Chlorothalonil

Carcinogenicity

Description of key information

NOAEL for preneoplastic tubular hyperplasia in the kidney was 1.8 mg/kg/day; in the forestomach, the NOAEL for preneoplastic effects and for tumours was 1.8 mg/kg/day; EPA OPP 83-2; Wilson, 1989

Key value for chemical safety assessment

Carcinogenicity: via oral route

Link to relevant study records

Reference

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 Dec 1985 to 06 May 1988

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPP 83-2 (Carcinogenicity)

GLP compliance:

yes

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: Approximately 4 weeks old when received; rats not more than 6 weeks old when placed on study.
- Weight at study initiation: males: 67 to 96 grams; females: 59 to 83 grams
- Housing: During the study, rats were housed individually in stainless steel cages.
- Diet: rodent chow, ad libitum
- Water: fresh potable water ad libitum
- Acclimation period: minimum 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23
- Humidity (%): 40 - 60
- Air changes (per hr): 8 - 12
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 13 Dec 1985 To: 6 May 1988

Route of administration:

oral: feed

Details on exposure:

DIET PREPARATION:
The test material for each group was weighed out into a properly labelled, tared plastic container of appropriate size. The individual weights to be used for each group were recorded in the study file. The non-treated rodent chow was weighed out into properly labelled, tared, plastic bags of appropriate size. The individual weights to be used for each group were also recorded in the study file. A premix was prepared by adding a small portion of the weighed compound (obtained from the correct group container) to a small portion of the weighed non-treated rodent chow (obtained from the correct group bag) in a mortar, and pestling until the compound was dispersed evenly throughout the chow with no clumps of compound visibly apparent. This procedure was continued until there was no compound left in the container. Before it was discarded the container was ”rinsed” with a few grams of chow into the mortar. Following the mortar and pestle blending, the premix was mixed in a Hobart mixer with additional rodent chow and a ”secondary” premix was prepared. The remaining portion of the non-treated lab chow was then placed in a twin shell mixer, along with the ”secondary” premix and allowed to mix for approximately 20 minutes. When the mixing was completed, the diet was placed back into the plastic bag. The same procedure was carried out for each group, mixing in ascending order of concentration. Upon completion of the mixing, all the apparatus and equipment used are cleaned thoroughly, and the appropriate equipment logs are filled in.

For the second revised procedure (used from week 37), all conditions were the same as the procedure used primarily except that the mixing time in the blender was increased to 30 minutes with the intensifier bar running only for 20 minutes.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Prior to the administration of the test material to the rats, analyses were conducted to assess the homogeneity and 14-day stability of the diets prepared according to the method used during the study. During the study, two samples of each dose level of the diets administered to the rats were collected immediately after the preparation of each batch. Diet samples were analyzed weekly for the first 14 weeks of the study and for even numbered weeks thereafter to confirm that the diets were prepared at their intended concentrations. In addition, the samples retained by the contract laboratory from various weeks were analyzed. At weeks 4, 8 and 12 and at 12 week intervals thereafter, diet samples in addition to those collected on the day of preparation were assayed after remaining four days under study room conditions. This was done to provide information about test material stability for the duration of the study. The concentrations of the test substance in diet were determined by gas chromatography.

Duration of treatment / exposure:

Up to 26 months for males and 29 months for females

Frequency of treatment:

Continuous treatment (in feed)

Dose / conc.:

2 mg/kg bw/day (nominal)

Remarks:

Low dose; dietary equivalent to 1.8 mg/kg/day for both males and females.

Dose / conc.:

4 mg/kg bw/day (nominal)

Remarks:

Low-mid dose: dietary equivalent to 3.8 mg/kg/day for both males and females.

Dose / conc.:

15 mg/kg bw/day (nominal)

Remarks:

High-mid dose: dietary equivalent to 15.3 and 15.2 mg/kg bw/day for males and females, respectively.

