Registration Dossier

Administrative data

Description of key information

Weight of evidence: The oral sub-chronic NOAEL of the test item is deemed to be ca. 662 mg/kg bw/d (worst-case scenario), based on the available information from supporting analogue substances:

 

- Read-across from supporting substance (structural analogue or surrogate). Source: Short-term repeated dose toxicity study, method similar to OECD 407, GLP study. The NOAEL of the analogue substance citicoline was found to be 1500 mg/kg bw/day in rats, after 30-day oral administration (Romero et al. 1983). Based on the read-across approach, the NOAEL of the target substance is 993 mg/kg bw/day.

 

- Read-across from supporting substance (structural analogue or surrogate). Source: Sub-chronic oral toxicity study, method according to OECD 408, GLP study. The NOAEL of the analogue substance citicoline was found to be 1000 mg/kg bw/day in rats, after 90-day oral administration (Schauss et al. 2009). Based on the read-across approach, the NOAEL of the target substance is 662 mg/kg bw/day.

 

- Read-across from supporting substance (structural analogue or surrogate). Source: Chronic oral toxicity study, method similar to OECD 452, GLP study. The NOAEL of the analogue substance citicoline was found to be 1500 mg/kg bw/day in dogs, after 6-month oral administration (Romero et al. 1983). Based on the read-across approach, the NOAEL of the target substance is 993 mg/kg bw/day.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1983
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 452 (Chronic Toxicity Studies)
Deviations:
yes
Remarks:
only two doses
GLP compliance:
not specified
Limit test:
yes
Species:
dog
Strain:
Beagle
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: from own quarters
- Age at study initiation: 6 months of age
- Diet: Animals were fed once a day with "Pan-lab-dog" standard diet.
- Water: ad libitum
Route of administration:
oral: gavage
Details on route of administration:
esophageal cannulation
Vehicle:
other: agar
Details on oral exposure:
- VEHICLE
- Concentration in vehicle: 25% test item in 0.25% agar suspension
- Amount of vehicle (if gavage): 6 ml/kg
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
6 months
Frequency of treatment:
Daily
Dose / conc.:
1 500 mg/kg bw/day (nominal)
Remarks:
of CDP-choline
No. of animals per sex per dose:
3/sex in the test item group
1/sex in the control group
Control animals:
yes, concurrent vehicle
Positive control:
No
Observations and examinations performed and frequency:
BODY WEIGHT: Yes
- Time schedule for examinations: weekly

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the begining and at the end of the test.
- Anaesthetic used for blood collection: Yes
- Animals fasted: Not specified.
- Parameters checked: hematocrit, hemoglobin, RBC, WBC, leukocyte formula.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the begining and at the end of the test.
- Animals fasted: Not specified.
- Parameters checked: glucose, GOT, GPT, uric nitrogen (BUN), chloride, protein, globulin, lipids, cholesterol, bilirubin

URINALYSIS: Yes
- Time schedule for collection of urine: At the begining and at the end of the test.
- Metabolism cages used for collection of urine: not specified
- Animals fasted: Not specified.
- Parameters checked: pH, urinary density and qualitative determination
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
All animals included in the study were submitted to a complete necropsy. A careful gross examination of organs was made and their weight recorded.
The following organs were examined and prepared for their histologic study:
Organs: liver (lower left and central lobules), kidney (both), heart, spleen, lung, ovary, testicle, mesenteric lymphatic ganglia (in some animals).

HISTOPATHOLOGY: Yes
The previously mentioned organs were examined. They were fixed in 10% neutral formol, then in paraffin and cut into 6 micrometers pieces. Dying of every organ was made with hematoxylin-eosin abd Perls' in the spleen for hemosiderin identification.
Statistics:
The number on animals used prevented from carrying out a stadistical study and comparison between both groups.The results shown are individually considered and restricted to the appraisal whether respective values fall within normal ranges.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Liver: No signs of toxicity. The alterations found were characteristic of liver parenchyma and not related to the test item.They appear with similar frequency and intensity in control animals. Among the most frequent onces can be mentioned small granulomas ands infiltrations of inflamatory cells, necrosis of isolated liver cells and small areas of necrosis, and other alterations such as cumulations of bilirubin, multinucleate eosinophilic bodies and rather irregular distribution of liver glycogen from a group of animals to another.

Kidney: No nephrotoxic signs were found in any of the animals. It was observed numerous cortical granulomas of probable parasitic origin in one control animal and one animal from the test item group, problable provoked by Toxcara canis. Some cortical areas of intersticial infoltration were also appreciated.

Heart: One female from CDP-choline group showed a noticeable myocardial necrosis area. None of the other animals showed any increase of necrosis, hence the non-toxic character of the mentioned lesion os assumed.

Lung: The existence of an intersticial pneumonic process in various development degrees was observedd in all animals under study; one of them had various granulomatous foci, with remains of hemosiderin from small old hemorrhages provoked by this process.

Testicle: Some multinucleate giant cells were found sporadically in the tubular openings, without bearing any relationship with the test item.
Histopathological findings: neoplastic:
no effects observed
Other effects:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
1 500 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
clinical biochemistry
gross pathology
histopathology: non-neoplastic
mortality
organ weights and organ / body weight ratios
urinalysis
Key result
Critical effects observed:
no

Table 1: Weight data (g):

Weeks

Control (male)

CDP-choline (male)

 

Control (female)

CDP-choline (female)

 

4

9.5

12.2

9.8

12.9

6

9.5

12.3

9.3

12.3

8

9.6

13.3

9.5

12.7

10

9.6

14.0

9.6

13.0

12

9.8

14.3

9.8

12.2

14

9.8

13.8

9.4

12.9

12

9.7

14.1

9.6

12.9

18

9.0

13.9

9.5

12.2

20

9.0

15.1

10.4

13.5

22

9.2

14.9

10.4

13.7

24

8.9

15.2

10.6

14.6

Weights for CDP-choline group (male and female) correspond to three animals' average

Table 2: Hematology parameters, 6 months

Parameters

Control (male)

Control (female)

CDP-choline

(male 1)

CDP-choline

(male 2)

CDP-choline

(male 3)

CDP-choline

(female 1)

CDP-choline

(female 2)

CDP-choline

(female 3)

Hematocrit

43

42

42

47

39

51

40

44

Hemoglobin

15.7

15.5

12.8

12.1

11.6

12.6

9.8

10.3

RBC

8430000

8040000

8070000

7800000

7480000

7230000

8960000

8550000

WBC

6000

6500

6700

11950

5100

10400

4200

6700

Lymphocite

 

41

44

45

30

38

42

48

42

Neutrophyl

58

55

54

70

61

57

52

58

Basophyl

0

0

0

0

0

0

0

0

Eosinophyl

1

1

0

0

0

1

0

0

Monocyte

0

0

1

0

1

0

0

0

Table 3: Chemistry parameters, 6 months.

