Registration Dossier

Administrative data

Description of key information

Classification for acute oral toxicity was performed based on a weight of evidence analysis, since no key study of high quality (Klimisch 1) is available. The results of these studies should be used cautiously. However, the overall quality of the data base is appropriate for risk assessment, which was confirmed by RAC in its decision process on current harmonised classification.

The studies for acute inhalation and acute dermal toxicity are both of good quality (Klimisch 1(dermal) and Klimisch 2 (inhalation)) and GLP compliant, therefore no further testing is needed.

Key value for chemical safety assessment

Acute toxicity: via oral route

Link to relevant study records

Referenceopen allclose all

Endpoint:
acute toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study was performed in accordance with standard test protocols (OECD 425) in a quality controlled laboratory. The study is valid according to criteria mentioned in the test protocols.
Qualifier:
according to
Guideline:
OECD Guideline 425 (Acute Oral Toxicity: Up-and-Down Procedure)
Deviations:
yes
Remarks:
95% Confidence intervals could not be calculated.
GLP compliance:
yes (incl. certificate)
Test type:
up-and-down procedure
Limit test:
no
Species:
mouse
Strain:
NMRI
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: TOXI COOP ZRT. Cserkesz u. 90., 1103 Budapest, Hungary
- Age at study initiation: 8 weeks old in first, second and third step and 9 weeks old in fourth, fifth, sixth, seventh, eighth and ninth step
- Weight at study initiation: +/- 20% of mean weight
- Fasting period before study: The day before treatment the animals were fasted. The food, but not water was withheld for 3-4 hours.
- Housing: 3 animals/cage during acclimatization, individually after the treatment
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 days in first step, 7 days in second step, 9 days in third step, 11 days in fourth step, 12 days in fifth step, 13 days in sixth step, 14 days in seventh step, 16 days in eighth step and 17 days in ninth step

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30 - 70 %
- Air changes (per hr): 10-15 air exchanges/hour by central air-condition system
- Photoperiod (hrs dark / hrs light): Artificial light, from 6 a.m. to 6 p.m.

IN-LIFE DATES: From: Day 0 To: Day 14
Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
VEHICLE
- Concentration in vehicle: The test item was applied in a concentration of 1.75, 5.5, 17.5 and 55 mg/mL
- Amount of vehicle (if gavage): Concentration of formulations was adjusted to maintain a treatment volume of 10 mL/kg bw



Doses:
17.5; 55; 175; 550 mg/kg bw. The first animal was dosed a step below the best preliminary estimate of the LD50. The dose progression factor was 3.2.
No. of animals per sex per dose:
Group 1 (17.5mg/kg bw): 3 animals
Group 2 (55mg/kg bw): 3 animals
Group 3 (175mg/kg bw): 2 animals
Group 4 (550mg/kg bw): 1 animal
Control animals:
no
Details on study design:
- Duration of observation period following administration: 14 days (see below)
- Necropsy of survivors performed: yes (see below)

Mortality
Animals were observed individually after dosing at least once during the first 30 minutes, then 1 h, 2 h, 3 h, 4 h after the treatment and twice each day for 14 days

General state, external appearance, behavior and clinical symptoms
Individual observations were performed on the skin and fur, eyes and mucous membranes and also respiratory, circulatory, autonomic and central nervous system, somatomotor activity and behaviour pattern. Particular attention was directed to observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.

Body weight
The body weights were recorded on day 0 (just before the treatment), on day 7 and on day 15 with a precision of 1 g.