Dose / conc.:

175 mg/kg bw/day (nominal)

Remarks:

High dose: dietary equivalent to 183 mg/kg bw/day for both males and females.

No. of animals per sex per dose:

Sixty five animals per dose per sex

Control animals:

yes, plain diet

Details on study design:

- Interim necropsy: 10 rats/sex/group (those selected for 12 month clinical pathology test) at 12 month interval. All rats at 12 month interim were examined for absolute and relative (to body weight) weight of brain, and absolute and relative (to body and brain weights) weights of kidneys and liver.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily, once each in the morning and afternoon

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly, from one week prior to the initiation of treatment through termination

BODY WEIGHT: Yes
- Time schedule for examinations: weekly, from one week prior to the initiation of treatment through week 14, and biweekly thereafter to termlnation

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, measured over a four-day period once per week for the first 14 weeks and biweekly thereafter to termination

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
The food efficiency was calculated for the first 14 weeks of the treatment.

OPHTHALMOSCOPIC EXAMINATION: Yes, An ophthalmologic examination of all surviving high dose males was conducted prior to termination of this group during month 23. An ophthalmologic examination of the survivors in all other groups was conducted at 24 months.

HAEMATOLOGY: Yes, blood samples collected from the orbital sinus of animals under ether anesthesia
- Time schedule for collection of blood: at 12, 18 and 24 months, and termination of the study
- Anaesthetic used for blood collection: Yes , ether
- Animals fasted: No data
- How many animals: 10 rats/sex/group randomly selected
- Parameters checked: Hemoglobin, hematocrit, erythrocyte count, mean corpuscular hemoglobin (MCH) mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC), leukocyte count (total and differential) and platelet count.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: 18 and 24 months and at termination of study
- Animals fasted: No data
- How many animals: 10 rats/sex/group randomly selected
- Parameters checked: Alkaline phosphatase, blood urea nitrogen, lactic dehydrogenase, serum glutamic pyruvic transaminase, serum glutamic oxaloacetic transaminase, glucose, total protein, albumin, globulin, A/G ratio, inorganic phosphate, calcium, sodium, potassium, chloride, creatine, total bilirubin and total cholesterol.

URINALYSIS: Yes
- Time schedule for collection of urine: 18 and 24 months and at termination of study, overnight collection
- Parameters checked: Color, volume, appearance, specific gravity, occult blood, protein, pH, bilirubin, ketones, glucose, nitrites, urobilinogen and microscopic examination of sediment

Sacrifice and pathology:

GROSS PATHOLOGY: Yes,
- Macroscopic pathology is done for all rats found dead, sacrificed in extremis and at scheduled sacrifices
- Tissues preserved: adrenals, aorta, bone (femur), bone marrow (sternum), brain (fore, mid and hind), clitoral gland (2), epididymis (2), esophagus, eye (with Rarderian gland) (2), stomach (forestomach and fundus), duodenum, ileum, jejunum, cecum, colon, rectum, heart, kidney (2), liver, lung with bronchi, lymph nodes (mesenteric, renal, gastric and cervical), mammary gland (females only), ovary (2), pancreas, pituitary, preputial gland, prostate, salivary gland (submaxillary), sciatic nerve, seminal vesicle (2), skeletal muscle (rectus femoris), skin, skull (skin removed), spinal cord (cervical, thoracic and lumbar), spleen, testis (2), thymus, thyroid/parathyroid complex, tongue, trachea, urinary bladder, uterus (corpus and cervix), gross lesions, tissue masses with regional lymph nodes.

HISTOPATHOLOGY: Yes
- A histopathological evaluation of haematoxylin and eosin stained sections of the kidney, stomach, mesenteric lymph node, and renal lymph node from male and female Fischer 344 rats receiving the test substance in their diet for up to 29 months was conducted. A similar set of tissues from male and female rats receiving laboratory chow for the same periods of time were also examined microscopically and served as controls. An interim necropsy of 10 rats per sex in each group was conducted at one year of the study. The tissues from all rats necropsied at one year and from all rats which died during the first year were examined microscopically. Tissues from all other animals were examined after termination of the study.

