6-amino-1,3-dimethyluracil

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1997-07-15 - 1997-11-12 (in-life phase)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1998

Report date:

1998

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

EC Directive 87/302/EEC, B.26 90-day repeated oral dose, rodents, 1988.

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

OECD Guideline 408, Rodent: 90-day study, 1981

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

OECD Guideline 408, 90-day Oral Toxicity Study in Rodents, Draft 1996

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

6-amino-1,3-dimethyluracil

EC Number:

229-662-0

EC Name:

6-amino-1,3-dimethyluracil

Cas Number:

6642-31-5

Molecular formula:

C6H9N3O2

IUPAC Name:

6-amino-1,3-dimethyl-1,2,3,4-tetrahydropyrimidine-2,4-dione

Test material form:

solid: particulate/powder

Remarks:

off-white to yellow

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature in the dark, stable under storage conditions
- Stability under test conditions: Stability in the diet was determined over 23 weeks at room temperature

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Wistar Crl:{WI) BR (outbred, SPF-Quality)

Details on species / strain selection:

Recognised by international guidelines as the recommended test system (e.g. EPA, FDA, OECD, EEC)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: Approximately 6 weeks (start of treatment)
- Weight at study initiation: approx. 190 g (males), approx. 160 g (females)
- Fasting period before study: no
- Housing: Group housing of 5 animals per sex per cage in stainless steel suspended cages with wire mesh floors. During activity monitoring, animals were housed overnight per 2 or 3 animals in Macrolon plastic cages with sterilised sawdust (B.M.I. Helmond, The Netherlands) provided as bedding. Results of bedding analyses for contaminants are examined and archived.
- Diet (e.g. ad libitum): Free access to prepared diets. During the pretest period, animals had free access to standard pelleted laboratory animal diet (from Carfil Quality BVBA, Oud-Turnhout, Belgium).
- Water (e.g. ad libitum): Free access to tap water.
- Acclimation period: Acclimatisation period was at least 5 days before start of treatment under laboratory conditions.

DETAILS OF FOOD AND WATER QUALITY:
Each batch of powder and pelleted diet from the supplier is analysed for nutrients and contaminants are analysed on a regular basis. Results are examined and archived.
Certificates of tap water analysis (performed quarterly) were examined and archived-

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21°C
- Humidity (%): 50%
Fluctuations from these optimal conditions were noted, but were considered not to have affected study integrity.
- Air changes (per hr): Air-conditioned room with approximately 15 air changes per hour and the environment control led with optimal conditions.
- Photoperiod (hrs dark / hrs light): Lighting was 12 hours artificial fluorescent light and 12 hours dark per day.

Administration / exposure

Route of administration:

oral: feed

Details on route of administration:

Oral, by inclusion in the diet

Vehicle:

other: feed

Details on oral exposure:

DIET PREPARATION
The amount of test substance incorporated into the diet was kept at a constant level in terms of ppm, throughout the study period. All dose levels presented in this report are expressed as nominal dose levels.
After termination, the actual test substance intake was estimated based on the body weight and food consumption values.
In the calculations for diet preparation, the concentrations were corrected to 100%.
The test substance was mixed with powder feed. Water (approximately 11%) was added to aid pelleting, The pellets were dried for approximately 24 hours at 35°C before storage. The control animals received similarly prepared pellets but without the test substance. Stability was determined over 23 weeks at room temperature
Analysis of the prepared diets revealed that they were homogeneous, but overall mean values for accuracy of preparation were 59, 64 and 75% of nominal for the 500, 2000 and 10000 ppm dose groups due to partial hydrolysis of the test compound in the diet. After correction for these analytical results, test substance intake was ± 25, 110 and 700 mg/kg body weight/day respectively.
- Rate of preparation of diet (frequency): Diets were prepared once weekly
- Mixing appropriate amounts with (Type of food): Standard laboratory animal diet (R-03-18-M from Carfil Quality BVBA, Oud-Turnhout, Belgium).
- Storage temperature of food: Diet was kept in the diet store room in the animal house.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis of formulations: Duplicate samples of diets prepared in weeks 1, 6 and 12 were analysed to check accuracy of preparation (all concentrations) and stability in the food. In addition, homogeneity was analysed in weeks 1 and 12. Samples of week 1 were stored at room temperature, and samples of weeks 6 and 12 were stored at -20°C until analysis. Stability was assessed after 23 weeks at room temperature in week 1 samples. In addition, random samples of 100 grams from all diet preparations were taken and stored at -20°C. Any remaining samples will be discarded after finalisation of the study report.

