Barium 3-hydroxy-4-[(4-methyl-2-sulphonatophenyl)azo]-2-naphthoate

Repeated dose toxicity: oral

Administrative data

Endpoint:

chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1977 -1982

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1978 - 1981

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: US FDA requirements for feeding studies of D&C colours

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 451 (Carcinogenicity Studies)

Deviations:

no

GLP compliance:

yes

Remarks:

part of the study was performed prior to GLP.

Specific details on test material used for the study:

- Name of test material (as cited in study report): D&C red no 6
- Physical state: dark orange-red powder
- Analytical purity: 95%
- Purity test date: no data
- Lot/batch No.: AA 3473
- Expiration date of the lot/batch: no data
- Stability under test conditions: yes
- Storage condition of test material: yes
- Other: Purchased from H. Kohnstamm and Company, New York (USA)

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: The Charles River Breeding Laboratories, Portage, Michigan (USA)
- Age at study initiation: 6 weeks
- Weight at study initiation: 24 - 30 g (males) and 20 - 24 g (females)
- Fasting period before study: not applicable
- Housing: individually in suspended wire-mesh cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 2 weeks


ENVIRONMENTAL CONDITIONS
- Temperature (°C): controlled
- Humidity (%): controlled
- Air changes (per hr): controlled
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: February 14, 1978 To: February 28, 1980

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:


DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Mixing appropriate amounts with (Type of food): basal laboratory diet (mixing for 10 minutes in a twin-shell blender)
- Storage temperature of food: no data

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Each batch of prepared test diet was sampled (three subsamples representing three distinct areas of the batch) and assayed weekly
during the first 13 weeks and every 4 weeks thereafter. On day 7 of week 1, 4 and 12 and every 3 months thereafter, random samples of feed
remaining in the feeder jars were collected from the top, middle and bottom levels of selected animal racks for each group and subsequently assayed.

Homogeneity of D & C Red #6 in diet preparation from June 20, 1980 was determined.

The diet sampled on Day 0 of study week 97 was shipped to Robert Lukey at Elizabeth Arden, Inc. for assay.
A 5 gram colorant sample collected on February 22, 1980 was shipped to Robert Lukey at Elizabeth Arden, Inc. for assay. A 10 gram sample of colorant was shipped to H. Kohnstanm Company for assay on February 27, 1979.
The stability of the test material of this study was also conducted as part of the concurrent rat study with D & C Red #6. Analysis of test material samples in the rat study was performed for samples collected weeks 42 and 93. These study weeks correspond approximately with weeks 16 and 69 of this study.

Duration of treatment / exposure:

24 months

Frequency of treatment:

daily

Post exposure period:

none

Remarks:

Doses / Concentrations:
0, 0.05, 1 and 5 % in the diet
Basis:
analytical conc.

Remarks:

Doses / Concentrations:
70/85; 1421/1712, 7148/8868 mg/kg bw (males/females)
Basis:
actual ingested
average

No. of animals per sex per dose:

60

Control animals:

yes, plain diet

Details on study design:

After 24 months of oral compound administration, all surviving mice were sacrificed by carbon dioxide asphyxiation.

Method for assigning animals to groups:
First, the mean body weight and standard deviation were calculated by sex, and a computer-generated edit developed a listing of those animals whose body weights were within + 1.5 standard deviations of the mean. From the qualifying animals, the randomization procedure selected and assigned the required number of animals. Bartlett’s Chi-square test for homogeneity of variances was applied to the groups. If the groups were not judged to be homogeneous, new randomizations were applied until homogeneity was established.

Positive control:

not applicable

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: three times daily (Mo-Fr), twice daily (Sa/So, holidays)
- Cage side observations checked in table [No.?] were included.