Statistics:

Survival of groups was analyzed using life-table (actuarial) methods. Body weights, food consumption, food efficiency, clinical pathology parameters and organ weights, analyzed using analysis of variance and Bartlett's test for homogeneity of variances. Treatment groups compared to the control group using the appropriate t-statistic (equal or unequal variance), using Dunnett's multiple comparison tables. Nonparametric analysis, when appropriate, by rank transformation. All statistical tests were two-tailed, using p<0.05 and p<0.01 as levels of significance.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Discolored urine (dark yellow) was noted for all 175.0 mg/kg/day males and females. This was apparent beginning week 5 and generally persisted throughout the study; however as the study progressed the incidence decreased for females and was no longer evident for males during the last five months of study. This sign is thought to be test article related. Females at the 4.0, 15.0 and 175.0 had a higher incidence of yellow anogenital staining as compared to control females.
Corneal opacity, area around eye/eyelids red and enlarged testis for the males were signs seen during the study that are normally seen in aging or moribund rats and are signs normally seen in this strain of rats and do not appear to be treatment related.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

Fifty-four control (23 male, 31 female), forty-eight 2.0 mg/kg/day (22 male, 26 female), fifty-eight 4.0 mg/kg/day (22 male, 36 female), sixty-nine 15.0 mg/kg/day (34 male, 35 female) and 89 of the 175.0 mg/kg/day animals (44 male, 45 female) died/sacrificed in extremis prior to scheduled sacrifice. After approximately 550 days of study there was an increase in the mortality rate for the 175.0 mg/kg/day male animals and due to the low percentage of animals surviving in this group, the group was sacrificed after 99 weeks of study. The remaining male groups were sacrificed at study week 111 and the females were sacrificed at week 125.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Significant reductions in mean body weight values relative to those of the control group were noted for the 175 mg/kg/day males for weeks 1-98 and for the 175 .0 mg/kg/day females weeks 1 and 6 - 124. The 15 mg/kg/day males were noted with statistically significantly reduced mean body weights when compared with the control group on several occasions during the study. The differences were less than they were for the 175 mg/kg/day animals (when compared to the control group). The 2.0 and 4.0 mg/kg/day males and the 15.0 mg/kg/day females were also noted with statistically significantly differences (both increases and decreases) in mean body weights when compared with the control group on a few occasions throughout the study. However, the magnitude of these differences was very slight and this was considered to have no biological significance.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Mean food consumption values on a g/animal/day basis showed statistically significant decreases from those of the control group for the 175.0 mg/kg/day males, and increases and decreases for females beginning with study week one and continuing throughout the study. Statistically significant differences in g/animal/day mean food consumption values were noted for the 2.0, 4.0, and 15.0 mg/kg/day males (decreased) and females (increased) when compared to the control group on several occasions throughout the study. The 175.0 mg/kg/day animals were noted with statistically significantly increased mean food consumption values (on a g/kg/day basis) when compared to the control group on most occasions throughout the study. Mean food consumption values on a g/kg/day basis also showed scatistically significant differences (both increases and decreases) from those of the control group for the 2.0, 4.0 and 15.0 males and females on several occasions throughout the study. The magnitude of the differences in food consumption values (g/animal/day and g/kg/day) were very slight at the 2.0, 4.0 and 15.0 mg/kg/day dosage levels and were considered to have no biological significance.

Food efficiency:

no effects observed

Description (incidence and severity):

Food efficiency values for the treated groups fluctuated relative to the control values; no dose-related trend was evident.