Parameters

Control (male)

Control (female)

CDP-choline

(male 1)

CDP-choline

(male 2)

CDP-choline

(male 3)

CDP-choline

(female 1)

CDP-choline

(female 2)

CDP-choline

(female 3)

Glucose

70

66

66

78

75

79

102

79

GOT

63

35

43

55

30

43

76

41

GPT

81

95

68

31

35

35

37

27

BUN

16.5

12.5

19.5

18.3

32

19

18

23.5

Chloride

355

461.5

532.5

461.5

426

461.5

532.5

426

Lipids

574.1

629.6

751.9

796.3

592.6

685.2

703.7

777.8

Cholesterol

155.5

168.2

177.3

170.9

135.5

181.8

157.3

191.8

Bilirubin

0.4

0.3

0.1

0.1

0.3

0.2

0.1

0.2

Proteins

7.5

6.0

0.1

6.7

6.0

6.5

7

6.9

Globulins

6.3

5.6

5.5

4

4.6

5.1

6

4.6

Table 4: Urinalysis, 6 months.

Parameters

Control (male)

Control (female)

CDP-choline

(male 1)

CDP-choline

(male 2)

CDP-choline

(male 3)

CDP-choline

(female 1)

CDP-choline

(female 2)

CDP-choline

(female 3)

Urobilinogen

0.1

0.1

0.1

 

0.1

0.1

0.1

0.1

Blood

+ weak

+ weak

+ weak

+ weak

+ weak

+ +

NF

NF

Bilirubin

NF

NF

NF

NF

NF

NF

NF

NF

Ketones

NF

NF

NF

NF

NF

NF

NF

NF

Glucose

NF

NF

NF

NF

NF

NF

NF

NF

Proteins

NF

NF

NF

NF

NF

NF

NF

NF

pH

7

6

6

6

6

6

6

6

Density

1.06

1.02

1.00

1.03

1.00

1.02

1.00

1.00

Aspect

normal

normal

normal

normal

turbid

normal

normal

normal

NF = not found

Table 5: Weight of organs (g) and weight rate (%) after necropsy

Animal/ Organ

Liver

Spleen

Heart

R. kidney

L. kidney

Lung

R testicle

R. ovary

L. testicle

L. ovary

Control (female)

8.9 kg

38.1

4.3%

64.6

0.7%

69.3

0.8 %

21.1

0.2 %

20.7

0.2 %

81.7

0.9 %

0.3

0.003 %

5.4

0.1 %

Control (male)

10.6 kg

474.4

45.5%

69.9

0.7%

71.1

0.7 %

30.8

0.3 %

25.3

0.2

85.5

0.8 %

1.84

0.2 %

-

-

CDP-choline (male 1)

17.3 kg

496.5

4%

93

0.5%

93.3

0.5 %

42.1

0.2 %

44.3

0.3 %

165.2

1 %

12

0.1 %

12.5

0.1 %

CDP-choline (male 2)

16.7 kg

646.2

3.9%

92.5

0.6%

132.1

0.8%

42.2

0.3%

41.6

0.2%

137.9

0.8%

11.2

0.1%

10.6

0.1%

CDP-choline (male 3)

11.7 kg

427.0

3.6%

46.8

0.4%

94.5

0.8%

26.9

0.2%

27.7

0.2%

92.2

0.8%

10.4

0.1%

7.9

0.1%

CDP-choline (female 1)

15.6 kg

544.8

3.5%

84.8

0.5%

94.2

0.6%

31.0

0.2%

31.0

0.2%

22.2

0.1%

0.3

0.002%

1.2

0.01%

CDP-choline (female 2)

12.3 kg

655.0

5.3%

140.6

1.1%

79.0

0.6%

31.2

0.3%

31.3

0.3%

110.8

0.9%

1.4

0.01%

1.1

0.01%

CDP-choline (female 3)

15.8 kg

785.0

5%

107.0

0.7%

106.0

0.7%

32.2

0.2%

33.0

0.2%

125.9

0.8%

0.7

0.004%

1.6

0.01%

Conclusions:
As no toxic effects were observed related to the test item, it can be concluded that citicoline has a NOAEL of 1500 mg/kg bw/day in dogs, after a 6 months oral administration.
Executive summary:

A 6-months repeated dose toxicity study was performed on Beagle dogs following a method similar to OECD 452. Citicoline was orally administered to 5 male and 5 female dogs, at doses of 1500 mg/kg/day and one control animal per sex which only received the vehicle. All animals were thoroughly observed daily for the onset of any toxic signs. Body weight changes were evaluated weekly, and haematology, clinical chemistry and urine parameters were also evaluated at the beginning and at the end of the test. After finalization of treatment, a gross pathology and histopathology analysis were conducted. No mortality or other effects were observed, except for some histopathological findings not related to the test item administration. The study did not reveal any clinical signs of toxicity. Under these conditions, Citicoline was found to have a NOAEL of 1500 mg/kg bw/day in dogs.

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1983
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 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Deviations:
yes
Remarks:
only two doses
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Comming from an own colony from the original CD strain from Charles River. The colony of animals was subjected to quality controls every two months, evaluating the quality of the flora in the digestive and respiratory systems, as well as the generic derivation of the morphometric parameters.
- Age at study initiation: 5-6 weeks
- Housing: The ground used was of treated wood shavings, sieved with powder removal and sterilized with gamma rays.
- Diet: ad libitum, the diet used was standard one, type 004 (maintenance) of the U.A.R. trade-mark.
- Water: ad libitum (automatic valves).

DETAILS OF FOOD AND WATER QUALITY: Daily control of diet consumption. Quality of water constant and controlled.

ENVIRONMENTAL CONDITIONS :
- Temperature (°C): Constant within the recommendations and coincident with GLP's rules.
- Humidity (%): Constant within the recommendations and coincident with GLP's rules.
- Air changes (per hr): Constant within the recommendations and coincident with GLP's rules.
- Other: All the material used in the environment close to the animal as well as the general facilities, met with the OCDE's regulations.
Route of administration:
oral: gavage
Details on route of administration:
Gastric catheter
Vehicle:
water
Details on oral exposure:
- PREPARATION OF DOSING SOLUTIONS: The product was solved in distilled water, until concentrations of 1% and 10%, respectively, were obtained.

