Flucytosine

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2004-06-29 to 2004-12-06

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

adopted 27 July 1995

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Version / remarks:

Commission Directive 96/54/EC

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Flucytosine

EC Number:

217-968-7

EC Name:

Flucytosine

Cas Number:

2022-85-7

Molecular formula:

C4H4FN3O

IUPAC Name:

flucytosine

Test material form:

solid: particulate/powder

Test animals

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:CD® (SD) BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation:approximately 5 - 7 weeks
- Weight at study initiation: males: 131-177 g, females: 118-156 g
- Housing: animals were housed in groups of five by sex in polypropylene grid-floor cages suspended over trays lined with absorbent paper
- Diet (e.g. ad libitum): ad libitum, pelleted diet (Rodent 5LF2 (Certified) Diet, BCM IPS Limited, London, UK)
- Water (e.g. ad libitum): ad libitum, mains drinking water
- Acclimation period: 7 days

DETAILS OF FOOD AND WATER QUALITY:
The diet was analysed for contaminants. The diet and drinking water were considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 1
- Humidity (%): 55 ± 15
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

arachis oil

Remarks:

BP

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: For the purpose of this study the test material was prepared at the appropriate concentrations as a suspension in Arachis oil BP. The formulations to be stable for at least fourteen days. Formulations were therefore prepared weekly and stored at approximately +4ºC in the dark.

VEHICLE
- Justification for use and choice of vehicle (if other than water): An aqueous formulation of the test material was not possible, therefore Arachis Oil BP was chosen for formulation
- Concentration in vehicle: 37.5, 100, 250 mg/mL
- Amount of vehicle (if gavage): 4 mL/kg

Analytical verification of doses or concentrations:

yes

Remarks:

HPLC

Details on analytical verification of doses or concentrations:

The stability and homogeneity of the test material formulations were determined by the testing facility. Results show that the formulations were stable for at least 14 days. Samples were taken of each test material formulation and were analysed for concentration of 5-fluorocytosine at the testing facility. HPLC was used for analysis of formulations and the results obtained indicate that the prepared formulations were within ± 8% of the nominal concentration.
The test material formulations were mixed thoroughly and samples were taken from the top, middle and bottom of the container, shaking between sampling. Sampling was performed in triplicate.

Duration of treatment / exposure:

28 days

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

150 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

400 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose selection was based on a preliminary dose range finding test.
- Rationale for animal assignment (if not random): The animals were randomly allocated to treatment groups using random letter tables and the group mean bodyweights were then determined to ensure similarity between the treatment groups.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Before dosing, 1 and 5 h after dosing during the working week. Before dosing and 1 h after dosing at weekends

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Before dosing, 1 and 5 h after dosing during the working week. Before dosing and 1 h after dosing at weekends

BODY WEIGHT: Yes
- Time schedule for examinations: Individual bodyweights were recorded on Day 0 (the day before the start of treatment) and on Days 7, 14, 21 and 28. Bodyweights were also recorded at terminal kill.

FOOD CONSUMPTION:
- Food consumption was recorded for each cage group at weekly intervals throughout the study.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION: Yes
- Time schedule for examinations: Water intake was observed daily, for each cage group, by visual inspection of the water bottles for any overt changes.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At day 28 blood samples were obtained from the lateral tail vein or by cardiac puncture prior to necropsy on day 29.
- Anaesthetic used for blood collection: Not specified for days 28, for day 29 animals were sacrificed by an intravenous overdose of sodium pentobarbitone.
- Animals fasted: No
- How many animals: all
- Parameters checked in table [No.1] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:At day 28 blood samples were obtained from the lateral tail vein or by cardiac puncture prior to necropsy on day 29.
- Animals fasted: No
- How many animals: all
- Parameters checked in table [No.1] were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Prior to the start of treatment and on Days 5, 12, 19 and 26
- Dose groups that were examined: all
- Battery of functions tested: sensory activity / grip strength / motor activity / behavioural assessments: Gait, Hyper/Hypothermia, Tremors, Skin colour, Twitches, Respiration, Convulsions, Palpebral closure, Bizarre/Abnormal/Stereotypic behaviour, Urination, Salivation, Defecation, Pilo-erection, Transfer arousal, Exophthalmia, Tail elevation, Lachrymation

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Statistics:

Haematological, blood chemical, organ weight (absolute and relative to terminal bodyweight), weekly bodyweight gain and quantitative functional performance and sensory reactivity data were assessed for dose response relationships by linear regression analysis, followed by one way analysis of variance (ANOVA) incorporating Levene’s test for homogeneity of variance. Where variances were shown to be homogenous, pairwise comparisons were conducted using Dunnett’s test. Where Levene’s test showed unequal variances the data were analysed using non-parametric methods: Kruskal-Wallis ANOVA and Mann-Whitney ‘U’ test.