Necropsy
All survivor mice were sacrificed under isofluran anaesthesia at the end of the observation period. After examination of the external appearance, the cranial, thoracic and abdominal cavities were opened and the appearance of the tissues and organs was observed, and any abnormality was recorded with details of its location, colour, shape and size.
Statistics:
The using doses were given by a software program: AOT425StatPgm.
Calculation of the results was performed with the help of validated software. The estimation of the biological data was accomplished using the Probit analysis by SPSS+software.
The 95%-confidence limits were not calculated by statistical test (Probit analysis by SPSS+software), because their range is too wide.
Sex:
female
Dose descriptor:
LD50
Effect level:
ca. 77.83 mg/kg bw
Based on:
test mat.
Mortality:
The animal treated with 550 mg/kg bw dose (group 4) died on the treatment day 10 sec after the treatment.
The animal treated with 175 mg/kg bw dose (group 3) died on the treatment day 10 sec after the treatment. The other animal of this dose group survived until the end of the 14-day observation period.
Two animals treated with 55 mg/kg bw dose (group 2) died on the treatment day 20 sec and 30 minutes after the treatment, respectively. The third animal of this dose group survived until the end of the 14-day observation period.
There was no deaths in 17.5 mg/kg bw dose (group 1), all three animals survived until the end of the 14-day observation period.
The deaths were related to the systemic toxic effect of the test item.

The LD50 value is 77.83 mg/kg bw on basis of statistical test (Probit analysis by SPSS+software). The approximate 95 % confidence limits were not calculated because their range is too wide.
Clinical signs:
In the group 1 (17.5 mg/kg bw), CNS - and emotion symptoms (decreased activity, tremor, clonic convulsion), disturbance of coordination (abnormal gait) and disturbance of the autonomic functions (dyspnoea) were observed in animals on the treatment day between 30 min. and 3 hours after the treatment.

In the group 2 (55 mg/kg bw), CNS - and emotion symptoms (decreased activity, tremor, tonic convulsion, clonic convulsion, closed eyes) and disturbance of the autonomic functions (dyspnoea) were observed in animals on the treatment day between 30 min. and 4 hours after the treatment.

In the group 3 (175 mg/kg bw), CNS - and emotion symptoms (decreased activity, tremor, closed eyes, clonic convulsion) and disturbance of the autonomic functions (decreased respiration rate, dyspnoea) were observed in animals on the treatment day between 30 min. and 4 hours after the treatment, except clonic convulsion, which appeared 10 seconds after the treatment.

In the group 4 (550 mg/kg bw), CNS - and emotion symptoms (tonic convulsion, clonic convulsion) were observed in animals on the treatment day 10 seconds after the treatment.
Body weight:
Body weight loss was observed in one female treated with 55 mg/kg bw dose (group 2) between Day 7 and Day 15 and in one female treated with 175 mg/kg bw (group 3) between Day 0 and Day 7. These body weight losses were below 10 % (approx. 3.5 % and 3.4%, respectively) and the body weight of animal exceeded the original body weight by the end of study, thus it can be evaluated as an individual variation without toxicological meaning.
The body weight and body weight gain data in two animals of group 2 (55 mg/kg bw), in one animal of group 3 (175 mg/kg bw) and in the animal of group 4 (550 mg/kg bw) could not be evaluated during the study, because these animals died on Day 0.
The body weight and body weight gain of all animals of group 1 treated with 17.5 mg/kg bw dose corresponded to its species and age throughout the study.
Gross pathology:
Altogether 9 mice were treated with the test item. Four animals died spontaneously during the study. Five animals survived until the end of study.

No pathological changes were found related to the effect of the test item during the macroscopic examination of animals.

Summary of Lethality - Post-treatment observation period (14 days), Females
Groups Test Item Dose (mg/kg bw) Lethality
 1 Nicotine  17.5 0/3
 2 Nicotine  55  2/3 
 3 Nicotine 175 1/2
 4 Nicotine  550  1/1 
Remark: LD50= 77.83 mg/kg bw (based on Probit analysis by SPSS+software). [95%Confidence intervals could not be calculated (too wide).]
Interpretation of results:
Toxicity Category III
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
It can be concluded, that the LD50 value of test item Nicotine is 77.83 mg/kg bw, given to mice orally for single application on basis of statistical test (Probit analysis by SPSS+software). However, the 95 % confidence limits could not calculated because their range is too wide.
Executive summary:

The Up-and-Down-Procedure (OECD 425) was performed to determine the acute oral toxicity of the test item Nicotine in mice.

