6-amino-1,3-dimethyluracil

Endpoint summary

Description of key information

Repeated Dose Toxicity: Subchronic (90 day + 28 day recovery) study oral (feed), Wistar rat m/f, 0, 500, 2000, 10000 ppm in diet, corresponding to approx. 0, 25, 110, 700 mg/kg bw/d (OECD 408, GLP):

NOEL = 2000 ppm / ca. 110 mg/kg bw/d (based on clinical signs, body weight (gain), food consumption, clinical biochemistry, organ weights, histopathology)

NOAEL = 2000 ppm / ca. 110 mg/kg bw/d (based on clinical biochemistry, histopathology)

Total protein and globulin were decreased and albumin/globulin ratio was increased in males and females receiving 10000 ppm. These changes were still observed for males of this group after 17 weeks.

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). 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

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

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

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

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

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

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

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.

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

Dose / conc.:

0 ppm

Remarks:

control

Dose / conc.:

500 ppm

Remarks:

corresponding to ± 25 mg/kg bw/d

Dose / conc.:

2 000 ppm

Remarks:

corresponding to ± 110 mg/kg bw/d

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

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).

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

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

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

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

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

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

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

Key result

Critical effects observed:

no

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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

110 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

There is a valid OECD 408 repeated dose study on the registered substance available, the tonnage-driven data requirements are fully met, so the database is of high quality.

System:

other: no single system could be identified, overall effects were noted

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: inhalation

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Justification for type of information:

JUSTIFICATION FOR DATA WAIVING
According to REACH Annex IX column 1 (8.6.2), the following study for repeated dose toxicity is required: Sub-chronic toxicity study (90-day), one species, rodent, male and female, most appropriate route of administration, having regard to the likely route of human exposure.
There is a suitable Klimisch 1 GLP OECD 408 guideline study available, assessing the toxicological properties of 1,3-Dimethyl-4-aminouracil after oral application in feed over 90 days. In general, the oral route is the most suitable one to assess systemic effects in humans, which is the main aim of this endpoint. The dermal or inhalative route is only scientifically relevant in case of considerable exposure, any route-specific toxicological mode of action or local effects, whereas sufficient information on the latter can be gained via irritation tests (REACH No. 8.1. or 8.2).
According to REACH Annex IX column 2 (8.6.2), testing by the inhalation route is appropriate if exposure of humans via inhalation is likely taking into account the vapour pressure of the substance and/or the possibility of exposure to aerosols, particles or droplets of an inhalable size.
The substance melts at 299°C, the boiling point could not be determined with the most sensitive method, i.e. vapour pressure measurement by effusion method, weight loss, according to OECD Guideline for the Testing of Chemicals 104. It could not be determined because the vapour pressure is too low. The vapour pressure itself was determined as very low, i.e. 1 * 10exp(-7) hPa at 25°C (calculated using the Modified Watson Correlation, OECD 104, GLP). So, the exposure to 1,3-dimethyl-4-aminouracil via inhalation of vapour can be disregarded as no vapour will be formed during handling. Also, the substance is a solid, so the formation of inhalable droplets or aerosols can furthermore be excluded.
The particles of 1,3-Dimethyl-4-aminouracil form various size agglomerates. The measurement of the single particles was not possible. The amount of test item with a particle size below 250 µm was only about 2 %. This amount is too small and not relevant for the particle size distribution. The main agglomerate size was found in the range of 250 µm to 2000 µm. 50 % of the test item agglomerates exceeded 500 µm. 0.00% were < 75µm. According to ECHA’s guidance on data requirements Chapter R.7c, In humans, particles with aerodynamic diameters below 100 μm have the potential to be inhaled. Particles with aerodynamic diameters below 50 μm may reach the thoracic region and those below 15 μm the alveolar region of the respiratory tract. As obvious, only less than 2% of the particles in general have the potential to be inhaled, and none of the particles have the potential to reach the part of the lung in which no ciliary clearance occurs. Also, direct dust exposure is excluded during handling, hence not fulfilling the above-mentioned criteria for the necessity of testing via the inhalative route.
In consequence, the available OECD 408 study (oral exposure route) is sufficient to cover this endpoint, no repeated dose testing via inhalation route needs to be performed and can consequently be waived due to animal welfare.

