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Description of key information

Repeated dose toxicity: Oral

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 is considered to be 14.8 mg/Kg/day using female Wistar Han rats for 4 weeks.

Repeated inhalation study:

According to Annex IX of the REACH regulation, testing by the inhalation route is appropriate only if exposure of humans via inhalation is likely. Taking into account the low vapour pressure of the substance Nicotine dihydrogen ditartrate (65-31-6) which is reported as 9.26E-22 mmHg at 25 C. Thus, exposure to inhalable dust, mist and vapour of the chemical Nicotine dihydrogen ditartrate is highly unlikely. Therefore this study is considered for waiver.

Repeated dermal study;

The acute toxicity value for Nicotine dihydrogen ditartrate (65-31-6) (as provided in section 7.2.3) is >2000 mg/kg body weight. Also, given the use of the chemical; repeated exposure by the dermal route is unlikely since the use of gloves is common practice in industries. Thus, it is expected that Nicotine dihydrogen ditartrate shall not exhibit 28 day repeated dose toxicity by the dermal route. In addition, there is no data available that suggests that Nicotine dihydrogen tartrate shall exhibit repeated dose toxicity by the dermal route. Hence this end point was considered for waiver.

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
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
Qualifier:
according to
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
Species:
rat
Strain:
Wistar
Remarks:
Han
Details on species / strain selection:
No data
Sex:
female
Details on test animals 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
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
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
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.
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
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
Critical effects observed:
not specified

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)

Consumptionb(g/kg BW/day)

Day 0

Day of sacrificea

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 groupa

3

7b

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.

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.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LOAEL
14.8 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Data is from K2 publication

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Repeated dose toxicity: Oral

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.

Combined repeated dose & carcinogenicity study was performed to determine the toxic nature of the test chemical. The study was performed using 20 female C57BI/6 mice per dose for 4 weeks. The test chemical was mixed with drinking water and used at dose level of 0 or 209 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. Mice treated with nicotine showed significantly (p ≤ 0.05) decreased body weight after week 1 and continuing until sacrifice. At sacrifice there was an 7.3% decrease in body weight gain in mice. Nicotine treatment caused no difference in food and water. 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 mice. Mild simple hyperplasia of the urinary bladder epithelium was observed in 4 of 10 mice with nicotine treatment. Kidney tissue from treated animals was normal histopathologically, including no evidence of kidney pelvis urothelial hyperplasia.Nicotine treatment in mice did not induce a significant increase in BrdU labeling index. In nicotine-treated mice, 5 bladders were Class I and 4 were Class II, indicating no major changes as a result of nicotine treatment. Based on the observations made, the Low observed adverse effect level (LOAEL) for the test chemical is considered to be 209 mg/Kg/day using female C57BI/6 mice for 4 weeks.

As above studies are carcinogenicity and single dose studies on rodent. No significant repeated dose toxicity were observed on rodents. Thus based on the data available for the target chemical, Nicotine hydrogen tartrate is not likely to exhibit adverse effect upon repeated exposure. Hence the substance is not classified for repeated oral route.

 

Repeated inhalation study:

According to Annex IX of the REACH regulation, testing by the inhalation route is appropriate only if exposure of humans via inhalation is likely. Taking into account the low vapour pressure of the substance Nicotine dihydrogen ditartrate (65-31-6) which is reported as 9.26E-22 mmHg at 25 C. Thus, exposure to inhalable dust, mist and vapour of the chemical Nicotine dihydrogen ditartrate is highly unlikely. Therefore this study is considered for waiver.

Repeated dermal study;

The acute toxicity value for Nicotine dihydrogen ditartrate (65-31-6) (as provided in section 7.2.3) is >2000 mg/kg body weight. Also, given the use of the chemical; repeated exposure by the dermal route is unlikely since the use of gloves is common practice in industries. Thus, it is expected that Nicotine dihydrogen ditartrate shall not exhibit 28 day repeated dose toxicity by the dermal route. In addition, there is no data available that suggests that Nicotine dihydrogen tartrate shall exhibit repeated dose toxicity by the dermal route. Hence this end point was considered for waiver.

 

Based on the data available for the test chemical not likely to exhibit toxic nature upon repeated exposure by oral, inhalation and dermal route of exposure and hence is not likely to classify as per the criteria mentioned in CLP regulation.

Justification for classification or non-classification

Based on the data available for the test chemical not likely to exhibit toxic nature upon repeated exposure by oral, inhalation and dermal route of exposure and hence is not likely to classify as per the criteria mentioned in CLP regulation.