Duration of treatment / exposure:

90 days, followed by a 28-day recovery period

Frequency of treatment:

continously in the diet

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 ppm

Remarks:

control

Doses / concentrations 2

Dose / conc.:

500 ppm

Remarks:

corresponding to ± 25 mg/kg bw/d

Doses / concentrations 3

Dose / conc.:

2 000 ppm

Remarks:

corresponding to ± 110 mg/kg bw/d

Doses / concentrations 4

Dose / conc.:

10 000 ppm

Remarks:

corresponding to ± 700 mg/kg bw/d

No. of animals per sex per dose:

10 / sex / dose, both main test and recovery groups (control & high dose)

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: Based on results of a 14-day study in rats.
- Rationale for animal assignment (if not random): At least 5 days before study start, by computer -generated random algorithm according to body weight, with all animals within ± 20% of the sex mean. A health inspection was performed prior to commencement of treatment to ensure that the animals were in a good state of health.
- Rationale for selecting satellite groups: control & high dose groups
- Post-exposure recovery period in satellite groups: 28 days

Positive control:

not required

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Mortality I Viability was checked twice daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily from day 1 onwards. The time of onset, degree and duration were recorded. All symptoms were recorded and graded according to fixed scales:
Maximum grade 4: grading slight (1) to very severe (4).
Maximum grade 3: grading slight (1) to severe (3).
Maximum grade 1: presence is scored (1).

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly

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

WATER CONSUMPTION
Subjective appraisal was maintained during the study but no quantitative investigation introduced as no effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Following instillation of tropicamide solution (5 mg/ml), both eyes were examined by means of an ophthalmoscope.
at pretest: All animals
at week 13: Groups 1 and 4
Since no treatment-related findings were noted in Groups 1 (control) and 4 (high dose), Groups 2 (low dose) and 3 (intermediate dose) were not examined.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: See clinical chemistry
- Anaesthetic used for blood collection: Yes (identity) / No / Not specified
- Animals fasted: Yes
- How many animals:
- The following haematology parameters were determined from blood prepared with EDTA as an anti-coagulant, using the instruments 1 isted：
Parameter/Abbreviation Unit Instrumentation
Erythrocyte count/RBC T/l Sysmex K-1000
Haemoglobin/HB mol/l Sysmex K-1000
Haematocrit/HCT l/l Sysmex K-1000
Mean corpuscular volume/MCV fl Sysmex K-1000
Mean corpuscular haemoglobin/MCH fmol Sysmex K-1000
Mean corpuscular haemoglobin concentration/MCHC mmol/l Sysmex K-1000
Platelet count G/l Sysmex K-1000
Red cell distribution width/RDW % Sysmex K-1000
Total leucocyte count/WBC G/l Sysmex K-1000
Differential leucocyte count/SEG (Neutrophils) EO (Eosinophils), BASO (Basophils), LYMPH (Lymphocytes), MONO (Monocytes) l(rel) Manual (microscope)
The following haematology parameters were determined from blood prepared with citrate as an anti-coagulant, using the instruments listed:
Parameter/Abbreviatio Unit Instrumentation/Method
Prothrombin time/PT sec Sysmex CA-5000
Partial thromboplastin time/PTT sec Sysmex CA-5000