General information

The first animal was dosed a step below the best preliminary estimate of the LD50. The initial dose was 17.5 mg/kg bw. The first animal survived until 48 hours after the treatment, so the second animal received a higher dose as 55 mg/kg bw. The second animal survived until 48 hours after the treatment, so the third animal received a higher dose as 175 mg/kg bw. The third animal survived until the 48 hours after the treatment, so the fourth animal received a higher dose as 550 mg/kg bw. The fourth animal died on the treatment day, so the fifth animal received a lower dose as 175 mg/kg bw. The fifth animal died on the treatment day, so the sixth animal received a lower dose as 55 mg/kg bw. The sixth animal died on the treatment day, the seventh animal received a lower dose as 17.5 mg/kg bw. The seventh animal survived until the 48 hours after the treatment, so the eighth animal received a higher dose as 55 mg/kg bw dose. The eighth animal died on the treatment day, so the ninth animal received a lower dose as 17.5 mg/kg bw. The ninth animal survived until the end of study. Dosing was stopped, because the stopping criteria according to the Guideline was met: LR criterion (if the likelihood-ratios calculated exceed the critical likelihood-ratio, the LR stopping criterion is satisfied and testing stops). The dose progression factor was 3.2. The first, second, third and seventh survivor animal was observed for up to 48 hours before the next animal treated. Dosing was stopped, because the maximum number of animals tested. The doses were given by a software program: AOT425StatPgm. All animals were periodically weighed, observed for clinical and behavioral symptoms. Gross pathological examination was carried out on the treatment day and 15th day after the treatment.

Lethality, Clinical symptoms and Body weight

The mouse treated with 550 mg/kg bw dose of test item died on the treatment day 10 seconds after the treatment. One out of two mice treated with 175 mg/kg bw dose died on the treatment day 10 seconds after the treatment. Two out of three mice treated with 55 mg/kg bw dose died on the treatment day 20 seconds and 30 minutes after the treatment, respectively. No lethality was noted at single oral dose (gavage) of 17.5 mg/kg bw. In the group 1 (17.5 mg/kg bw), CNS - and emotion symptoms (decreased activity, tremor, clonic convulsion), disturbance of coordination (abnormal gait) and disturbance of the autonomic functions (dyspnoea) were observed in animals on the treatment day between 30 min. and 3 hours after the treatment. In the group 2 (55 mg/kg bw), CNS - and emotion symptoms (decreased activity, tremor, tonic convulsion, clonic convulsion, closed eyes) and disturbance of the autonomic functions (dyspnoea) were observed in animals on the treatment day between 30 min. and 4 hours after the treatment.

In the group 3 (175 mg/kg bw), CNS - and emotion symptoms (decreased activity, tremor, closed eyes, clonic convulsion) and disturbance of the autonomic functions (decreased respiration rate, dyspnoea) were observed in animals on the treatment day between 30 min. and 4 hours after the treatment, except clonic convulsion, which appeared 10 seconds after the treatment. In the group 4 (550 mg/kg bw), CNS - and emotion symptoms (tonic convulsion, clonic convulsion) were observed in animals on the treatment day 10 seconds after the treatment. The body weight development was undisturbed in all survivor animals.

Gross pathology

The animal of group 4, one animal of group 3 and two animals of group 2 died spontaneously on the treatment day. One animal of group 3, one animal of group 2 and all animals of group 1 survived until the end of the 14-day observation period. No pathological changes were found related to the effect of the test item during the macroscopic examination of the animals.

Endpoint:
acute toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
significant methodological deficiencies
Qualifier:
no guideline followed
Principles of method if other than guideline:
Study was performed before OECD test guidelines and principles of GLP were available. Study design is not in line with the OECD guidelines.

