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Administrative data

Description of key information

Repeat dose studies by the oral route have been conducted ( 59 day, 13 week (according to OECD guideline 408) and 104 weeks (according to EU Method B.33)  on cyanuric acid or sodium cyanurate monohydrate.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Deviations:
yes
Remarks:
histopathology limited to kidneys and urinary tract
Principles of method if other than guideline:
The 59 day study was designed to evaluate the toxicity of s-triazinetriol monosodium salt (cyanuric acid, monosodium salt) to rats and to establish dosage levels to be used in a 13 week toxicity study.
GLP compliance:
yes
Species:
rat
Strain:
other: Charles River CD® rats
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc., Portage, Michigan, USA
- Age at study initiation: ~ 3 weeks
- Weight at study initiation: Males: 84 – 100 g, Females: 84 – 101 g
- Housing: wire-mesh cages
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 9 days


ENVIRONMENTAL CONDITIONS
- Temperature (°F): 68-70
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 12/12


Route of administration:
oral: drinking water
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: A suitable amount of the test compound was dissolved in tap water, on a stir plate to yield test solutions. The pH of the test solutions was adjusted to 7.2-7.6 (using sodium hydroxide and glacial acetic acid) and the solutions aged for 4 days in vented containers at room temperature. Before the test solutions were offered to the animals, the pH was readjusted to 7.2 - 7.6, if necessary. Fresh test solutions were prepared twice a week, 4 days prior to administration.


Duration of treatment / exposure:
28 days extended to 59 days
Frequency of treatment:
Continuous
Dose / conc.:
0 ppm
Dose / conc.:
400 ppm
Remarks:
males - 49 mg/kg bw/day; females - 65 mg/kg bw/day
Dose / conc.:
1 200 ppm
Remarks:
males - 141 mg/kg bw/day; females - 264 mg/kg bw/day
Dose / conc.:
2 000 ppm
Remarks:
males - 260 mg/kg bw/day; females - 370 mg/kg bw/day
Dose / conc.:
4 000 ppm
Remarks:
males - 521 mg/kg bw/day; females - 717 mg/kg bw/day
No. of animals per sex per dose:
5
Control animals:
yes, concurrent no treatment
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily/7 days a week


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly


BODY WEIGHT: Yes
- Time schedule for examinations: Twice weekly


FOOD CONSUMPTION: Yes
- Time schedule for examinations: Weekly



WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Three times a week


OPHTHALMOSCOPIC EXAMINATION: No


HAEMATOLOGY: Yes
- Time schedule for collection of blood: end of study (week 9)
- How many animals: all animals
- Parameters checked: haematocrit, haemoglobin concentration, erythrocyte count, total and differential leukocyte count, platelet count, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration and reticulocyte count.


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: end of study (week 9)
- How many animals: all animals
- Parameters checked: glucose, cholesterol, blood urea nitrogen, total bilirubin, creatinine, total protein, albumin, alkaline phosphatase, serum glutamic oxaloacetate transamine, serum glutamic pyruvic transaminase, calcium, lactic dehydrogenase, phosphorous, sodium, potassium, chloride, globulin, (calculated) and osmolarity


URINALYSIS: Yes
- Time schedule for collection of urine: end of study (week 9)
- Metabolism cages used for collection of urine: Yes
- Parameters checked: colour and appearance, volume, osmolality, specific gravity, pH, protein, glucose, occult blood ketones, bilirubin, nitrite, urobilinogen, microscopic examination of sediment, sodium, potassium, chloride, phosphorous, calcium, creatinine, urea nitrogen


Sacrifice and pathology:
GROSS PATHOLOGY: : All gross lesions, adrenals, eye, trachea, duodenum, jejunum, ileum, cecum, colon, ureters, brain, spinal cord, pituitary, thyroid, parathyroid, thymus, oesophagus, submaxilliary salivary glands, stomach, small and large intestines, liver, pancreas, kidneys, spleen, heart, lungs with bronchi, aorta, gonads, uterus, prostate, urinary bladder, gall bladder (mouse), mesenteric lymph node, peripheral nerve, bone marrow, skin, mesenteric lymph node, sternum, skeletal thigh muscle, peripheral nerve

HISTOPATHOLOGY: Microscopic examination of hematoxylin and eosin stained sections of ureters, urinary bladders and Buoin’s fluid fixed kidneys for all animals in the control group, s-triazinetriol monosodium salt treated groups. Histopathology was not performed on terminal sacrifice animals treated with the chlorinated compounds.
Statistics:
All statistical analyses compared the treatment groups of each compound with the control group, by sex.
Body weights (weeks 4 and 8), water consumption (weeks 4 and 8), haematological, biochemical and urinalysis parameters (day 59) and absolute and relative organ weights (terminal sacrifice) were compared by ANOVA. Dunnett’s multiple comparison tables were used to judge significance of difference.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Dose descriptor:
LOAEL
Effect level:
> 521 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: No evidence of toxicity reported
Dose descriptor:
LOAEL
Effect level:
717 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: No evidence of toxicity reported
Dose descriptor:
NOAEL
Effect level:
521 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: No evidence of toxicity reported
Dose descriptor:
NOAEL
Effect level:
717 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: No evidence of toxicity reported
Critical effects observed:
not specified

Haematology:

All values were within the normal range.  Occasional values were statistically significant, although the absolute differences were small. Albumin, globulin and serum protein values were slightly elevated in high dose males which may be suggestive of hemoconcentration, although water consumption and urine volume were normal.

Clinical chemistry:

All values were within the normal range. Occasional values were statistically significant, although the absolute differences were small.  Glucose values for males and females at the 4000 ppm dosage level were significantly greater than the control values, although the difference between the high dose and control values was not.  All values were within the normal range for this laboratory

Urinlaysis:

Urine values were within the normal range, except for urea nitrogen which was significantly decreased in female rats at 1200 ppm and in both male and female rats at 4000 and 8000 ppm, by around 50%.

