Registration Dossier

Diss Factsheets

Administrative data

Description of key information

Acute toxicity: oral route.

Weight of evidence:

 

- Read-across from analogue substance. Source: Method similar to OECD 423. GLP study. The oral LD50 of Citicoline was determined to be higher than 2000 mg/kg bw in rats (Schauss et al 2009). Based on the available information for the read-across approach, the target substance is deemed to have an LD50 higher than 1324 mg/kg bw.

- Read-across from analogue substance. Source: Method similar to OECD 420. The LD50 of Citicoline is higher than 8000 mg/kg body weight by oral route in the rat (Grau et al. 1983). Based on the available information for the read-across approach, the target substance is deemed to have an LD50 higher than 5295 mg/kg bw.

- Read-across from analogue substance. Source: Method similar to OECD 401. The LD50 of Citicoline is 18501 mg/kg body weight by oral route in male rats (Kanabayashi et al. 1980). Based on the available information for the read-across approach, the target substance is deemed to have an LD50 of 12245 mg/kg bw.

- Read-across from analogue substance. Source: Method similar to OECD 420. The LD50 of Citicoline is higher than 8000 mg/kg body weight by oral route in the mice (Grau et al. 1983). Based on the available information for the read-across approach, the target substance is deemed to have an LD50 higher than 5295 mg/kg bw.

- Read-across from analogue substance. Source: Method similar to OECD 401. The LD50 of Citicoline is 27142 mg/kg body weight by oral route in male mice (Kanabayashi et al. 1980). Based on the available information for the read-across approach, the target substance is deemed to have an LD50 of 17964 mg/kg bw.

- Read-across from analogue substance. Source: Method similar to OECD 401. The LD0 of Citicoline is 14000 mg/kg body weight by oral route in mice (Agut et al. 1983). Based on the available information for the read-across approach, the target substance is deemed to have an LD0 of 9266 mg/kg bw.

 

Based on the available information for the read-across approach and in a weight of evidence scenario, the target substance Cytidine 3'-(dihydrogen phosphate) has an LD50 higher than 2000 mg/kg bw in rats and mice.

Acute toxicity: dermal route

- Key study: OECD 402 and EU B.3. GLP study. The dermal LD50 of Cytidine 3'-(dihydrogen phosphate) in rats is greater than 2000 mg/kg bw.

Acute toxicity: inhalation route

- Data waiving (other justification): According to Regulation (EC) No. 1907/2006, Annex VIII, 8.5.3, column 2, the acute toxicity by inhalation route is not required, as both oral and dermal studies are available.

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:
key study
Study period:
1980
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 401 (Acute Oral Toxicity)
Principles of method if other than guideline:
- Principle of test: To evaluate the oral acute toxicity of a substance.
- Short description of test conditions: The test substance is administered orally by gavage in graduated doses to several groups of experimental animals, one dose being used per group. After administration, observations of effects and deaths are made for 7 days.
- Parameters analysed / observed: Mortality and adverse effects produced after the administration of the substance.
GLP compliance:
not specified
Test type:
standard acute method
Limit test:
no
Species:
rat
Strain:
not specified
Sex:
male
Route of administration:
oral: gavage
Vehicle:
not specified
Doses:
11520, 14400, 18000 and 22500 mg/kg.
No. of animals per sex per dose:
10
Control animals:
yes
Key result
Sex:
male
Dose descriptor:
LD50
Effect level:
18 501 mg/kg bw
Based on:
test mat.
95% CL:
> 16 590 - < 21 213
Mortality:
After 7 days, deaths were observed at doses 14400 (1), 18000 (5) and 22500 (8) mg/kg bw. In most animals, death occurred between 2 and 48 hours after administration.
Clinical signs:
Toxicological signs were sedation, convulsion, dyspnea, lacrimation, and collapse. This signs were severer in the dead animals and some of them continued until the death.

Table 1. Acute toxicity of citicoline at day 7 after single administration on male rats.

Dosage (mg/kg) p.o.

Distribution of dead animals

Mortality

LD50 Values (mg/kg) (95% confidence limits)

30 min.

1

2

3

4

5

6

7 days

22500

0

8

0

0

0

0

0

0

8/10

18501

(16590 – 21213)

18000

0

4

1

0

0

0

0

0

5/10

14400

0

0

1

0

0

0

0

0

1/10

11520

0

0

0

0

0

0

0

0

0/10

Interpretation of results:
GHS criteria not met
Conclusions:
The LD50 of Citicoline is 18501 mg/kg body weight by oral route in male rats.
Executive summary:

An acute toxicity study of Citicoline has been published. Following a method similar to OECD Guideline 401, 10 male rats were administered orally with 11520, 14400, 18000 and 22500 mg/kg bw of citicoline. After 7 days, mortality was observed at all doses except for the lowest. In most animals, death occurred between 2 and 48 hours after administration. Toxicological signs were sedation, convulsion, dyspnea, lacrimation, and collapse, which were severer in the dead animals and some of them continued until the death. According to the results, the LD50 of Citicoline is 18501 mg/kg body weight by oral route in male rats.

