Nitrapyrin

Repeated dose toxicity: oral

Administrative data

Endpoint:

chronic toxicity: oral

Remarks:

combined repeated dose and carcinogenicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 October 1986 to 26 December 1989

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline, and the study was conducted under GLP conditions.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1989

Report date:

1989

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EPA OPP 83-1 (Chronic Toxicity)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EPA OPP 83-2 (Carcinogenicity)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Nitrapyrin

EC Number:

217-682-2

EC Name:

Nitrapyrin

Cas Number:

1929-82-4

Molecular formula:

C6H3Cl4N

IUPAC Name:

2-chloro-6-(trichloromethyl)pyridine

Test material form:

solid: crystalline

Details on test material:

- Appearance: white crystals

Test animals

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 4 weeks old on arrival; 5 to 6 weeks old at start of dosing
- Weight at study initiation: Group means on Study Day -1: 117.2 - 118.7 g (males); 97.9 - 98.1 g (females)
- Housing: The animals were housed singly in stainless-steel, suspended cages. Catch pans under the cages were lined with absorbent, antibiotic-impregnated paper sheets.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 7 - 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 72 ± 5 °F
- Humidity (%): 40 - 60 %
- Air changes (per hr): 13 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours of darkness / 12 hours of light

IN-LIFE DATES: From 07 October 1986 (Study Day 1) To: necropsy, performed 11 - 18 October 1988 (Study Days 736 - 743)

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): diets were prepared at 1 - 2 week intervals. The test diets were prepared weekly through the first 13 weeks of this study; then the test diets were prepared every two weeks throughout the remainder of the study. Initial concentrations of test material in the diets were calculated from pre-test body weights and feed consumption. Thereafter, the mean body weight and feed consumption data determined during treatment were used to adjust the concentration of test material in the diets in order to maintain constant dose levels on a mg/kg bw/day basis. The adjustments were made for each diet period with projected body weights.
- Mixing appropriate amounts with (Type of food): Diet preparation was done according to the following scheme: A premix was prepared by ball-milling the test material with rodent chow overnight. Test diets were prepared by serial dilution beginning with the premix. Paddle-type mixers were used to blend the diets.
- Stability of diet preparations: The stability of test material in rodent chow for up to 14 days had previously been determined. A check for homogeneity of mixing was performed on representative samples of mixed diet at the beginning of the study. The mixing procedure used was found to result in homogeneous dispersion of test material in the feed samples.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses to confirm test material concentrations were performed on samples of premixes, control feed, and all dose-level diets at the start of the study and at approximately 3-month intervals until the end of the study.
For analysis, approximately 2 grams of each sample submitted was weighed in duplicate or triplicate into extraction thimbles. The samples were extracted over a 14 hour period using the soxhlet method and 100 mL of benzene. The soxhlet apparatus heated the benzene and allowed it to reflux through the extraction thimbles. At the end of the extraction period, the extracts were allowed to cool and transferred to 100 mL volumetric flasks, brought to volume with benzene, then put into 4 ounce amber bottles for storage. The bottles were taped and stored in the refrigerator until diluted prior to analysis. All the extracts were diluted to an approximate range of 0.1 to 1.0 µg/mL with toluene. Standards were also prepared in toluene and diluted to approximately 0.1 to 1.0 µg/mL. The standards and samples were analysed by gas chromatography using an electron capture detector. Duplicate injections were made of each sample and standard and concentrations were calculated using external standard calibration.
Overall, the analytically determined dietary concentrations were sufficiently comparable to targeted concentrations for meaningful interpretation of the study.

Duration of treatment / exposure:

2 years

Frequency of treatment:

Continuous (in diet)

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

5 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

20 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

60 mg/kg bw/day (nominal)

No. of animals per sex per dose:

60 males and 60 females per dose

Control animals:

yes, plain diet

Details on study design:

- Rationale for animal assignment (if not random): During the acclimation period, the rats (identified by cage number) were weighed and ranked by body weights. Rats at the extremes of the rankings were eliminated from the lists until only the numbers required for the study remained. Using a computer, random numbers were used to allocate the rats to the various treatment groups. At this time, 10 rats/sex/group were randomly allocated for the 12-month interim sacrifice.

Positive control:

not applicable

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- All rats were observed daily for signs of toxicity or changes in demeanour. These observations included observing the animals' movement in the cage, a check for availability of feed and water, the presence of faeces, and excessive feed wastage. Specific attention was also given to changes in skin and fur, eyes, visible mucous membranes, respiration, and behaviour.