- VEHICLE
- Concentration in vehicle: 1% and 10%
- Amount of vehicle (if gavage): The administered volumes related to animal's weight were 10 and 15 ml/kg. Control group received 15 ml/kg but of distilled water only.
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
30 days
Frequency of treatment:
Daily and on 7 days weekly
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
of CDP-choline
Dose / conc.:
1 500 mg/kg bw/day (nominal)
Remarks:
of CDP-choline
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Positive control:
No
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes. The health condition of the animals was controlled though a visual examination and palpation if necessary.
- Time schedule: Daily

DETAILED CLINICAL OBSERVATIONS: Yes.
- Time schedule: Daily

BODY WEIGHT: Yes
- Time schedule for examinations: twice a week during the whole experiment.

FOOD CONSUMPTION AND COMPOUND INTAKE
- Food consumption: Controlled daily

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the begining and at the end of the test.
- Anaesthetic used for blood collection: Yes
- Animals fasted: Yes, 18h fasting, animals remained in indivudual metabolic cages.
- Parameters checked: red blood corpuscles, leukocytes, hematocrit, hemoglobin, whiite-blood count

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the begining and at the end of the test.
- Animals fasted: Yes, 18h fasting, animals remained in indivudual metabolic cages.
- Parameters checked: glucose, GOT, GPT, uric nitrogen (BUN), creatinine, total proteins, total lipids, bilirubin

URINALYSIS: Yes
- Time schedule for collection of urine: At the begining and at the end of the test.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Not specified
- Parameters checked: volume, pH, density, urobilinogen, blood, bilirubin, ketonic bodies, glucose, proteins.

BEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Daily
- Dose groups that were examined: all
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
All animals included in the study were submitted to a complete necropsy, including the outer examination of the whole animal surface, eyes and natural holes, as well as inner cavities and the organs placed there.
Organs: brain, lung, heart, liver, thymus, spleen, stomach, duodenum, kidney, adrenal glands, gonads, as well as those organs and tissues showing abnormalities either macroscopic or related to weight changes.

HISTOPATHOLOGY: Yes
Organs were weighed in a fresh state and introduced in a fixing agent (formol) for their later histopathologic examination.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
No morphological changes related to test item administration were found. The histological alterations not attributed to the treatment were the followng:

Respiratory tract: In lung some emphysematous changes and viral interstitial pneumonitis in general of chronic type were seen. They were very small with no quantitative or qualitative differences between the control and treated animals.

Cardiocirculatory system: In the myocardium tiny infiltration foci of inflamatory cell of chronic type, very sporadic and of viral origin were found.

Liver: Both in the control and treated animals small foci of inflamatory infiltration of chronic and/or acute type were found, with usual frequency and intensity.

Lymphatic system: normal deposits of hemosiderin in variable amounts and related to the sex of the animals were ofund, as well as different degrees of hematopoietic activity, all of them within normal limits.

Digestive system: Both in the stomach and duodenum the changes were minimal and consisted in inflitrates of isolated eosinophils or lymphocytes, in mucosa or submucosa.
Histopathological findings: neoplastic:
no effects observed
Other effects:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
1 500 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
clinical biochemistry
clinical signs
food consumption and compound intake
gross pathology
histopathology: non-neoplastic
mortality
organ weights and organ / body weight ratios
urinalysis
Key result
Critical effects observed:
no

Table 1: Control of weights with time:

Weeks

Control

CDP-choline

100 mg/kg

CDP-choline

1500 mg/kg

Males

1

2

3

4

 

223.6 g

272.6 g

311.2 g

337.4 g

 

195.8 g

260.7 g

300.4 g

330.6 g

 

205.6 g

265.3 g

307.4 g

340.2 g

Females

1

2

3

4

 

208.2 g

229.6 g

242.5 g

251.7 g

 

192.3 g

210.1 g

235.3 g

248.1 g

 

199.9 g

220.6 g

237.5 g

245.4 g

Each value is the average of 2 weekly weights.

Table 2: Control of consumed diet with time:

Weeks

Control

CDP-choline

100 mg/kg

CDP-choline

1500 mg/kg

Males

1

2

3

4

 

26.2 g

27.3 g

26.6 g

27.7 g

 

25.3 g

26.2 g

27.1 g

27.4 g

 

25.0 g

26.3 g

26.4 g

27.1 g

Females

1

2

3

4

 

18.8 g

17.2 g

19.5 g

19.4 g

 

19.1 g

18.8 g

18.9 g

19.2 g

 

18.0 g

18.7 g

19.4 g

19.0 g

Values show the weekle averages of the daily consumptions of diet per animal.

 

 Table 3: Average values at 30 days in male animals

Determination

Unit

Control

CDP-choline

100 mg/kg

CDP-choline

1500 mg/kg

Weight

g

337.4± 13.90

330.6± 21.18

340.2± 12.80

Hematocrit

%

44.0± 1.58

40.62± 3.19

42.14± 4.38

Hemoglobin

g/100

12.96± 0.70

13.10± 1.99

12.37± 1.14

Erythrocytes

n/nm³

7195000± 609017.8

7515000± 1053586

705100± 604953.86

Leukocytes

n/nm³

8690± 2765.33

7990± 1213.3

82000± 1234.2

Glucose

mg/100

103.6± 11.22

110.1± 15.33

97.85± 26.89

GOT

IU/l

130.96± 22.77

97.37± 25.40

95.00± 15.06

GPT

IU/l

23.62± 2.94

25.12± 3.04

27.71± 6.26

BUN

mg/100

13.42± 1.10

15.91± 3.04

12.83± 1.99

Proteins

g/100

6.39± 0.13

6.61± 0.20

6.36± 0.18

Lipids

mg/100

388.8± 26.39

361.3± 67.78

350.72± 37.97

Creatinine

mg/100

0.85± 0.01

1.2± 0.36

0.97± 0.16

Urine volume

ml/181

25.85± 5.58

19.20± 3.90

20.00± 5.07

Urine pH

-

7.05± 0.59

7.50± 0.55

7.50± 1.13

Urine density

g/ml

1.01± 0.01

0.97± 0.07

0.89± 0.14

 