Endpoint:
acute toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2009
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)
Deviations:
no
GLP compliance:
yes
Remarks:
Certificate not included in the publication.
Test type:
acute toxic class method
Limit test:
yes
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source: Kyowa Hakko Bio, Chiyoda-ku, Tokyo, Japan
- Lot/batch No.of test material: I43026
- Purity: 99.85 - 99.9%

OTHER SPECIFICS:
The heavy metal content (as lead) was not more than 10 ppm, ammonium not more than 0.05%, free phosphoric acid not more than 0.1%, 5'-cytidylic acid not more than 1%, and arsenic not more than 2 ppm.
Species:
rat
Strain:
Sprague-Dawley
Remarks:
SPF Crl:CD BR
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Hungary, Budapest, Hungary
- Females (if applicable) nulliparous and non-pregnant: Not specified
- Age at study initiation: 7-8 weeks
- Weight at study initiation: 211-227 g (males), 180-193 g (females)
- Fasting period before study: overnight
- Housing: 5 rats were housed per cage in type II macrolone cages.
- Diet: S8106-S011 SM R/M-Z+H (Ssniff Spezialdiäten, Soest, Germany) ad libitum
- Water: Tap water (supplied by Waterworks of Budapest, average hardness 148 mg CaO/L) ad libitum. Checked monthly for selected microbiological parameters at PCDL. The results met the requirements for potable water.
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ºC ± 3
- Humidity (%): 30% - 70%
- Air changes (per hr): 15 times/hour
- Photoperiod (hrs dark / hrs light): 12-hour light-dark cycle

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
VEHICLE
- Concentration in vehicle: 10 g of test item in 50 mL distilled water (0.2 g/mL)
- Amount of vehicle (if gavage): 10 mL/Kg
- Justification for choice of vehicle: Three samples of 5 mL each were taken from the formulated test item immediately before the dosing procedure and transferred for analysis by Wessling Chemical Laboratory GmbH (Budapest, Hungary). The actual concentration was ± 4% of the theoretical value.

MAXIMUM DOSE VOLUME APPLIED: 10 mL/kg

Doses:
2000 mg/kg
No. of animals per sex per dose:
10 animals/sex/dose
Control animals:
no
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing:
Observations: Twice daily at the beginning and end of the workday on weekdays and once on weekend days.
Body weight was measured on days 1, 2, 8 and 15.
- Necropsy of survivors performed: yes
- Other examinations performed: The animals were checked for mortality, general state, external appearance, behaviour and clinical symptoms. Tissues changes were observed and recorded.
Key result
Sex:
male/female
Dose descriptor:
LD50
Effect level:
> 2 000 mg/kg bw
Based on:
test mat.
Mortality:
No mortality was observed at 2000 mg/kg.
Clinical signs:
There were no treatment-related effects on clinical signs and behavior.
Body weight:
No treatment-related effects on body weights were observed.
Gross pathology:
No gross lesions were noted in any organs of the animals on day 15.
Interpretation of results:
GHS criteria not met
Conclusions:
The LD50 of Citicoline is higher than 2000 mg/kg body weight by oral route in the rat.
Executive summary:

In order to determine the acute oral toxicity of Citicoline, a limit test was performed similar to OECD 423, following GLP (certificate not available). The test item was administered to a group of 10 male and 10 female Sprague-Dawley rats at the dose of 2000 mg/kg body weight by oral gavage. All animals were thoroughly observed twice daily at the beginning and the end of the workday and once on the weekend days. Survival, clinical symptoms, behaviour and body weight were monitored. No mortality occurred during the study and the body weight evolution of the animals remained normal during the study. There were no treatment-related effects on clinical signs and behaviour. No gross lesions were noted in any organs of the animals on day 15. The LD50 of the test item is higher than 2000 mg/kg body weight by oral route in the rat.

Endpoint:
acute toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1980
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 401 (Acute Oral Toxicity)
Principles of method if other than guideline:
- Principle of test: To evaluate the oral acute toxicity of a substance.
- Short description of test conditions: The test substance is administered orally by gavage in graduated doses to several groups of experimental animals, one dose being used per group. After administration, observations of effects and deaths are made for 7 days.
- Parameters analysed / observed: Mortality and adverse effects produced after the administration of the substance.
GLP compliance:
not specified
Test type:
standard acute method
Limit test:
no
Species:
mouse
Strain:
not specified
Sex:
male
Route of administration:
oral: gavage
Vehicle:
not specified
Doses:
15625, 19531, 24414 and 30518 mg/kg.
No. of animals per sex per dose:
10
Control animals:
yes
Key result
Sex:
male
Dose descriptor:
LD50
Effect level:
27 142 mg/kg bw
Based on:
test mat.
95% CL:
> 24 138 - < 33 581
Mortality:
After 7 days, deaths were observed at doses 15625, 19531, 24414 and 30518 mg/kg bw. In most animals, death occurred between 2 and 48 hours after administration.
Clinical signs:
Toxicological signs were sedation, convulsion, dyspnea, lacrimation, and collapse. This signs were severer in the dead animals and some of them continued until the death.

