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Administrative data

Description of key information

-Skin irritation: Irritating but non-corrosive effects on the treatment site of rabbits in both sexes could be observed in an acute dermal toxicity study (OECD 402). Erythrema were fully reversible within 3 days in females and 14 days in males. No other dermal effects noted.

-Eye irritation: Highly corrosive to the eye in rats. irritating effects of tissues were observed in vitro (OECD492) after 30 minutes. Irreversible redness (grade 1), haemorrhage (grade 2) and opacity (grade 2) were observed in an in vivo study of high quality (OECD 405).

All studies were GLP compliant and of high quality (Klimisch 1).

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015-05-26 to 2015-08-10
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was performed in accordance with standard test protocols (OECD 402) in a quality controlled laboratory. The study is valid according to criteria mentioned in the test protocols.
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
other: OECD Guideline 402
Deviations:
no
GLP compliance:
yes (incl. certificate)
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
cross-reference: section 7.2.3
Type of coverage:
semiocclusive
Preparation of test site:
shaved
Vehicle:
unchanged (no vehicle)
Controls:
no
Amount / concentration applied:
Preliminary study: 1000, 200, 50 and 5 mg/kg bw dose in females
Main study: 200, 100 and 50 mg/kg bw dose in females and 50 mg/kg bw dose in males
Duration of treatment / exposure:
24-hour exposure period
Observation period:
14-day observation period
Number of animals:
Preliminary study: n=2 animals/dose
Main study: n=5 animals/sex/dose
Irritation parameter:
erythema score
Remarks on result:
other: see "irritant/corrosive response data" and for detailed information please see "Any other information on results incl. tables".
Irritation parameter:
edema score
Remarks on result:
other: see "irritant/corrosive response data" and for detailed information please see "Any other information on results incl. tables".
Irritant / corrosive response data:
Dermal irritation symptoms were observed on the treatment site in both sexes.
In males treated with 50 mg/kg bw dose (group 1), very slight redness (score 1), well defined redness (score 2) and moderate to severe redness (score 3) was observed in one animal between Day 1 and Day 7. Moderate to severe redness (score 3) and severe redness (score 4) was detected in two animals between Day 1 and Day 7. Very slight redness (score 1) and well defined redness (score 2) was recorded in one animal between Day 1 and Day 7. Very slight redness (score 1) occurred in one animal between Day 1 and Day 5. Another sign as desquamation appeared in four animals on Day 6 and in one animal on Day 7. Crusting was recorded in four animals. It was observable between Day 7 and Day 14. Wound was found in two animals. It was observable between Day 11 and Day 14.
One male became free of skin irritation symptoms by Day 10.
In females treated with 50 mg/kg bw dose (group 1), very slight redness (score 1) was observed in two animals on Day 1. Well defined redness (score 2) and very slight redness (score 1) was detected in one animal on Day 1 and on Day 2, respectively. Moderate to severe redness (score 3) and well defined redness (score 2) was recorded in one animal on Day 1 and on Day 2, respectively.
Very slight redness (score 1) occurred in one animal treated with 100 mg/kg bw dose (group 2) on Day 1.
The survivor females became free of skin irritation symptoms by Day 3.
There was no oedema observed during the 14-day observation period in both sexes, but it was observable in females of group 2 and 3 on necropsy day.

Local symptoms (dermal irritation)

Oe:0 = No oedema

R:0 = No redness

R:1 = Very slight redness

R:2 = Well defined redness

R:3 = Moderate to severe redness

R:4 = Severe redness

C = Crusting; W = Wound; D = Desquamation

Males - Group 1 (50 mg/kg bw)