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

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: dermal

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Justification for type of information:

JUSTIFICATION FOR DATA WAIVING
According to REACH Annex IX column 1 (8.6.2), the following study for repeated dose toxicity is required: Sub-chronic toxicity study (90-day), one species, rodent, male and female, most appropriate route of administration, having regard to the likely route of human exposure.
There is a suitable Klimisch 1 GLP OECD 408 guideline study available, assessing the toxicological properties of 1,3-Dimethyl-4-aminouracil after oral application in feed over 90 days. In general, the oral route is the most suitable one to assess systemic effects in humans, which is the main aim of this endpoint. The dermal or inhalative route is only scientifically relevant in case of considerable exposure, any route-specific toxicological mode of action or local effects, whereas sufficient information on the latter can be gained via irritation tests (REACH No. 8.1. or 8.2).
According to REACH Annex IX column 2 (8.6.2), the appropriate route shall be chosen on the following basis: Testing by the dermal route is appropriate if:
(1) skin contact in production and/or use is likely; and
(2) the physicochemical properties suggest a significant rate of absorption through the skin; and
(3) one of the following conditions is met:
— toxicity is observed in the acute dermal toxicity test at lower doses than in the oral toxicity test, or
— systemic effects or other evidence of absorption is observed in skin and/or eye irritation studies, or
— in vitro tests indicate significant dermal absorption, or
— significant dermal toxicity or dermal penetration is recognised for structurally-related substances.
Although inhalation of 1,3-Dimethyl-4-aminouracil to any toxicologically relevant amount is unlikely, the latter conditions do not apply:
(1) Due to appropriate precautionary measures, skin contact in production and/or use is minimized.
(2) The physicochemical and toxicological properties do not suggest potential for a significant rate of absorption through the skin. Skin absorption is influenced by several factors, i.a.:
- Molecular weight: Less than 100 favors dermal uptake. Above 500 the molecule may be too large. With a molecular weight of 155.15 g/mol, absorption is in theory possible.
- LogPow: In order to cross the skin, a compound must first penetrate into the stratum corneum and may subsequently reach the epidermis, the dermis and the vascular network. The stratum corneum provides its greatest barrier function against hydrophilic compounds, whereas the epidermis is most resistant to penetration by highly lipophilic compounds. logPow values between 1 and 4 hence favour dermal absorption. 1,3-Dimethyl-4-aminouracil has a low logPow, i.e. -0.4 at 20°C and pH 6.2-6.9 (flask method, OECD 107). Hence, it is unlikely that the substance could pass the stratum corneum, and dermal absorption is unlikely to occur.
- Water solubility: with a water solubility of 5.46 g/L at 20°C the substance is slightly lipophilic, and is hence presumably slightly hindered to pass the stratum corneum.
- Skin irritation / corrosion: If the substance is a skin irritant or corrosive, damage to the skin surface may enhance penetration. 1,3-Dimethyl-4-aminouracil is not classified as irritant to the skin. Further, the substance does not need to be classified as irritating to the eye, which is in general considered to be more sensitive than the skin. Therefore, no additional penetration enhancement must be considered.
So summarizing, absorption cannot be excluded, but a significant rate of absorption is not expected.
(3) None of the following conditions are met:
- Both acute oral toxicity studies were performed as limit test (5000 mg/kg for oral, 2000 mg/kg for dermal testing), and no deaths occurred, no abnormalities were noted. Hence, toxicity in the acute dermal toxicity test at lower doses than in the oral toxicity test can neither be confirmed nor excluded as no toxicity was observed at dose levels relevant for classification.
- no systemic effects or other evidence of absorption were observed in skin the available eye irritation studies
- no data in in vitro absorption tests or structurally related substances is available
In consequence, the available OECD 408 study (oral exposure route) is sufficient to cover this endpoint, no repeated dose testing via dermal route needs to be performed and can consequently be waived due to animal welfare.

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Mode of Action Analysis / Human Relevance Framework

A mode of action analysis in its classic sense cannot be performed for repeated dose toxicity as there is only one repeated dose study available in one species. However, the rat is an established model for human risk assessment, interspecies differences are well studied and allow transfer conclusions to humans for most chemicals exhibiting certain effects in the rat. Further, subchronic exposure is a sufficient time span to detect most systemic effects in rats; a carcinogenic MoAA which requires a longer duration can be reasonably excluded as the substance is not a directly acting mutagen, and in the 90 study there were no effects observed which may indicate tumour promoting effects.

In the only available OECD 408 study, 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).

After a 28-day recovery period, most of the effects had reversed completely or partially.

It is hence considered that the observed effects are only minor and are most likely mainly due to an adaptive response as well as systemically connected. So, there is no indication given that the substance may potentially be a specific organ toxicant.

The noted effects are as such plausible taking into account the connection of the affected organ systems. There is no indication given that the observed effects are not in general relevant for humans, no indication of a species-specific response was given. However, the observed effects are mainly adaptive and occur at rather high doses. It is evident that the substances exhibits here a threshold mode of action, so very like a sufficiently high exposure of humans is not given to lead to the observed effects at all.

Justification for classification or non-classification

There is only one OECD 408 oral repeated dose study available adressing this endpoint, which serves so as the basis for assessing the necessity for classification.

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.