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Animals of either sex treated with 1000 mg/kg/day showed increased salivation up to ten minutes after dosing from Day 6 onwards and on one occasion, one female up to one hour after dosing on Day 21. Observations of this nature are often reported following oral administration of an unpalatable or slightly irritant test material formulation and, in isolation, are not indicative of systemic toxicity. Males treated with 1000 mg/kg/day showed generalised fur loss from Day 9 onwards. This finding is occasionally seen in laboratory maintained rats and in isolation can be regarded as incidental. One female treated with 1000 mg/kg/day had a damaged tail tip from Day 12 onwards. This was a physical injury and was considered unrelated to test material toxicity.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

A reduction in bodyweight gain was detected for 1000 or 400 mg/kg/day males during weeks 1, 2 and 3 with statistical significance being achieved during weeks 1 and 2. Females treated with 1000 mg/kg/day showed a statistically significant reduction in bodyweight gain during week 1 only. Males treated with 150 mg/kg/day showed a statistically significant reduction in bodyweight gain during week 2 only. The dose response was unconvincing and in isolation, was considered of no toxicological importance.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Animals of either sex treated with 1000 mg/kg/day showed a reduction in food consumption during weeks 2 and 3 and also for females during week 4. Males treated with 400 mg/kg/day showed a reduction in food consumption during week 2 whilst females showed a reduction in food consumption during weeks 3 and 4.

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

Food efficiency (the ratio of bodyweight gain to dietary intake) for animals of either sex treated with 1000 mg/kg/day was slightly reduced during week 1 and also during weeks 2 and 3 for males.

Water consumption and compound intake (if drinking water study):

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Animals of either sex treated with 1000 mg/kg/day showed evidence of a mild anaemia, which was haemolytic in nature, with statistically significant reductions for females in haemoglobin, haematocrit and erythrocyte count. Reductions were also evident for males but statistical significance was only achieved in erythrocyte count. Males from this treatment group also showed statistically significant increases in platelet count, mean corpuscular haemoglobin and mean corpuscular volume. The anaemic condition extended to the 400 mg/kg/day dose group with reductions in erythrocyte count, haemoglobin and haematocrit evident in females and increases in mean corpuscular haemoglobin and mean corpuscular volume evident in males.

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Males treated with 1000 mg/kg/day showed a statistical significant increase in relative spleen weight compared with controls. No such effects were detected in 1000 mg/kg/day females or animals of either sex treated with 400 or 150 mg/kg/day. Animals of either sex from all treatment groups showed a statistically significant reduction in absolute heart weight. Males from all treatment groups showed a statistically significant reduction in liver weight whilst females from all treatment groups also showed a statistically significant reduction in absolute ovary and thymus weight. The dose response was unconvincing and in the absence of any histopathological correlates, the intergroup differences were considered to be of no toxicological importance. Animals of either sex treated with 1000 mg/kg/day and males treated with 400 mg/kg/day showed a statistically significant reduction in absolute kidney weight. In the absence of a similar effect for relative organ weights or any histopathological correlates, the intergroup differences were considered not to be toxicologically significant.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

One female treated with 1000 mg/kg/day and one male treated with 150 mg/kg/day incurred damage to an eye upon removal at necropsy. This was a physical injury that occurred after termination and was considered unrelated to test material toxicity. Males treated with 1000 mg/kg/day showed fur loss at necropsy. This finding is occasionally seen in laboratory maintained rats and was considered unrelated to test material toxicity.

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

SPLEEN: Marginally higher grades of severity of extramedullary haemopoiesis were observed in the spleen of males treated with 1000 or 400 mg/kg/day. All remaining morphological changes were those commonly observed in laboratory maintained rats of the age and strain employed, and there were no differences in incidence or severity between control and treatment groups that were considered to be of toxicological significance.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

150 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

haematology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Target system / organ toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

400 mg/kg bw/day (nominal)

System:

haematopoietic

Organ:

blood

spleen

Treatment related:

yes

Dose response relationship:

not specified

Relevant for humans:

yes

Any other information on results incl. tables

Table 2: Selected haematology, clinical chemistry and histopathological findings

Doses (mg/kg bw)	0	150	400	1000	0	150	400	1000
	male				female
Number of animals/group	5	5	5	5	5	5	5	5
Haematology(day 28)
- Hb (g/dL)	15.0 ± 0.6	15.1 ± 0.7	14.9 ± 0.5	14.2 ± 0.6	15 ± 0.4	14.1 ± 0.5	13.5** ± 0.7	13.8** ± 0.4
- RBC (TERA/L)	7.33±0.29	7.16± 0.29	6.89± 0.49	6.42**± 0.44	7.41 ± 0.3	6.91* ± 0.35	6.45***± 0.25	6.72** ± 0.24
- HCT (L/L)	42.3± 1.9	42.7± 1.5	42.0± 2.1	39.7± 1.7	42.1 ± 1.1	39.6 ± 1.6	37.4***± 1.7	38.8** ± 1.1
- MCH (pg)	20.4± 0.2	21.1± 0.2	21.6*± 0.9	22.2**± 0.8	20.3 ± 0.5	20.4 ± 0.5	20.9 ± 0.5	20.6 ± 0.7
- MCV (FL)	58± 1	60± 1	61**± 2	62***± 2	57 ± 2	58 ± 1	58 ± 1	58 ± 2
- MCHC (g/dL)	35.4± 0.6	35.4± 0.4	35.4± 0.6	35.9± 0.6	35.7 ± 0.1	35.6 ± 0.3	36.1 ± 0.4	35.7 ± 0.2
- WBC (GIGA/L)	10.2± 1.0	7.4± 1.7	7.9± 2.3	7.9± 1.4	9.2 ± 2.9	7.8 ± 2	7.5 ± 1.6	7.7 ± 3.8
- Retics	5.5± 0.4	2.5± 1.4	5.6± 0.4	5.2± 0.9	5.1 ± 0.7	2.5 ± 1.1	2.5 ± 1.1	4.2 ± 0.9
- Neutr	1.76 ± 0.78	1.13 ± 0.27	1.06 ± 0.35	1.09 ± 0.16	1.24 ± 0.54	1.22 ± 0.61	0.51 ± 0.24	0.64 ± 0.27
- Lymph	8.38 ± 0.58	6.24 ± 1.50	6.83 ± 2.09	6.75 ± 1.36	7.88 ± 2.38	6.51 ± 1.49	6.96 ± 1.53	7.03 ± 3.79
- Mono	0 ± 0	0 ± 0	0.02 ±0.04	0 ± 0	0 ± 0	0 ± 0	0.02 ±0.04	0 ± 0
- Eos	0.04 ± 0.09	0.07 ± 0.07	0.04 ± 0.04	0.09 ± 0.09	0.04 ± 0.06	0.09 ± 0.06	0.07 ± 0.10	0.05 ± 0.07
- Bas	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0
- CT (secs)	17.6± 0.6	17.9± 0.6	19± 0.6	18.4± 0.6	20.2 ± 1.5	19.2 ± 3.1	18.8 ± 2.3	19.1 ± 3.4
- PLT (GIGA/L)	780± 0.6	879± 0.6	837± 0.6	1016*± 0.6	819 ± 83	900 ± 73	927 ± 102	883 ± 126
APTT (secs)	13.5± 0.6	13.4± 0.6	12.7± 0.6	14.1± 0.6	13.9 ± 0.4	13.1 ± 1.5	14 ± 1.7	13.5 ± 1.8
Blood chemistry(day x)
- sodium (MMOL/L)	147 ± 1	146 ± 2	147 ± 2	146 ± 2	148 ± 2	148 ± 2	147 ± 2	148 ± 1
- potassium (MMOL/L)	5.04 ± 0.19	4.81 ± 0.30	5.07 ± 0.13	5.17 ± 0.30	5.00 ± 0.44	4.92 ± 0.21	4.98 ± 0.24	4.97 ± 0.24
- chloride (MMOL/L)	106 ± 1	105 ± 1	105 ± 1	105 ± 2	108 ± 2	110 ± 3	108 ± 1	108 ± 1
- Albumin (G/dL)	4.42 ± 0.11	4.58 ± 0.27	4.48 ± 0.15	4.61 ± 0.25	4.56 ± 0.15	4.41 ± 0.26	4.34 ± 0.36	4.51 ± 0.25
- cholesterol (MMOL/L)	61 ± 8	63 ± 8	64 ± 12	58 ± 9	62 ± 7	61 ± 8	69 ± 20	62 ± 12
- Bili (mg/dL)	0.06 ± 0.02	0.09 ± 0.04	0.09 ± 0.01	0.08 ± 0.03	0.07 ± 0.02	0.07 ± 0.03	0.08 ± 0.02	0.11 ± 0.02
Histopathological findings
Bone marrow
- Adipose infiltration	4/5 minimal, 1/5 slight	5/5 no data	5/5 no data	5/5 minimal	4/5 minimal, 1/5 slight	5/5 no data	5/5 no data	5/5 minimal
Heart
- Focal myocarditis	5/5 absent	5/5 no data	5/5 no data	4/5 absent; 1/5 minimal	4/5 absent, 1/5 minimal	5/5 no data	5/5 no data	4/5 absent, 1/5 minimal
Kidneys
- Groups of basophilic tubules	2/5 absent, 3/5 minimal	5/5 no data	5/5 no data	4/5 absent, 1/5 minimal	5/5 absent	5/5 no data	5/5 no data	4/5 absent, 1/5 minimal
- Hydronephrosis	4/5 absent, 1/5 minimal	5/5 no data	5/5 no data	5/5 minimal
Spleen
- Extramedullary haemopoiesis	5/5 no data	5/5 no data		2/5 minimal, 2/5 slight,   1/5 moderate	5/5 minimal	5/5 minimal	5/5 minimal	5/5 minimal

Statistics: Anova; Levene’s test or Mann-Whitney ‘U’ test

* = significantly different from control group p <0.05

** significantly different from control group p <0.01

*** significantly different from control group p <0.001

Applicant's summary and conclusion

Conclusions:

In the present study conducted according to OECD guideline 407 male and female Wistar rats were administered 0, 150, 400 and 1000 mg/kg bw 5-Fluorocytosine for 28 consecutive days. Adverse effects were observed for the 400 and 1000 mg/kg bw groups, consisting of haematological changes, changes in organ weights and histopathological changes of the spleen, thus the NOAEL was determined to be 150 mg/kg bw.