Organ weights:

No statistically significant organ weight variations of livers and kidneys were noted

Gross and histopathology:

No gross or microscopic lesions of treatment-related significance were noted

Conclusions:
LOAEL (CYA) >521 mg/kg bw/d (males); >717 mg/kg bw/d (females)
NOAEL (CYA) 521 mg/kg bw/d (males); 717 mg/kg bw/d (females)
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Charles River CD
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Portage, Michigan, USA
- Age at study initiation: Males ~3weeks; Females ~3 weeks
- Weight at study initiation: Males 82 – 106 g; Females 78 – 96 g
- Fasting period before study:
- Housing: Suspended wire mesh cages
- Diet: Provided ad libitum
- Water: Provided ad libitum



ENVIRONMENTAL CONDITIONS
- Photoperiod (hrs dark / hrs light): 12h/12h


Route of administration:
oral: drinking water
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Appropriate amounts of the test article and tap water were added together to obtain the proper dosage levels. Sodium hippurate and tap water were similarly mixed. The pH of the control, treated and sodium control solutions were adjusted to pH 7.4 (using glacial acetic acid) prior to offering the solutions to the rats. fresh solutions were preapred twice weekly, 4 days prior to adminsitration and stored under ambient conditions

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Duplicate (100 ml) samples from the control, treated and sodium control groups were collected on days 3 and 6 of study weeks 1-13.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
continuous
Dose / conc.:
0 ppm
Dose / conc.:
896 ppm
Remarks:
males - 109 mg/kg bw/day; females - 137 mg/kg bw/day based on total s-triazinetriol content
Dose / conc.:
1 792 ppm
Remarks:
males - 231 mg/kg bw/day; females - 276 mg/kg bw/day based on total s-triazinetriol content
Dose / conc.:
5 375 ppm
Remarks:
males - 711 mg/kg bw/day; females - 914 mg/kg bw/day based on total s-triazinetriol content
No. of animals per sex per dose:
24
Control animals:
other: Vehicle control, sodium control (7812 ppm sodium hippurate)
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily (detailed observations were made every week)



DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly


BODY WEIGHT: Yes
- Time schedule for examinations: weekly



FOOD EFFICIENCY: Yes



WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: daily


OPHTHALMOSCOPIC EXAMINATION: No



HAEMATOLOGY: Yes
- Time schedule for collection of blood: 2, 4, 6, 8 and 10 weeks of study
- Anaesthetic used for blood collection: Yes (identity) / No / No data
- How many animals: 10 male and 10 female
- Parameters checked: haematocrit, haemoglobin concentration, erythrocyte count, total leukocyte count, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, reticulocyte count, differential leucocyte count and platelet count


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: 2, 4, 6, 8 and 10 weeks
- How many animals: 10 male and 10 female
- Parameters checked: sodium, potassium, chloride, serum glutamic oxaloacetic transaminase (SGOT), serum glutamic pyruvic transaminase (SGPT), lactic dehydrogenase (inadvertently not conducted for the interim sacrifice rats at study week 2) glucose, albumin, blood urea nitrogen, total bilirubin, alkaline phosphatase, total protein, calcium, phosphorous, cholesterol, creatinine, globulin, osmolality.


URINALYSIS: Yes
- Time schedule for collection of urine:2, 4, 6, 8 and 10 weeks
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked: colour and appearance, volume, osmolality, microscopic examination of sediment, sodium, potassium, chloride, calcium, phosphorous.Dipstick tests: protein, glucose, occult blood, nitrites, urobilinogen, ketones, bilirubin, urea nitrogen, creatinine and pH


NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:
Gross pathology: After 2, 4, 6, 8 and 10 weeks 4 rats per sex from control and high dose groups were necropsied. All rats surviving to 13 weeks were also necropsied. Rats found dead during the study were processed in the same way.
The external surface, all orifices, cranial cavity, the external and cut surfaces of the brain and spinal cord, thoracic, abdominal and pelvic cavities and viscera of the cervical tissue and organs and the entire urinary system. The lungs were inflated with formalin via he trachea
Kidneys were sectioned longitudinally. One half of each kidney and both eyes were stored in ethanol after fixing and representative sections of the following tissues were collected and preserved in phosphate-buffered neutral 10% formalin:

Histopathology: hematoxylin and eosin stained paraffin sections of the kidneys, ureters and urinary bladder were conducted in the control, sodiumcontrol, and high-dose rats sacrificed at the 2, 4, 6, 8 and 10 week interim sacrifices and rats found dead during the course of the study. Sections were prepared and examined from the following tissues of both control groups and the high dose group rats sacrificed at the end of the study:
All gross lesions, both adrenals, eye, trachea, oesophagus, stomach, duodenum, jejunum, ileum, cecum, colon, liver (2 sections), spleen, urinary bladder, testes/ovaries, pancreas, brain (forebrain, midbrain and hindbrain), heart, lungs and mainstem bronchi, pituitary, thyroid and parathyroid, thymus, mesenteric lymph node, sternum bone marrow, spinal cord, submaxillary salivary gland, skeletal thigh muscle, both kidneys, prostate / corpus and cervix uteri, peripheral sciatic nerve and ureters.
Sections were prepared and examined from the heart, liver, urinary bladder, kidneys, ureters and all gross lesions of terminal sacrifice in both mid dose groups.
Statistics:
All statistical analyses compared the treatment groups with the control group by sex. Body weights (week 13), haematological, biochemical, and urinalysis parameters (weeks 6 and 13 of main study, and 2, 4, 6, 8, 10 week interim sacrifices) and absolute and relative organ weights (weeks 2, 4, 6, 8 and 10 week terminal sacrifices) were compared by analysis of variance (one –way classification), Bartlett’s test for homogeneity of variance and the appropriate T-test (for equal or unequal variances) as described by Steel and Torrie using Dunnett’s multiple comparison tables to judge significance of difference.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No signs considered compound related were noted for rats that received s-triazinetriol, monosodium salt at dosages of 896, 1792 and 5373 pm. Infrequent signs for treated rats included swelling of the head and neck regions, scabbed areas of the shoulder and neck and a white material protruding from the nasal corner of the eye (probably due to blood collection). No trend in mortality suggestive of compound related effects occurred.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One female rat from the each of the control groups died following blood collection
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
No changes considered compound related occurred. There were statistically significant increases in mean body weights of doubtful biological significant for the 896 ppm females and 1792 ppm males at 13 weeks.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
effects observed, non-treatment-related
Description (incidence and severity):
No changes considered compound related occurred. Occasional increases in food consumption somewhat paralleled those increases in group mean body weights
Water consumption and compound intake (if drinking water study):
effects observed, non-treatment-related
Description (incidence and severity):
No trends in group mean water consumption values were considered compound related, although mean water increases were noted for the 896 and 1792 ppm females and were of doubtful biological significance.
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
In the interim sacrifices there were scattered statistically significant differences between the non-treated control, sodium hippurate control and test compound treated mean values. However, these differences were well within the normal range, showing no discernable trends and were not considered test compound related.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Total protein and globulin levels appeared to decrease with increasing sodium intake. There was no significant difference between total protein and globulin levels for 5375 ppm treated males and sodium control males and sodium control males; however there was a significant difference between 5375 ppm treated females and sodium control females at 6 weeks, but not 13 weeks. The contribution of the test compound to protein alterations was equivocal.
Calcium levels decreased with sodium intake in time for both males and females. No consistent changes were observed at 6 weeks; but, at 13 weeks there were statistically significant dose related differences between the compound treated test groups and the untreated controls, but not between the 5375 ppm group and the sodium control group. Decreased calcium did not appear to be test compound related.
Chloride levels in males of all test groups and in females in the 5375 ppm showed small but statistically significant decreases which appeared to be compound related.
Occasional statistically significant alterations were seen randomly during the test, but did not appear compound related. All changes were of a minor degree and were within acceptable normal limits and therefore the biological significance is equivocal.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
No compound related effects were noted, although scattered statistically significant alterations were identified with numerous tests.
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Statistically significant increases in relative kidney weights of males in the 5375 ppm group occurred at the 4 and 10 week interim sacrifices. Statistically significant variation occurred in some absolute and /or relative weights of liver, kidney, testes, heart and brain at several dose levels and in either sex. There were no dose relationships or statistically significant variations in females of the 5375 ppm dose group when compared with either control group. There was a statistically significant decrease in relative weight of testes and heart for the males of the 5375 group when compared to the sodium control group, which was not dose related to the compound.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Non-specific changes unrelated to the compound were observed.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Very slight to slight hyperplasia of the urinary bladder epithelium occurred in some males at the 5375 ppm dosage and in 1 male at 1792 ppm at terminal sacrifice, this was not found in females. Hyperplasia of the urinary bladder was also found at the 5375 ppm dosage level in 1of 4 males at both the 6 and 8 week interim sacrifices and 2 of 4 at the 10 week sacrifice. 1 of 4 females had hyperplasia of the bladder epithelium in the sodium control group at the 8 week sacrifice. Therefore, hyperplasia of the urinary bladder epithelium was compound related in males, but not females.
Dose descriptor:
NOAEL
Effect level:
109 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
histopathology: non-neoplastic
Dose descriptor:
NOAEL
Effect level:
914 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: No treatment related changes in females
Dose descriptor:
LOAEL
Effect level:
231 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
histopathology: non-neoplastic
Dose descriptor:
LOAEL
Effect level:
> 914 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: No treatment related changes in females
Critical effects observed:
not specified