- Principle of test:
Nicotine, in its undiluted form, was dropped orally on the tongue or between the lips and gums of a total of 19 dogs.

GLP compliance:
not specified
Test type:
standard acute method
Limit test:
no
Specific details on test material used for the study:
not specified
Species:
dog
Strain:
not specified
Sex:
not specified
Details on test animals and environmental conditions:
not specified
Route of administration:
other: dropped on the tongue or between the lips and gums
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
MAXIMUM DOSE VOLUME APPLIED: 20 mg/kg bw
Doses:
Group 1: 4.6-5.0 mg/kg bw
Group 2: 9.2-10.3 mg/kg bw
Group 3: 12.0 mg/kg bw
Group 4: 20.0 mg/kg bw
No. of animals per sex per dose:
Group 1: 2 dogs
Group 2: 14 dogs
Group 3: 1 dog
Group 4: 2 dogs
Control animals:
no
Details on study design:
not specified
Statistics:
data allowes for an estimation of the LD50.
Key result
Sex:
not specified
Dose descriptor:
LD50
Effect level:
9.2 mg/kg bw
Based on:
test mat.
Mortality:
Group 1: 0 deaths
Group 2: 8 deaths
Group 3: 1 death
Group 4: 2 deaths
Clinical signs:
not specified
Body weight:
not specified
Gross pathology:
not specified
Interpretation of results:
Category 2 based on GHS criteria
Conclusions:
While the study design is not in line with the OECD guidelines, RAC considered that the number of animals is sufficient, the dosing method seems relevant and the data allows for an estimation of the LD50.
Executive summary:

In the oral toxicity study of Franke and Thomas (1932) nicotine was dropped undiluted on the tongue or between the lips and gums of a total of 19 dogs.

After a single oral administration of undiluted nicotine alkaloid in the mouth, the LD50 was 9.2 mg/kg.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LD50
Value:
9 mg/kg bw
Quality of whole database:
LD50 values of nicotine in mice and dogs (most sensitive species) from available studies ranged from 3.34 mg/kg to 77.83 mg/kg. The most appropriate LD50 value was derived on a weight of evidence analysis, considering the most reliable study and the most sensitive species (RAC opinion on nicotine, 20th CARACAL meeting 2016).

Acute toxicity: via inhalation route

Link to relevant study records
Reference
Endpoint:
acute toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Remarks:
Conducted according to the up and down procedure (UDP) recommended by the US EPA Health Effects Test Guidelines (EPA, 2002). However, the duration of exposure to nicotine mist (20 minutes) was 12-fold shorter than 4 hours (240 minutes).
Qualifier:
equivalent or similar to
Guideline:
other:
Version / remarks:
The study was performed equivalent or similar to EPA Health Effects Test Guidelines: Acute inhalation toxicity (1998).
Deviations:
not applicable
Principles of method if other than guideline:
Different exposure time of 20 min (cf. 4h) than recommended in relevant inhalation guidelines.
GLP compliance:
not specified
Remarks:
No statement about the GLP status of the study was published.
Test type:
other: Up and down procedure
Limit test:
no
Specific details on test material used for the study:
-(s)-(-)-nicotine freebase (liquid, 99%), ordered from Alfa Aesar Co.
-pH 7.4 and 8.0.
Species:
rat
Strain:
Sprague-Dawley
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
-8–11-week-old
-body weight 250–400g
-housed in the vivarium under a 12-hr light/dark cycle
-ad libitum access to food and water.

