Cytidine 3'-(dihydrogen phosphate)

Repeated dose toxicity: oral

Administrative data

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

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

1983

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

Cytidine 5'-(trihydrogen diphosphate), mono[2-(trimethylammonio)ethyl] ester, hydroxide, inner salt, monosodium salt

EC Number:

251-689-1

EC Name:

Cytidine 5'-(trihydrogen diphosphate), mono[2-(trimethylammonio)ethyl] ester, hydroxide, inner salt, monosodium salt

Cas Number:

33818-15-4

Molecular formula:

C14H26N4O11P2.Na

IUPAC Name:

Sodium[(2R,3S,4R,5R)-5-(4-amino-2-oxo-1,2-dihydropyrimidin-1-yl)-3,4-dihydroxyoxolan-2-yl]methyl({[2-(trimethylazaniumyl)ethyl]phosphanato}oxy)phophonate

Test material form:

solid

Test animals

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

Administration / exposure

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

Doses / concentrations

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

Examinations

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.

Results and discussion

Results of examinations

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

Effect levels

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

Target system / organ toxicity

Key result

Critical effects observed:

no

Any other information on results incl. tables

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%

Applicant's summary and conclusion

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.