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly


BODY WEIGHT: Yes
- Time schedule for examinations: weekly (first 14 weeks), bi-weekly (week 14 - 36) and monthly (7days on the third of each month) thereafter


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
Schedule: weekly (first 14 weeks), bi-weekly (week 14 - 36) and monthly (7days on the third of each month) thereafter
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data


FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No data



OPHTHALMOSCOPIC EXAMINATION: No



HAEMATOLOGY: Yes
- Time schedule for collection of blood: 6, 12 and 18 months
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: 10 per sex and group
- Parameters examined: hemoglobin, hematocrit, differential leucocyte counts, differential erythrocyte counts, mean corpuscular volume, mean corpuscular hemoglobin and MCHV


CLINICAL CHEMISTRY: No



URINALYSIS: No


NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table 1)
HISTOPATHOLOGY: Yes (see table 1)

Other examinations:

Organ weight determination was performed for brain, kidney (both), liver and spleen

Statistics:

All pairwise statistical comparisons, unless noted otherwise, were two-tailed with the individual probability level for significance at 0.01.

Body weights (weeks 0 through 14 and quarterly thereafter), food consumption (weeks 1-13, 14-26, 30-39, 43-52, 56-64, 69-78, 82-91, and 95-104), clinical pathology parameters (3, 6, 12 and 18 months) and absolute and relative organ weights (terminal sacrifice) were compared by analysis of variance (one-way classification), Bartlett’s test for homogeneity of variances and the appropriate t-test (for equal or unequal variances) as described by Steel and Torrie8 using Dunnett’s multiple comparison tables to judge significance of differences.
The two control groups were compared against each other. If there was no significant difference, then they were combined and the treated groups were compared against the combined controls. If the two control groups were significantly different, then each control group was compared against each treated group separately.
Survival indices (week 91 and 104) were compared using Chi-square test criterion with Yates correction on 2 x 2 contingency tables as described by Siegel to judge significance of differences.

Various analyses for relating proportions to dose were performed, using procedures outlined in Thomas, Breslow, and Gart. Data for animals with malignant tumors, with benign tumors, and all tumors combined were analyzed separately by sex.

For each data set, life table curves were computed using both Kaplan-Meler and standard methods. Homogeneity of curves was tested
using Cox's test for life table data and the Gehan-Breslow generalized Kruskal-Wallis test. For both tests, exact and conservative approximations
are reported, and all pairwise comparisons of groups are given.
Finally, for each data set, unadjusted and time-adjusted tests for linear trend in the proportions were performed. For both methods, the exact test for trend and approximate tests for homogeneity and departure.

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not specified

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

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:

A. GENERAL OBSERVATIONS
I. Appearance and Behavior
From the first week of study, the hair and exposed skin areas appeared light red for mice at the 1.0% dosage level and red for mice while the feces (when sectioned and smeared) appeared red and bright red for mice at these two dosage levels, respectively. From the second week of study, the urine from the mice at these two dosage levels appeared orange. A reddish-brown discoloration ventral abdomen and/or anogenital region was observed occasionally for mice at the 1.0 and 5.0% dosage levels until week 86.
Beginning in study week 86, the hair on the anogenital region of males at the 1.0 and 5.0% dosage levels was observed to be reddish-brown in
color. Beginning in study week 90, the hair on the anogenital region of males at the 0.05% dosage level was observed to be orange in color.
A hunched posture was noted occasionally for treated mice but not for control mice, however, the observation was infrequent. Palpable masses
for both treated and control mice were observed in numbers expected for this species at this age.

2. Mortality
Beginning about week 64, the appearance of a possibly compound-related increase of mortality in males was evident in the 5.0% dosage group
During the last 6 months of the study, there was also a slight increase in mortality in the males at the 0.05% and 1.0% dosage levels
Statistical analyses of the survival of males at 91 and 104 weeks revealed significantly lower survival of the animals in the 5.0% dosage
level at 104 weeks.

3. Body weights
Group mean body weights were generally similar compared with combined control values. Before week 11, there were occasional statistically significant differences considered to be of no biological significance.

4. Food consumption
Food consumption was similar for control and treated mice. There were no statistically significant differences at any of the tested intervals.

5. Haematology
No compound-related effects were observed

6. Gross pathology and Histopathology
There were no compound-related changes observed during postmortem examination of animals dying during the study or sacrificed at the termination of the study.