Table 1. Acute toxicity of citicoline at day 7 after single administration on male mice.

Dosage (mg/kg) p.o.

Distribution of dead animals

Mortality

LD50 Values (mg/kg) (95% confidence limits)

30 min.

1

2

3

4

5

6

7 days

30518

0

7

0

0

0

0

0

0

7/10

27142

(24138 – 33581)

24414

0

2

1

0

0

0

0

0

3/10

19531

0

0

1

0

0

0

0

0

1/10

15625

0

0

0

0

0

0

0

0

0/10

Interpretation of results:
GHS criteria not met
Conclusions:
The LD50 of Citicoline is 27142 mg/kg body weight by oral route in male mice.
Executive summary:

An acute toxicity study of Citicoline has been published. Follwoing a method similar to OECD 401, 10 male mice were administered orally with 15625, 19531, 24414 and 30518 mg/kg bw of citicoline. After 7 days, mortality was observed at all doses except for the lowest. In most animals, death occurred between 2 and 48 hours after administration. Toxicological signs were sedation, convulsion, dyspnea, lacrimation, and collapse, which were severer in the dead animals and some of them continued until the death. According to the results, the LD50 of Citicoline is 27142 mg/kg body weight by oral route in mice.

Endpoint:
acute toxicity: oral
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH (see "Attached justification")

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The target substance is a breakdown product of the analogue substance.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
- Source: Citicoline.
- Target: Cytidine 3'-(dihydrogen phosphate) (see "Test material" for further information).

3. ANALOGUE APPROACH JUSTIFICATION
Please find Reporting Format in "Attached justification".

4. DATA MATRIX
Please find data Matrix included in "Attached justification".
Reason / purpose for cross-reference:
read-across source
Key result
Sex:
male
Dose descriptor:
LD50
Effect level:
17 964 mg/kg bw
Based on:
test mat.
Remarks on result:
other: Read across from analogue substance, LD50 of 27142 mg/kg bw
Mortality:
After 7 days, deaths were observed at all doses except to the lowest. In most animals, death occurred between 2 and 48 hours after administration.
Clinical signs:
Toxicological signs were sedation, convulsion, dyspnea, lacrimation, and collapse. This signs were severer in the dead animals and some of them continued until the death.
Interpretation of results:
GHS criteria not met
Conclusions:
Based on the read-across approach, the LD50 of Cytidine 3'-(dihydrogen phosphate) is 17964 mg/kg body weight by oral route in male mice.
Executive summary:

Read-across from supporting substance (structural analogue or surrogate): An acute toxicity study of Citicoline has been published. Follwoing a method similar to OECD 401, 10 male mice were administered orally with 15625, 19531, 24414 and 30518 mg/kg bw of citicoline. After 7 days, mortality was observed at all doses except for the lowest. In most animals, death occurred between 2 and 48 hours after administration. Toxicological signs were sedation, convulsion, dyspnea, lacrimation, and collapse, which were severer in the dead animals and some of them continued until the death. According to the results, the LD50 of Citicoline is 27142 mg/kg body weight by oral route in mice. Based on the available information for the read-across approach, the target substance has an oral LD50 of 17964 mg/kg body weight in mice.

Endpoint:
acute toxicity: oral
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH (see "Attached justification")

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The target substance is a breakdown product of the analogue substance.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
- Source: Citicoline.
- Target: Cytidine 3'-(dihydrogen phosphate) (see "Test material" for further information).

3. ANALOGUE APPROACH JUSTIFICATION
Please find Reporting Format in "Attached justification".

4. DATA MATRIX
Please find data Matrix included in "Attached justification".
Reason / purpose for cross-reference:
read-across source
Key result
Sex:
male
Dose descriptor:
LD50
Effect level:
12 245 mg/kg bw
Based on:
test mat.
Remarks on result:
other: Read across from analogue substance, LD50 of 18501 mg/kg bw
Mortality:
After 7 days, deaths were observed at all doses except for the lowest. In most animals, death occurred between 2 and 48 hours after administration.
Clinical signs:
Toxicological signs were sedation, convulsion, dyspnea, lacrimation, and collapse.
Interpretation of results:
GHS criteria not met
Conclusions:
Based on the read-across approach, the LD50 of Cytidine 3'-(dihydrogen phosphate) is 12245 mg/kg body weight by oral route in male rats.
Executive summary:

Read-across from supporting substance (structural analogue or surrogate): An acute toxicity study of Citicoline has been published. Following a method similar to OECD Guideline 401, 10 male Fisher 344 rats were administered orally with 11520, 14400, 18000 and 22500 mg/kg bw of citicoline. After 7 days, mortality was observed at all doses except for the lowest. In most animals, death occurred between 2 and 48 hours after administration. Toxicological signs were sedation, convulsion, dyspnea, lacrimation, and collapse. According to the results, the LD50 of Citicoline is 18501 mg/kg body weight by oral route in male rats.