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

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No test article related ophthalmoscopic abnormalities were detected; the observations noted were representative of pathology that could be expected for this group of rats considering age, sex and strain

Haematological findings:

no effects observed

Description (incidence and severity):

There were no discernable test article related hematological changes at any interval tested. Statistical significance was noted for a few parameters when comparing the treated and control groups; however, the differences were generally not dose responsive and were considered to be related to normal animal variation

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

There was a great deal of variation among animals for several parameters; however, a definitive pattern of the test article related changes was apparent. At 175 mg/kg/day, animals of both sexes had elevated phosphorus and cholesterol and had decreased alkaline phosphatase, alanine aminotransferase and albumin at various intervals. In addition males at this level had elevated urea nitrogen and creatinine levels at 18 and 23 months. There were no changes at dosage levels of 15 mg/kg/day or lower that are unequivocally related to test article administration. Decreased alanine aminotransferase in the 15 mg/kg/day females at 24 months and increased urea nitrogen the 15 mg/kg/day males at 24 months may have been test article related but the values were within normal biochemical ranges and may have been due to animal variation.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

The only test article related urological finding that occurred in the 175 mg/kg/day males where decreased specific gravity and increased volume that were apparent at 18 and 23 months. All other values in treated animals were considered to be comparable to control values.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

- 12 Months: Male mean body weight was significantly decreased in the 175 mg/kg/day group. Female mean body weight differences were not statistically significant. Male and female mean absolute and relative (to body and brain weights) kidney weights were significantly increased in the 175 mg/kg/day group. Statistically significant increases were observed in kidney:body ratios in males at 4 and 15 mg/kg/day and absolute kidney weights and kidney:brain weight ratios in females at 4 and 15 mg/kg/day. However, these differences were small and their relationship to the the test material is unclear. Female mean absolute and relative (to body and brain weights) and male relative (to body and brain weights) hepatic weights were significantly increased in the 175 mg/kg/day group. A relationship to the test article cannot be determined.
Statistically significant variations in male and female relative brain weights and male relative hepatic weight are functions of body weight.
- Termination: A statistically significant decrease in absolute body weight was observed in males of the 15.0 mg/kg/day group and in males and females of the 175.0 mg/kg/day group. Females in the 175.0 mg/kg/day group showed a statistically significant increase in absolute kidney weight. Males in the 15.0 mg/kg/day group and males and females in the 175.0 mg/kg/day group showed a statistically significant increase in relative kidney weight. These changes may reflect a test article dose related effect.
Statistically significant changes in various relative organ weights in 15.0 mg/kg/day males and in 175.0 mg/kg/day males and females were noted and believed to be a reflection of the lower absolute body weight of animals in those groups.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

