Cytidine 3'-(dihydrogen phosphate)

Endpoint summary

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

Reference

Reference 1

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.

Reference 2

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.

Reference 3

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
Mortality^a	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
Hematology^b
Red blood cell count, 10^12/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, 10^9/L	5.16±1.34	5.70±1.48	5.46±1.26	6.40±1.14^c
Lymphocyte count, 10^9/L	4.31±1.14	4.77±1.27	4.66±1.12	5.47±0.98^c
Neutrophil granulocyte count, 10^9/L	0.76±0.25	0.81±0.28	0.69±0.15	0.78±0.20
Platelet count, 10^9/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
Urine^d
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	1/10^a; 0.1^e	1/10^a; 0.1^e	2/10^a; 0.2^e	2/10^a; 0.2^e
Protein negative^a	10/10	10/10	10/10	10/10
Blood negative^a	10/10	10/10	10/10	10/10
Serum chemistry^b
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±0.94^f	6.46±0.85^c	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± 5.72^c
Potassium, mmol/L	4.01±0.46	3.80±0.31	3.74±0.39	3.68±0.29^c
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 weights^b
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±0.079^c	0.735±0.066
Histopathology^b
Kidney intratubular (mineral) deposits,	4/20^a;0.2^e	8/20^a;0.4^e	10/20^a;0.65^e	19/20^a;1.1^e,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
Mortality^a	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±1.56^b	26.7±1.98
Week 13	28.1±1.52	27.5±2.17	29.2±2.61	28.1±2.29
Hematology^c
Red blood cell count, 10^12/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, 10^9/L	12.0±2.7	12.3±2.5	12.2±2.8	11.7±2.1
Lymphocyte count, 10^9/L	10.3±2.3	10.7±2.3	10.5±2.5	10.0±1.8
Neutrophil granulocyte count, 10^9/L	1.3±0.43	1.3±0.26	1.2±0.33	1.2±0.27
Platelet count, 10^9/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
Urine^d
Volume, mL
0-1 h	4.37±1.73	2.27±0.57^e	2.2±0.67^e	1.4±0.58^e
0-3 h	5.99±1.84	3.80±0.41^e	3.47±0.93^e	2.12±0.80^e
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	2/10^a; 0.2^f	6/10^a; 0.6^f	7/10^a; 0.9^f	6/10^a; 0.6^f
Protein negative^a	10/10	10/10	10/10	10/10
Blood negative^a	10/10	10/10	10/10	10/10
Clinical chemistry^c
Alanine aminotransferase, U/L	37.7±6.14	42.1±44.7^b	31.8±3.97^b	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±0.007^b	0.062±0.01^b
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±0.24^b	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 weights^c
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
Histopathology^c
Kidney intratubular (mineral) deposits, grade I^a,f	0/20^a;0^f	0/20^a;0^f	0/20^a;0^f	0/20^a;0^f
Kideney, peritubular focal lymphohistiocytic infiltration	1/20^a;0.05^f	4/20^a;0.2^f	5/20^a;0.25^f	1/20^a;0.05^f

^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.

Reference 4

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.

Reference 5

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.

Reference 6

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.