No clinical signs considered compound related through 13 weeks of the study. Corenal opacity was noted frequently for control and treated rats. No changes considered compound related occurred in survival, body weights, food or water consumption throughout the study.

Although there were statsitically significant changes in some biochemical values, these were of a random nature and therefore were of equivocal biological significance and not considered related to the compound.

There were no macroscopic changes which could be attributed to the compound. There were no compound related variations in organ weights. Histologically, hyperplasia of the urinary bladder epithelium resulted from compound treatment in males but not the females. This lesion was present at the 6, 8 and 10 week interim sacrifice, and at terminal sacrifice. Based on the incidence pattern found in terminal sacrifice and interim sacrifice rats, it is concluded that hyperplasia of the urinary bladder epithelium was compound related in the male but not the female rats in this study.

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Principles of method if other than guideline:
The test material was evaluated for subchronic toxicity when administered in the drinking water to mice for 13 weeks for the purposes of setting dose levels for an oncogenicity study.
GLP compliance:
yes
Species:
mouse
Strain:
B6C3F1
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Portage, Michigan, USA
- Age at study initiation: Six weeks old
- Weight at study initiation: Males: 11.7-22.1 g, Female: 13.1-18.5 g
- Diet: Provided ad libitum
- Water: Provided ad libitum
- Acclimation period: 2 weeks


ENVIRONMENTAL CONDITIONS
- Temperature (°F): 63-81
- Humidity (%): 38-69
- Photoperiod (hrs dark / hrs light): 12/12


Route of administration:
oral: drinking water
Vehicle:
other: Tap water, adjusted to pH 7.4
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The bulk compound was ground and screened to form a fine powder. An appropriate amount of monosodium cyanuarate needed to prepare the high dose level was of 5375 ppm was weighed and added to a 2.5 gallon glass carboy, prefilled with tap water. The solution was mixed until the compound was completely dissolved (~ 2 hours) with a Lightnin stirrer. The solution was adjusted to pH 7.4 with glacial acetic acid. Solutions for the lower dose groups were prepared as described for the high dose solutions. Fresh solutions were prepared twice weekly.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of each dosing solution were taken at weeks 1a, 1b, 2a, 2b, 3a, 3b, 4a, 4b, 6a, 6b, 9a, 9b, 12a and 12b to determine the stability of the test solution (a) = three day portion of week, (b) = four day portion of week.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
ad libitum/ continuous
Dose / conc.:
0 ppm
Dose / conc.:
896 ppm
Remarks:
males - 252 mg/kg bw/d; females - 298 mg/kg bw/d; based on cyanurate content
Dose / conc.:
1 792 ppm
Remarks:
males - 522 mg/kg bw/d; females - 610 mg/kg bw/d; based on cyanurate content
Dose / conc.:
5 375 ppm
Remarks:
males - 1994 mg/kg bw/d; females - 2200 mg/kg bw/d; based on cyanurate content
No. of animals per sex per dose:
25
Control animals:
other: Sodium intake control - sodium salt of hippuric acid at a level of 7796 ppm and a tap water control l
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: 3 times daily for mortality and moribundity



DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly


BODY WEIGHT: Yes
- Time schedule for examinations: Weekly



FOOD EFFICIENCY: Yes


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: - twice weekly then summed to yield a weekly value.


OPHTHALMOSCOPIC EXAMINATION: No


HAEMATOLOGY: Yes
- Time schedule for collection of blood: End of study
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes
- How many animals: 10 mice/sex/group;
- Parameters checked: Hematocrit, hemoglobin, erythrocyte count, leukocyte count, leukocyte differential count, platelet count, reticulocyte count.



CLINICAL CHEMISTRY: No


URINALYSIS: Yes
- Time schedule for collection of urine: End of study
- Metabolism cages used for collection of urine: No
- Animals fasted: Yes
- Parameters checked: First priority: volume, colour and appearance, pH, osmolarity, glucose, proteins, ketones, bilirubin, microscopic examination of sediments (particular vigilance for crystals). Second priority: cyanuric acid concentration. Third priority: chloride, inorganic phosphate, calcium, urea nitrogen, potassium, sodium, urobilinogen . Fourth priority: zinc, copper, magnesium. Due to the small volume of urine produced the parameters were prioritized



NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:
ORGAN WEIGHTS: After six weeks of treatment, 5 animals/sex/group were chosen at random. After 13 weeks of treatment, all remaining animals were sacrificed. From the animals terminated at 6 weeks, the kidneys were excised and weighed. From all terminally sacrificed animals the following were weighed:
Brain (including brainstem), heart, liver, kidneys, testes with epididymides and ovaries (weight obtained after fixation). Relative organ weights were calculated for all organs weighed.