DETAILED CLINICAL OBSERVATIONS: Yes
- All rats were given a careful clinical examination by a staff veterinarian at least once weekly.
Moribund animals or those which appeared unlikely to survive over a weekend or holiday were brought to the attention of the study director for a decision about removal. Rats found dead outside normal working hours were refrigerated until necropsy.
All animals were palpated for externally detectable masses during the pre-study period, prior to the 12-month interim kill, and monthly thereafter for the duration of the test period. Individual "palpable mass" records were maintained for each animal indicating the size, location, date of onset, and progression or disappearance of each mass.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were determined weekly for the first 3 months and monthly thereafter.

FOOD CONSUMPTION: Yes
- Feed consumption was determined weekly for the first 3 months and monthly thereafter. Feed consumption was determined only for 20 rats/sex/dose level in order to adjust the concentration of test material in the diet. In the event that one of these 20 rats/group died, it was replaced by another animal in the same dose group so as to maintain the sample size as close to 20 as practicable.

WATER CONSUMPTION: No data

OPHTHALMOSCOPIC EXAMINATION: No data:

CLINICAL LABORATORY DETERMINATIONS
All clinical laboratory procedures scheduled for 6 and 12 months were performed on rats designated for the 12-month interim sacrifice (10/sex/dose). Clinical laboratory tests at 18 and 24 months were done on rats designated for the terminal sacrifice. An effort was made to sample the same animals at 18 and 24 months.

HAEMATOLOGY: Yes
- Blood samples for haematology from 10 rats/sex/dose level were obtained by orbital sinus puncture under light methoxyflurane anaesthesia at 6 and 18 months and immediately prior to sacrifice at the 12 month necropsy. Twenty rats/sex/dose level were bled for haematology samples by orbital sinus puncture under light anaesthesia immediately prior to sacrifice at the 24 month necropsy.
- Haematology determinations consisted of: packed cell volume (PCV), haemoglobin concentration (HGB), erythrocyte count (RBC), total (WBC), differential leukocyte counts, and platelet count (PLAT)
- Complete blood smear examinations included: differential white blood cell counts (100 cells) and an assessment of erythrocyte, leukocyte, and platelet morphology. Any morphological abnormality was reported.

CLINICAL CHEMISTRY: Yes
Serum from 10 fasted rats/sex/dose level for clinical chemistry determinations were obtained by orbital sinus puncture at 6, 12, and 18 months; serum from 20 fasted rats/sex/dose level for clinical chemistry determinations were obtained by orbital sinus puncture at the 24 month period.
- The following clinical biochemical parameters were measured: alkaline phosphatase (ALP), alanine transaminase (ALT), aspartate transaminase (AST), blood urea nitrogen (BUN), cholesterol (CHOL), creatine phosphokinase (CK), glucose (GLU), total bilirubin (TBIL), albumin (ALB), total protein (TPRO), calcium (CA), phosphorus (PHOS), sodium (NA), potassium (K), and chloride (Cl). Globulin (GLOB) values were calculated from the total protein and albumin values.

URINALYSIS: Yes
Urine samples were obtained from 10 rats/sex/dose level at approximately 6 and 18 months and the week prior to the 12-month necropsy; urine samples were obtained from 20 rats/sex/dose level the week prior to the 24-month necropsy.
- The following parameters were determined: specific gravity, pH, protein, glucose, ketones, bilirubin, occult blood, and urobilinogen.
- Sediment was examined microscopically from a pooled specimen/sex/dosage.

Sacrifice and pathology:

POSTMORTEM EXAMINATIONS AND MEASUREMENTS
At the two scheduled sacrifice intervals, rats were presented for necropsy after an overnight fast. They were weighed, anaesthetised, and the tracheas were exposed and clamped to prevent aspiration of blood prior to decapitation. Each received a complete postmortem examination by a veterinary pathologist, including an in situ examination of the eyes with a moist microscope slide under fluorescent light, and the tissues were preserved in neutral, phosphate-buffered 10 % formalin.
Following examination, the liver, kidneys, brain, testes (males), ovaries (females), and adrenal glands were weighed, and the organ weight to final fasted body weight ratio was calculated. The lungs were distended to their approximate normal inspiratory volume by tracheal infusion of formalin, and the nasal cavities were flushed with formalin to ensure rapid fixation of the nasal mucosa. Hollow organs were incised for internal examination.
Rats which died or were submitted moribund were necropsied in a similar manner; however, final body weight and organ weights were not obtained. Due to the rapid onset of postmortem corneal artifacts, the detailed eye examination was not performed on animals that were found dead.