- 0 to 12 months: Granular kidneys were seen with high incidence (7/10) in the male 175 mg/kg/day group. This lesion did not appear in any
other male group, nor in any female group. Gastric, non-glandular, mucosal thickening was seen with high incidence in the male (10/10) and female (7/10) 175 mg/kg/day group. It also occurred in one female and two males from the 15 mg/kg/day group but did not appear in the 2.0 or 4.0 mg/kg/day groups or in the control group of either sex. All other macroscopic lesions were considered to be spontaneous changes.
- 12 Months to termination: Granular kidneys were observed in all dose groups, as well as control animals, in both male and female animals at terminal sacrifice and in those that died or were sacrificed prior to study termination. There was, however, an increased incidence of granular kidneys observed in the 175.0 mg/kg/day males and females in both terminally sacrificed animals and in those that died on study.
There was also an increased incidence of enlargement of the parathyroid glands noted in the 175.0 mg/kg/day group females at terminal sacrifice and in the corresponding dose group of males and females that died prior to termination.
Various lesions of the non-glandular stomach were also observed throughout this study. An increased incidence of thickened mucosa, granular mucosa or irregular mucosa of the non-glandular stomach was noted in the 15.0 mg/kg/day and 175.0 mg/kg/day males and females at the terminal sacrifice and in those animals that died on study between 12 months and termination. The incidence of ulcers or incidence of ulcers or erosions of the non-glandular stomach was increased in females that died on study and in terminally sacrificed males and females of the 15.0 mg/kg/day and 175.0 mg/kg/day groups.
Additionally, an increased incidence of erosions of the glandular stomach was noted in males of the 175.0 mg/kg/day group at terminal sacrifice. An increase in the incidence of this lesion did not occur in females from this group or in rats which died or were sacrificed in extremis during the course of the study. The significance of this observation is unclear but may reflect a test article related effect. All of the other above mentioned observations are apparently test article dose-related and of possible toxicologic significance. All other lesions noted are believed to be spontaneous in nature and incidental or due to post mortem change.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related microscopic alterations were seen in the kidneys from the male and female rats receiving either 15.0 or 175.0 mg/kg/day of the test substance in their diet. In addition, treatment-related microscopic alterations were seen in the kidneys from the female rats receiving 4.0 mg/kg/day of the test substance.
In addition, there was a dose-related increased incidence and severity of focal epithelial hyperplasia in the proximal tubules in both male and female rats receiving either 15.0 or 175.0 mg/kg/day of the test substance. Focal epithelial hyperplasia was also slightly increased in incidence and severity in the female rats receiving 4.0 mg/kg/day dose. A direct relationship between the presence of primary renal neoplasms and focal epithelial hyperplasia is evidenced by the presence of hyperplasia in 58 of 62 animals with renal neoplasms in this study. In the remaining four animals with renal neoplasms, autolysis and/or chronic progressive nephropathy prevented evaluation of the presence of epithelial hyperplasia. The severitv of chronic progressive nephtopathy increased in a dose-related manner in the male and female rats receiving the test substance in their diet at levels of 4.0 mg/kg/day and greater. The incidence of chronic progressive nephropathy was increased in the female rats receiving 175.0 mg/kg/day of the test substance. Clear cell hyperplasia was associated with the dietary administration of the test substance in the male and female rats receiving 175.0 mg/kg/day and female rats receiving 15.0 mg/kg/day. An increased incidence of cortical cysts occurred in the male and female rats receiving 175.0 mg/kg/day of the test substance. An increase in pelvic epithelial hyperplasia was seen in male rats receiving 175 mg/kg/day of the test substance. The increased number of cortical cysts and the increased incidence of pelvic epithelial hyperplasia in this high dose male group was related to the increased severity of chronic progressive nephropathy. Microscopic examination of the renal lymph nodes and mesenteric lymph nodes did not reveal treatment-related microscopic changes. Pigment deposition was frequently seen in the renal lymph nodes. The incidence and severity of this microscopic alteration were comparable among the control and treated rats. In male rats, hemorrhage of the renal lymph node was more frequently observed in the groups receiving 4.0, 15.0, or 175.0 mg/kg/day of the test substance. The association of this microscopic alteration with the dietary administration of the test substance is unclear. The majority of animals exhibiting this lesion were among the moribund sacrificed or death groups. The incidence of hemorrhage of the renal lymph node was slightly increased in the female rats receiving 175.0 mg/kg/day of the test substance.

Associated with the forestomach neoplasms was evidence of direct irritation to the forestomach mucosa illustrated by a dose-related increased incidence and severity of epithelial hyperplasia and hyperkeratosis in male and female rats receiving either 4.0, 15.0, or 175.0 mg/kg/day of the test substance. In addition, ulcers or erosions of the nonglandular mucosa were increased in incidence and correlated with the presence of forestomach neoplasms. In the glandular mucosa, erosions were increased in incidence and severity in the male and female rats receiving 175.0 mg/kg/day of the test substance, providing additional evidence of direct irritation to the gastric mucosa. Inclusion cysts were seen in the forestomach in one female rat receiving 15.0 mg/kg/day and six female rats receiving 175.0 mg/kg/day of the test substance.

