Nicotine dihydrogen ditartrate

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

Data is from peer reviewed publication

Data source

Reference

Reference Type:

publication

Title:

Repeated dose oral toxicity study of the test chemical

Author:

Dodmane et al

Year:

2014

Bibliographic source:

Toxicology

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: Refer below principle

Principles of method if other than guideline:

Combined repeated dose & carcinogenicity study was performed to determine the toxic nature of the test chemical

GLP compliance:

not specified

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Nicotine dihydrogen ditartrate

EC Number:

200-607-2

EC Name:

Nicotine dihydrogen ditartrate

Cas Number:

65-31-6

Molecular formula:

C10-H14-N2.2C4-H6-O6

IUPAC Name:

nicotine dihydrogen ditartrate

Details on test material:

- Name of test material: Nicotine dihydrogen tartrate
- Molecular formula: C10H14N2.2C4H6O6
- Molecular weight: 462.4054 g/mol
- Substance type: Organic
- Physical state: No data
- Impurities (identity and concentrations): ≤ 2% (≥ 98% pure)

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Han

Details on species / strain selection:

No data

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Portage, MI
- Age at study initiation: 8 weeks
- Weight at study initiation:
- Fasting period before study:
- Housing: Two or 3 rats per cage were housed in polycarbonate cages with dry corn cob bedding. Mice cages had micro-isolator tops. Nylabones for rats were placed inside the cages for environmental enrichment.
- Diet (e.g. ad libitum): Certified Lab Diet 5002 was pelleted and used as the basal diet
- Water (e.g. ad libitum): Drinking water treated by reverse osmosis was provided to all animals
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22˚C
- Humidity (%): 50%
- Air changes (per hr): No data
- Photoperiod (hrs dark / hrs light): light/dark cycle of 12 h (0600lights on/1800 lights off, CST)

IN-LIFE DATES: From: To: No data

Administration / exposure

Route of administration:

oral: drinking water

Details on route of administration:

No data

Vehicle:

water

Remarks:

Drinking water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: The test chemical was mixed with distilled water at dose level of 0 or 148 ppm (0 or 14.8 mg/Kg/day)

DIET PREPARATION
- Rate of preparation of diet (frequency): No data
- Mixing appropriate amounts with (Type of food): No data
- Storage temperature of food: No data

VEHICLE
- Justification for use and choice of vehicle (if other than water): RO water
- Concentration in vehicle: 0 or 148 ppm (0 or 14.8 mg/Kg/day)
- Amount of vehicle (if gavage): No data
- Lot/batch no. (if required): No data
- Purity: No data

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

No data

Duration of treatment / exposure:

4 weeks

Frequency of treatment:

Daily

Doses / concentrations

Remarks:

0 or 148 ppm (0 or 14.8 mg/Kg/day)

No. of animals per sex per dose:

Total: 50
0 mg/Kg/day: 25 females
14.8 mg/Kg/day: 25 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: No data
- Rationale for animal assignment: Following quarantine, rats were randomized into 2 groups each (10 animals/group) using a weight stratification method
- Rationale for selecting satellite groups: No data
- Post-exposure recovery period in satellite groups: No data
- Section schedule rationale (if not random): No data

Positive control:

No data

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: No data
- Cage side observations checked in table [No.?] were included. Clinical signs and mortality

DETAILED CLINICAL OBSERVATIONS: No data
- Time schedule: No data

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed the day after arrival, just prior to randomization, at the end of the consumption periods and just prior to sacrifice.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes, 7 days beginning on study day 0 and study day 14in rats
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: 7 days beginning on study day 0 and study day 14in rats

OPHTHALMOSCOPIC EXAMINATION: No data
- Time schedule for examinations: No data
- Dose groups that were examined: No data

HAEMATOLOGY: No data
- Time schedule for collection of blood: No data
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: No data
- Parameters checked in table [No.?] were examined. No data

CLINICAL CHEMISTRY: No data
- Time schedule for collection of blood: No data
- Animals fasted: No data
- How many animals: No data
- Parameters checked in table [No.?] were examined. No data

URINALYSIS: No data
- Time schedule for collection of urine: No data
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data
- Parameters checked in table [No.?] were examined. No data

NEUROBEHAVIOURAL EXAMINATION: No data
- Time schedule for examinations: No data
- Dose groups that were examined: No data
- Battery of functions tested: sensory activity / grip strength / motor activity / other: No data

OTHER: No data

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, All animals were sacrificed by an overdose of Nembutal®(150 mg/kg bodyweight). One hour prior to sacrifice, all animals were injected with bromodeoxyuridine (BrdU) (100 mg/kg BW intraperitoneally). At necropsy, the urinary bladder was inflated in situ with Bouin’s fixative, while the animal was under deep anesthesia and still alive.

HISTOPATHOLOGY: Yes, small section of intestine was removed, and both were placed in Bouin’s fixative. The kidneys were removed, weighed and fixed in 10% neutral buffered formalin (NBF). One half of the bladder was processed for SEM, examined and classified as described previously. The other half of the bladder, intestine and kidneys were paraffin embedded in the Tissue Sciences Facility, UNMC. Approximately 4–5 micron sections were stained with hematoxylin and eosin and examined histopathologically. Unstained slides of bladder and intestinal tissue were obtained for immune-histochemical determination of the BrdU labeling index. The intestinal tissue on each slide served as the positive control for the BrdU immunohistochemistry procedure. A diagnosis of mild simple hyperplasia was made if the bladder epithelium wasfour to five cell layers thick.