Table 4: Average values at 30 days in female animals

Determination

Unit

Control

CDP-choline

100 mg/kg

CDP-choline

1500 mg/kg

Weight

g

230.7± 11.10

220.14± 11.29

225.14± 14.31

Hematocrit

%

39.67± 4.05

39.44± 2.11

43.67±4.55

Hemoglobin

g/100

12.69± 1.05

13.31± 1.03

14.00± 1.28

Erythrocytes

n/nm³

7233888± 934309.07

7452222.2± 733842.6

7551666.6± 785470.5

Leukocytes

n/nm³

7900± 1911.26

6866.67± 1190.81

7933.3± 901.13

Glucose

mg/100

97.78± 6.01

94.11± 10.96

103.50± 16.14

GOT

IU/l

106.11± 11.29

107.67± 17.66

85.67± 15.39

GPT

IU/l

19.89± 1.28

23.31± 3.00

22.63± 1.82

BUN

mg/100

15.89±1.28

19.31± 3.00

15.63± 1.82

Proteins

g/100

6.71± 0.12

6.60± 0.13

6.31± 0.12

Lipids

mg/100

325.93± 33.35

350.06± 30.36

339.71± 47.67

Creatinine

mg/100

0.72± 0.07

0.72± 0.10

0.91± 0.07

Urine volume

ml/181

13.17± 4.49

14.61± 7.63

12.33±9.88

Urine pH

-

6.72± 0.82

7.70± 1.08

6.17± 0.27

Urine density

g/ml

0.92± 0.04

0.91± 0.08

0.85± 0.12

Conclusions:
As no toxic effects were observed related to the test item, it can be concluded that citicoline has a NOAEL of 1500 mg/kg bw/day in rats, after 30-day oral administration.
Executive summary:

A 30-day repeated dose toxicity study was performed on Sprague-Dawley rats similar to OECD 407 (GLP study, certificate not available). Citicoline was orally administered to 5 male and 5 female Sprague-Dawley rats at doses of 0 (control), 100 and 1500 mg/kg/day. All animals were thoroughly observed daily for the onset of any toxic signs, clinical effects and food consumption. Body weight changes were also evaluated twice a week, and haematology, clinical chemistry and urine parameters were also evaluated at the beginning and at the end of the test. After finalization of treatment, gross pathology and histopathology analysis were conducted. No mortality or other effects were observed, except for some histopathological findings not related to the test item administration. The study did not reveal any clinical signs of toxicity. Under these conditions, Citicoline was found to have a NOAEL of 1500 mg/kg bw/day in rats.

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2009
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
yes
Remarks:
absence of use of a functional observational battery
GLP compliance:
yes
Remarks:
Certificate not included in the publication.
Limit test:
no
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: 6 weeks
- Weight at study initiation: 120-157 g (males) and 120-153 g (females)
- Fasting period before study: overnight
- Housing: 2 rats per cage in type II macrolone cages
- Diet: S8106-S011 SM R/M-Z+H (Ssniff Spezialdiäten, Soest, Germany) ad libitum
- Water: Tap water ad libitum.
- Acclimation period: 5 days

DETAILS OF FOOD AND WATER QUALITY: The tap water was supplied by Waterworks of Budapest (average hardness 148 mg CaO/L) and checked monthly for selected microbiological parameters at PCDL. The results met the requirements for potable water.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ºC ± 3 ºC
- 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:
PREPARATION OF DOSING SOLUTIONS: Dosing suspensions were formulated by mixing 1, 3.5 or 10 g of citicoline free-base with 100 mL of destilled water for the 100, 350 and 1000 mg/kg/d test articles, respectively. Solutions were stirred continuosly during the treatment with a magnetic stirrer.

VEHICLE
- Concentration in vehicle: 0.01 g/mL, 0035 g/mL and 0.1 g/mL
- Amount of vehicle (if gavage): 10 mL/kg, adjusted weekly for changes in the animals' body weight.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples were analysed by Wessling Chemical Laboratory immediately preceding the first dosing procedurem and the actual cocentrations were reported as being ± 5% of the theoretical.
Duration of treatment / exposure:
90 days
Frequency of treatment:
Once daily
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
350 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
20
Control animals:
yes, concurrent vehicle
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At the beginning and end of the workday on weekdays and once on weekend days until the morning of the 91st day
- Parameters checked: mortality, general state, external appearance and behaviour

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At the beginning and end of the workday on weekdays and once on weekend days until the morning of the 91st day

BODY WEIGHT: Yes
- Time schedule for examinations: Day 1 and weekly thereafter


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

OPHTHALMOSCOPIC EXAMINATION: Yes
Direct ophtalmoscopic examination was conducted on 10 males and 10 females of each dose group on the day before the first treatment and on all high-dose and control animals during week 11 (on days 75 or 76 for females and days 77 or 78 for males) by a veterinary ohptalmologist using a Welch Allyn Pan Optic Ophthalmoscope (Skaneateles Falls, NY).

HAEMATOLOGY: Yes
- Time schedule for collection of blood: day 91, prior to euthanasia
- Anaesthetic used for blood collection: Yes (diethyl ether)
- Animals fasted: Yes (overnight)
- How many animals: All animals
- Parameters checked: red blood cell count, hematocrit, hemoglobin, white blood cell count, lymphocyte count, neutrophil granulocyte count, platelet count, prothrombin time.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: day 91
- Animals fasted: Yes
- How many animals: All animals
- Parameters checked. aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, total choresterol, total protein, blood glucose, albumin, blood urea nitrogen, sodium, chloride, total calcium and creatinine.

URINALYSIS: Yes
- Time schedule for collection of urine: once during the 12th treatment week (on days 82 or 83 of the study), 10 males and 10 females of each dose group
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No
- Parameters checked: appearance, volume, and specific gravity (by refractometry), pH, protein, glucose and blood were anlyzed using test strips (Hepta phan, Lachema, Brno, Czech Republic)

Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Tissues and organs were observed, collected and preserved in 8% neutral buffered formalin.
Liver, kidneys, adrenals, testicle (left only), spleen, brain, spinal cord, eye (left only), thymus gland, heart, mesenteric liimph node, submandibular limph node, sternum, sciatic nerve with skeletical muscle, stomach, duodenum with pancreas, lungs, pituitay glan, submandibular salivary gland (left), trachea, esophagus, thyroid (including parathyroids), epididymis (right only), protate, female mammary gland, uterus, ovaries, thymus, spleen, brain, and heart.