HISTOPATHOLOGY
Representative sections of the tissues listed below were examined histologically for all rats in the control and high dose level groups from both sacrifice intervals, as well as for any animals in the low and middle dose groups which did not survive until scheduled sacrifice. Histopathologic examination of tissues from the low and middle dose groups at the 12-month sacrifice was limited to the liver, kidneys, and tissues with gross lesions.
At the 24-month sacrifice, the following tissues were routinely examined from all surviving rats in the low and middle dose groups: liver, kidneys, spleen, lungs, testes, pituitary, thyroid/parathyroid, adrenal glands, and all significant gross lesions. All routine histopathologic examinations were performed on paraffin-embedded tissues stained with haematoxylin and eosin. Kidneys, from male animals designated for the 12-month sacrifice, were stained with Lee's methylene blue - basic fuchsin to assess protein droplets in the proximal convoluted tubules. Kidneys from male and female rats designated for the 12-month sacrifice were stained by the immunoperoxidase method for the presence of alpha 2u-globulin in the cytoplasm of cells lining the proximal convoluted tubules.

- Tissues examined included: adrenal glands, aorta, bone, bone marrow, brain, cecum, cervix, coagulating glands, colon, duodenum, epididymides, eyes, gross lesions, heart, ileum, jejunum, kidneys, lacrimal/Harderian glands, larynx, liver, lungs, lymph nodes (mediastinal, mesenteric) mammary gland, mediastinal tissue, mesenteric tissue, nasal tissue, oesophagus, oral tissues, ovaries, oviducts, pancreas, parathyroid glands, peripheral nerve (sciatic), pituitary gland, prostate, salivary glands, seminal vesicles, skeletal muscle, skin and subcutis, spinal cord (cervical, thoracic, lumbar), spleen, stomach, testes, thymus, thyroid gland, tongue, trachea, urinary bladder, uterus and vagina.

Statistics:

Descriptive statistics (mean and SD) are reported for feed consumption and white blood cell differential counts. Body weights, clinical chemistry, haematology, organ weights, and urine specific gravity were evaluated by Bartlett's test. Based upon the outcome of Bartlett's test, exploratory data analysis was performed by a parametric or non-parametric analysis of variance (ANOVA), followed by Dunnett's test or the Wilcoxon Rank-Sum test with Bonferroni's correction for multiple comparisons. Statistical outliers were identified by a sequential test.
Differences in mortality patterns were tested by the Gehan-Wilcoxon procedure for all animals scheduled for the 2-year sacrifice.
For tissues with complete histopathologic examination (terminal sacrifice only), the incidences of specific observations were first tested for linearity using ordinal spacings of the doses. If linearity was not rejected, the data were then tested for a dose response relationship using the Cochran-Armitage trend test. If the trend was statistically significant, or if a significant deviation from linearity was found, incidences for each dose were compared to that of the control using a pairwise chi-square test with Yates correction.
When significant mortality differences existed between dose groups, mortality adjusted statistics were used to supplement the routine statistics. The adjusted statistics (Peto et al. 1980) were applied to those tumours that were observed in an early mortality context. The adjusted statistics in this study were only applied to data on the male rats and only for tumour data and chronic progressive glomerulonephropathy. In addition, adjusted statistics were only applied to those tissues that are completely sampled.
For tissues which were evaluated from all control and high dose level rats but only from selected rats from the low and middle dose level groups, statistical analysis was limited to the pairwise comparison of control and high dose.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

See "Details on results" for information

Mortality:

mortality observed, treatment-related

Description (incidence):

See "Details on results" for information

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

See "Details on results" for information

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

See "Details on results" for information

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

See "Details on results" for information

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

See "Details on results" for information

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

See "Details on results" for information

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

See "Details on results" for information

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

See "Details on results" for information

Details on results:

MORTALITY
Male rats did demonstrate a statistical difference in mortality. Examination of each dose versus the control identified the high dose group as statistically different. Neither the low dose group or middle dose group males or any female dose groups were identified as statistically different from the control group. As expected for Fischer 344 rats, the most common causes of death or morbidity for most dose groups were pituitary neoplasms and Fischer rat leukaemia (LGL leukaemia; large granular lymphocyte leukaemia). The most frequent cause of death for male rats in the high dose group (60 mg/kg bw/day) was severe chronic progressive glomerulonephropathy.