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples were collected under light ether anaesthesia immediately prior to sacrifice, between ± 7.30 and 10.00 a.m. The animals were fasted overnight before blood sampling, but water was provided. Blood samples were drawn from the retro-orbital sinus of all rats/sex/group and collected into tubes prepared with EDTA for haematological parameters (0.6 ml), with citrate for clotting tests (1.0 ml) and untreated tubes for clinical biochemistry parameters (>2.0 ml).
- Animals fasted: Yes
- How many animals:
The following clinical biochemistry parameters were determined from serum samples using the instruments listed. Serum samples were stored at -80°C for maximally 2 days prior to analysis.
Parameter/Abbreviation Unit Instrumentation
Alanine aminotransferase (ALAT/GPT) µkat/l Epos selective analyser 5060 (Eppendorf)
Aspartate aminotransferase (ASAT/GOT) µkat/l Epos selective analyser 5060 (Eppendorf)
Bilirubin, total / BILI T. µmol/l Epos selective analyser 5060 (Eppendorf)
Cholesterol total / CHOLEST.T. mmol/l Epos selective analyser 5060 (Eppendorf)
Creatinine µmol/l Epos selective analyser 5060 (Eppendorf)
Glucose mmol/l Epos selective analyser 5060 (Eppendorf)
Urea mmol/l Epos selective analyser 5060 (Eppendorf)
Protein, total / PROTEIN T. g/l Epos selective analyser 5060 (Eppendorf)
Protein, albumin/ ALBUMIN g/l Epos selective analyser 5060 (Eppendorf)
Protein, globulin / GLOBULIN g/l Calculation [PROTEIN T.-ALBUMIN]
Albumin Globulin ratio A/G RATIO 1 Calculation [ALBUMIN/GLOBULIN]
Alkaline phosphatase / ALP µkat/l Epos selective analyser 5060 (Eppendorf)
Sodium mmol/l Eppendorf Flame Photometer MFM 6350
Potassium mmol/l Eppendorf Flame Photometer MFM 6350
Chloride mmol/l Jenway PCLM3
Calcium mmol/l Eppendorf Flame Photometer MFM 6350
Phosphorus / INORG. PHOSPH mmol/l Epos selective analyser 5060 (Eppendorf)


URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during week 13 of treatment
- Dose groups that were examined: all animals
- Battery of functions tested:
hearing ability
pupillary reflex
static righting reflex
hanging wire test (grip strength)
activity test (based on hourly data per 2 or 3 animals, using a computerised motor activity monitoring system, Pearson Technical Services, Debenham, Stowmarket, England)

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
NECROPSY
All animals surviving to the end of the observation period and all moribund animals were deeply anaesthetised using ether vapour and subsequently exsanguinated. All animals assigned to the study were necropsied and descriptions of all macroscopic abnormalities recorded.
Samples of the following tissues and organs were collected from all animals at necropsy and fixed in neutral phosphate buffered 4% formaldehyde solution:
Adrenal glands
Aorta
Brain
Caecum
Cervix
(Clitbral gland)
Colon
Duodenum
Epididymides
Eyes with optic nerve and Harderian gland
Female mammary gland area
Femur including knee joint
Heart
Ileum
Jejunum
Kidneys
(Larynx)
(Lacrimal gland, exorbital)
Liver
Lung, infused with formalin
Lymph nodes - mandibular, mesenteric
(Nasopharynx)
Oesophagus
Ovaries
Pancreas
Peyer's patches (jejunum, Ileum) if detectable
Pituitary gland
(Preputial gland)
Prostate gland
Rectum
Salivary glands - mandibular, sublingual
Sciatic nerve
Seminal vesicles
Skeletal muscle
Skin
Spinal cord -cervical, midthoracic, lumbar
Spleen
Sternum with bone marrow
Stomach
Testes
Thymus
Thyroid including parathyroid
(Tongue)
Trachea
Urinary bladder
Uterus
Vagina
All gross lesions, tissue masses and tumours

ORGAN WEIGHTS
The following organ weights (and terminal body weight) were recorded from the surviving animals on the scheduled day of necropsy:
Adrenal glands
Brain
Epididymides
Heart
Kidneys
Liver
Spleen
Testes
Thymus