Relevance of carcinogenic effects / potential:

not carcinogenic

Dose descriptor:

NOEL

Effect level:

1 421 mg/kg bw/day (actual dose received)

Sex:

male

Basis for effect level:

other: 1% in the diet; higher incidence of mortality at 5% in the diet

Remarks on result:

other: Effect type: toxicity (migrated information)

Dose descriptor:

NOEL

Effect level:

1 712 mg/kg bw/day (actual dose received)

Sex:

female

Basis for effect level:

other: see above

Remarks on result:

other: Effect type: toxicity (migrated information)

Dose descriptor:

NOEL

Effect level:

>= 7 148 mg/kg bw/day (actual dose received)

Sex:

male

Basis for effect level:

other: 5% in the diet

Remarks on result:

other: Effect type: carcinogenicity (migrated information)

Dose descriptor:

NOEL

Effect level:

>= 8 868 mg/kg bw/day (actual dose received)

Sex:

female

Basis for effect level:

other: 5% in the diet

Remarks on result:

other: Effect type: carcinogenicity (migrated information)

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

1981

Report date:

1981

Reference 2

Reference Type:

other: Amendment to study report

Title:

Unnamed

Year:

1982

Report date:

1982

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

Deviations:

yes

Remarks:

Designed to meet US FDA requirements for long-term feeding studies, combines a fertility element, in-utero-exposure and long-term exposure. Histopathology of kidneys assessed for mid and low dose group as follow-up investigation and reported in amendment

GLP compliance:

no

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Disodium 3-hydroxy-4-[(4-methyl-2-sulphonatophenyl)azo]-2-naphthoate

EC Number:

227-497-9

EC Name:

Disodium 3-hydroxy-4-[(4-methyl-2-sulphonatophenyl)azo]-2-naphthoate

Cas Number:

5858-81-1

Molecular formula:

C18H12N2Na2O6S

IUPAC Name:

disodium 3-hydroxy-4-[(4-methyl-2-sulfonatophenyl)diazenyl]-2-naphthoate

Test material form:

solid

Test animals

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. Wilmington, Massachusetts (USA)
- Age at study initiation: no information
- Weight at study initiation: 75 - 155 g (males) and 75 - 144g (females)
- Fasting period before study: not applicable
- Housing: individually, except for mating period and nursing period
- Diet (e.g. ad libitum): yes (Supplier Ralston Purina company)
- Water (e.g. ad libitum): yes
- Acclimation period: 15 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): controlled at 71° Fahrenheit (recorded weekly starting week 31)
- Humidity (%): controlled at 53% (recorded weekly starting week 31)
- Air changes (per hr): controlled, no further data
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: Sept 2, 1977 To: January 6, 1978 (F0-Generation) and December 1977 To:June 3, 1980 (F1-Generation)

Individual ear tags were used for identification. Rats were examined for physical abnormalities prior to entering the study. Rats were then randomly assigned to groups.

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The main batch of feed for each group was sampled (three sub-samples representing three distinct areas of the batch) and assayed weekly during the first 13 weeks and every 4 weeks thereafter. Concentrations were determined to be 95 - 99% of target values.
Homogenicity was also confirmed.
Chemical analysis was performed at the sponsor's laboratories.

Duration of treatment / exposure:

F0-generation (not relevant for the endpoint chronic toxicity):
Rats were given the test item in the feed for 60 days prior to mating, during mating (maximum 7 days), during gestation and during lactation (21 days).F0-animals were sacrificed after weaning.

F1-animals (chronic toxicity study part)
exposure started in-utero. After weaning, rats received the control diet for two weeks and were then exposed via the diet until a survival rate of 20% was reached for the highest dose group; this was 95 weeks for males and 126 weeks for females.