 Animal No. Day 1  Day 3   Day 7  Day 14
 #1  R:1; Oe:0 R:2; Oe:0  R:1; Oe:0; D  R:0; Oe:0; C 
 #2  R:3; Oe:0 R:4; Oe:0  R:4; Oe:0; D; C  R:0; Oe:0; C 
 #3  R:1; Oe:0 R:1; Oe:0  R:2; Oe:0; D; C  R:0; Oe:0; W 
 #4  R:3; Oe:0 R:3; Oe:0  R:4; Oe:0; D; C  R:0; Oe:0; W 
 #5  R:1; Oe:0 R:1; Oe:0  R:0; Oe:0; D  R:0; Oe:0 

Females - Group 1 (50 mg/kg bw)

Animal Number Day 1  Day 2  Day 3 
#1 R:1; Oe:0  R:0; Oe:0  R:0; Oe:0 
#2  R:1; Oe:0  R:0; Oe:0  R:0; Oe:0
#3 
#4   R:2; Oe:0 R:1; Oe:0  R:0; Oe:0 
#5   R:3; Oe:0 R:2; Oe:0  R:0; Oe:0 

Females - Group 2 (100 mg/kg bw)

Animal Number Day 1  Day 2  Day 3 
#1 
 #2
 #3
 #4 R:1; Oe:0  R:0; Oe:0  R:0; Oe:0 
 #5

Females - Group 3 (200 mg/kg bw)

No data available due to lethality.

The survivor females were free of skin irritation symptoms from Day 4 until the end of observation period.

Interpretation of results:
Category 2 (irritant)
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
Nicotine caused dermal irritation symptoms on the treatment site in both sexes in an acute toxicity study by dermal route in rabbits exposed to 3.0-3.2 mg/cm². No corrosion symptoms, according to CLP-regulation, Annex I, were observed, thus the substance is classified as Skin Irrit.2
Executive summary:

Data for skin irritation potential were obtained from the acute dermal toxicity test (OECD 402, section 7.2.3.).

The test item caused dermal irritation symptoms on the treatment site in both sexes.

In males, very slight to severe erythema was observed between Day 1 and Day 7 and other signs as desquamation, crusting and wound were detected between Day 6 and the necropsy day, as well.

In females, very slight to severe erythema was recorded in 50 mg/kg bw dose group between treatment day and Day 2. Very slight to severe erythema and slight oedema was found in 100 mg/kg bw dose group between treatment day and Day 1. Moderate to severe erythema and slight to moderate oedema was detected in 200 mg/kg bw dose group on the treatment day.

Justification of the classification category (Skin Irrit. 2) is based on calculation of the amount of test substance per cm² and comparison to the test substance concentration of 80 mg/cm² employed in the EU B.4/OECD TG 404 (according to the recommended approach of testing and assessment strategy for skin corrosion/irritation in the Guidance on Information Requirements and Chemical Safety Assessment Chapter R.7a, Version 4.0, July 2015, Section R.7.2.6.2 (d)):