HISTOPATHOLOGY: Yes
HISTOTECHNOLOGY
All organ and tissue samples, as defined under Histopathology (following), were processed, embedded and cut at a thickness of 2-4 micrometers and stained with haematoxylin and eosin.
All tissues collected from all recovery group animals were processed and sectioned.
HISTOPATHOLOGY
SIides of all tissues collected at the scheduled sacrifice from all Main group animals of the control and the highest dose group, as well as from all animals of all dose groups which died spontaneously or were terminated jn extremis, all gross lesions and lungs of all animals (all dose groups) were examined by a pathologist.
Based on the treatment related morphologic changes, liver and adrenal glands were also examined from animals of the intermediate dose groups and the recovery groups. All abnormalities were described and included in the report.
Tissues mentioned between brackets were only examined if indicated by signs of toxicity or target organ involvement. An attempt was made to correlate gross observations with microscopic findings.

Statistics:

STATISTICAL ANALYSIS
The following statistical methods were used to analyse the data:
Univariate one-way analysis of variance was used to assess the significance of intergroup differences.
If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
The Steel-test (many-to-one rank test) was applied when the data could not be assumed to follow a normal distribution.
The exact Fisher-test were applied to the ophthalmoscopic observations.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.
Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables.
Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.
References:
C.W. Dunnett, A Multiple Comparison Procedure for Comparing Several Treatments with a Control J. Amer. Stat. Assoc. 50, 1096-1121 (1955).
R.G. Miller, Simultaneous Statistical Inference, Springer Verlag, New York (1981).
R.A. Fisher, Statistical Methods for Research Workers, Oliver and Boyd, Edinburgh (1950).

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Hunched posture and piloerection were evident among 10000 ppm treated males and females. The incidences of these findings were clearly increased when compared to control animals. The observations were mainly seen during the treatment period.
There were no clinical signs of toxicity or behavioural changes noted in the 500 and 2000 ppm dose groups over the treatment or recovery period, that were considered to be related to treatment.
Other findings which were noted among control and/or treated animals included erythema, alopecia, scabs, brown discolouration of the skin, red staining around the eye and an injured eye.
These observations were considered to be within the normal biological variation for rats of this age and strain, and/or incidences did not suggest a treatment-related effect.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

No treatment-related mortality occurred during the study period.
One control male (animal 16) died accidentally during blood sampling prior to necropsy. Two intercurrent deaths occurred among 2000 ppm treated females: animal 98 died in week 6, animal 91 was killed in a moribund condition in week 11. Microscopy revealed abnormalities in the urinary bladder and kidneys, which were considered to have contributed to the cause of death or morbidity. No relationship with treatment was suspected on the basis of these findings.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Throughout the treatment period, a statistically significant decrease in body weights and body weight gain was noted in males and females receiving 10000 ppm in the diet. During the course of the recovery period, differences diminished and did not reach statistical significance.
No effects were noted on body weights of 500 or 2000 ppm treated animals.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

FOOD CONSUMPTION
Food consumption was decreased in the first week of treatment only in 10000 ppm treated males and females, and normal values were attained during the remaining part of the treatment period. During the first week of the recovery period, a slight increase in food consumption could be noted among these animals.
The values for relative food consumption (gram food/kg body weight/day) were decreased in the first week of treatment in 10000 ppm treated males and females, and increased during the rest of treatment and recovery, as a consequence of the lower body weights.
Food consumption and relative food consumption in 500 and 2000 ppm treated animals were considered to be normal.

TEST SUBSTANCE INTAKE
Mean values over the 90-day period were calculated on the basis of body weights, food consumption and nominal dietary inclusion levels and were as follows:
Control: 0
500 ppm: 39 and 47 mg/kg/day for males and females, respectively
2000 ppm: 162 and 189 mg/kg/day for males and females, respectively
10000 ppm: 861 and 1004 mg/kg/day for males and females, respectively
After correction for the overall mean above) the test substance intake was recalculated on the basis of body weights inclusion levels and were as follows:
Control: 0
500 ppm: 23 and 28 mg/kg/day for males and females, respectively
2000 ppm: 104 and 121 mg/kg/day for males and females, respectively
10000 ppm: 646 and 753 mg/kg/day for males and females, respectively

Food efficiency:

not examined

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

not specified

Description (incidence and severity):

Subjective appraisal was maintained during the study but no quantitative investigation introduced as no effect was suspected.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