CLINICAL SIGNS AND PALPABLE MASSES
There were no clinical signs for individual animals which suggest a relationship to exposure to test material. Female rats, in all dose groups including control, demonstrated rough haircoats/alopecia, that was judged to be unrelated to test material administration and which appeared histologically to be a lack of active hair shaft production by the hair follicles.
There is no indication from the monthly palpation examinations of any excess of masses, unusual tumours, or early onset in any treatment group versus respective controls. The type and incidence of cutaneous and subcutaneous "masses" noted were within the normal range of neoplasms for the Fischer 344 rat.

BODY WEIGHT AND WEIGHT GAIN
Throughout the first 12 months of this study, there were no appreciable differences in male body weights for any treatment groups relative to controls. Male group mean body weights were shown to be statistically different from control by study day 483 for rats given 60 mg/kg bw/day, whereas for rats administered 20 mg/kg bw/day, group mean body weights were shown to be different from control by study day 595.
Female group mean body weights were shown to be statistically different from control by study day 49 for rats given 60 mg/kg bw/day. These lower body weight values were attributed to test material administration. Group mean body weight for female rats administered 5 or 20 mg/kg bw/day and male rats administered 5 mg/kg bw/day showed no appreciable difference from control attributable to test material administration throughout the study.

FOOD CONSUMPTION
There was no appreciable difference in the amount of feed consumed by any treatment group relative to controls.

HAEMATOLOGY
In males, RBC, HGB, and PCV were consistently lower for rats administered 60 mg/kg bw/day, although only shown to be statistically different from control at the 6, 12, and 18 month (except RBC) sampling intervals. In this study, these minor differences from control values are judged not to be a primary effect of test material on the erythron, but rather a secondary effect of the test material, possibly relating to the nutritional status of the animal. Statistically identified differences in platelet counts were shown in males at the 6 and 18 month sampling intervals for rats administered 60 mg/kg bw/day and at the 6 month interval for rats administered 20 mg/kg bw/day and judged to reflect biologic variation since these differences were not reproducible between sampling intervals and no correlative lesion was seen in platelet morphology in blood smears or abnormal condition in megakaryocytes of the spleen or bone marrow was seen in histologic sections.
Female haematologic parameters were unaffected by test material administration at all sampling intervals. There was a decrease in WBC at the 12 month period in rats administered 5 mg/kg bw/day and a decrease in platelet numbers at the 24 month period in rats administered 60 mg/kg bw/day. Due to their isolated occurrence, no association with test material administration was made.

CLINICAL CHEMISTRY
There were a number of parameters which were statistically identified as different from the control means at various dose levels and sampling periods. However, in male rats, the only parameters which were consistently different and appeared to be associated with the treatment regimen at all sampling periods were decreases in creatine phosphokinase activity (CK) (20 and 60 mg/kg bw/day) and increased blood urea nitrogen concentration (BUN) (60 mg/kg bw/day). Parameters associated with test material administration for rats given 60 mg/kg bw/day during the first 12 months of the study were decreases in alanine transaminase (ALT) and aspartate transaminase activity (AST), and an increase in total bilirubin concentration (TBIL). These decreases in ALT, AST, and CK activities did not appear to indicate any direct organ toxicity, but are most likely secondary effects of altered metabolism, possibly in conjunction with histomorphologic alterations in the liver. At the 18 and 24 month sampling periods, in addition to the increase in BUN concentration, rats administered 60 mg/kg bw/day had lower albumin (ALB) and total protein (TPRO) concentrations and increased phosphorus (PHOS) concentrations; these were judged to reflect the increase in severity of renal toxicity seen in this group of rats and not in male rats fed 5 or 20 mg/kg bw/day.
In female rats, no parameters were consistently different from controls or associated with test material treatment at all sampling periods. Only CK activity was shown to be affected by treatment at the 18 and 24 month sampling period for female rats administered 60 mg/kg bw/day; as in male rats, the decrease in CK activity was considered not to indicate any direct organ toxicity, but rather secondary effects of altered metabolism. At the terminal sampling period (24 month), ALT, AST, BUN, CHOL, and TBIL were increased, reflecting test material effects on the liver and kidneys of female rats administered 60 mg/kg bw/day. ALB concentration was shown to be statistically decreased in this group of rats and TPRO was lower in this group of rats, again reflecting the greater severity of renal disease in this group of rats and not in female rats given 5 or 20 mg test material/kg bw/day.
All other statistically identified values in males and females for clinical chemistry and electrolyte parameters were considered incidental and due to random variability because they were either not dose-related or were inconsistent over time as to occurrence or direction of change relative to control.