HISTOPATHOLOGY: Yes
Following dehydration and embedding in paraffin, tissues were sectioned at microns, stained with hematoxilin and eosin, and then examined microscopically. Histopathological examination was performed on all collected tissues from all males and females from the 0 and 1000 mg/kg/d groups. Because increased degrees of renal mineral deposits were found in the 1000 mg/kg/d female group, preparation of slides and histopathological examination of the kidneys were also performed in the 100 and 300 mg/kg/d groups of both sexes.
Liver, heart, and kidney sections of the 0 and 1000 mg/kg/d groups and kidney sections of the 100 and 350 mg/kf/d groups of both sexes were stained using periodic acid-Schiff (PAS) reaction combined with diastase digestion. Sections of liver, heart and kidney, previously fixed in formalin, were cut on a freezing microtome (Cryostate) at 10 to 15 micrometers and stained for fat with Fat Red.
Statistics:
Statistical analysis were performed with Statistica 5.5 (edition 99, StatSoft, Tulsa, Okla). Data were analyzed for homogeneity of variance by the Barlett test. If the variances proved to be homogeneous, 1-way analysis of variance (ANOVA) was performed. If ANOVA detected significant differences (P<0.05), the Tukey test was used to compare the treatment groups versus the control group. In the case that the variances failed the Bartlett's homogeneity test, the Kurskal-Wallis nonparametric 1-way ANOVA was performed followed by the Kolmogorov-Smirnov test. In the case of nonparametric values, the Kruskal-wallis nonparametric 1-way ANOVA was performed followed by the Kolmogorov-Smirnov test.
Clinical signs:
no effects observed
Description (incidence and severity):
No treatment related changes in either the general condition or external appearance were observed in any of the male and female dose groups treated with citicoline free-base.
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no significant differences in body weight or body weight gain among groups treated with citicoline free-base compared with the control group at any time point during the experimental period.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
All male and female groups that were treated with citicoline free-base consumed similar amounts of food compared with the corresponding control groups throughout the study.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
No ophtalmological changes were observed in males or females from the 0 or 1000 mg/kg/d groups at the 12th week of treatment.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
In females, slight but statistically significant increases in WBC and absolute lymphocyte counts were noted in the 1000 mg/kg/d group; corresponding elevation did not occur in the male treatment groups. No other treatment-related changes in hematology parameters were noted in males or females (Tables 1 and 2).
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
The serum creatinine levels in the 350 and 1000 mg/kg/d males exhibited statistically significant increases (11% and 15%, respectively), but the levels were unchanged in the females. Blood urea nitrogen levels exhibited statistically significant increases in the 100 and 350 mg/kg/d females (19% and 10%, respectively) but were not elevated in the 1000 mg/kg/d females or in any male groups. There was a very mild, statistically significant decrease in the 1000 mg/kg/d female serum sodium level (-2%) (Tables 1 and 2).
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
A dose-related decreased in urine volume was found in all treated groups (Table 1). The differences were statistically significant in all groups during the first hour of urine collection as well as during the 0- to 3-hour period. Although dose-related increases in specific gravity were noted in all treated male groups, these increases were not statistically significant. There were no differences in the urine pH, protein, glucose, or blood in any of the treated male groups compared with control. Brownish-colored urine samples were more often noted in the treated males of all dose groups than in the controls (Table 1). In females, no treatment-related differences in urinalysis parameters were noted (Table 2).
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related differences in the organ weights or relative organ weights were found. Mineralization occurred with dose-related increased incidence and severity in the kidney of female rats (Table 2). In males, similar deposits were seen only in the kidneys of two rats from the 1000 mg/kg/d treated group (Table 1). Mineralization that consisted of lamellated basophilic concretions located intraluminally in the descending proximal tubules (corticomedullary junction region) were seen without apparent degenerative of inflammatory reaction (Figures 1 and 2). No other treatment-related changes were noted in the kidneys or other tissues and organs. Special stains (PAS, Fat Red) of kidneys, livers, and/or hearts did not reveal any treatment-related changes. Sporadic focal lymphohistiocytic infiltration between the tubuli of the renal cortex was noted in a few control and treated males but was not considered treatment related (Table 1).
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
A minimal (6%) yet statistically significant increase in water consumption that occurred in the male group treated with 350 mg/kg/d was attributed to sporadic deviation within the groups and has no biological significance.
Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
behaviour (functional findings)
body weight and weight gain
clinical signs
food efficiency
gross pathology
mortality
ophthalmological examination
Key result
Critical effects observed:
no

Table 1.Selected Data From the Subchronic Study of Male Crl:CD BR Sprague-Dawley Rats Exposed to Citicoline Free-Base By Gavage

Dose Group (mg/kg/d by Gavage)

Parameter

0

100

350

1000

General

 

 

 

 

Mortalitya

0/20

0/20

0/20

0/20

Cumulative body weight gain, g

 

 

 

 

Days 1-29

82.8±11.4

90.7±16.8

84.0±12.4

84.7±11.6

Days 1-90

167.6±24.8

181.5±31.3

164.5±23.3

170.6 ±23.8

Water consumption g/rat/d,

 

 

 

 

Week 4

35.3±8.18

37.5±8.31

32.0±4.65

36.5±6.15

Week 13

39.9±6.68

39.6±8.02

37.8±8.05

40.1±10.9

Food consumption, g/rat/d

 

 

 

 

Week 4

20.0±1.38

20.8±1.07

20.3±1.13

20.6±1.72

Week 13

20.6±1.92

21.8±1.22

21.7±3.08

21.3±2.41

Hematologyb

 

 

 

 

Red blood cell count, 1012/L

8.00±0.42

8.18±0.38

8.10±0.35

8.05±0.29

Hematocrit, %

43.6±1.89

44.3± 1.66

44.0±1.80

45.1±1.64

Hemoglobin, g/L

148±7.58

149±6.19

149±6.34

150±5.39

White blood cell count, 109/L

5.16±1.34

5.70±1.48

5.46±1.26

6.40±1.14c

Lymphocyte count, 109/L

4.31±1.14

4.77±1.27

4.66±1.12

5.47±0.98c

Neutrophil granulocyte count, 109/L

0.76±0.25

0.81±0.28

0.69±0.15

0.78±0.20

Platelet count, 109/L

918±131

996±122

981±108

1007±167

Prothrombine time, s

18.8±1.40

18.5±1.35

18.2±1.08

17.7±0.85

Urined

 

 

 

 

Volume, mL

 

 

 

 

0-1 h

3.08±0.99

3.29±0.65

3.01±0.90

2.98±0.80

0-3 h

4.27±1.05

4.34±0.77

4.21±0.97

4.16±0.79

pH

7.3±0.823

7.5±0.707

7.3±0.823

7.3±0.675

Specific gravity

1024±0.014

1025±0.005

1024±0.007

1021±0.004

Appearance: brownish discoloration

1/10a; 0.1e

1/10a; 0.1e

2/10a; 0.2e

2/10a; 0.2e

Protein negativea

10/10

10/10

10/10

10/10

Blood negativea

10/10

10/10

10/10

10/10

Serum chemistryb

 

 

 

 