There were no ophthalmology findings after 13 weeks of treatment that were attributed to dietary administration of the test item. The abnormalities which were observed at pretest or after 13 weeks were considered to be within the biological range of variation for rats in this type of study.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Haematological parameters of treated rats were considered not to have been affected by-treatment.
A number of statistically significant differences were detected among treated males and females when compared to control animals, especially in red blood cell parameters. However, no relationship with the dose was established for any of the parameters. Moreover, evaluation of these values against the historical data of untreated animals of this age and strain revealed that nearly all values remained within the expected range.
Outside the range were the high values for mean corpuscular volume among males and females at 10000 ppm but these were not supported by clear changes in RDW, and the high value for partial thromboplastin time in females at 2000 ppm but the opposite effect was seen at 10000 ppm.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

After 13 weeks, total protein and globulin were decreased in males and females receiving 10000 ppm. As a consequence, albumin/globulin ratio was increased. These changes were still observed for males of this group after 17 weeks.
Other findings which may have been related to treatment with the test substance were decreased serum levels for glucose and urea in 10000 ppm treated females after 13 weeks.
Several other statistically significant differences were found after 13 or 17 weeks for treated animals when compared to control animals. However, in nearly all cases, values remained within the range established in the historical data. Moreover, no dose-effect relationship could be detected or changes were found to be inconsistent between the two sexes.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

No abnormalities were noted in the animals during the functional observation tests in week 13 that were considered to be treatment-related.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

After 13 weeks, autopsy body weights were decreased in males and females receiving 10000 ppm, but reached statistical significance for females only. In addition, kidney:body weight ratios were increased in both males and females of this dose group and testes weights were decreased,
Other changes in organ weights or organ:body weight ratios were considered to be of no relevance as they occurred secondary to body weight effects (brain) or did not show a dose relationship (liver).
After 17 weeks, no toxicologically significant changes in organ weights were detected.
Increased organ:body weight ratios were considered not to be treatment-related but to have resulted from slightly low control values (testes) or to have occurred incidentally (epididymides).

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Macroscopic observations of the intercurrent animals and the animals surviving up to the scheduled necropsy did not reveal any alterations that were considered to have arisen as a result of treatment.
Incidental findings which were found at post mortem examination in treated and/or control animals were considered to be within the range of biological variation for rats of this age and strain and not to represent a change of toxicological significance.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Hepatocyte apoptosis was recorded at a minimal to moderate degree in 7/10 females treated at 10000 ppm at the end of the main study. In some of these rats this finding was accompanied by minimal to moderate degrees of hepatocyte karyomegaly (5/10) and minimal or slight degrees of hepatocyte mitoses (3/10). Two females of this dose group also had minimal basophilic foci of cellular alteration in the liver. The only finding in 10000 ppm treated males was a single case of a minimal degree of karyomegaly. The intermediate dose groups as well as controls were free of these alterations.
Apoptosis is an active process of cellular self destruction in response to growth and regression and thereby serves to control the hepatocellular population. It may be postulated that this finding represents an increased rate of hepatocyte turnover in 10000 ppm treated females.
Following the recovery period, minimal apoptosis was seen in 4/10 females accompanied by minimal mitoses in 2/4 females of the 10000 ppm dose group. Minimal mitoses were also recorded in 1/10 control females. Minimal basophilic foci of cellular alteration were noted in one case each from 10000 ppm treated males and females, and control females.
In the cortex of the adrenal glands an increased incidence of minor (minimal or slight) degrees of vacuolation of the zona glomerulosa was recorded in 10000 ppm treated rats of both sexes (males 8/10, females 10/10) compared to the control animals (males 3/10, females 1/10). The degree and incidence of this finding in the intermediate dose groups was similar to those in the control group animals. The presence of these fine lipid vacuoles can be associated with increased cellular activity in the production of mineralocorticoids which regulate electrolyte and water balance.
Following the recovery period, this finding was still present at minimal or slight degrees in 5/10 males and 6/10 females of the 10000 ppm treated groups versus 1/10 males and 2/10 females of the control group.
In both unscheduled deaths in group 3 females there was inflammation of the urinary bladder coupled with inflammation or degenerative lesions in the kidneys, which were considered to have contributed to the cause of death or morbidity in these rats. However, these alterations were not considered related to treatment with the test article.
Minimal degrees of epidermal hyperplasia in some of the animals with alopecia was the only microscopic correlate to this gross finding. This was considered to be a non-specific reaction.
The remainder of microscopic findings recorded were considered to be spontaneous in nature and within the range of background morphological alterations encountered in Wistar rats of this age and strain. They occurred at similar incidences and severity in both control and treated rats.