URINALYSIS
Urine specific gravity was consistently lower in male rats of the high dose group at all sampling periods. This persistently lower specific gravity value appears to be treatment-related and was interpreted to be a reflection of the greater renal pathology in the high dose group males. The statistically significant lower urinary specific gravity value recorded for male rats in the 20 mg/kg bw/day dose group at the 18 month sampling period appears to be a reflection of abnormally high specific gravity values in the low dose and control groups rather than a treatment-related depression.
Female urinary specific gravity values were not different from control dose group values for any dose level group or any sampling period. All other urinary parameters in either males or females appeared to be unaffected by test material administration.

ORGAN WEIGHTS
The calculated "relative" organ weights are the ratio of the absolute organ weight to the final fasted body weight (g/100 g body weight). Parameters affected by treatment with test material were limited to liver and kidney in male (20 and 60 mg/kg bw/day) and female (60 mg/kg bw/day) rats.
In male rats, the final fasted body weights were not significantly different from control values for any dose level group at the 12-month interim sacrifice; however, final fasted body weights were significantly lower in male rats administered 20 or 60 mg/kg bw/day at the terminal sacrifice. Absolute and relative liver weight parameters were increased in high dose male rats at the 12-month necropsy; at the 24-month necropsy, absolute and relative liver weight values were statistically increased in male rats administered 20 or 60 mg test material/kg bw/day. Absolute and relative kidneys weight parameters were increased in male rats given 20 or 60 mg/kg bw/day at the 24-month necropsy, indicative of the increased renal effect of test material administration on these dose level groups. The increase in absolute and relative adrenal gland weights in male rats (20 or 60 mg/kg bw/day) at the 24-month necropsy was judged to be a secondary phenomena to stress in animals with advanced renal disease. The increase in relative brain weight is a reflection of the lower body weight in male rats of the 60 mg/kg bw/day dose group and therefore secondary to administration with test material.
Final fasted body weights for female rats administered 60 mg/kg bw/day were significantly lower at the 24-month necropsy but not at the 12-month interim sacrifice. Absolute and relative liver weight parameters were increased in high dose level female rats at both the 12 and 24-month sacrifice intervals. The relative kidney weight value for the high dose group female rats was elevated at 12 months; both the absolute and relative kidney weight parameters were statistically increased at the 24-month necropsy. Similar to the high dose male rats, the increase in relative brain weight in the high dose group females was determined to be a reflection of the lower body weight of this dose group and not a primary effect of test material administration.

GROSS PATHOLOGY
There was no obvious pattern of changes in any dose group at either sacrifice interval suggestive of a treatment-related effect. All gross lesions were examined histologically (with the obvious exception of certain superficial changes such as perineal staining, facial soiling, decreased mesenteric fat, etc.), and the final interpretation of these was based on histopathologic examination.

