Nicotine

Skin irritation / corrosion

Administrative data

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.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

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

Guideline:

OECD Guideline 402 (Acute Dermal Toxicity)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.3 (Acute Toxicity (Dermal))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test type:

standard acute method

Limit test:

no

Species:

rabbit

Strain:

New Zealand White

Sex:

male/female

Details on test animals or test system 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:

other: 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 pos

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	1	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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 402

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Nicotine

EC Number:

200-193-3

EC Name:

Nicotine

Cas Number:

54-11-5

Molecular formula:

C10H14N2

IUPAC Name:

3-(1-methylpyrrolidin-2-yl)pyridine

Test material form:

liquid

Test animals

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

cross-reference: section 7.2.3

Test system

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

Results and discussion

In vivo

Results

Results 1

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".

Results 2

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.

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.