The back of animals was shaven (approximately 10 % area of the total body surface, approx. 36 cm²) 24 hours prior to the treatment. Group 1 (males) was treated with 50 mg nicotine per kg bw. The body weight range in main study at starting (males, group 1) was 2210 - 2310 g, thus the dose was 110.5-115.5 mg/36 cm². Therefore the animals were exposed to 3.0-3.2 mg/cm² nicotine. Compared to the standard test design treatment of 80 mg/cm², this small amount of nicotine per cm² resulted in clear dermal irritation symptoms on the treatment site in both sexes, especially in males: slight to severe redness, desquamation, crusting and wound. No corrosion symptoms (CLP,Annex I: 3.2.1.1. “irreversible damage of the skin […] typified by ulcers, bleeding, bloody scabs and, by the end of observation at 14 days, by discolouration due to blanching of the skin, complete areas of alopecia, and scars”) were observed, thus the substance is classified as Skin Irrit. 2.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-12-27 to 2018-03-27
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Deviations:
yes
Remarks:
A temperature higher than 23°C was registered on 08 January 2018. The maximum value measured was 25°C. This deviation is considered as without impact on the conclusion of the study.
Principles of method if other than guideline:
At the request of the Ethics Committee, the following procedure, slightly different from the
recommendation of the OCDE guideline, has been followed:
• Sixty minutes prior to test substance application (TSA), buprenorphine (batch No.: H011) 0.01
mg/kg was administered by subcutaneous injection (SC) to provide a therapeutic level of systemic
analgesia.
• Five minutes prior to TIA, one or two drops of a topical ocular anesthetic (e.g. Tetracaïne 1% batch
No.: 7D79A) were applied to each eye. The eye of each animal that was not treated with a test article,
but which was treated with topical anesthetics, served as a control.
• Eight hours after TSA, buprenorphine 0.01 mg/kg SC and meloxicam (batch No.: 16A271)
0.5 mg/kg SC were administered to provide a continued therapeutic level of systemic analgesia.
• About 24 hours after treatment, after the observations of the animal, if the animal presents with
marked lesions or if suffering of the animal was noted, buprenorphine 0.01 mg/kg SC and meloxicam
0.5 mg/kg SC were administered (Cf Analgesia administration appendix 2).
Remark: the lesions are considered as marked if the score obtained is ≥ 2 for at least two parameters
chemosis, redness or opacity of the cornea.
• Up to the end of the study and after each observation of the animal, buprenorphine 0.01 mg/kg SC
and meloxicam 0.5 mg/kg SC were administered if suffering or marked lesions were observed.
GLP compliance:
yes (incl. certificate)
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
Animals
One female albino New Zealand rabbit was supplied by Hypharm (F-49450 La Renaudiere). The
animal was kept during a minimal 5-day acclimatisation period.
At the beginning of the test, the animal was 15 weeks old.
It was identified prior to inclusion in the test by means of a numbered ring on the edge of one ear.

Housing
The animal was kept in an individual box installed in conventional air conditioned animal husbanding:
The temperature and relative humidity of the main test were controlled to remain within target ranges
of 17°C to 23°C and 30% to 70%, respectively.
The rate of air exchange was at least ten changes per hour and the lighting was controlled by a time
switch to give twelve hours continuous light (07.00 to 19.00) and twelve hours darkness.

Food and drink
Drinking water (tap-water from public distribution system) and foodstuff (ENVIGO – 2930C) were
supplied ad libitum.
Microbiological and chemical analyses of the water were carried out once every six months by Bureau
Veritas – Eurofins (FRANCE).
Vehicle:
unchanged (no vehicle)
Controls:
no
Amount / concentration applied:
0.1 mL of the test item was instilled, as supplied
Duration of treatment / exposure:
n.a.
Observation period (in vivo):
Ocular examinations were performed on both right and left eyes 1 hour, 24, 48 and 72 hours following
treatmentIf no reaction is observed 72 hours after instillation, the study is terminated. In case of persistent reactions,
additional observations can be carried out from D7 to D21 in order to determine the reversible character of the
lesions observed.
Number of animals or in vitro replicates:
One female
Details on study design:
Treatment
0.1 mL of the test item was instilled, as supplied, into the conjunctival sac of one eye after gently
pulling the lower lid away from the eyeball. The lids were then gently held together for about one
second in order to prevent loss of the test item. The other eye remained untreated serving as control.
Initially, a single animal was treated. After consideration of the responses in the first animal, no
additional animal was treated.
Irritation parameter:
chemosis score
Basis:
animal #1
Time point:
24/48/72 h
Score:
3.3
Max. score:
4
Reversibility:
not fully reversible within: 21 d
Remarks on result:
positive indication of irritation
Remarks:
for detailed information please see "Any other information on results incl. tables"
Irritation parameter:
overall irritation score
Basis:
animal #1
Time point:
21 d
Score:
24
Max. score:
90
Reversibility:
not fully reversible within: 21d
Remarks on result:
positive indication of irritation
Remarks:
for detailed information please see "Any other information on results incl. tables"
Irritation parameter:
conjunctivae score
Basis:
animal #1
Time point:
21 d
Score:
4
Max. score:
20
Reversibility:
not fully reversible within: 21d
Remarks on result:
positive indication of irritation
Remarks:
for detailed information please see "Any other information on results incl. tables"
Irritation parameter:
iris score
Basis:
animal #1
Time point:
24/48/72 h
Score:
2
Max. score:
2
Reversibility:
fully reversible within: 21d
Remarks on result:
positive indication of irritation
Remarks:
for detailed information please see "Any other information on results incl. tables"
Irritation parameter:
cornea opacity score
Basis:
animal #1
Time point:
24/48/72 h
Score:
3
Max. score:
3
Reversibility:
not fully reversible within: 21d
Remarks on result:
positive indication of irritation
Remarks:
for detailed information please see "Any other information on results incl. tables"
Irritant / corrosive response data:
The ocular reactions observed during the study have been moderate to severe and partially reversible:
- at the conjunctivae level: an important redness was noted 1 hour after the test item instillation and
remaining on the last day of the test (day 21) with slight intensity (grade 1). This reaction was
associated with a severe chemosis noted 24 hours after the test item instillation and remaining on the
last day of the test (day 21) with slight intensity (grade 1).
- at the iris level: an injection noted 1 hour after the test item instillation and haemorrhage was noted
between days 1 and 7 (grade 2).
- at the corneal level: an important opacity, noted 24 hours after the test item instillation and remaining
on the last day of the test (day 21) with moderate intensity (grade 2).
Withe secretions requiring a physiological saline rinse were noted between days 1 and 7.
A corneal-neovascularisation was noted between days 14 and 21.
Brown coloration on eyelids and nictitating membrane was noted between days 0 and 7.