HISTOPATHOLOGY
At the 12-month sacrifice, histopathologic alterations associated with test material administration were found in kidneys and liver. Control male rats, at the 12-month sacrifice, had a moderate number of small (approximately 1 µ) protein droplets in the cytoplasm of renal proximal convoluted tubules (P2 segment); male rats given 20 or 60 mg/kg bw/day demonstrated alterations in protein accumulation in these tubule segments (Protein Droplet Nephropathy). Histologically, P2 segments of rats affected at 20 or 60 mg/kg bw/day showed an increased variability in the size of protein droplets, an increase in the quantity of protein droplets within the epithelial cell cytoplasm, and an increase in the number of P2 segments containing protein droplet accumulations. The use of the term "slight" refers to the quantity and distribution of affected tubules, i.e., there is a greater than control amount of protein droplets, most of which are larger than protein droplets present in the cytoplasm of control male rats, and there appears to be a greater number of tubule segments containing protein droplets. The use of the term "moderate" refers again to the quantity and distribution of affected tubules, i.e., proximal tubule epithelial cells contain numerous variably-sized protein droplets (in some cases margination of the nucleus is present) and most of the proximal tubule segments of the outer cortex appear to be affected. Immunoperoxidase staining for α2u-globulin of kidney tissue from male rats administered 60 mg/kg bw/day (12-month sacrifice) demonstrated a marked retention of α2u-globulin within tubules containing protein droplets. Male rats administered 60 mg test material/kg bw/day also demonstrated mineralisation, confined primarily to the thin loops of Henle deep in the renal papilla. Other renal diagnoses used at the 12 month interim sacrifice (Degeneration/Regeneration, Tubule(s) - Slight and Dilated with Proteinaceous Casts, Tubule(s), Multifocal) showed no dose-response relationship in male rats and were judged to reflect the ongoing renal disease in the Fischer 344 rat as commonly seen in a variety of strains of laboratory rat. The only renal lesion in female rats attributable to test material administration was the presence of renal tubules dilated with proteinaceous casts - very slight in females administered 60 mg/kg bw/day, suggesting an exacerbation of naturally occurring renal disease in this sex. Female rats did not demonstrate protein droplet accumulation in segments of the proximal convoluted tubules or α2u-globulin accumulation.
Histopathologic examination of renal tissue from male rats of the high dose designated for the terminal sacrifice revealed an increase in the severity of chronic progressive glomerulonephropathy (CPG-severe) and an increase in primary renal tumours; an increase in the severity of centrilobular hepatocellular hypertrophy.
Supplemental analysis (mortality adjusted), treating the primary renal tumours as incidental findings demonstrated a positive trend test (α = 0.05) and a negative pairwise comparison. Although these tumours are small, they suggest that the test material is a weak renal carcinogen in the male Fischer 344 rat. Clinical chemistry data confirm the severe nature of the chronic progressive glomerulonephropathy in the high dose level male rats at the 18 month and 24 month sampling periods. The lack of a discernible protein droplet nephropathy at the 24 month terminal sacrifice was due to the severe, diffuse nature of the chronic progressive glomerulonephropathy.
Hepatocellular alterations seen in male and female rats administered 60 mg/kg bw/day (12-month sacrifice) consisted of hypertrophy (moderate) and vacuolation (slight to moderate) of centrilobular hepatocytes. Male rats given 20 mg/kg bw/day, but not female rats, had a slight hypertrophy of centrilobular hepatocytes without concomitant fatty vacuolation of affected hepatocytes. Histopathologic examination of liver sections from rats at the high dose of the terminal sacrifice revealed: an increase in the severity of centrilobular hypertrophy; an increase in fatty vacuolation of centrilobular hepatocytes; and an increased incidence of biliary hyperplasia (male only). Clinical chemistry parameters, at the 12-month sacrifice, were consistent with a histomorphologic lesion in the liver; clinical chemistry parameters, at the 24 month terminal sacrifice, did not reflect a similar liver response but may have been artificially lower due to the severity of the renal lesion in these animals with loss of serum enzymes in the urine. The statistical significance of areas of altered cells and foci of altered cells in male rats is more a reflection of the conservative nature by which the pathologist diagnosed these hepatocellular alterations, i.e., when areas and foci are combined there is no statistical or biological difference between controls and any treatment group.
Cystic thyroid follicles, more precisely coalescence of adjoining thyroid follicles, were shown to be statistically significant in male rats administered 60 mg/kg bw/day. The biologic or toxicologic significance of this histopathologic alteration is unknown; it was not judged to be a primary effect of test material administration due to the lack of alterations in the thyroid glands of male rats sacrificed at 1 year. It may have represented a physiologic response on the part of the thyroid gland to the loss of protein-bound thyroid hormone in the urine of animals with severe renal disease.
At the terminal sacrifice, there were no additional histopathologic changes in any dose level other than the 60 mg/kg bw/day level which were interpreted as effects of test compound administration.

Effect levels

1

Dose descriptor:

NOEL

Effect level:

5 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Mortality, bodyweight, haematology, clinical chemistry, urinalysis, organ weights, neoplastic and non-neoplastic histopathology.

2

Dose descriptor:

NOEL

Effect level:

20 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: Bodyweight, clinical chemistry, organ weights, non-neoplastic histopathology.

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Under the conditions of the study, the No Observed Effect Level for all parameters in male rats was 5 mg/kg bw/day and for females was 20 mg/kg bw/day.