Alanine aminotransferase, U/L

30.6±4.68

31.7±5.54

29.9±6.29

28.8±6.71

Aspartate aminotransferase, U/L

97.7±10.5

94.8±15.6

92.8±19.6

94.4±20.0

Alkaline phosphatase, U/L

129±19.1

138±33.8

125±22.1

127±24.4

Total protein, g/L

61.2±5.26

62.0±4.26

60.4±5.45

58.5±3.75

Blood glucose, mmol/L

4.88±0.57

5.14±0.66

4.83±0.66

5.19±5.19

Cholesterol, mmol/L

1.87±0.38

2.06±0.38

2.06±0.51

2.08±0.38

Blood urea nitrogen, mmol/L

5.89±0.51

7.02±0.94f

6.46±0.85c

6.30±1.04

Creatinine, mmol/L

0.072±0.013

0.076±0.011

0.068±0.008

0.070±0.011

Sodium, mmol/L

143±4.28

143±4.50

142±2.08

140± 5.72c

Potassium, mmol/L

4.01±0.46

3.80±0.31

3.74±0.39

3.68±0.29c

Chloride, mmol/L

97.2±3.84

96.4±2.28

97.0±2.1

95.9±4.66

Calcium, mmol/L

2.96±0.19

2.87±0.10

2.92±0.15

2.90±0.17

Organ weightsb

 

 

 

 

Liver weight, absolute, g

7.85±0.85

8.09±0.77

8.06±0.94

8.00±0.88

Liver-to-body-weight ratio, g/100 g

2.85±0.20

2.80±0.16

2.94±0.20

2.88±0.27

Kidney weight, absolute, g

1.98±0.24

1.96±0.24

2.04±0.22

2.05±0.27

Kidney-to-body-weight ratio, g/100 g

0.721±0.061

0.676±0.10

0.749±0.079c

0.735±0.066

Histopathologyb

 

 

 

 

Kidney intratubular (mineral) deposits,

4/20a;0.2e

8/20a;0.4e

10/20a;0.65e

19/20a;1.1e,f

Kidney, peritubular focal lymphohistiocytic infiltration

0/20

0/20

0/20

0/20

aIncidence of animals affected/number of animals examined.

bSampling performed on day 91 of study.

cSignificantly different from the control group±standard deviation,P.05.

dSampling and determination performed on week 12 of study (day 82 or day 83 of the study).

eAverage degree of severity given for urine appearance/kidney intratubular deposits/kidney peritubular infiltration: grade 0 = negative/none; grade I = bright brownish/slight: 1-10 deposits per kidney section/slight, grade 2 = intermediate brownish/moderate: 11-20 deposits per kidney section/moderate, grade 3 = dark brown/marked: 11-20 deposits per kidney section/marked.

fSignificantly different from the control group±standard deviation,P.01.

 

Table 2.Selected Data From the Subchronic Study of Female Crl:CD BR Sprague-Dawley Rats Exposed to Citicoline Free-Base By Gavage

Dose Group (mg/kg/d by Gavage)

Parameter

0

100

350

1000

General

 

 

 

 

Mortalitya

0/20

0/20

0/20

0/20

Cumulative body weight gain, g

 

 

 

 

Days 1-29

188.6±20.1

192.4±16.8

198.6±20.2

188.5±22.9

Days 1-90

400.1±58.7

397.6±46.7

433.8±61.5

397.3±59.5

Water consumption g/rat/d,

 

 

 

 

Week 4

39.7±12.2

37.4±4.87

39.0±3.04

40.6±8.43

Week 13

39.8±2.92

41.3±7.73

35.6±8.11

37.6±8.35

Food consumption, g/rat/d

 

 

 

 

Week 4

27.3±2.14

26.6±1.88

29.0±1.56b

26.7±1.98

Week 13

28.1±1.52

27.5±2.17

29.2±2.61

28.1±2.29

Hematologyc

 

 

 

 

Red blood cell count, 1012/L

8.60±0.43

8.86±0.60

8.66±0.58

8.99±0.69

Hematocrit, %

45.2±2.0

46.8±3.5

45.6±2.3

47.3±3.3

Hemoglobin, g/L

155.0±7.16

160±10.8

156±8.12

161±10.4

White blood cell count, 109/L

12.0±2.7

12.3±2.5

12.2±2.8

11.7±2.1

Lymphocyte count, 109/L

10.3±2.3

10.7±2.3

10.5±2.5

10.0±1.8

Neutrophil granulocyte count, 109/L

1.3±0.43

1.3±0.26

1.2±0.33

1.2±0.27

Platelet count, 109/L

1018±140

1035±122

1014±101

1026±135

Prothrombine time, s

17.4±1.35

17.7±0.98

18.0±1.25

17.3±1.46

Urined

 

 

 

 

Volume, mL

 

 

 

 

0-1 h

4.37±1.73

2.27±0.57e

2.2±0.67e

1.4±0.58e

0-3 h

5.99±1.84

3.80±0.41e

3.47±0.93e

2.12±0.80e

pH

7.5±0.71

6.8±0.79

6.5±1.08

7.4±0.84

Specific gravity

1021±0.008

1026±0.007

1027±0.006

1028±0.006

Appearance: brownish discoloration

2/10a; 0.2f

6/10a; 0.6f

7/10a; 0.9f

6/10a; 0.6f

Protein negativea

10/10

10/10

10/10

10/10

Blood negativea

10/10

10/10

10/10

10/10

Clinical chemistryc

 

 

 

 

Alanine aminotransferase, U/L

37.7±6.14

42.1±44.7b

31.8±3.97b

35.0±6.20

Aspartate aminotransferase, U/L

128±52.6

122±49.4

125±34.4

120±30.5

Alkaline phosphatase, U/L

302±70.0

275±85.9

268±59.7

290±73.0

Total protein, g/L

62.6±4.25

65.2±5.23

63.6±4.72

62.5±3.25

Blood glucose, mmol/L

5.88±0.77

5.87±0.85

6.09±0.56

6.16±0.67

Cholesterol, mmol/L

1.82±0.48

1.98±0.49

1.99±0.36

1.78±0.36

Blood urea nitrogen, mmol/L

6.82±0.85

6.82±0.71

6.64±0.80

6.48±0.58

Creatinine, mmol/L

0.054±0.005

0.057±0.006

0.060±0.007b

0.062±0.01b

Sodium, mmol/L

144±1.10

143±3.29

144±2.95

144± 1.85

Potassium, mmol/L

4.65±0.39

4.67±0.32

4.94±0.24b

4.84±0.31

Chloride, mmol/L

96.2±4.41

99.0±8.47

98.4±3.73

97.5±3.85

Calcium, mmol/L

2.88±0.12

2.91±0.29

2.83±0.15

2.79±0.12

Organ weightsc

 

 

 

 

Liver weight, absolute, g

14.1±2.01

14.5±2.32

15.1±2.23

13.5±1.99

Liver-to-body-weight ratio, g/100 g

2.80±0.22

2.90±0.35

2.82±0.18

2.70±0.21

Kidney weight, absolute, g

3.18±0.29

3.39±0.49

3.32±0.46

3.19±0.48

Kidney-to-body-weight ratio, g/100 g

0.639±0.069

0.683±0.095

0.625±0.071

0.640±0.057

Histopathologyc

 