ENVIRONMENTAL CONDITIONS:
no data
Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
other: water
Mass median aerodynamic diameter (MMAD):
2.5 µm
Geometric standard deviation (GSD):
1.8
Details on inhalation exposure:
- 3-jet Collison nebulizer (BGI Inc.)
- air pressure gauge (Ashcroft@ filled gauge, 0–100 psi, Cole-Parmer)
- air flowmeter with a valve (150-mm Direct Reading, 23 LPM, Cole-Parmer) that regulated the airflow rate and the pressure entering the nebulizer
- homemade nose-only exposure chamber
- rat holders (Model #: CHT-250, CH Technologies Inc.). About 30ml nicotine solution was put in the nebulizer jar.
- Exposure of rats to the aerosol in a nose-only system, one at a time with a fixed air pressure (40 psi).
Analytical verification of test atmosphere concentrations:
yes
Remarks:
Mass concentrations measured with a one-stage cascade impactor (SKC Inc.).
Duration of exposure:
20 min
Concentrations:
Ordered concentration progression in a rage of 5%-56% nicotine. Concentration progression factor of antilog 0.25=1.78.
No. of animals per sex per dose:
7 male rats in sum
Control animals:
no
Details on study design:
- Duration of observation period following administration: 24 hours
- Necropsy of survivors performed: no
- Other examinations performed: Arterial blood sample collection and plasma nicotine level measurement
Statistics:
Computer program (AOT425StatPgm, US EPA) was used to calculate the LC50 and its confidence intervals
Key result
Sex:
male
Dose descriptor:
LC50
Effect level:
2.3 mg/L air
Based on:
test mat.
95% CL:
>= 1.24 - <= 4.07
Exp. duration:
20 min
Remarks on result:
other: Using the Habers law, according to point 3.1.2.1. (c) of the CLP Regulation (Cn x t = constant), the converted LC50 (4 hours) of nicotine is 0.19 mg/L.
Mortality:
Exposure to 2.3 mg/L nicotine in air for 20 min causes death in 50% of rats.
Some of the rats died within 1-3 min after the end of nicotine aerosol exposure period of 20 min.
Clinical signs:
other: Exposure in the high concentration range caused a Straub tail within 20-60 s. Within 1-2.5 min rats became apneic, interspersed with gasps.
Interpretation of results:
Category 2 based on GHS criteria
Conclusions:
An LC50 (20 min) of 2.3 mg/L air was reported for nicotine aerosol, with a confidence interval of 1.24 - 4.07 mg/L (pH 8.0), based on the mass concentration of aerosol at the breathing zone 7.17 mg/L at 40 psi.
Using the Habers law, according to point 3.1.2.1. (c) of the CLP Regulation (Cn x t = constant), the converted LC50 (4 hours) of nicotine is 0.19 mg/L.
Executive summary:

Nicotine was tested using the up and down procedure (UDP) recommended by the US EPA Health Effects Test Guidelines (2002). Seven rats were exposed to nicotine aerosol in a nose-only system. The mass median aerodynamic diameter (MMAD) was within the range of respirable droplet size recommended by USEPA. Exposure to 2.3 mg/L for 20 minutes caused death in 50% of rats.Signs and symptoms were observed. Furthermore, arterial and venous blood samples were collected and plasma level was measured after a 2 min exposure period.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LC50
Value:
190 mg/m³
Quality of whole database:
The study was performed using the up and down procedure (UDP) recommended by the US EPA Health Effects Test Guidelines (EPA, 2002) and is reliable with restrictions (20 minutes exposure instead of 4 hours).Conversion was made to 4 hours exposure by RAC, using Haber's law. The RAC considered this study the most recent and reliable study submitted during public consultation.

Acute toxicity: via dermal route

Link to relevant study records
Reference
Endpoint:
acute toxicity: dermal
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.
Qualifier:
according to
Guideline:
OECD Guideline 402 (Acute Dermal Toxicity)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.3 (Acute Toxicity (Dermal))
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test type:
standard acute method
Limit test:
no
Species:
rabbit
Strain:
New Zealand White
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: S&K LAP Kft.
2173 Kartal, Császár út 135, HUNGARY
- Age at study initiation: Young adult rabbits, nulliparous and non-pregnant, females were 11 weeks old, males were 11 weeks old
- Weight at study initiation: Body weight range in main study
at starting (males, group 1): 2210 - 2310 g
Body weight range in main study
at starting (females, group 1): 2420 - 2776 g
Body weight range in main study
at starting (females, group 2): 2606 - 2780 g
Body weight range in main study
at starting (females, group 3): 2500 - 2656 g
- Housing: Animals were housed individually in metal cages.
- Diet (e.g. ad libitum): CRLT/ny rabbit diet produced by Szindbád Kft., 2100 Gödöllő, Szárítópuszta, Hungary, ad libitum
- Water (e.g. ad libitum): tap water from watering bottles ad libitum
- Acclimation period: 5 days in females and 6 days in males