Histopathological evaluation was performed blinded with respect to knowledge of the treatment group. The other half of each bladder was processed for examination by SEM and classified in one of five categories. The categories have the following characteristics: Class I bladders contain polygonal superficial urothelial cells; class II bladders have occasional small foci of superficial urothelial necrosis, especially in the dome of the bladder; class III bladders have numerous small foci of superficial urothelial necrosis; class IV bladders have extensive superficial urothelial necrosis, especially in the dome of the bladder; class V bladders have necrosis and piling up(hyperplasia) of rounded urothelial cells. Bladders from normal animals are usually class I or II, or occasionally class III.

Other examinations:

No data

Statistics:

Comparison of all data collected on body weights, food and water consumption, bladder weights, and the labeling index were performed by the SAS general linear models procedure and Duncan’s multiple range test. All means were accompanied by calculation of standard errors. Histology results were analyzed using Fisher’s exact test (2-tail). SEM data were analyzed using 1-way non-parametric procedures followed by a chi square test. p values less than 0.05 were considered significant.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

No adverse clinical signs were shown by the treated animals

Mortality:

no mortality observed

Description (incidence):

No mortality was noted in the treated and control animals

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Rats treated with nicotine showed significantly (p ≤ 0.05) decreased body weight after week 1 and continuing until sacrifice. At sacrifice there was an 8.7% decrease in body weight gain in rats

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Nicotine treatment caused no difference in food consumption

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

effects observed, treatment-related

Description (incidence and severity):

Nicotine treatment caused a significant (p ≤ 0.05) decrease in water consumption in rats

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There was no change in relative weights of urinary bladders, but the relative weight of kidneys in the nicotine-treated group was significantly increased compared to respective control groups in both rats

Gross pathological findings:

not specified

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Mild simple hyperplasia of the urinary bladder epithelium was observed in 7 of 10 rats with nicotine treatment. Kidney tissue from treated animals was normal histopathologically, including no evidence of kidney pelvis urothelial hyperplasia.

Nicotine-treated rats showed a greater mean BrdU labeling index compared to untreated rats, but the increase was not statistically significant.

The bladder from nicotine-treated rats showed greater numbers of Class III changes (9/10) when examined by SEM, suggesting localized exfoliating changes induced by nicotine treatment

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Effect levels

Dose descriptor:

LOAEL

Effect level:

14.8 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: Decrease in body weight and urothelial hyperplasia was noted

Remarks on result:

other: Low adverse effect were observe

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Table 1: General findings. After administration of nicotine hydrogen tartrate (NT) in drinking water for a total of 4 weeks, rats were sacrificed on study day 29. Whole body, kidney and urinary bladder weights were recorded. Water and feed consumption were measured during week 3 in rats.

Treatment	Body weight (BW)		Relative bladder weight (mg/g BW)	Relative kidney weight (mg/g BW)	Consumption^b(g/kg BW/day)
	Day 0	Day of sacrifice^a			Water (n)	Food (n)
Control	166 ±2	206 ± 3	0.31 ± 0.02	7.6 ± 0.2	129 ±22 (4)	101 ± 19 (4)
Test group	167 ±2	188 ± 2c	0.34 ±0.03	8.3 ± 0.2c	80 ± 5 (4)	147 ± 42 (3)

b Consumption measured for rats during week 3 and for mice during week 4.

c Significantly different from respective control group, p ≤ 0.05.

 

Table 2: Effects of nicotine on the urothelium of rats. Rats administered nicotine for 4 weeks in drinking water. Histologically hyperplasia was investigated by hematoxylin and eosin staining. The urothelial cell BrdU labeling index was determined by immunohistochemistry and the luminal surface of the urothelium was visualized by SEM.

Treatment	Histopathology		BrdU Labeling Index (%)	SEM classification
	Normal	Simple hyperplasia	Mean ± S.E. (n)	I	II	III	IV	V
Control	10	0	0.10 ± 0.03 (7)	6	4	0	0	0
Test group^a	3	7^b	0.11 ± 0.09 (9)	0	0	9	1	0

a SEM classification significantly different compared to respective control group, p < 0.05.

b Significantly different compared to respective control group, p ≤ 0.05.

Applicant's summary and conclusion

Conclusions:

The Low observed adverse effect level (LOAEL) for the test chemical is considered to be 14.8 mg/Kg/day using female Wistar Han rats for 4 weeks.

Executive summary:

Combined repeated dose & carcinogenicity study was performed to determine the toxic nature of the test chemical. The study was performed using 25 female Wistar Han rats per dose for 4 weeks. The test chemical was mixed with drinking water and used at dose level of 0 or 14.8 mg/Kg/day. During the study period, the treated animals were observed clinical signs, mortality, changes in body weight, food and water consumption. The animals were also subjected to gross pathology and histopathology. No adverse clinical signs and no mortality were shown by the treated animals. Rats treated with nicotine showed significantly (p ≤ 0.05) decreased body weight after week 1 and continuing until sacrifice. At sacrifice there was an 8.7% decrease in body weight gain in rats. Nicotine treatment caused no difference in food consumption but a significant (p ≤ 0.05) decrease in water consumption was noted in the treated rats. There was no change in relative weights of urinary bladders, but the relative weight of kidneys in the nicotine-treated group was significantly increased compared to respective control groups in both rats. Mild simple hyperplasia of the urinary bladder epithelium was observed in 7 of 10 rats with nicotine treatment. Kidney tissue from treated animals was normal histopathologically, including no evidence of kidney pelvis urothelial hyperplasia.Nicotine-treated rats showed a greater mean BrdU labeling index compared to untreated rats, but the increase was not statistically significant. The bladder from nicotine-treated rats showed greater numbers of Class III changes (9/10) when examined by SEM, suggesting localized exfoliating changes induced by nicotine treatment. Based on the observations made, the Low observed adverse effect level (LOAEL) for the test chemical is considered to be 14.8 mg/Kg/day using female Wistar Han rats for 4 weeks.