 

 

 

Kidney intratubular (mineral) deposits, grade Ia,f

0/20a;0f

0/20a;0f

0/20a;0f

0/20a;0f

Kideney, peritubular focal lymphohistiocytic infiltration

1/20a;0.05f

4/20a;0.2f

5/20a;0.25f

1/20a;0.05f

aIncidence of animals affected/number of animals examined.

bSignificantly different from the control group±standard deviation,P.05.

cSampling performed on day 91 of study.

dSampling and determination performed on week 12 of study (day 82 or day 83 of the study).

eSignificantly different from the control group±standard deviation,P.01.

fAverage degree of severity given for urine appearance/kidney intratubular deposits/kidney peritubular infiltration: grade 0 = negative/none; grade I = bright brownish/slight: 1-10 deposits per kidney section/slight, grade 2 = intermediate brownish/moderate: 11-20 deposits per kidney section/moderate, grade 3 = dark brown/marked: 11-20 deposits per kidney section/marked.

 

Conclusions:
Under the conditions of the test, it has been determined that Citicoline has a NOAEL of 1000 mg/kg bw/day in rats, after 90-day oral administration in rats.
Executive summary:

A 90-day repeated dose toxicity study was performed on Sprague-Dawley rats according to OECD 408 (GLP study, certificate not available). Citicoline was orally administered to 20 male and 20 female Sprague-Dawley rats, at doses of 0 (control), 100, 350 and 1000 mg/kg/day. All animals were thoroughly observed immediately after administration for the onset of any toxic signs and once daily thereafter. Mortality, feed intake, water consumption and body weight were monitored and urinalysis were performed. Animals were also evaluated for ophthalmological, haematological and biochemistry changes, in addition to behavioural and pathological effects. No effects of treatment were reported on mortality or general condition, appearance, body weight, food and water intake, ophthalmology, gross necropsy, or organ weights. Kidney-related changes were noted in both male and female animals despite an absence of apparent toxicity. In males, slight significant increases in serum creatinine (350 and 1000 mg/kg/day), and decreases in urine volume (all treated groups) were observed. In females, slight significant increases in total white blood cell and absolute lymphocyte counts (1000 mg/kg/day), and blood urea nitrogen (BUN) (100 and 350, but not 1000 mg/kg/day) were noted. A dose-related increase in renal tubular mineralisation, without degenerative or inflammatory reaction in rats (especially females), is influenced by calcium: phosphorus ratios in the diet. A high level of citicoline consumption resulted in increased phosphorus intake in the rats, and likely explain this result. Under these conditions, Citicoline was found to have a NOAEL of 1000 mg/kg bw/day in rats.

Endpoint:
chronic 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
Positive control:
No
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Liver: No signs of toxicity. The alterations found were characteristic of liver parenchyma and not related to the test item.They appear with similar frequency and intensity in control animals. Among the most frequent onces can be mentioned small granulomas ands infiltrations of inflamatory cells, necrosis of isolated liver cells and small areas of necrosis, and other alterations such as cumulations of bilirubin, multinucleate eosinophilic bodies and rather irregular distribution of liver glycogen from a group of animals to another.

Kidney: No nephrotoxic signs were found in any of the animals. It was observed numerous cortical granulomas of probable parasitic origin in one control animal and one animal from the test item group, problable provoked by Toxcara canis. Some cortical areas of intersticial infoltration were also appreciated.

Heart: One female from CDP-choline group showed a noticeable myocardial necrosis area. None of the other animals showed any increase of necrosis, hence the non-toxic character of the mentioned lesion os assumed.

Lung: The existence of an intersticial pneumonic process in various development degrees was observedd in all animals under study; one of them had various granulomatous foci, with remains of hemosiderin from small old hemorrhages provoked by this process.

Testicle: Some multinucleate giant cells were found sporadically in the tubular openings, without bearing any relationship with the test item.
Histopathological findings: neoplastic:
no effects observed
Other effects:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
ca. 993 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
clinical biochemistry
gross pathology
histopathology: non-neoplastic
mortality
organ weights and organ / body weight ratios
urinalysis
Remarks on result:
other: Read-across from analogue substance: NOAEL of 1500 mg/kg bw/day
Key result
Critical effects observed:
no
Conclusions:
Based on the read-across approach, Cytidine 3'-(dihydrogen phosphate) has a NOAEL of 993 mg/kg bw/day, after daily administration for 6 months in dog.
Executive summary:

Read-across from supporting substance (structural analogue or surrogate): The chronic oral toxicity of the analogue substance Citicoline was determined. A 6-months repeated dose toxicity study was performed on Beagle dogs following a method similar to OECD 452. Citicoline was orally administered to 5 male and 5 female dogs, at doses of 1500 mg/kg/day and one control animal per sex which only received the vehicle. All animals were thoroughly observed daily for the onset of any toxic signs. Body weight changes were evaluated weekly, and haematology, clinical chemistry and urine parameters were also evaluated at the beginning and at the end of the test. After finalization of treatment, a gross pathology and histopathology analysis were conducted. No mortality or other effects were observed, except for some histopathological findings not related to the test item administration. The study did not reveal any clinical signs of toxicity. Under these conditions, Citicoline was found to have a NOAEL of 1500 mg/kg bw/day in dogs. Based on the available information for the read-across approach, the target substance has a No Observed Adverse Effect Level of 993 mg/kg bw/day in dogs.

Endpoint:
short-term repeated dose 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
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Immunological findings:
not specified
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
No morphological changes related to test item administration were found. The histological alterations not attributed to the treatment were the followng:

Respiratory tract: In lung some emphysematous changes and viral interstitial pneumonitis in general of chronic type were seen. They were very small with no quantitative or qualitative differences between the control and treated animals.

Cardiocirculatory system: In the myocardium tiny infiltration foci of inflamatory cell of chronic type, very sporadic and of viral origin were found.

Liver: Both in the control and treated animals small foci of inflamatory infiltration of chronic and/or acute type were found, with usual frequency and intensity.

Lymphatic system: normal deposits of hemosiderin in variable amounts and related to the sex of the animals were ofund, as well as different degrees of hematopoietic activity, all of them within normal limits.