GROSS AND HISTOPATHOLOGY: Of the control and the high two dose group animals (groups 1, 4 and 5, respectively) sacrificed after six weeks of treatment, specimens of kidney, urinary bladder, ureter and any gross lesions were subjected to microscopic examination. From the animals in groups 1, 4 and 5 sacrificed after thirteen weeks of treatment, specimens of kidneys, urinary bladder, ureter, liver, heart, brain, testes or ovaries, lung and any gross lesions were examined. The kidneys, urinary bladder, ureter and any gross lesions were obtained from the terminally sacrificed low and middle dose animals. These specimens were prepared for examination by embedding in paraplast, sectioning and staining with hematoxylin and eosin.
The following tissues were preserved in 10% neutral buffered formalin:
all gross lesions (to include apparently normal contiguous tissue) brain (at least three levels from fore-, mid-, and hindbrain), pituitary, salivary gland, heart, thymus, thyroid (with parathyroid), lung, trachaea, esophagus, stomach, duodenum, jejunum, ileum, colon, cecum, adrenals, pancreas, gall bladder, liver (at least two lobes), kidneys, urinary bladder, ureter, testes with epididymides, prostate, ovaries, corpus and cervix uteri, spleen, lymph nodes, skin, sciatic nerve, mammary gland, Zymbal’s gland, bone (including marrow), skeletal muscle, spinal cord, three coronal sections through the head to include nasal cavity, paranasal sinuses, tongue, oral cavity, nasopharynx, and middle ear, eyes and carcass and remaining viscera. Specimens of kidneys, urinary bladder, ureter, and any gross lesions were subjected to microscopic examinations
Statistics:
The growth rates, total food consumption, water consumption, clinical pathology, some urine data and organ weight data of the control group were compared statistically to the data of the treated groups of the same sex by Bartlett’s test for homogeneity of variance (Bartlett, 1937). This analysis was followed by a one-way classification analysis of variance (ANOVA) if the variances proved to be homogeneous. If the variances proved to be heterogeneous, a log10 transformation was performed, followed by Bartlett’s test. If the log10 transformation was ineffective in removing variance heterogeneity, a loge transformation of the original data was performed followed by a Bartlett’s test. If homogeneity could not be achieved by transformation, ANOVA of the non-transformed data was completed. If ANOVA of homogenous data was significant, Scheffe’s (1953) multiple pairwise comparison procedure was used to compare the group mean values. If ANOVA of heterogeneous data was significant, Games' and Howell’s (1976) multiple pairwise comparison procedure was used to compare the group mean values. All analyses were evaluated at the 5.0% probability (one-tailed) level
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Administration of monosodium cyanurate at a level of 5375 ppm in the drinking water for 6 or 13 weeks failed to cause compound related histomorphologic alterations in the tissues examined, with the exception of changes in the urinary bladder resulting from the presence of calculi in two male mice in the high-dose monosodium cyanurate group
Details on results:
CLINICAL SIGNS AND MORTALITY
No unusual clinical signs were noted. Alopecia was noted in all groups at approximately equal frequencies.
No treatment related mortality occurred during the course of this study

BODY WEIGHT AND WEIGHT GAIN
Analysis of growth through weeks six and thirteen did not reveal any significant difference. No apparent trends were noted in the weekly mean. Means of the treated groups of either sex differed by one gram or less from the respective control group.

FOOD EFFICIENCY
Analysis of the total food consumption through weeks six and thirteen did not reveal any significant differences; weekly mean values of the treated groups were generally comparable to control values.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study)
Consistently higher water consumption than the control values was noted in 5375ppm animals of either sex

HAEMATOLOGY
No apparent differences were noted.

URINALYSIS
Statistical evaluation revealed incidental decreases in mean chloride levels in the 896ppm males and the urine volume of the 7769ppm (monosodium salt of hippuric acid control) males and an increase in urobilinogen in the 5375ppm females. No other apparent differences were noted.

ORGAN WEIGHTS
No significant differences were noted in the week six kidney weight data.
Dose related increases in absolute and relative ovarian weights were noted in the week thirteen data. Statistical evaluation revealed this difference to be significant for the 5375 and 7769 ppm (monosodium salt of hippuric acid control) animals. Since this finding also occurred in the sodium control, the increased sodium intake may be responsible for this effect.

GROSS PATHOLOGY
Administration of monosodium cyanurate at a level of 5375 ppm in the drinking water for 6 or 13 weeks failed to cause compound related histomorphologic alterations in the tissues examined, with the exception of changes in the urinary bladder resulting from the presence of calculi in two male mice in the high-dose monosodium cyanurate group
Dose descriptor:
NOAEL
Effect level:
522 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
histopathology: non-neoplastic
Dose descriptor:
NOAEL
Effect level:
2 200 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: No treatment related changes
Dose descriptor:
LOAEL
Effect level:
1 994 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
histopathology: non-neoplastic
Critical effects observed:
not specified
Conclusions:
Administration of monosodium cyanurate at a level of 5375 ppm (equivalent to cyanuric acid concentrations of 1523 mg/kg bw/day (males) and 1582 mg/kg bw/day (females) in the drinking water for thirteen weeks failed to cause compound-related changes
Endpoint:
chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
equivalent or similar to guideline
Guideline:
EU Method B.33 (Combined Chronic Toxicity / Carcinogenicity Test)
GLP compliance:
yes
Species:
rat
Strain:
other: Charles River CD
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc. Portage, Michigan, USA
- Age at study initiation: ~5 weeks old
- Weight at study initiation: males: 87.0-164.0 g; females 72.0 – 141.0 g
- Housing: Suspended wire mesh cages
- Diet: Provided ad libitum
- Water: Provided ad libitum



ENVIRONMENTAL CONDITIONS
- Temperature (°F): 71
- Humidity (%): 59-61
- Photoperiod (hrs dark / hrs light): 12/12


Route of administration:
oral: drinking water
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The appropriate amount of test material was dissolved in tap water and mixed using a motor driven propeller. The pH of the testing solutions was adjusted to pH 7.4 using glacial acetic acid and/or sodium hydroxide. Fresh drinking water solutions were prepared 3 times weekly.


Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of drinking water from all groups were analysed on days 1, 3, 5 and of weeks 1 through 15 and on day 1 of weeks 16 through 106
Duration of treatment / exposure:
104 weeks
Frequency of treatment:
ad libitum / continuous
Dose / conc.:
0 ppm
Dose / conc.:
400 ppm
Remarks:
males - 25 mg/kg bw/d; females - 42 mg/kg bw/d
Dose / conc.:
1 200 ppm
Remarks:
males - 76 mg/kg bw/d; females - 129 mg/kg bw/d
Dose / conc.:
2 400 ppm
Remarks:
males - 154 mg/kg bw/d; females - 266 mg/kg bw/d
Dose / conc.:
5 375 ppm
Remarks:
males - 371 mg/kg bw/d; females - 634 mg/kg bw/d
No. of animals per sex per dose:
Low dose group: 80/sex
Mid dose, mid-high dose and high dose groups: 100/sex/dose level
Tap water control: 100/sex
Sodium control: 80/sex
Control animals:
other: Vehicle (tap water), Sodium control (sodium hippurate)
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly


BODY WEIGHT: Yes
- Time schedule for examinations: Recorded weekly during the pretest and first 14 weeks of the study, every 2 weeks thereafter


FOOD CONSUMPTION: Yes
- Time schedule for examinations: recorded weekly for the first 14 weeks of the study, every 2 weeks thereafter

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Weekly


OPHTHALMOSCOPIC EXAMINATION: No


HAEMATOLOGY: Yes
- Time schedule for collection of blood: 6, 12, 18 and 24 months
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes
- How many animals: 10 rats/sex
- Parameters checked: Total and differential leukocyte count, erythrocyte count, haemoglobin, haematocrit, platelet count, reticulocyte count, haematological indices mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin concentration (MCH).


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: 6, 12, 18 and 24 months
- Animals fasted: Yes
- How many animals: 10 rats/sex
- Parameters checked: Sodium, potassium, chloride, calcium, phosphorous, osmolality, alkaline phosphatase, total bilirubin, aspartate aminotransferase, alanine aminotransferase, lactic dehydrogenase, blood urea nitrogen, creatinine, total protein, albumin, globulin (calculated), cholesterol and glucose.


URINALYSIS: Yes
- Time schedule for collection of urine: 6, 12, 18 and 24 months
- Metabolism cages used for collection of urine: No
- Animals fasted: Yes
- Parameters checked: Colour and appearance, microscopic examination of sediment, volume, osmolality, pH, protein, glucose, occult blood, nitrites, bilirubin, ketones, urobilinogen, sodium, potassium, chloride, magnesium, phosphorous, calcium, urea nitrogen, creatinine


NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes all animals received a complete post mortem.
Organs: external examination; contents of the abdominal, thoracic and cranial cavities were examined both in situ and after removal and dissection. Adrenal, all gross lesions, all tissue masses, brain (fore-, mid-, and hind-), cecum, colon, duodenum, esophagus, eye and contiguous Harderian gland, heart with coronary vessels, ileum, jejunum, kidneys, liver (3 sections) lung and mainstream bronchi, lymph nodes (medistinal, mesenteric and regional), mammary gland, ovaries, pancreas, peripheral nerve (sciatic), pituitary, prostate / corpus and cervix uteri, mandibular salivary gland, skeletal thigh muscle, skin, spinal cord (cervical and thoracic), spleen, sternum bone marrow, stomach, testis with epididymus, thymus, thyroid and parathyroid, tongue, trachea, ureter, urinary bladder, whole head (including nasal, oral and otic tissue) and Zymbal’s gland.

HISTOPATHOLOGY: Yes a full tissue compliment was prepared for all animals in control and high dosage groups. Sections of the following organs from animals in the 400 ppm, 1200 ppm, 2400 ppm groups sacrificed at 6, 12and 18 months and all animals which died or were sacrificed in extremis between 0 and 24 months of study: adrenal, heart, kidneys, liver, ovaries, spleen, testes, ureter, urinary bladder, tissue masses and gross lesions. Gross lesions and tissue masses were microscopically examined for animals in the 400 ppm, 1200 ppm, 2400 ppm dose groups which survived to terminal sacrifice
Statistics:
Body weights and food consumption (weekly for weeks 1 – 4, quarterly thereafter) organ weights, (absolute and relative to body weights, interim and terminal sacrifices) clinical laboratory values (6, 12, 18 and 24 months prior to interim and terminal sacrifices) were analysed by Bartlett’s test for homogeneity of variance and analysis of variance (one-way classification). Treatment groups were compared to the control group and to the sodium control group by sex, using the appropriate t-statistic (equal or unequal variance) as described by Steel and Torrie and Ostle. Dunnett’s multiple comparison tables were used to determine significance. All statistical tests were two-tailed. Survival data and data on time to neoplastic lesion were analysed using the computer program of Thomas, Breslow and Gart. Statistical procedures included in this program are the Kaplan-Meier and standard methods for computing survival curves, Cox’s test for linear trend in proportions and both Cox’s test and Gehan-Breslow’s generalized Kruskall-Wallis test for comparing survival distributions. Data on time to neoplastic lesion were analysed for all benign tumors, all malignant tumors, all tumors combined and for individual type that appeared in two or more animals in the high dose group
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Red urine was observed for 5375 ppm group males (week 1 – 62) and appeared to be test article related and correlated with the high incidence of urinary bladder calculi. Decreased defecation and / or no faeces occurred more frequently for 5375 ppm group males (week 1 – 62) and a slightly higher incidence of yellow material (anogenital region, ventral surface, ventral abdomen, hindlimb) for 2400 and 5375 ppm female groups (weeks 14 – 39) and for male rats in the 5375 ppm group (weeks 1 – 52). In weeks 14 – 52 some male rats in the 5375 ppm group were noted to feel cold to the touch.
Mortality:
mortality observed, treatment-related
Description (incidence):
Survival was a little lower for high dose males (5375 ppm) as compared to control animals in weeks 26 – 104. Females given 1200 ppm and 2400 ppm had a lower survival rate at 104 weeks compared to control animals
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Male control and treatment withdrawal rats were similar in weeks 63 – 104. The body weight of the withdrawal rats was also similar to their respective male groups not placed on compound withdrawal. Group mean body weights of female rats receiving 2400 and 5375 ppm after withdrawal were higher compared to group mean body weights of female control withdrawal animals and non withdrawal females receiving 2400 and 5375 ppm
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
Water consumption of male and female treatment groups and the sodium control group was higher than those of the control group. The increased water consumption appeared dose related. No definite trend in water consumption of the compound withdrawal group was evident. Average water consumption (weeks 63 – 104) of male and female 2400 ppm and 5375 ppm withdrawal groups was higher than those of male and female control groups during the recovery period. Male and female 5375 ppm withdrawal groups showed decreased average water consumption as compared to non withdrawal male and female 5375 ppm groups in weeks 63 – 104
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Group mean food consumption values for both males and females were similar in all groups including the untreated controls. Overall. the with drawal animals in all groups consumed more food (g/rat/day) during the recovery period than the animals in the main study.