Based on the available information for the read-across approach, the target substance has an oral LD50 of 12245 mg/kg body weight in male rat.

Endpoint:
acute toxicity: oral
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH (see "Attached justification")

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The target substance is a breakdown product of the analogue substance.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
- Source: Citicoline.
- Target: Cytidine 3'-(dihydrogen phosphate) (see "Test material" for further information).

3. ANALOGUE APPROACH JUSTIFICATION
Please find Reporting Format in "Attached justification".

4. DATA MATRIX
Please find data Matrix included in "Attached justification".
Reason / purpose for cross-reference:
read-across source
Key result
Sex:
male/female
Dose descriptor:
LD50
Effect level:
> 1 324 mg/kg bw
Based on:
test mat.
Remarks on result:
other: Read across from analogue substance, LD50 > 2000 mg/kg bw
Mortality:
No mortality was observed.
Clinical signs:
There were no treatment-related effects on clinical signs and behavior.
Body weight:
No treatment-related effects on body weights were observed.
Gross pathology:
No gross lesions were noted in any organs of the animals on day 15.
Interpretation of results:
GHS criteria not met
Conclusions:
Based on the available information for the read-across approach, the LD50 of Cytidine 3'-(dihydrogen phosphate) is higher than 1324 mg/kg body weight by oral route in the rat.
Executive summary:

Read-across from supporting substance (structural analogue or surrogate): The acute oral toxicity of the analogue substance Citicoline was determined. A limit test was performed similar to OECD 423, following GLP (certificate not available). The test item was administered to a group of 10 male and 10 female Sprague-Dawley rats at the dose of 2000 mg/kg body weight by oral gavage. All animals were thoroughly observed twice daily at the beginning and the end of the workday and once on the weekend days. Survival, clinical symptoms, behaviour and body weight were monitored. No mortality occurred during the study and the body weight evolution of the animals remained normal during the study. There were no treatment-related effects on clinical signs and behaviour. No gross lesions were noted in any organs of the animals on day 15. The LD50 of the test item is higher than 2000 mg/kg body weight by oral route in the rat. Based on the available information for the read-across approach, the target substance has an oral LD50 higher than 1324 mg/kg body weight in rat.

Endpoint:
acute toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1983
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 420 (Acute Oral Toxicity - Fixed Dose Method)
GLP compliance:
not specified
Remarks:
Animals were kept under GLP regulations and standard conditions
Test type:
fixed dose procedure
Limit test:
yes
Species:
rat
Strain:
Sprague-Dawley
Remarks:
CD-2 holoxenic
Sex:
male/female
Route of administration:
oral: unspecified
Vehicle:
other: agar
Details on oral exposure:
VEHICLE
- Concentration in vehicle: CDP-choline was administered as 16% suspension in 0.25% agar.

MAXIMUM DOSE VOLUME APPLIED: 50 ml

CLASS METHOD (if applicable)
- Rationale for the selection of the starting dose: After a dose ratio test, the most suitable doses were selected under geometric progression.
Doses:
8000 mg/kg
No. of animals per sex per dose:
10
Control animals:
no
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: regular times
- Other examinations performed: behavioural changes
Statistics:
Mortality rate was calculated using the method by Reed-Muench on cumulative values at increasing doses. The statistical analysis of standard error was made according to Pizzi's method.
Preliminary study:
After a dose ratio test, the most suitable dose was selected under geometric progression (8 g/kg • 50 ml of CDP-choline).
Key result
Sex:
male/female
Dose descriptor:
LD50
Effect level:
> 8 000 mg/kg bw
Based on:
act. ingr. (total fraction)
Remarks on result:
not determinable due to absence of adverse toxic effects
Mortality:
No deaths occurred.
Clinical signs:
Slight sedation with substantial lethality, and tremors in a small number of animals.
Interpretation of results:
GHS criteria not met
Conclusions:
The LD50 of Citicoline is higher than 8000 mg/kg body weight by oral route in the rat.
Executive summary:

In order to determine the acute oral toxicity of Citicoline, a limit test was performed following a method similar to OECD 420.

After a dose ratio test, the most suitable dose was selected. The test item was administered by oral route to a group of 10 male and female CD-2 holoxenic Sprague-Dawley rats at the dose of 8000 mg/kg body weight. All animals were thoroughly observed regularly during 14 days. Survival, clinical symptoms and behaviour were monitored. No mortality occurred during the study, but slight sedation and tremors were observed .The LD50 of the test item was undeterminable, due to the absence of deaths, but is higher than 8000 mg/kg body weight by oral route in the rat.

Endpoint:
acute toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1983
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 420 (Acute Oral Toxicity - Fixed Dose Method)
Principles of method if other than guideline:
The acute toxicity of the test item was evaluated on mouse. The LD5O values were determined according to the cumulative method by Reed-Muench for mortality rate, and Pizzi's method for calculatiob of standard error.
GLP compliance:
not specified
Remarks:
Animals were kept under GLP regulations and standard conditions
Test type:
standard acute method
Limit test:
yes
Species:
mouse
Strain:
CD-1
Remarks:
Swiss mice
Sex:
male/female
Route of administration:
oral: unspecified
Vehicle:
other: agar
Details on oral exposure:
VEHICLE
- Concentration in vehicle: CDP-choline was administered as 16% suspension in 0.25% agar.