Digestive system: Both in the stomach and duodenum the changes were minimal and consisted in inflitrates of isolated eosinophils or lymphocytes, in mucosa or submucosa.
Histopathological findings: neoplastic:
no effects observed
Other effects:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
ca. 993 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
clinical biochemistry
clinical signs
food consumption and compound intake
gross pathology
histopathology: non-neoplastic
mortality
organ weights and organ / body weight ratios
urinalysis
Remarks on result:
other: Read-across from analogue substance: NOAEL 1500 mg/kg bw/day
Key result
Critical effects observed:
no
Conclusions:
Based on the read-across approach, Cytidine 3'-(dihydrogen phosphate) has a NOAEL of 993 mg/kg bw/day, after 30-day oral administration.
Executive summary:

Read-across from supporting substance (structural analogue or surrogate): The short-term repeated dose oral toxicity of the analogue substance Citicoline was determined. A 30-day repeated dose toxicity study was performed on Sprague-Dawley rats similar to OECD 407 (GLP study, certificate not available). Citicoline was orally administered to 5 male and 5 female Sprague-Dawley rats, at doses of 0 (control), 100 and 1500 mg/kg/day. All animals were thoroughly observed daily for the onset of any toxic signs, clinical effects and food consumption. Body weight changes were also evaluated twice a week, and haematology, clinical chemistry and urine parameters were also evaluated at the beginning and at the end of the test. After finalization of treatment, gross pathology and histopathology analysis were conducted. No mortality or other effects were observed, except for some histopathological findings not related to the test item administration. The study did not reveal any clinical signs of toxicity. Under these conditions, Citicoline was found to have a NOAEL of 1500 mg/kg bw/day in rats. Based on the available information for the read-across approach, the target substance has a No Observed Adverse Effect Level of 993 mg/kg bw/day in rats.

Endpoint:
sub-chronic 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
Clinical signs:
no effects observed
Description (incidence and severity):
No treatment related changes in either the general condition or external appearance were observed in any of the male and female dose groups treated with citicoline free-base.
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
effects observed, treatment-related
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, non-treatment-related
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
ca. 662 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
behaviour (functional findings)
body weight and weight gain
clinical signs
food efficiency
gross pathology
mortality
ophthalmological examination
Remarks on result:
other: Read-across from analogue substance: NOAEL of 1000 mg/kg bw/day
Key result
Critical effects observed:
no
Conclusions:
Based on the read-across approach, Cytidine 3'-(dihydrogen phosphate) has a NOAEL of 662 mg/kg bw/day in rats, after 90-day oral administration in rats.
Executive summary:

Read-across from supporting substance (structural analogue or surrogate): The sub-chronic oral toxicity of the analogue substance Citicoline was determined. A 90-day repeated dose toxicity study was performed on Sprague-Dawley rats according to OECD 408 (GLP study, certificate not available). Citicoline was orally administered to 20 male and 20 female Sprague-Dawley rats, at doses of 0 (control), 100, 350 and 1000 mg/kg/day. All animals were thoroughly observed immediately after administration for the onset of any toxic signs and once daily thereafter. Mortality, feed intake, water consumption and body weight were monitored and urinalysis were performed. Animals were also evaluated for ophthalmological, haematological and biochemistry changes, in addition to behavioural and pathological effects. No effects of treatment were reported on mortality or general condition, appearance, body weight, food and water intake, ophthalmology, gross necropsy, or organ weights. Kidney-related changes were noted in both male and female animals despite an absence of apparent toxicity. In males, slight significant increases in serum creatinine (350 and 1000 mg/kg/day), and decreases in urine volume (all treated groups) were observed. In females, slight significant increases in total white blood cell and absolute lymphocyte counts (1000 mg/kg/day), and blood urea nitrogen (BUN) (100 and 350, but not 1000 mg/kg/day) were noted. A dose-related increase in renal tubular mineralisation, without degenerative or inflammatory reaction in rats (especially females), is influenced by calcium: phosphorus ratios in the diet. A high level of citicoline consumption resulted in increased phosphorus intake in the rats, and likely explain this result. Under these conditions, Citicoline was found a NOAEL of 1000 mg/kg bw/day in rats. Based on the available information for the read-across approach the target tes item has a NOAEL of 662 mg/kg bw/day in rats.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
662 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
All studies available have a Klimisch score of 2.

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

Weight of evidence: The substance is considered to be non-toxic, with an oral sub-chronic NOAEL of ca. 662 mg/kg bw/d (worst-case), based on the available information from supporting analogue substance. The target substance is a degradation product of the analogue substance sharing a common metabolic pathways.

- Read-across from supporting substance (structural analogue or surrogate). Source: Short-term repeated dose toxicity study, method similar to OECD 407, GLP study. The analogue substance Citicoline was orally administered to 5 male and 5 female Sprague-Dawley rats at doses of 0 (control), 100 and 1500 mg/kg/day. The study did not reveal any clinical signs of toxicity, and no mortality or other effects were observed, except for some histopathological findings not related to the test item administration. The NOAEL of the analogue substance Citicoline is considered to be1500 mg/kg bw/day in rats (Romero et al. 1983). Based on the read-across approach, the NOAEL of the target substance is ca. 993 mg/kg bw/day.

- Read-across from supporting substance (structural analogue or surrogate). Source: Sub-chronic oral toxicity study, method according to OECD 408, GLP study. The analogue substance Citicoline was orally administered to 20 male and 20 female Sprague-Dawley rats, at doses of 0 (control), 100, 350 and 1000 mg/kg/day for 90 days. No effects of treatment were reported on mortality or general condition, appearance, body weight, food and water intake, ophthalmology, gross necropsy, or organ weights. Kidney-related changes were noted in both male and female animals despite an absence of apparent toxicity. A dose-related increase in renal tubular mineralisation, without degenerative or inflammatory reaction in rats (especially females), is influenced by calcium: phosphorus ratios in the diet. The increased phosphorus intake in the rats likely explain this result. Under these conditions, Citicoline was found to have a NOAEL of 1000 mg/kg bw/day in rats (Schauss et al. 2009). Based on the read-across approach, the NOAEL of the target substance is ca. 662 mg/kg bw/day.

- Read-across from supporting substance (structural analogue or surrogate). Source: Sub-chronic oral toxicity study, method similar to OECD 452, GLP study. The analogue substance Citicoline was orally administered to 5 male and 5 female Beagle dogs, at doses of 1500 mg/kg/day and one control animal per sex which only received the vehicle. No mortality or other effects were observed, except for some histopathological findings not related to the test item administration. The study did not reveal any clinical signs of toxicity. Under these conditions, Citicoline was found to have a NOAEL of 1500 mg/kg bw/day in dogs (Romero et al. 1983). Based on the read-across approach, the NOAEL of the target substance is ca. 993 mg/kg bw/day.

Justification for classification or non-classification

Based on the available information (NOAEL ca. 662 mg/kg bw/d in rats), the substance is not classified for repeated dose toxicity (STOT RE) in accordance with CLP Regulation (EU) No. 1272/2008.