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

The dietary administration of the test substance at 175.0 mg/kg/day resulted in an increase in renal neoplasms in both male and female rats. An increase in renal neoplasms was also evident in male rats receiving 15.0 mg/kg/day of the test substance. These neoplasms were classified as either tubular adenomas or tubular carcinomas and were characterized by a neoplastic proliferation of the epithelial cells lining the proximal tubules. In several instances, these neoplasms were multiple and occasional metastases were seen in the regional lymph nodes. No primary renal neoplasms were seen in the control females or in female rats receiving 2.0, 4.0, or 15.0 mg/kg/day of the test substance. Single primary renal neoplasms were seen in a control male rat and in one male rat from each of the 2.0 or 4.0 mg/kg/day dose groups. Because a primary renal neoplasm was observed in a control male rat, the single neoplasms seen in male rats in the 2.0 and 4.0 mg/kg/day groups were considered unrelated to treatment.

Treatment-related microscopic alterations were seen in the stomachs from male and female rats receiving 4.0, 15.0, or 175.0 mg/kg/day of the test substance. An increase in the incidence of forestomach neoplasms was present in male rats receiving 4.0, 15.0, or 175.0 mg/kg/day and in female rats receiving 15.0 and 175.0 mg/kg/day of the test substance.

Other effects:

not examined

Relevance of carcinogenic effects / potential:

The progression from preneoplastic lesions to tumors in the kidney and forestomach of the test substance-treated rats apparently occurs through a nongenetic mechanisms. For nongenetic mechanisms, no tumors attributable to the compound would be expected at or below the no-effect level (NOEL) for preneoplastic toxicity. During the blinded histopathologic examination of kidneys in this tumorigenicity study, renal tumors and preneoplastic tubular epithelial hyperplasia were observed at dose levels of 15 and 175 mg/kg/day. As expected, no treatment related renal tumors were observed at the NOEL for the preneoplastic hyperplasia, 1.8 mg/kg/day.
In the forestomach of animals in this study, tumors and preneoplastic lesions (e.g. squamous hyperplasia and hyperkeratosis) were observed and were associated with the irritative properties of the test substance at dose levels of 3.8, 15 and 175 mg/kg/day. The NOEL for irritation was 1.8 mg/kg/day. In the absence of the irritation-related, preneoplastic lesions, no treatment related tumors were observed in the forestomach at 1.8 mg/kg/day.

Key result

Dose descriptor:

NOAEL

Remarks:

Carcinogenicity (originally reported NOELs reevaluated to yield NOAELs, see study and endpoint summaries)

Effect level:

2 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: neoplastic

Remarks on result:

other: Dietary equivalent to 1.8 mg/kg/day for both males and females.

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

3.8 mg/kg bw/day (nominal)

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

3.8 mg/kg bw/day (nominal)

System:

gastrointestinal tract

Organ:

stomach

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Diet Analyses

Analysis of diet samples collected routinely on the day of diet preparation during this study indicated that the diets were prepared at or near the intended concentrations. The animals were fed fresh diet no less frequently than every four days throughout the study and analysis of diet samples collected during the study after four days of storage at room temperature indicated that the amount of test substance extractable from the diets with lower concentrations decreased over the four day period. In previously conducted studies in rats and mice,a similar decrease in the amount of test substance extractable from diets with low concentrations was determined to be the result of binding of the compound to the diet which prevented extraction by routine methods. In those studies, analysis of diet samples stored under frozen conditions indicated that freezing of the diets after preparation prevented the binding.

Because of the binding of low concentrations to the feed, the dose levels for the low and low-mid group were corrected for the measured average binding of test substance to the feed. The mid-high and high dose levels are expressed as the nominal levels in the results.