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0, 0.05, 0.3 and 2.0 %
Basis:
other: in the diet (measured)

Doses / concentrations 2

Remarks:

Doses / Concentrations:
0, 26, 161 and 1117 mg/kg bw
Basis:
other: average calculated for males from food consumption and body weight

Doses / concentrations 3

Remarks:

Doses / Concentrations:
0, 31, 189 and 1315 mg/kg bw
Basis:
other: average calculated for females from food consumption and body weight

No. of animals per sex per dose:

70 rats per sex per dose group for the F1-Generation (chronic toxicity study):
of these 10 per sex per dose group were sacrified at the age of 12 months (interim sacrifice)

Control animals:

yes, plain diet

Details on study design:

The F1 generation was used for the long-term part of the study. Parental animals were used for the fertility element and for in-utero exposure. F1 pups were counted and sexed on days 0,4,14 and 21 of lactation. Litters were housed with their dams until 21 days after birth. After that pups were housed together for two weeks and during that time, they received control diets and were selected for the long-term feeding part of the study.

Positive control:

none

Examinations

Observations and examinations performed and frequency:

Clinical signs: Daily (pup stage), twice daily (adult stage)
Body weight and food consumption: weekly for the first 14 weeks, biweekly for the next 12 weeks and monthly thereafter. At the pup stage, weighing of the litter was done on days 0, 4 and 14
Hematology, biochemistry and urinalysis were performed for 10 animals per sex and dose group at 3, 6, 12, 18 and 21 months for both genders, for females, also after 24 months. Rats were fasted overnight prior to blood and urine collection.
At 20 months of study, blood was obtained from 2 unfasted rats/sex/group (one rat in apparent good health and one rat exhibiting signs of pulmonary distress). Blood was collected and sent to Microbiological Associates, Rockville, Maryland for viral serology analysis. Lung and trachea tissue from three male rats (two control and one high dose) were sent to Microbiological Associates in an attempt to isolate Sendai virus and mycoplasmas, respectively. Tissue samples from these rats were delivered to Kar Laboratory, Kalamazoo, Michigan for growth, isolation and characterization of the bacterial populations. At 20 months of study, additional blood sampling involving five rats/sex/groups was conducted in an attempt to identify the etiological agent responsible for the numbers of deaths which were occurring.


Hematology parameters: hemoglobin, hematocrit, total and differential leucocyte counts, total erythrocyte counts and total reticulocyte counts; for the 20,21 and 24 month investigation also mean corpulscular volume, mean corpuscular hemoglobin and mean corpuscular hemoglobin concentration

Biochemistry parameters: glucose, blood urea nitrogen, serum SGOT, SGPT, ALP, creatinine and total protein

Urinalysis parameters: pH, specific gravity, qualitative tests for protein, glucose, bilirubin, ketones and occult blood; description of color and appearance, microscopic analysis of sediments. Volumes were inadvertently recorded at 3, 6, 12 and 24 months.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (both interim and final sacrifice)
HISTOPATHOLOGY: Yes (both interim and final sacrifice: any gross lesion plus for control and highest dose group: abdominal aorta, adrenals, bone and bone marrow(femur), blood smear, brain (3 sections), esophagus, eye, ovaries, testes with epididymides, heart, colon, cecum, duodenum, ileum, kidneys, liver, lung and mainstream bronchi, lymph nodes (mediastinal and mesenteric), mammary gland, mandibular salivary gland, sciatic nerve, pancreas, pituitary, seminal vesicles, skeletal muscle, skin, spinal cord, spleen, stomach, thymus, trachea, thyroid/parathyroid, urinary bladder, uterus/prostate
Kidney tissues from the low and mid dose groups (0.05% and 0.3%) were sectioned and examined microscopically (reported in study amendment).

Organ weights: brain, kidney, liver, spleen, testes, thyroid, heart, uterus, ovaries, adrenals

Other examinations:

ophthalmoscopic examinations (after pupillary dilation with 1% tropicamide, using binocular indirect ophthalmoscope):
-Schedule: month 3, 6 ,12 ,18 and 21 for both genders and month 24 for females

Statistics:

For fertility indices, gestation, pup survival indices: Chi-square test criterion with Yates correction on 2x2 contingency tables and/or Fisher's exact probability test. For mean number of liveborn pups per litter and litter mean pup weight: analysis of variance (one-way classification), Bartlett's test for homogeneity of variances and the appropriate t-test as described by Steel and Torrie (1960) using Dunnett's multiple comparison tables (1964).
The same tests were used for the long-term study as appropriate. Life time table curves were computed using both Kaplan-Meier and standard methods. Homogenicity of curves was tested using Cox's test for life table data and the Gehan-Breslow generalized Kruskal-Wallis test.
The data on time to neoplastic lesion was analyzed by methods described by Thomas, Breslow and Gart (1977).