MAXIMUM DOSE VOLUME APPLIED: 50 ml

CLASS METHOD (if applicable)
- Rationale for the selection of the starting dose: After a dose ratio test, the most suitable doses were selected under geometric progression.
Doses:
8g/kg
No. of animals per sex per dose:
10
Control animals:
no
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: regular times
- Other examinations performed: behavioural changes
Statistics:
Mortality rate was calculated using the method by Reed-Muench on cumulative values at increasing doses. The statistical analysis of standard error was made according to Pizzi's method.
Preliminary study:
After a dose ratio test, the most suitable dose was selected under geometric progression (8 g/kg • 50 ml of CDP-choline).
Key result
Sex:
male/female
Dose descriptor:
LD50
Effect level:
> 8 000 mg/kg bw
Based on:
act. ingr. (total fraction)
Remarks on result:
not determinable due to absence of adverse toxic effects
Mortality:
No deaths occurred.
Clinical signs:
Slight sedation with substantial lethality, and tremors in a small number of animals.
Interpretation of results:
GHS criteria not met
Conclusions:
The LD50 of Citicoline is higher than 8000 mg/kg body weight by oral route in mice.
Executive summary:

In order to determine the acute oral toxicity of Citicoline, a limit test was performed with a method similar to OECD 420. After a dose ratio test, the most suitable dose was selected. The test item was administered by oral route to a group of 10 male and female CD-1 Swiss mice at the dose of 8000 mg/kg body weight. All animals were thoroughly observed regularly during 14 days. Survival, clinical symptoms and behaviour were monitored. No mortality occurred during the study, but slight sedation and tremors were observed .The LD50 of the test item was undeterminable, due to the absence of deaths, but is higher than 8000 mg/kg body weight by oral route in mice.

Endpoint:
acute toxicity: oral
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH (see "Attached justification")

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The target substance is a breakdown product of the analogue substance.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
- Source: Citicoline.
- Target: Cytidine 3'-(dihydrogen phosphate) (see "Test material" for further information).

3. ANALOGUE APPROACH JUSTIFICATION
Please find Reporting Format in "Attached justification".

4. DATA MATRIX
Please find data Matrix included in "Attached justification".
Reason / purpose for cross-reference:
read-across source
Key result
Sex:
male/female
Dose descriptor:
LD50
Effect level:
> 5 295 mg/kg bw
Based on:
act. ingr. (total fraction)
Remarks on result:
other: Read across from analogue substance, LD50 of 8000 mg/kg bw
Mortality:
No deaths occurred.
Clinical signs:
Slight sedation with substantial lethality, and tremors in a small number of animals.
Interpretation of results:
GHS criteria not met
Conclusions:
Based on the available information for the read-across approach, the LD50 of Cytidine 3'-(dihydrogen phosphate) is higher than 5295 mg/kg body weight by oral route in mice.
Executive summary:

Read-across from supporting substance (structural analogue or surrogate): The acute oral toxicity of the analogue substance Citicoline was determined with a method similar to OECD 420. After a dose ratio test, the most suitable dose was selected. The test item was administered by oral route to a group of 10 male and female CD-1 Swiss mice at the dose of 8000 mg/kg body weight. All animals were thoroughly observed regularly during 14 days. Survival, clinical symptoms and behaviour were monitored. No mortality occurred during the study, but slight sedation and tremors were observed .The LD50 of the test item was undeterminable, due to the absence of deaths, but is higher than 8000 mg/kg body weight by oral route in mice. Based on the available information for the read-across approach, the target substance has an oral LD50 higher than 5295 mg/kg body weight in mice.

Endpoint:
acute toxicity: oral
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH (see "Attached justification")

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The target substance is a breakdown product of the analogue substance.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
- Source: Citicoline.
- Target: Cytidine 3'-(dihydrogen phosphate) (see "Test material" for further information).

3. ANALOGUE APPROACH JUSTIFICATION
Please find Reporting Format in "Attached justification".

4. DATA MATRIX
Please find data Matrix included in "Attached justification".
Reason / purpose for cross-reference:
read-across source
Key result
Sex:
male/female
Dose descriptor:
LD50
Effect level:
> 5 295 mg/kg bw
Based on:
act. ingr. (total fraction)
Remarks on result:
other: Read across from analogue substance, LD50 of 8000 mg/kg bw
Mortality:
No deaths occurred.
Clinical signs:
Slight sedation with substantial lethality, and tremors in a small number of animals.
Interpretation of results:
GHS criteria not met
Conclusions:
Based on the available information for the read-across approach, the LD50 of Cytidine 3'-(dihydrogen phosphate) is higher than 5295 mg/kg body weight by oral route in the rat.
Executive summary:

Read-across from supporting substance (structural analogue or surrogate): The acute oral toxicity of the analogue substance citicoline was determined following a method similar to OECD 420. After a dose ratio test, the most suitable dose was selected. The test item was administered by oral route to a group of 10 male and female CD-2 holoxenic Sprague-Dawley rats at the dose of 8000 mg/kg body weight. All animals were thoroughly observed regularly during 14 days. Survival, clinical symptoms and behaviour were monitored. No mortality occurred during the study, but slight sedation and tremors were observed .The LD50 of citicoline was undeterminable, due to the absence of deaths, but is higher than 8000 mg/kg body weight by oral route in the rat. Based on the available information for the read-across approach, the target substance has an oral LD50 higher than 5295 mg/kg body weight in rat.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
LD50
Value:
2 000 mg/kg bw
Quality of whole database:
The studies used for read-across have a Klimisch score of 2.

Acute toxicity: via inhalation route

Link to relevant study records
Reference
Endpoint:
acute toxicity: inhalation
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
the study does not need to be conducted because exposure of humans via inhalation is not likely taking into account the vapour pressure of the substance and/or the possibility of exposure to aerosols, particles or droplets of an inhalable size
Justification for type of information:
JUSTIFICATION FOR DATA WAIVING
Study scientifically not necessary / other information available: In accordance with column 2 of REACH Annex VIII, the study does not need to be conducted because exposure of humans via inhalation is likely taking into account the vapour pressure of the substance and/or the possibility of exposure to aerosols or droplets of an inhalable size.
Endpoint conclusion
Endpoint conclusion:
no study available

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:
May 10, 2017 - May 24, 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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:
fixed dose procedure
Limit test:
yes
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: yes
Species:
rat
Strain:
Sprague-Dawley
Remarks:
SPF Caw
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Elevage JANVIER LABS (53940 Le Genest St Isle - France)
- Females nulliparous and non-pregnant: yes
- Age at study initiation: male animals of the treated group were 7 weeks old and the female were 8 weeks old
- Weight at study initiation: males= 258.8 ± 13, females= 215.4 ± 10.9
- Fasting period before study: no
- Housing: during the treatment, the animals were in individual cages. On D1, the animals were put into their cage by 5. The rats were kept in solid-bottomed clear polycarbonate cages with a stainless steel mesh lid. Each cage contains dust free weed shavings which were changed at least 2 times a week. Each cage was installed in conventional air conditioned animal husbandry.
- Diet (e.g. ad libitum): foodstuff (ENVIGO 2016) ad libitum
- Water (e.g. ad libitum): tap-water from public distribution system ad libitum. Microbiological and chemical analyses of the water were carried out once every six months by Bureau Veritas - Eurofins (France).
- Acclimation period: at least five days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19ºC - 25ºC
- Humidity (%): 30% - 70% (a temperature lower than 19ºC was registered on 10, 13, 16, 17, 23 and 24 May 2017, the minimum valued measured was 18ºC. A temperature higher than 25ºC was registered on 18 May 2017, the maximum value mesured was 27ºC)
- Air changes (per hr): at least ten changes cycles per hour
- Photoperiod (hrs dark / hrs light): twelve hours continuous light (07:00 to 19:00) and telve hours darkness.

IN-LIFE DATES: From: To: 10 May 2017 - 24 May 2017
Type of coverage:
other: non-occlusive
Vehicle:
water
Details on dermal exposure:
TEST SITE
- Area of exposure: dorsal area of the trunk of the animal
- % coverage: 10% of the body surface area
- Type of wrap if used: non-occlusive porous gauze dressing (50x50 mm2 non woven swab, 4-layer patch, MEDISTOCK) secured in position with a strip of surgical adhesive tape (50 mm wides hypoallergenic micropore TM adhesive tape from 3M)

REMOVAL OF TEST SUBSTANCE
- Washing (if done): after removing the gauze dressings, the treated area was rinsed with destilled water and liquid paraffin
- Time after start of exposure: 24 h

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 mL/kg bw
- Concentration (if solution): 0.2 g/ml
- Constant volume or concentration used: yes
- For solids, paste formed: no