EVALUATION OF OCULAR IRRITATION

Eye examinations are carried out using the scale of lesion scores in the following order:

 

CHEMOSIS (A)

·       No swelling.............................................................................................................  0

·       Slight swelling, including the nictitating membrane.................................................  1

·       Swelling with eversion of the eyelid.........................................................................  2

·       Swelling with eyelid half-closed...............................................................................  3

·       Swelling with eyelid more than half-closed..............................................................  4

 

 

DISCHARGE (B)

·     No discharge...........................................................................................................  0

·     Slight discharge (normal slight secretions in the inner corner not to be taken into account).................................. 1

·     Discharge with moistening of the eyelids and neighbouring hairs..............................  2

·     Discharge with moistening of the eyelids and large areas around the eye...................  3

 

 

REDNESS (C)

·     Blood vessels normal...............................................................................................  0

·     Vessels significantly more prominent than normal...................................................  1

·     Vessels individually distinguishable with difficulty

-...Generalised red coloration...................................................................................  2

-...Generalised deep red coloration...........................................................................  3

 

 

Iris (D)

·     Normal...................................................................................................................  0

·     Iris significantly more wrinkled than normal, congestion, swelling of the iris which continues to react to light, even slowly.............................  1

·     No reaction to light, haemorrhage, significant damage (any or all of these characteristics)...........................................................................  2

 

 

Cornea:DEGREE OF OPACITY (E)

·     No modification visible either directly or after instillation of fluorescein (no loss of glint or polish)........................  0

·     Translucent areas (diffuse or disseminated), iris details clearly visible........................  1

·     Easily identifiable translucent area, iris details slightly obscured...............................  2

·     Opalescent area, no iris details visible, pupil outline scarcely distinguishable.............  3

·     Total corneal opacity, completely obscuring the iris and pupil...................................  4

 

 

Cornea:EXTENT OF OPACITY (F)

·     Opaque area present but covering one quarter or less................................................  1

·     Between one quarter and half..................................................................................  2

·     Between half and three quarters...............................................................................  3

·     Between three quarters and the entire surface...........................................................  4