Executive summary:

The two year repeated dose toxicity of the test material, via the oral route, was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guidelines EPA OPP 83-1 and 83-2 in Fischer 344 rats.

During the study sixty rats/sex/dose level were fed test material in their diets at concentrations formulated to provide 0 (control), 5, 20, or 60 mg/kg bw/day. Ten rats/sex/dose level were randomly designated at the start of the study for an interim sacrifice at 12 months. The remaining animals were continued on test until 24 months or they died or were killed moribund. Parameters measured during the study were clinical observations; body weights; feed consumption; haematology; clinical chemistries and electrolytes; urinalyses; and gross and histopathologic examination.

The primary effects of administration of 60 mg test material/kg bw/day to male rats for up to 2 years were a decrease in body weight gain relative to controls in the second year of the study, a statistically significant increase in early mortality, increased relative and absolute kidney weights (24-month sacrifice only), histopathologic changes in the kidneys characterised at the 12-month sacrifice as protein droplet accumulation (α2u-globulin) in the epithelial cells of the proximal convoluted tubules and at the 24-month sacrifice as an increase in the severity of chronic progressive glomerulonephropathy and the presence of primary renal tumours, increased relative and absolute liver weights, and histopathologic changes in the liver characterised as hypertrophy and fatty vacuolation of centrilobular hepatocytes and an increased incidence of biliary hyperplasia. Other effects of the treatment regimen, which may be secondary to the aforementioned changes, included lower alanine transaminase and aspartate transaminase activity and increased total serum bilirubin concentration during the first 12-months; increased blood urea nitrogen (all sampling periods); decreased albumin and total protein concentrations; decreased urine specific gravity; increased absolute and relative adrenal gland weight (24-month sacrifice only); and coalescence of thyroid follicles. Mechanistically, the altered clinical chemistry parameters reflect the presence of toxicologic lesions in the liver and kidneys of these animals. Adrenal gland and thyroid gland changes most likely are physiologic responses to stress in animals with severe renal disease and ongoing hepatic alterations.

Altered parameters in females at the high dose level were a decrease in body weight gain relative to control within the first 50 days on study, increased absolute and relative kidney weights (12-month sacrifice only), histopathologic changes in the kidney consisting of an increase in the severity of the chronic progressive glomerulonephropathy which occurs in this strain of rat, increased absolute and relative liver weights, and histopathologic changes in the liver characterised as hypertrophy and fatty vacuolation of centrilobular hepatocytes. The severity and incidence of these lesions were generally less in females than in males. As in male rats, several clinical chemistry parameters were elevated in relation with the histopathologic identified organ toxicity, however this occurred only at the 24-month sampling period. Survival in high dose level female rats was unaffected by test material treatment.

At the 20 mg/kg bw/day treatment level, the effects of treatment seen in males consisted of a decrease in body weight gain relative to control during the final 4 months of this study, increased relative and absolute kidney weights (24-month sacrifice only), histopathologic changes in the kidneys characterised at the 12-month sacrifice as protein droplet accumulation in the epithelial cells of the proximal convoluted tubules, increased relative and absolute liver weights, and histopathologic changes in the liver characterised as hypertrophy and fatty vacuolation of centrilobular hepatocytes and an increased incidence of biliary hyperplasia. Other effects of the treatment regimen, which may be secondary to the aforementioned changes, included: increased blood urea nitrogen (18 and 24-month sampling periods); decreased albumin and total protein concentrations (18 and 24 month sampling periods); and increased absolute and relative adrenal gland weight (24-month sacrifice only). The altered clinical chemistry parameters reflect the presence of toxicologic lesions in the liver and kidneys of these animals. Adrenal gland changes, as in the high dose level male rats, most likely are a physiologic response to stress in animals with ongoing renal and ongoing hepatic disease.

In female rats administered 20 mg/kg bw/day, no effects of treatment were seen in the two years of dietary administration.

The chronic toxicity noted at the high dose in both male and female rats in this study was considered indicative of a sufficient challenge for the assessment of oncogenicity. Male rats administered 60 mg/kg bw/day had a sex-specific incidence of primary renal tumours which were not seen in any other male dose level or in any female dose level. Besides the male renal tumours, there was no increase in tumours of any type in any other organ or tissue at any of the dose levels tested which was attributable to test material administration.

The No-Observed-Effect-Level (NOEL) for all parameters in males was 5 mg/kg bw/day and for females was 20 mg/kg bw/day.