Results and discussion

Results of examinations

Details on results:

No compound-related effects were seen in behavior, survival, food consumption, ophthalmoscopic examinations, hematological, biochemical or urinalysis parameters.

Throughout the study, significant body weight depressions were seen for males at the 2.0% dosage level. When compared to control means, these decreases were considered moderate by 52 weeks and marked by 78 weeks of study. When analyzed statistically, significance was seen (when compared to combined control means) at all intervals analyzed between study weeks 10-91. Statistical significant decreases were also seen (when compared to combined control means) for females at the 2.0% dosage level at study weeks 2-4, 8, 9, 11-14 and 65. However, these decreases were not considered biologically significant.
The changes in body weight were reflected in changes in relative organ weights.

For animals that died between the interim and the terminal sacrifice, males of the highest dose group had a higher incidence of chronic nephritis, renal tubular epithelial hyperplasia, myocardial fibrosis, reticular hyperplasia of the spleen, atrophy/degeneration of the testicular tubules and pigment in the spleen (Table 3 and 4 for kidney histopathology). These lesions are common spontaneous or aging lesions in rats. There appeared to be an exacerbation of this spontaneous renal disease in aged rats in the mid and high-dose group male rats and in the high-dose group female rats (Table 2).
At the interim sacrifice, no test-item related histophathology findings were noted (Table 5).

After one year, males at the 2 % dose level had a slightly higher incidence of deaths (or sacrificed in extremis). The 20% survival rate for (2.0% dosage level) was reached at week 95 for males and at week 126 for females. (see table 1). Based on virology examinations, it was concluded that these deaths were of microbiological origin and not related to treatment with the test item. According to the veterinarian at The Charles River Breeding Laboratories, North Wilmington, Massachusetts, the PVM and KEV titers were higher than expected for rats at this age. in the two rats examined at 12 months.
No Sendal virus was isolated from the lung samples. Trachea samples from these ssme rats for mycoplasma isolation revealed that all three
samples were positive for M. Pulmonis. The most predominant organism was a species of Pseudomonas.

A dose-dependent orange coloration of urine and feces was caused by the colored test item and indicates elimination without destruction of the chromophore. Coloration was also observed for hair and skin.
No adverse effects were noted during the pup stage (day 0-23).

Effect levels

1

Dose descriptor:

NOEL

Effect level:

0.3 other: % in the diet (analytically verified)

Sex:

female

Basis for effect level:

other: increased incidence of chronic nephritis in aged rats

2

Dose descriptor:

NOEL

Effect level:

0.05 other: % in the diet (analytically verified)

Sex:

male

Basis for effect level:

other: Increased incidence of chronic nephritis in aged rats; reduced body weight observed at 2.0 %

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Table 1

dose	Number of surviving rats at terminal sacrifice*
% diet	Male (week 95)	Female (week 126)
0	29	15
0	17	16
0.05	18	16
0.3	24	15
2	9	11
*Initial number: 60 rats after interim sacrifice

