5 ruffled fur. On day 3, the animals treated with a test item concentration of 2% showed an erythema of the ear skin (Score 1). Animals treated with 0.5 and 1% test item concentration did not show any signs of local skin irritation and toxicity.

Body Weights

The body weight of the animals, recorded prior to the first application and prior to treatment with 3HTdR, was within the range commonly recorded for animals of this strain and age.

Calculation and Results of Individual Data

Test item concentration %

Group

Measurement DPM

Calculation

Result

DPM-BGa)

number of lymph nodes

DPM per lymph nodeb)

S.I.

---

BG I

14

---

---

---

---

---

BG II

14

---

---

---

---

0

1

5899

5885.0

8

735.6

1.00

0.5

2

6251

6237.0

8

779.6

1.06

1

3

5382

5368.0

8

671.0

0.91

2

4

5451

5437.0

8

679.6

0.92

1    = Control Group

2-4= Test Group

a)   = The mean value was taken from the figures BG I and BG II

b)    = Since the lymph nodes of the animals of a dose group were pooled, DPM/node was determined by dividing the measured value by the number of lymph nodes pooled

The EC3 value could not be calculated, since all S.I.´s are below the threshold value of 3.

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
No observations were made which might cause doubts concerning the validity of the study outcome. The test is considered valid. The test item nicotine was not a skin sensitiser and is therefore not classified according to Regulation (EC) No 1272/2008.
Executive summary:

In order to study a possible skin sensitising potential of nicotine, three groups each of four female mice were treated once daily with the test item at concentrations of 0.5, 1, and 2% (w/v) in acetone/olive oil (4+1, v/v) by topical application to the dorsum of each ear for three consecutive days. The highest concentration tested was the highest concentration that could be achieved whilst avoiding systemic toxicity and excessive local skin irritation as confirmed by three pre-experiments. A control group of four mice was treated with the vehicle (acetone/olive oil (4+1, v/v)) only. Five days after the first topical application the mice were injected intravenously into a tail vein with radio-labelled thymidine (3H-methyl thymidine). Approximately five hours after intravenous injection, the mice were sacrificed, the draining auricular lymph nodes excised and pooled per group. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes, which were subsequently washed and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of 3H-methyl thymidine measured in a beta-scintillation counter.

All treated animals survived the scheduled study period. From day 1 to 3, the animals treated with 2% test item concentration showed reduced spontaneous activity and on days 1 (here only animal no. 15), 4, and 5 ruffled fur. On day 3, the animals treated with a test item concentration of 2% showed an erythema of the ear skin (Score 1). Animals treated with 0.5 and 1% test item concentration did not show any signs of local skin irritation and toxicity.

A test item is regarded as a sensitiser in the LLNA if the exposure to one or more test concentration resulted in a 3-fold or greater increase in incorporation of 3HTdR compared with concurrent controls, as indicated by the Stimulation Index (S.I.). The estimated concentration of test item required to produce a S.I. of 3 is referred to as the EC3 value.

In this study Stimulation Indices of 1.06, 0.91, and 0.92 were determined with the test item at concentrations of 0.5, 1, and 2% (w/v) in acetone/olive oil (4+1, v/v). A dose response was not observed. The EC3 value could not be calculated, since none of the tested concentrations induced a S.I. greater than the threshold value of 3.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

The provided in vivo study (LLNA) is GLP compliant and of high quality (Klimisch 1). Nicotine was not a skin sensitiser and the EC3 vlue could not be calculated since none of the tested concentrations induced a S.I greater than the threshold of 3.

There is no information available for respiratory sensitisation. There is no internationally accepted animal model for respiratory sensitisation, In case human data emerges, this will be taken into account.

Based on the available data, nicotine is not classified for sensibilisation and no further testing is needed.