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Toxicological information

Acute Toxicity: oral

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Administrative data

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.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guideline
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

Test animals

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

Administration / exposure

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.

Results and discussion

Effect levels
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.

Any other information on results incl. tables

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).]

Applicant's summary and conclusion

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.