Table 1

 Animal No     Time after treatment     Conjunctivae    Iris  Cornea
 Chemosis (A) Redness (C)  Lesion (D)  Opacity (E) 
 #1 (A6838)              24 h  4  3  2  3
 48 h  3  3  2  3
 72 h  3  3  2  3
 TOTAL  10  9  6  9
 MEAN  3.3  3  2  3

Table 2

 Observation time    CONJUNCTIVAE          IRIS     CORNEA        Individual irritation index    
 A (A+B+C)x2  Dx5  ExFx5 
         X=   Y=      Z=  X+Y+Z= 
 1 hour (D0)  2  3  3 16  20  41 
 24 hours (D1)  3  20 10  60  90 
 48 hours (D2)  3  1  14 10  60  84 
 72 hours (D3)  1  14 10  60  84 
 Day 7 (D7)  2  0  2  12 10  3 45  67 
 Day 14 (D14)  2  0  2  8 20  28 
 Day 21 (D21)  1  0  1  4 20  24 

FromT0 to T0+30 sec: increase of salivation associated with tremors

T0+1h: decrease of spontaneous activity

D1 to D7:haemorrhage

D1 to D7: brown coloration on lids and nictitating membrane

D1 to D7:withe secretions requiring a physiological saline rinse,cornealoedema.

D2 to D7:scab on internal and external eyelids.

D14 to D21:corneal-neovascularisation

Interpretation of results:
Category 1 (irreversible effects on the eye) based on GHS criteria
Conclusions:
The results obtained, under these experimental conditions, enable to conclude that the test item (S)-Nicotine has to be classified in category 1 “irreversible effects on the eye” in accordance with the Regulation (EC) No. 1272/2008, the test item.
The signal word “Danger” and hazard statement H318 “Causes serious eye damage” are required.
Executive summary:

The test item (S)-Nicotine was instilled, as supplied, into the eye of one New Zealand rabbit at the dose of 0.1 mL. The experimental protocol was established on the basis of the official method as defined in the O.E.C.D. Test Guideline No. 405 dated October 9th, 2017.

An increase of salivation associated with tremors was noted immediately after the test item instillation and this during approximately 30 seconds. A decrease of spontaneous activity was noted at 1 hour after the test item instillation. The animal recovered a normal activity on day 1.

The ocular reactions observed during the study have been moderate to severe and partially reversible:

- at the conjunctivae level: an important redness was noted 1 hour after the test item instillation and

remaining on the last day of the test (day 21) with slight intensity (grade 1). This reaction was

associated with a severe chemosis noted 24 hours after the test item instillation and remaining on the

last day of the test (day 21) with slight intensity (grade 1).

- at the iris level: an injection noted 1 hour after the test item instillation and haemorrhage was noted

between days 1 and 7 (grade 2).

- at the corneal level: an important opacity, noted 24 hours after the test item instillation and remaining

on the last day of the test (day 21) with moderate intensity (grade 2).

Withe secretions requiring a physiological saline rinse were noted between days 1 and 7.

A corneal-neovascularisation was noted between days 14 and 21.

Brown coloration on eyelids and nictitating membrane was noted between days 0 and 7.

In conclusion, the results obtained, under these experimental conditions, enable to conclude that the

test item (S)-Nicotine has to be classified in category 1 “irreversible effects on the eye” in

accordance with the Regulation (EC) No. 1272/2008, the test item.

The signal word “Danger” and hazard statement H318 “Causes serious eye damage” are required.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

Nicotine has proven to be irritant to skin in a suitable acute dermal toxicity test, hence it is classified accordingly and no further testing was required.

For an assessment of the irritant potential on the eye a BCOP assay (OECD 437) was performed as an initial step within the testing strategy, but the result was not suitable for classification purposes (3<IVIS<55). Eye irritant effects and irreversible damage on the eye were observed in a second in vitro OECD TG 492 study and an in vivo OECD TG 405 study.

The studies were GLP compliant and of high quality (Klimisch 1).

Therefore, nicotine has to be classified as Skin Irrit. 2 and Eye Dam. 1.