Table 2

Incidence of non-neoplastic microscopic observations in kidney (females)
Terminal Sacrifice (TS) and Death and unscheduled sacrifices (DOS) between interim and terminal sacrifice
	Control 1		Control 2		0.05%		0.30%		2%
Lesion	TS	DOS	TS	DOS	TS	DOS	TS	DOS	TS	DOS
	43	15	43	16	39	17	43	15	48	11
abscess	1	1	3	0	4	0	4	0	2	0
aurolysis	0	0	0	0	0	0	1	0	2	0
cystic dilatation	0	3	0	1	1	0	2	0	2	1
chronic nephritis	10	7	16	10	22	12	26	15	31	6
cyst	0	0	0	1	1	1	0	0	1	0
degeneration	1	0	1	0	8	0	2	0	0	0
extramedullary hematopoiesis	0	0	0	0	0	0	0	0	1	0
granuloma	0	0	0	0	0	0	0	0	1	0
hematocyst	0	0	1	0	0	0	0	0	0	0
hydronephrosis	2	1	1	0	2	1	1	0	1	0
hyperplasia of pelvic epithelium	0	0	2	0	0	0	0	0	0	0
interstitial lymphocytic infiltrates	0	0	0	0	0	0	1	0	0	0
mineralization	9	3	7	2	2	2	6	1	1	0
necrosis	0	0	0	0	0	1	0	0	0	0
nephrosis	0	0	0	0	1	0	1	0	0	0
papillitis	0	0	0	0	0	0	2	0	0	0
pigmentation	0	0	0	0	0	0	1	0	0	0
pyelonephritis	0	0	1	0	1	0	2	0	2	1
pyelitis	1	1	1	0	4	3	4	1	3	0
within normal limits	22	4	20	3	5	2	5	3	0	0

Table 3

Incidence of non-neoplastic microscopic observations in kidney (males)
Death and unscheduled sacrifices (DOS) at 12 month to termination
	Control 1	Control 2	0.05%	0.30%	2%
Number examined	30	42	40	35	49
abscess	0	0	0	0	1
cystic dilatation, tubules	2	5	2	0	5
chronic nephritis	18	28	36	34	48
cortical cyst	0	0	2	3	3
hydronephrosis	1	0	0	0	0
hyperplasia of pelvic epithelium	0	1	0	1	2
interstitial lymphocytic/mononuclear/inflammatory cell infiltrates	3	6	0	1	0
tubular mineralization	0	0	0	1	2
nephrosis	1	0	0	0	0
necrotic papillitis	0	0	0	1	0
yellow-brown pigment, tubular epithelium	1	1	0	2	0
pyelonephritis/purulent exudate, pelvis	1	1	0	0	0
microcalculi pelvis	1	3	0	0	0
epithelial hyperplasia, tubules	0	0	0	1	8
arterial mineralization	0	0	0	0	1
hyaline droplet degeneration, tubules	0	0	0	0	1
cellular debris, lumina	0	0	0	0	1

Table 4

Incidence of non-neoplastic microscopic observations in kidney
Terminal sacrifice
Males
	Control 1	Control 2	0.05%	0.30%	2%
number of kidneys examined	29	17	18	25	9
cystic dilatation, tubules	1	0	0	0	0
chronic nephritis	22	16	15	22	9
cyst	0	0	2	1	0
hyperplasia of pelvic epithelium	1	0	0	0	0
interstitial lymphocytic/mononuclear/inflammatory cell infiltrates	2	0	0	0	0
pyelonephritis/pyelitis	0	0	2	2	0
microcalculi pelvis	1	0	0	0	0
interstitial fibrosis	1	0	0	0	0
ectatic capillaries	0	0	0	1	0

Table 5

Incidence of non-neoplastic microscopic observations in kidney
12-Months interim sacrifice and unscheduled sacrifice between 0-12 months
	Control 1		Control 2		0.05%		0.30%		2%
Lesion	M	F	M	F	M	F	M	F	M	F
number examined	11	12	11	11	11	10	10	10	12	11
chronic nephritis	6	4	10	3	7	3	7	2	10	3
cyst	0	0	0	0	0	0	1	0	0	0
hydronephrosis	0	0	1	0	1	1	0	0	0	0
hyperplasia of pelvic epithelium	0	0	2	0	0	0	0	0	0	0
interstitial lymphocytic infiltrates	3	2	0	1	0	0	0	0	0	0
mineralization	1	1	0	3	1	2	0	1	0	0
hydronephrosis	0	0	1	0	1	1	0	0	0	0
pyelonephritis, pyelitis	1	0	0	0	1	2	0	2	0	0
concretions, pelvis	0	1	0	0	0	0	0	0	0	0
hemorrhage	1	0	0	0	0	0	0	0	0	0
papillary necrosis	1	0	1	0	0	0	0	0	0	0