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 ± 3 °C
- Humidity (%): 30 - 70 %
- Air changes (per hr): 10-15 air exchanges/hour by central air-condition system
- Photoperiod (hrs dark / hrs light): 12 hours daily, from 6.00 a.m. to 6.00 p.m.

IN-LIFE DATES: From: 0 To: 15
Type of coverage:
semiocclusive
Vehicle:
unchanged (no vehicle)
Details on dermal exposure:
TEST SITE
- Area of exposure: back of animals
- % coverage: 10%
- Type of wrap if used: The entire trunk of the animal was wrapped with semi occlusive plastic wrap

REMOVAL OF TEST SUBSTANCE
- Washing (if done): any residual test item was removed using body temperature water
- Time after start of exposure: 24-hour exposure period

Duration of exposure:
24-hour exposure period
Doses:
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
No. of animals per sex per dose:
Preliminary study: n=2 animals/dose
Main study: n=5 animals/sex/dose
Control animals:
no
Details on study design:
- Duration of observation period following administration: 14 days

Observations

Mortality
Inspection for signs of morbidity and mortality were made twice daily at the beginning and end of the working day.

Clinical observations
Careful clinical observation was made at the following intervals: 1h, 5h after the treatment and once each day for 14 days thereafter in preliminary study and 30 min., 1h, 2h, 3h, 4h and 5h after the treatment and once each day for 14 days thereafter in main study.
Individual observations included the skin and fur, eyes and mucous membranes, and also respiratory, circulatory, autonomic and central nervous system, somatomotor activity and behaviour pattern as well. Particular attention was directed to the observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.

Body weight
The body weight was recorded on day 0 (shortly before the treatment) in preliminary study and on day -1, on day 0 (just before the treatment), on day 1, on day 7 and on day 15 in main study. The precision was 1 g.