Histopathological findings: neoplastic:

no effects observed

Effect levels

1

Key result

Dose descriptor:

NOEL

Effect level:

2 000 ppm

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

histopathology: non-neoplastic

organ weights and organ / body weight ratios

2

Key result

Dose descriptor:

NOEL

Remarks:

calculated from feed intake

Effect level:

ca. 110 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

histopathology: non-neoplastic

organ weights and organ / body weight ratios

3

Key result

Dose descriptor:

NOAEL

Effect level:

2 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

histopathology: non-neoplastic

4

Key result

Dose descriptor:

NOAEL

Remarks:

calculated from feed intake

Effect level:

ca. 110 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

histopathology: non-neoplastic

5

Key result

Dose descriptor:

LOAEL

Effect level:

10 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

histopathology: non-neoplastic

6

Key result

Dose descriptor:

LOAEL

Remarks:

calculated from feed intake

Effect level:

ca. 700 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

histopathology: non-neoplastic

Target system / organ toxicity

Key result

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

The study was conducted according to OECD 408 (1981 / Draft 1996) under GLP without deficiencies on 1,3-Dimethyl-4-aminouracil itself. Hence, the study can be considered as sufficiently reliable to assess the repeated dose toxicity (subchronic) of the test item to rats.
Rats were treated with the test item via the diet for 90 days with nominal dietary inclusion levels of 500, 2000 and 10000 ppm, the corresponding test substance intakes were ± 25, 110 and 700 mg/kg body weight/day respectively. Treatment-related effects were evident at the dose level of 10000 ppm only. At a dose level of 2000 ppm, there were no changes in any of the investigated parameters that were considered to be an effect of treatment. Therefore, a definitive No Observed Effect Level (NOEL) of 2000 ppm in the diet was concluded. Due to the effects seen in the 10000 ppm group without showing complete recovery the No Observed Adverse Effect Level was established to be also 2000 ppm in the diet, which corresponds to a NOAEL = 110 mg/kg and a LOAEL = 700 mg/kg.
Clinical features noted in the 10000 ppm group included hunched posture, piloerection and depressed growth during the treatment phase, and slightly low food intake in week 1. Low serum values for glucose and urea may be related to alterations in metabolic status.
Histopathology revealed that the morphology of the liver and adrenal glands was altered after treatment with 1,3-Dimethyl-4-aminouracil. Hepatocyte apoptosis, karyomegaly and mitoses were increased in incidence in 10000 ppm treated females. It was postulated that this represented an increased rate of hepatocyte turnover. The decreased serum levels for proteins may be related to this phenomenon, since the liver is the major site of protein production. This may be however also indicative for a non-adverse adaptive response, commonly associated with xenobiotic metabolism.
Following the recovery period, minimal apoptosis was seen in 4/10 females accompanied by minimal mitoses in 2/4 females of the 10000 ppm dose group. Minimal mitoses were also recorded in 1/10 control females. Minimal basophilic foci of cellular alteration were noted in one case each from 10000 ppm treated males and females, and control females. So it can be concluded that the effect is reversible upon termination of treatment. Whether the incidence of minimal apoptosis/mitoses over control should be considered permanent or whether the recovery period of 28 did not suffice for complete remission cannot be concluded definitively, but complete recovery is likely.
In the cortex of the adrenal glands, vacuolation of the zona glomerulosa was recorded in 10000 ppm treated rats of both sexes. This change was considered to be adaptive in nature, and may be associated with increased cellular activity in the production of mineralocorticoids. It is unclear if this was also related to the slightly higher kidney:body weight ratios in males and females. Following the recovery period, this finding was still present at minimal or slight degrees in 5/10 males and 6/10 females of the 10000 ppm treated groups versus 1/10 males and 2/10 females of the control group.
After 13 weeks, total protein and globulin were decreased in males and females receiving 10000 ppm. As a consequence, albumin/globulin ratio was increased. These changes were still observed for males of this group after 17 weeks.
Although weight and function of the gonads is usually maintained in the case of depressed growth, testes weights were noted to be decreased at the end of treatment. After 17 weeks, no toxicologically significant changes in organ weights were detected.
Increased organ:body weight ratios were considered not to be treatment-related but to have resulted from slightly low control values (testes) or to have occurred incidentally (epididymides).
Due to the fact that the observed effects are only minor and are considered to be mainly due to an adaptive response as well as systemically connected, there is no indication given that the substance may potentially be a specific organ toxicant.
According to Regulation 1272/2008, 3.9.2.9.7. Classification in Category 2 (STOT RE) is applicable, when significant toxic effects observed in a 90-day repeated-dose study conducted in experimental animals are seen to occur within the guidance value ranges as indicated in Table 3.9.3. In Table 3.9.3 “Guidance values to assist in Category 2 classification” the range of 10 < C ≤ 100 mg/kg bw/d. Hence, with an LOAEL = 700 mg/kg bw/d, those guidance values are clearly exceeded, even the NOAEL lies with 110 mg/kg bw/d above the limit value. Hence, no classification of 1,3-Dimethyl-4-aminouracil as STOT RE Cat. 2 is triggered.