HAEMATOLOGY
No significant treatment related effects were observed.

CLINICAL CHEMISTRY
No results of pathologic significance were observed

URINALYSIS
No significant treatment related effects were observed

ORGAN WEIGHTS
There were no test article related organ weight variations in the treatment groups at any of the interim or terminal sacrifices

GROSS PATHOLOGY
Calculi in the kidney (pelvis) or urinary bladder, hydronephrosis, hydroureter, blood in urine and distention of urinary bladder were test article related in male rats from the 5375 ppm group sacrificed at the 6 month interim, died / sacrificed in extremis during the 0 – 12 month period of the study. Urolithiasis was the primary lesion. None of the rats at 12, 18 or 24 months or any rat that died / sacrificed in extremis during the 12 – 24 month periods showed any test article effects.

HISTOPATHOLOGY: NON-NEOPLASTIC
No evidence of a test article related carcinogenic effect was observed in any tissues or organs examined microscopically from male or female rats from any of the experimental groups. A non neoplastic change of questionable toxicological significance occurred in the kidneys of a small number of female rats from the 5375 ppm group which died or were sacrificed in extremis during the period 6 – 12 months.
Urinary tract lesions were probably related to the presence of urinary tract calculi. These lesions included hyperplasia of the urinary bladder epithelium, acute cystitis and hemorrhage in the bladder wall, which correlated with macroscopic observations of bladder calculi. Almost all test article related urinary tract lesions were limited to male rats from the 5375 ppm group which died, were sacrificed in extremis or were electively sacrificed in the first 12 months of study with most occurring in animals which died or were sacrificed in extremis. Hydroureter was sometimes associated with bladder calculi, some dilated ureters were also inflamed and hemorrhagic. Test article related kidney lesions consisted of tubular nephrosis in some rats from the 5375 ppm group which died or were sacrificed in extremis during the period 6 - 12 months. Calculi were noted in the bladders of some affected males, no calculi were found in any of the affected females. Test article related urinary tract lesions only occurring in the first 12 months of study suggests that some males by the nature of their urinary tract structure were more prone to obstruction and succumbed early. Occurrence of bladder epithelial hyperplasia and associated test article related bladder lesions in rats which did not have grossly evident calculi is consistent with a calculus based mechanism. Calculi which were transiently present but which were small enough to pass on the urine stream would cause irritation during their residence in the bladder. Uremia due to urinary tract obstruction by calculi was the probable cause of death in animals in which calculi could not be passed.
Heart lesions in 5375 ppm males which died or were sacrificed in extremis during the first 12 months of study also had urinary bladder calculi and or distention at necroscopy suggesting that the heart lesions were of uremic etiology, secondary to urinary tract obstruction.
An increased incidence of splenic hemosiderosis in male rats of the 5375 ppm group was possibly test article related but did not correlate with any alteration in clinical pathologic parameters.
Dose descriptor:
NOAEL
Effect level:
154 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
histopathology: non-neoplastic
Dose descriptor:
NOAEL
Effect level:
266 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
histopathology: non-neoplastic
Dose descriptor:
LOAEL
Effect level:
371 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
histopathology: non-neoplastic
Dose descriptor:
LOAEL
Effect level:
634 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
histopathology: non-neoplastic
Critical effects observed:
not specified
Executive summary:

Sodium cyanurate was administered in the drinking water in a two year toxicity and oncogenicity study in rats.  Non-neoplastic lesions were observed in urinary tracts of males in the 5375 ppm group sacrificed at the 6 and 12 month interims.  Heart and urinary tract lesions occurred in 5375 ppm males which died or were sacrificed in extremis during the first 12 months of the study.  The only possible substance related changes in females were in the urinary tract of the 5375 ppm animals which died or were sacrificed in extremis during the 6 – 12 month period of study.  In the 18 month interim sacrifice and the 24 month terminal sacrifice no lesions were detected which could be attributed to treatment with the test article.

No evidence of test article related carcinogenic effect was observed in any tissues or organs examined microscopically from male or female rats of any test group.

Endpoint:
chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
equivalent or similar to guideline
Guideline:
EU Method B.33 (Combined Chronic Toxicity / Carcinogenicity Test)
GLP compliance:
yes
Species:
mouse
Strain:
B6C3F1
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc. Kingston, New York, USA.
- Age at study initiation: 6 weeks
- Weight at study initiation: males 15.5-21.1 g; females 14.6-18.5 g
- Housing: Stainless steel wire mesh cages
- Acclimation period: 13 days


ENVIRONMENTAL CONDITIONS
- Temperature (°F): 70± 4
- Humidity (%): 40-60
- Photoperiod (hrs dark / hrs light): 12/12


Route of administration:
oral: drinking water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
For the treatment groups monosodium cyanurate was screened and double ground in a mortar and pestle to achieve a fine powder. The required amount of compound for the high dose group was weighed on an Arbor 126 electronic balance and transferred to a carboy which was then filled to the required volume with water. The mixture was stirred for approximately 2.5 hours until complete dissolution was achieved The solution mixed at the high dose level was used as the stock solution for dilutions required for the lower dose group. The pH was adjusted to 7.4 for all study groups using glacial acetic acid or sodium hydroxide as necessary. Fresh test and control solutions were prepared and presented twice weekly throughout the study.
Details on analytical verification of doses or concentrations:
Monosodium cyanurate was analyzed for cyanuric acid concentration. Two samples were analyzed weekly from approximately 1 through 7 and one sample was analyzed weekly from approximately weeks 9 through 105.
Duration of treatment / exposure:
104 weeks
Frequency of treatment:
continuous
Dose / conc.:
0 ppm
Dose / conc.:
100 ppm
Remarks:
males - 24 mg/kg bw/day; females - 26 mg/kg bw/day as cyanuric acid
Dose / conc.:
400 ppm
Remarks:
males - 97 mg/kg bw/day; females - 100 mg/kg bw/day as cyanuric acid
Dose / conc.:
1 200 ppm
Remarks:
males - 307 mg/kg bw/day; females - 315 mg/kg bw/day as cyanuric acid
Dose / conc.:
5 375 ppm
Remarks:
males - 1520 mg/kg bw/day; females - 1580 mg/kg bw/day as cyanuric acid
No. of animals per sex per dose:
Low dose group: 80/sex
Mid dose, mid-high dose and high dose groups : 100/sex/dose level
Tap water control: 100/sex
Sodium control: 80/sex
Control animals:
other: Vehicle (tap water), Sodium control (sodium hippurate)
Details on study design:
- Dose selection rationale: 5375 ppm was the maximum solubility for sodium cyanurate in water.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Three times daily, although on rare occasions this was reduced to twice daily.
- Cage side observations checked included. Mortality and moribundity


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly


BODY WEIGHT: Yes
- Time schedule for examinations: Recorded weekly from study initiation through week 14, and biweekly thereafter.


FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: recorded weekly from study initiation through week 14, and biweekly thereafter.


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Twice each week at four and three-day intervals


OPHTHALMOSCOPIC EXAMINATION: No


HAEMATOLOGY: Yes
- Time schedule for collection of blood: Prior to initiation and after weeks 52 and 104.
- Anaesthetic used for blood collection: Yes (identity) / No / No data
- Animals fasted: Yes (overnight)
- How many animals:10 mice per sex
- Parameters checked: Hematocrit, haemoglobin concentration, erythrocyte count, total and differential leukocyte count, platelet count, erythrocyte morphology, reticulocyte count, myeloid / erythroid ratio (bone marrow smear).


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: prior to initiation and after weeks 52 and 104
- Animals fasted: Yes (overnight)
- How many animals:10 mice per sex
- Parameters checked: blood urea nitrogen.


URINALYSIS: Yes
- Time schedule for collection of urine: 26, 52, 78 and 104 weeks
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes (overnight)
- Parameters checked: Colour and appearance, volume, osmolality, pH, protein, glucose, blood, ketones, bilirubin, microscopic examination of sediment, zinc, copper, magnesium, chloride, inorganic phosphate, calcium, urea nitrogen, potassium, sodium, urobilinogen.


NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:
GROSS PATHOLOGY: mice used for clinical pathology including urinalysis and the weeks 27, 53 and 79 interim sacrifices. All surviving animals at week 105 were euthanized and examined. Animals which were found dead or sacrificed in extremis during the study were necropsied.

ORGANS: the external surface, all orifices, cranial cavity, carcass, external surface of the brain and spinal cord (performed post fixation), the nasal cavity and paranasal sinuses, the thoracic, abdominal and pelvic cavities and their viscera, the cervical tissues and organs.

HISTOPATHOLOGY: all preserved tissues from animals that were found dead or sacrificed in extremis as well as all control and high dose animals at terminal sacrifice were examined. Preserved tissues included all gross lesions and tumours, brain (at least 3 levels from the fore- mid- and hind-brain), pituitary, salivary gland, heart, thymus, thyroid with parathyroids, lung (2 corneal sections including all lobes and mainstream bronchi), trachea, esophagus, stomach, duodenum, jejunum, ileum, colon, cecum, adrenals, pancreas, gallbladder, liver (at least 2 lobes), urinary bladder, ureter with left and right identified, testes with epididymides, prostate, ovaries, corpus and cervix uteri, spleen, lymph nodes (all grossly enlarged, or otherwise abnormal nodes, nodes draining known and suspected tumour sites and nodes which show no gross abnormalities from other sites) skin, sciatic nerve, mammary gland, Zymbal’s gland (if present), bone (including marrow) from sternum or femur, skeletal muscle attached to tibio-femoral joint, spinal cord (2 levels), three coronal sections though the head (to include nasal cavity, paranasal sinuses, tongue, oral cavity, nasopharynx and middle ear, carcass and remaining viscera.
For animals surviving to study termination coronal sections of the head were examined microscopically from 10 animals per sex from each group. The kidneys, ureters, urinary bladder and gross lesions from 10 mice per sex from high dose and control groups at interim sacrifices were also examined microscopically. The tissues were processed by embedding in Paraplast®, sectioning and staining with hematoxylin and eosin.
Statistics:
Cumulative survival data through week 104 were analyzed using the National Cancer Institute Package. Groupwise comparisons were based on an analysis of all groups using the highest concentration of sodium hippurate that occurred on study.
Food and water consumption, body weight and growth rates, haematology (except leukocyte differentials and morphology), serum chemistry, urine chemistry and organ weight data were tested using Levene’s test for homogeneity of variance. If the variances proved to be homogenous, the data were analyzed by one-way ANOVA. If the variances proved to be heterogeneous, a series of transformations was performed until variance homogeneity was achieved, namely log10, square (X2), square root (X½), reciprocal (1/X), angular (arcsine X½) and rank, in that order. If one of the transformations was effective in achieving variance homogeneity, ANOVA of the transformed data was performed. If ANOVA of either untransformed or untransformed data was significant, the Games and Howell modification of the Tukey-Kramer honestly significant difference test was used for group mean comparisons. If ANOVA was not significant, the analysis was complete. If none of the transformations were effective in achieving variance homogeneity, the Terpstra-Jonckheere nonparametric test for trend and Kruskall-Wallis nonparametric one-way ANOVA and pairwise group comparisons were performed. Levene’s test, ANOVA, the Terpstra-Jonckheere test and the Kruskall-Wallis test were evaluated at the 5.0% one tailed probability level. Group comparisons by the Games and Howell test or the Kruskall-Wallis test were evaluated at the 5.0% and 1.0% two-tailed probability levels.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
effects observed, treatment-related
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY:
Increased incidences of swollen / enlarged abdomens were noted for the 1200 or 5375 ppm and the sodium control males beginning week 15 and continuing throughout the study. The incidences in the 5375 ppm males continued to be higher than the sodium control males. In the 100 and 400 ppm group males, the incidence of swollen / enlarged abdomens was similar to the control group.
In females, swollen / enlarged abdomens were observed less frequently than in the males, but after week 27 the incidences in groups treated with 1200, 5375 ppm and the sodium control were slightly higher than the control group females for several weeks.
A slightly increased number of occurrences of rough hair coat were noted in groups treated with 1200, 5375 ppm and the sodium control. Sporadic findings of tissue masses were observed in all groups of females except 1200 ppm, although the incidence was low and did not exceed 3.3% of the number of surviving females of groups at any given interval. Tissue masses were not observed in any of the male groups.