VEHICLE
- Amount(s) applied (volume or weight with unit): 10 mL/Kg bw
Duration of exposure:
24 h
Doses:
2000 mg/kg bw
No. of animals per sex per dose:
5 males and 5 females per dose
Control animals:
yes
Remarks:
Current control study (TAD-2017-001)
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: daily observations, the animals were weighed on day 0 (just before administering the test item) then on day 2, day 7, and day 14.
- Necropsy of survivors performed: yes.
At the end of the study, the animals were euthanized with sodium pentobarbital (Dolethal®). Macroscopic observations were entered on individual autopsy sheets. Only those organs likely to be modified in cases of acute toxicity were examined. No organ was removed and preserved in view to microscopic examinations.
- Other examinations performed:
Body weight and body weight gain: the animals were weighted on D0 (just before administering the test item) then on D2, D7, and D14.
Clinical signs: Spontaneous activity, Preyer's reflex (noise), Respiratory rate, Convulsions, Tremors, Body temperature, Muscle tone, Palpebral opening, Pupil appearance, Salivation, Lachrymation, Righting reflex, Treatment site, Mortality.
Gross pathology: On D14, the animals were anaesthetised with sodium pentobarbital and administration continued to fatal levels. Macroscopic observations were entered on individual autopsy sheets. Only those organs likely to be modified in cases of acute toxicity were examined. Parameters examined: Oesophagus, Stomach, Duodenum, Jejunum, Ileon, Caecum, Colon, Rectum, Spleen, Liver, Thymus, Trachea, Lungs, Heart, Kidneys, Urinary Bladder, Ovaries, Uterus, Treatment Area, Adrenals and Pancreas.
Key result
Sex:
male/female
Dose descriptor:
LD50
Effect level:
> 2 000 mg/kg bw
Based on:
test mat.
Remarks on result:
no indication of skin irritation up to the relevant limit dose level
Mortality:
No mortality occurred during the study.
Clinical signs:
Neither cutaneous reactions nor systemic clinical signs related to the administration of the test item were observed.
Body weight:
The body weitght evolution of the animals remained normal throughout the study.
Gross pathology:
The macroscopic examination of the animals at the end of the study did not reveal treatment-related changes.

All clinical observations were normal.

Macroscopical examinations: Nothing to report

                       

 Table 1. Test item at 2000 mg/kg bw. Body weight and weight gain in grams.

MALES

D0

D2

D2-D0

D7

D7-D0

D14

D14-D0

Rm 1465

268

270

2

310

42

334

66

Rm 1466

247

251

4

294

47

348

101

Rm 1467

261

283

22

336

75

359

98

Rm 1468

274

284

10

332

58

369

95

Rm 1469

244

258

14

294

50

329

85

MEAN

258.8

269.2

10.4

313.2

54.4

347.8

89.0

Standard deviation

13.0

14.7

8.0

20.1

12.9

16.7

14.2

FEMALES

D0

D2

D2-D0

D7

D7-D0

D14

D14-D0

Rf 1470

211

219

8

239

28

300

89

Rf 1471

227

229

2

248

21

278

51

Rf 1472

222

226

4

243

21

284

62

Rf 1473

199

203

4

210

11

269

70

Rf 1474

218

219

1

229

11

256

38

MEAN

215.4

219.2

3.8

233.8

18.4

277.4

62.0

Standard deviation

10.9

10.1

2.7

15.0

7.3

16.5

19.3

 

 

Table 6. Necropsy data sheet of males Rm1106 to Rm1110 (28 February 2017)

 

 

Found dead:   

 

 

Euthanasia: X

 

 

At term: X

 

GENERAL APPEARANCE BEFORE AUTOPSY: Normal

 

 

Observed Organs

 

Observations

 

* OESOPHAGUS

X

N.t.R.

* STOMACH

X

N.t.R.

* DUODENUM

X

N.t.R.

* JEJUNUM

X

N.t.R.

* ILEON

X

N.t.R.

* CAECUM

X

N.t.R.

* COLON

X

N.t.R.

* RECTUM

X

N.t.R.

* SPLEEN

X

N.t.R.

* LIVER

X

N.t.R.

* THYMUS

X

N.t.R.

* TRACHEA

X

N.t.R.

* LUNGS

X

N.t.R.

* HEART

X

N.t.R.

* KIDNEYS

X

N.t.R.

* URINARY BLADDER

X

N.t.R.

* OVARIES

X

N.t.R.

* UTERUS

X

N.t.R.

* TREATMENT AREA (Skin)

X

N.t.R

* ADRENALS

X

N.t.R.

* PANCREAS

X

N.t.R.

PARTICULARS. None

 

N.t.R.: Nothing to report

 

 

Table 7. Necropsy data sheet of females Rf1111 to Rm1115 (28 February 2017)

 

 

Found dead:   

 

 

Euthanasia: X

 

 

At term: X

 

GENERAL APPEARANCE BEFORE AUTOPSY: Normal

 

 

Observed Organs

 

Observations

 

* OESOPHAGUS

X

N.t.R.

* STOMACH

X

N.t.R.

* DUODENUM

X

N.t.R.

* JEJUNUM

X

N.t.R.

* ILEON

X

N.t.R.

* CAECUM

X

N.t.R.

* COLON

X

N.t.R.

* RECTUM

X

N.t.R.

* SPLEEN

X

N.t.R.

* LIVER

X

N.t.R.

* THYMUS

X

N.t.R.

* TRACHEA

X

N.t.R.

* LUNGS

X

N.t.R.

* HEART

X

N.t.R.

* KIDNEYS

X

N.t.R.

* URINARY BLADDER

X

N.t.R.

* OVARIES

X

N.t.R.

* UTERUS

X

N.t.R.

* TREATMENT AREA (Skin)

X

N.t.R

* ADRENALS

X

N.t.R.

* PANCREAS

X

N.t.R.