Pathology
All animals were subjected to gross pathology in main study. All survivor rabbits were sacrificed under EUTHANIMAL 40% anaesthesia (approx. 0.25 ml/kg). After examination of the external appearance, the cranial, thoracic and abdominal cavities were opened and the appearance of the tissues and organs was observed, and any abnormality was recorded with details of its location, colour, shape and size.
Animals of preliminary study were humanely sacrificed on Day 14.
Statistics:
Calculation of the results was performed with the help of validated software. The estimation of the biological data was accomplished using the Probit analysis by SPSS+software.
Sex:
female
Dose descriptor:
LD50
Effect level:
70.4 mg/kg bw
Based on:
test mat.
95% CL:
>= 28.3 - <= 131.2
Sex:
female
Dose descriptor:
other: LOAEL
Effect level:
50 mg/kg bw
Based on:
test mat.
Mortality:
In males, no mortality observed over a 14-day period following a 24-hour dermal exposure to Nicotine of group 1 treated with 50 mg/kg bw dose.
One out of five females treated with 50 mg/kg bw dose died on the treatment day 2 hours after the treatment. Four out of five females treated with 100 mg/kg bw dose died during the study. One animal died on Day 1. On the treatment day, one animal died 5 hours after the treatment, one animal died 1 hour after the treatment and one animal died 2 hours after the treatment. All females treated with 200 mg/kg bw dose died on the treatment day. Four animals died 1 hour after the treatment and one animal died 2 hours after the treatment, as well.
All deaths seemed to be consequences of systemic toxic effect of the test item.
The males were not more sensitive than females on the basis of this study.
Clinical signs:
Males: Group 1 (50 mg/kg bw): No treatment related systemic toxic symptoms were observed throughout the post-treatment period.
Females: Group 1 (50 mg/kg bw):Decreased activity (1 case of 82 observations), tremor (1/82), incoordination (1/82), lateral position (1/82) and dyspnoea (1/82). Decreased activity (score -3), tremor (score +1), incoordination (score +1), lateral position (score +3) and dyspnoea (score +2) were found in one animal (treatment day 1 hour after the treatment).
Group 2 (100 mg/kg bw): decreased activity (16/35), tremor (9/35), abnormal gait (7/35), closed eyes (3/35), increased respiration rate (2/35), clonic convulsion (1/35) and dyspnoea (1/35). Decreased activity (score -1, -2, -3, -4) occurred in all animals. Tremor (score +1, +2) was observed in four animals. Abnormal gait (score +1, +2) was detected in three animals. Closed eyes (score +1, +2) was recorded in two animals. Increased respiration rate (score +1) was observed in one animal. Clonic convulsion (score +2) and dyspnoea (score +2) were found in one animal.
Group 3 (200 mg/kg bw): Decreased activity (6/6), tremor (4/6), clonic convulsion (3/6), abnormal gait (3/6), closed eyes (3/6) and salivation (2/6). Decreased activity (score -1, -2, -3) occurred in all animals. Tremor (score +1, +4) was observed in three animals. Abnormal gait (score +2, +3) was detected in three animals. Closed eyes (score +2) was recorded in three animals. Clonic convulsion (score +1) was observed in two animals. Salivation (score +1, +2) was found in two animals (treatment day between 30 min. and 1 hour after the treatment).
Body weight:
Males
The mean body weight and the body weight gain of male animals treated with 50 mg/kg bw dose were in the normal range during the two weeks observation period.
Females
Body weight loss was observed in one animal treated with 50 mg/kg bw dose between Day 7 and Day 15. These body weight losses were below 10 % (approx. 8.6 %) and the body weight of animal exceeded the original body weight by the end of study, thus it can be evaluated as an individual variation without toxicological meaning.
In 100 mg/kg bw dose group, the body weight and body weight gain data between Day 7 and Day 15 and between Day 0 and Day 15 could not be evaluated in four animals, because of mortalities. The body weight gain of survivor animal corresponded to its species and age throughout the study.
In 200 mg/kg bw dose group, the body weight and body weight gain data between Day 7 and Day 15 and between Day 0 and Day 15 could not be evaluated, because of mortalities.
Gross pathology:
Altogether 10 female rabbits died spontaneously during the study. All male rabbits and five female animals survived until the scheduled necropsy on Day 15.
Males
External necropsy findings as crusting (2 animals) and wound (2 animals) on the treated site of skin was observed in group treated with 50 mg/kg bw dose
No gross pathology findings were found in one male during the macroscopic examination. These findings could be related to the test item irritant effect.
Females
In 50 mg/kg bw dose group, external finding as slight redness on the treated site was observed in one animal. It could be related to the test item irritant effect.
In 100 mg/kg bw dose group, an internal necropsy finding as autolysis was observed in one animal. It is normal physiological process after death. Severe hydrometra was observed in two animals. The hydrometra is physiological finding and connected to the cycle of the animal. External finding as slight redness on the treated site was observed in three animals and moderate redness on the treated site was recorded in one animal. Besides, slight oedema was detected in one animal. The redness and oedema could be related to the test item irritant effect.
In 200 mg/kg bw dose group, an internal necropsy finding as slight hydrometra was observed in two animals and moderate hydrometra was recorded in one animal. The hydrometra is physiological finding and connected to the cycle of the animal.
External finding as severe redness on the treated site was observed in four animals and moderate redness on the treated site was recorded in one animal. Slight oedema was detected in one animal and moderate oedema on the treated site was found in two animals. The redness and oedema could be related to the test item irritant effect.
Other findings:
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.