Executive summary:

Subchronic 90-day oral toxicity with1,3-Dimethyl-4-aminouracil was testedby dietary administration in the rat followed by a 28-day recovery period in a study under GLP. The nominal dose levels for the 90-day toxicity study were selected to be 0, 500, 2000 and 10000 ppm in the diet.

The study was based on the following guidelines:

- EC Directive 87/302/EEC, B.26 90-day repeated oral dose, rodents, 1988.

- OECD Guideline 408, Rodent: 90-day study, 1981.

- OECD Guideline 408, 90-day Oral Toxicity Study in Rodents, Draft 1996.

The test substance was administered via the diet for 91 days (males) or 92 days (females) to SPF-bred Wistar rats. One control group and three treated groups were tested, each consisting of 10 males and 10 females. An extra 10 animals per sex in the control and high dose group were allowed 28 days of recovery. The following parameters were evaluated: clinical signs daily; functional observation tests; body weight and food consumption weekly; clinical pathology and macroscopy at termination; organ weights and histopathology on a selection of tissues.

RESULTS

Diet preparations: Analysis of the prepared diets revealed that they were homogeneous, but overall mean values for accuracy of preparation were 59, 64 and 75% of nominal for the 500, 2000 and 10000 ppm dose groups due to partial hydrolysis of the test compound in the diet. After correction for these analytical results, test substance intake was ± 25, 110 and 700 mg/kg body weight/day respectively.

500 ppm: No treatment-related findings noted.

2000 ppm: No treatment-related findings noted.

10000 ppm:

1) Hunched posture and piloerection were observed.

2) Decreased body weights were noted throughout treatment.

3) Food consumption was decreased in the week 1 of treatment.

4) Total protein and globulin were decreased in males and females after 13 weeks, and in males after 17 weeks. Glucose and urea were decreased in females after 13 weeks.

5) After 13 weeks, autopsy body weights were decreased and kidney:body weight ratios were increased in males and females. Testes weights were decreased.

6) Hepatocyte apoptosis, karyomegaly and mitoses were increased in incidence in females. In the adrenal cortex, vacuolation of the zona glomerulosa was recorded in rats of both sexes. Following the recovery period, these findings had reversed partially.

CONCLUSION: From the results presented in this report a definitive No Observed Effect Level (NOEL) of 2000 ppm in the diet was established. Due to the effects seen in the 10000 ppm group without showing complete recovery The No Observed Adverse Effect Level was established to be also 2000 ppm in the diet.