PARTICULARS. None

 

N.t.R.: Nothing to report

Interpretation of results:
GHS criteria not met
Conclusions:
The dermal LD50 of the test item in rats is greater than 2000 mg/kg bw.
Executive summary:

A limit test was performed to determine the acute dermal toxicity of the test item in rats according to OECD 402 and EU method B.3, following GLP. The test item was applied onto the intact skin of 10 Sprague Dawley rats (5 males, 5 females) at a dose of 2000 mg/kg bw. All animals were observed once daily for 14 days, survival and body weight were monitored. There were no mortality ans systemic clinical signs related to the administration of the test irem during the study. Neither cutaneous reactions nor systemic clinical signs related to the administration of the test item were observed. Normal changes in body weights and none macroscopic findings were found. In conclusion, the test item was found to be non toxic by dermal route, with an LD50 > 2000 mg/kg bw in rats.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
LD50
Value:
2 000 mg/kg bw
Quality of whole database:
The study has a Klimisch score of 1.

Additional information

Acute toxicity: oral.

Weight of evidence: Based on studies from analogue substances, the oral LD50 of the target substance Cytidine 3'-(dihydrogen phosphate) is higher than 2000 mg/kg in rats and mice.

Studies on the analogue substance Citicoline:

- Key study. In order to determine the acute oral toxicity of Citicoline, a limit test was performed similar to OECD 423, following GLP (certificate not available). The test item was administered to a group of 10 male and 10 female Sprague-Dawley rats at the dose of 2000 mg/kg body weight by oral gavage. All animals were thoroughly observed twice daily at the beginning and the end of the workday and once on the weekend days. Survival, clinical symptoms, behaviour and body weight were monitored. No mortality occurred during the study and the body weight evolution of the animals remained normal during the study. There were no treatment-related effects on clinical signs and behaviour. No gross lesions were noted in any organs of the animals on day 15. The LD50 of the test item is higher than 2000 mg/kg body weight by oral route in the rat (Schauss et al 2009).

- Key study: The acute oral toxicity of Citicoline was studied on rats following a method similar to OECD 420. Citicoline was administered by oral route to a group of 10 male and female CD-2 holoxenic Sprague-Dawley rats at the dose of 8000 mg/kg body weight. All animals were thoroughly observed regularly during 14 days. No mortality occurred during the study, but slight sedation and tremors were observed. The LD50 was higher than 8000 mg/kg body weight by oral route in the rat (Grau et al. 1983).

- Key study: An acute toxicity study of Citicoline has been published which followed a method similar to OECD 401. 10 male Fisher 344 rats were administered orally with 11520, 14400, 18000 and 22500 mg/kg bw of citicoline. After 7 days, mortality was observed at all doses except for the lowest. In most animals, death occurred between 2 and 48 hours after administration. Toxicological signs were sedation, convulsion, dyspnea, lacrimation, and collapse, which were severer in the dead animals and some of them continued until the death (Kanabayashi et al. 1980).

- Key study: The acute oral toxicity of Citicoline was studied following a method similar to OECD 420. The test item was administered by oral route to a group of 10 male and female CD-1 Swiss mice at the dose of 8000 mg/kg body weight. All animals were thoroughly observed regularly during 14 days. No mortality occurred during the study, but slight sedation and tremors were observed .The LD50 of the test item was higher than 8000 mg/kg body weight by oral route in mice (Grau et al. 1983).

- Key study: An acute toxicity study of Citicoline has been published which followed a method similar to OECD 401. 10 male mice were administered orally with 15625, 19531, 24414 and 30518 mg/kg bw of citicoline. After 7 days, mortality was observed at all doses except for the lowest. In most animals, death occurred between 2 and 48 hours after administration. Toxicological signs were sedation, convulsion, dyspnea, lacrimation, and collapse, which were severer in the dead animals and some of them continued until the death (Kanabayashi et al. 1980).

- Key study: The acute toxicity of CDP-choline (Citicoline) was studied on CD-1 mice following a method similar to OECD 401. After the oral administration of different doses (highest dose 14 g/kg body weight) with a maximum volume applied of 50 ml/kg, animals were observed up to 72h post-treatment. The LD50 was not determined due to absence of adverse toxic effects. However, it can be concluded an LD0 of 14000 mg/kg bw (Agut et al. 1983).

.

Acute toxicity : dermal.

- Key study: A limit test was performed to determine the acute dermal toxicity of the test item in rats according to OECD 402 and EU method B.3, following GLP. The test item was applied onto the intact skin of 10 Sprague Dawley rats (5 males, 5 females) at a dose of 2000 mg/kg bw. All animals were observed once daily for 14 days, survival and body weight were monitored. There were no mortality and systemic clinical signs related to the administration of the test item during the study. Neither cutaneous reactions nor systemic clinical signs related to the administration of the test item were observed. Normal changes in body weights and none macroscopic findings were found. In conclusion, the test item was found to be non toxic by dermal route, with an LD50 > 2000 mg/kg bw in rats.

Justification for classification or non-classification

Based on the available information (oral and dermal LD50 higher than 2000 mg/kg bw), the substance is not classified for acute toxicity according to CLP Regulation (EC) no. 1272/2008.