BODY WEIGHT AND WEIGHT GAIN
Female mean body weights of groups treated with 400, 1200 or 5375 ppm or the sodium control, were almost exclusively lower than control group for the first 24 weeks of the study. The mean body weight of the high dose (5375 ppm) group females were consistently lower than control through weeks 78, while by week 24, body weights in the 400 ppm and 1200 ppm groups were similar to the control group. Mean body weights were slightly higher in the sodium control group females than the control group females.
Significantly lower mean absolute body weights at weeks 13 and 26 in the 5375 ppm dose females compared to the control group were noted. Mean growth rates were significantly lower in the groups treated with 400, 1200, 5375 ppm and the sodium control compared to the control group from initiation to week 26.
At week 52 no statistically significant differences were noted in the body weights or growth rates of control and cyanurate treated females.
No significant differences were noted in the male body weights. The mean body weights of males in the high dose and sodium control groups fell below the mean body weight values of the control group for several weeks between weeks 28 and 78


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Food consumption: slightly decreased food consumption was noted most weeks during the first 26 weeks in the cyanurate treated females and in the cyanurate treated males during the first 13 weeks. Statistically significant lower mean total food consumption was observed for 5375 ppm dose males and females at 13 and 26 weeks and in males at 52 weeks.
The food consumption was higher for the 5375 ppm dose males than for the sodium control group males. For the females, food consumption was generally lower for the high dose group than for the sodium control group for the first 13 weeks and was comparable for those two groups after week 13. The food consumption in sodium control males was significantly lower than the control group.



WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study):
Mean water consumption was consistently higher for the 5375 ppm dose males and females compared to the control group. Water consumption was also generally increased for the sodium control group males and females. Water lost within the sipper tube as well as the additional handling / agitation contributed to the significantly increased mean water consumption values for the high dose group. 5375 ppm dose males mean water consumption was significantly higher than the control group mean value when the procedures were the same and 1200 ppm treated males were generally higher (occasionally significantly) than in the control group after week 10.



HAEMATOLOGY:
No significant differences between the control and treated groups and no indications of compound related effects were noted.


CLINICAL CHEMISTRY:
A statistically higher blood urea nitrogen level was noted in the sodium control females at termination. As the values for all female groups at week 53 and all male groups at weeks 53 and 105 were comparable this was not considered biologically important


URINALYSIS:
Mean urine sodium concentrations and excretion values were significantly higher in the 5375 ppm dose and sodium control females than the control group. At weeks 27, 79 and 105 the mean sodium values for the sodium control group females were generally higher than for the 5375 ppm dose females although at week 53 the mean sodium values for the two groups were similar. Slight to moderate increases were also noted in the mean sodiumconcentrations and excretion values of the males. The mean urine sodium excretion values of the 5375 ppm dose group were slightly lower than the sodium control males. The mean sodium concentrations were similar in the two groups at weeks 79 and 105, the high dose group values were less than the sodium control




ORGAN WEIGHTS:
Although there were statistically significant differences between the control group and the test groups on the ovary, heart, brain and kidney weights, these effects are considered incidental and unrelated to treatment due to lack of dose relationship and / or the effect at subsequent intervals.


GROSS PATHOLOGY:
No distinct compound related trends were observed

HISTOPATHOLOGY: NON-NEOPLASTIC
Microscopic evaluation failed to reveal compound related histomorphologic alterations. Spontaneous disease lesions and incidental findings were noted but were of the expected frequency for mice of this age and strain and occurred without relationship to treatment. In the urinary system, the kidneys from all groups had lesions consistent with chronic nephropathy. There was a significant decrease in focal pituitary hyperplasia in the 5375 ppm group and the sodium control group compared to the control group. There was increased congestion in the mesenteric lymph node in the 5375 ppm group and the sodium control group compared to the control group females although this probably has little biological significance. Microscopic evaluation of tissues from mice receiving high doses of monosodium cyanurate failed to reveal specific treatment related histopathologic alterations consistent with the results of the 27, 53 and 79 week interim sacrifices.

MORTALITY: No statistically significant differences between groups or trends were revealed at termination
Dose descriptor:
NOAEL
Effect level:
1 523 mg/kg bw/day (nominal)
Sex:
male
Basis for effect level:
other: No treatment related effects reported
Dose descriptor:
NOAEL
Effect level:
1 582 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: No treatment related effects reported
Critical effects observed:
not specified
Conclusions:
Based upon the conditions and findings of the study, no definitive treatment related effects were observed in any of the levels of monosodium cyanurate tested. In addition, monosodium cyanurate was not found to be oncogenic in mice. Possible effects on some toxicologic indices were observed but could not be conclusively attributed to monosodium cyanurate administration.
Endpoint conclusion
Dose descriptor:
NOAEL
154 mg/kg bw/day
Study duration:
chronic
Species:
rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Oral Exposure:

The NOAEL for sub-chronic effects (90-days) is 109 mg/kg bw/day for males based on hyperplasia in the urinary bladder observed in one male in the mid-dose group (Rajasekaran, D (1981) The hyperplasia in males observed in the sub-chronic study has been elucidated in the 2-year combined chronic toxicity and cytogenicity study (Blair, M 1985) where it was seen that male rats were more susceptible to dose related effects during the early stages of the study with reversal of effects over the full dosing period. A small group of male rats were found to be more sensitive to the test material at an early stage of the study, which was considered unusual with chronic toxicity testing. In high dose males the main effects observed were heart and urinary tract lesions. The No Observed Effect Level (NOEL) in males from the 2-year study was identified as 154 mg/kg bw/day and the Lowest Observed Adverse Effect Level in the male was 371 mg/kg bw/day. The low sub-chronic NOAEL (109 mg/kg bw/day, male) should be considered as redundant based on the findings of the 2-year chronic study. The NOEL for males of 154 mg/kg bw/day from the 2-year combined chronic toxicity/carcinogenicity study is applicable for risk characterisation as a precautionary approach.

CYA has shown no evidence of carcinogenicity via oral exposure in drinking water in doses up to 5375 ppm (equivalent to doses of 1523 mg/kg bw/day for males, and 1582 mg/kg bw/day for females), in carcinogenicity studies in both rat and mouse.

Dermal Exposure:

In vivo and in vitro studies dermal absorption have been performed which demonstrate that very little cyanuric acid is absorbed through the skin. An in vivo study of cyanuric acid exposure in swimming pool water demonstrated that during swimming uptake is largely by the oral route with dermal uptake being considerably lower (Duncan RC (1980). An in vitro study of rat, guinea pig and human skin exposed to swimming pool water containing 55 mg/l cyanuric acid showed that cyanuric acid is poorly absorbed through the skin (Moody RP et al 1993). Inokuchi, N et al (1978) demonstrated in an in vivo skin absorption study that very little radiolabelled isocyanuric acid (1-3%) was taken up by the skin.

Justification for classification or non-classification

No systemic toxicological findings could be detected after repeated administration of cyanuric acid by either the oral or dermal route. Therefore, a classification as STOT RE is not justified.