Summary of lethality - Post-treatment observation period (14 days)

Females

 Test item  Group  Dose (mg/kg bw)  Lethality
 Nicotine  1  50  1/5
 Nicotine  2  100  4/5
 Nicotine  3  200  5/5

Remark: LD50 (females)= 70.4 mg/kg bw [95% Confidence limits: 29.3; 131.2]

Males

 Test item Group  Dose (mg/kg bw)  Lethality 
 Nicotine 50  0/5 
Interpretation of results:
Toxicity Category II
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Under the experimental conditions, the acute dermal LD50 value of the test item Nicotine is 70.4 mg/kg bw in female NZW rabbits on basis of statistical test (Probit analysis by SPSS+software). The confidence interval is 28.3 and 131.2 mg/kg bw.
The males are not more sensitive than females based on mortality.
Executive summary:

An acute dermal toxicity study was performed with test item Nicotine in NZW rabbits. A full test was carried out on basis of a preliminary study. First, three groups of female animals (n=5 animals/dose) were exposed to Nicotine at 50, 100 and 200 mg/kg bw test item by dermal route. After completion of the study in female sex, one group of 5 males was dosed at 50 mg/kg bw test item by dermal route. Test item was applied in undiluted form and left in contact with the skin for 24 hours, followed by a 14-day observation period.

Mortality

All male rabbits dosed at 50 mg/kg bw test item survived until the end of the 14-day observation period. All female rabbits dosed at 200 mg/kg bw test item died on the treatment day between 1 and 2 hours after the treatment. Four female rabbits dosed at 100 mg/kg bw test item died between the treatment day and Day 1. One female rabbit dosed at 50 mg/kg bw test item died on the treatment day 2 hours after the treatment. The mortalities seemed to be consequences of systemic toxic effect of the test item and were correlated with the systemic clinical symptoms. The LD50 value is 70.4 mg/kg bw in females. The 95 % confidence limits as follows: lower is 28.3 mg/kg bw and upper is 131.2 mg/kg bw. The males were not more sensitive than females on the basis of this study.

Systemic clinical symptoms, local dermal symptoms

In males treated with 50 mg/kg bw dose, no systemic clinical symptoms were observed during the study. In females treated with 50 mg/kg bw dose, CNS symptoms (decreased activity, tremor), disturbances of coordination (incoordination, lateral position) and disturbance of the autonomic functions (dyspnoea) were observed on the treatment day 1 hour after the treatment. In females treated with 100 mg/kg bw dose, CNS symptoms (decreased activity, tremor, closed eyes, clonic convulsion), disturbance of coordination (abnormal gait) and disturbances of the autonomic functions (increased respiration rate, dyspnoea) were observed on the treatment day between 30 min. and 5 hours after the treatment. In females treated with 200 mg/kg bw dose, CNS symptoms (decreased activity, tremor, closed eyes, clonic convulsion), disturbance of coordination (abnormal gait) and disturbance of the autonomic functions (salivation) were observed on the treatment day between 30 min. and 1 hour after the treatment.

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.

Body weight, body weight gain

The body weight development was undisturbed in all survivor animals, except one female treated with 50 mg/kg bw dose on second week. This body weight loss can be evaluated as an individual variation without toxicological meaning.

Gross pathology

The macroscopic external necropsy findings on the treated site as erythema, oedema, crusting and wound were related to the local irritant effect of the test item. No macroscopic alterations of organs and tissues referred to the systemic toxic effect of the test item were seen during the necropsy.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LD50
Value:
70.4 mg/kg bw
Quality of whole database:
The study is a GLP compliant and has Klimisch score 1.

Additional information

Justification for classification or non-classification

Classification, based on the acute studies on nicotine, is in line with the current harmonised classification of nicotine:

Oral route of exposure: H300 Acute Tox. 2

Dermal route of exposure: H310 Acute Tox. 2

Inhalation route of exposure: H330 Acute Tox. 2