 

Table 1. Results of diet analyses conducted during the tumorigenicity study in rats with the test substance

Dose group	Intended dose level (mg/kg/bw)	Mean analytically-determined concentration
		Day 0 (% of nominal)^a		Day 4 (% of day 0)^b
		Males	Females	Males	Females
Low	2 (≥1.5)^c	95	97	79	79
Low-Mid	4 (≥ 3)^c	97	97	86	85
High-Mid	15	98	98	98	99
High	175	99	100	102	101

a) Mean for samples routinely collected on the day of diet preparation during the study.

b) Mean for samples routinely collected 4 days after diet preparation during the study.

c) Diets for the low and low-mid dose groups were prepared at concentrations designed to deliver dose levels of 2.0 and 4.0 mg/kg/day on the day of preparation so that the animals would receive dose levels of at least 1.5 and 3.0 mg/kg/day even if binding affected the availability of the test substance to the animals during the feeding period

Table 2. Mean compound consumption during the tumorigenicity study in rats with the test substance

Dose group	Mean compound consumption (mg/kg/day)
	Nominal		Analytical
			Complete availability		Partial availability
	Males	Females	Males	Females	Males	Females
Low	2.1	2.1	2	2	1.8	1.8
Low-Mid	4.2	4.2	4.1	4.1	3.8	3.8
High-Mid	15.8	15.7	15.5	15.3	15.3	15.2
High	181	182	181	182	183	183

Complete availability - Assumes availability of the test substance to the animals is unaffected by binding in the diet

Partial availability - Assumes binding of the test substance in the diet has some effect on its availability to the animals.

Table 3. Incidence of renal and tubular adenomas and carcinomas in the tumorigenicity study in rats.

	Dose level (mg/kg/day)
	0		1.8		3.8		15		175
	M	F	M	F	M	F	M	F	M	F
Number of tubular adenomas
One year exam	0/10	0/10	0/11	0/11	0/11	0/10	0/11	0/12	1/10	0/10
Terminal exam	1/55	0/55	1/54	0/54	1/54	0/55	3/54	0/53	17/55	24/55
Number of tubular carcinomas
One year exam	0/10	0/10	0/11	0/11	0/11	0/10	0/11	0/12	0/10	0/10
Terminal exam	0/55	0/55	0/54	0/54	0/54	0/55	1/54	0/53	7/55	11/55
Number of animals with tubular carcinoma/adenoma
One year exam	0/10	0/10	0/11	0/11	0/11	0/10	0/11	0/12	1/10	0/10
Terminal exam	1/55	0/55	1/54	0/54	1/54	0/55	4/54	0/53	23/55	32/55

Table 4. Animals with renal adenoma/carcinoma and tubular hyperplasia in the tumorigenicity study in rats.

	Dose level (mg/kg/day)
	0		1.8		3.8		15		175
	M	F	M	F	M	F	M	F	M	F
Number of animals with:
Tumors	1	0	1	0	1	0	4	0	24	32
Tumors associated with hyperplasia	1	-	1	-	1	-	4	-	22	30
Tumors in which autolysis and/or chronic nephropathy precluded diagnosis of hyperplasia	0	-	0	-	0	-	0	-	2	2

Table 5. Incidience of papillomas and carcinomas in the forestomachs from the tumorigenicity study in rats.

	Dose level (mg/kg/day)
	0		1.8		3.8		15		175
	M	F	M	F	M	F	M	F	M	F
Number of papillomas
One year exam	0/10	0/10	0/11	0/11	0/11	0/10	0/11	0/12	0/10	0/10
Terminal exam	0/55	1/55	0/54	1/54	3/54	2/55	2/54	4/53	5/55	7/55
Number of carcinomas
One year exam	0/10	0/10	0/11	0/11	0/11	0/10	0/11	0/12	0/10	0/10
Terminal exam	0/55	1/55	0/54	0/54	0/54	0/55	0/54	1/53	0/55	3/55
Number of animals with carcinoma/papilloma
One year exam	0/10	0/10	0/11	0/11	0/11	0/10	0/11	0/12	0/10	0/10
Terminal exam	0/55	1/55	0/54	1/54	3/54	2/55	2/54	5/53	5/55	9/55

Conclusions:

The NOAEL for renal tubular tumours was 3.8 mg/kg/day (measured dose) and the NOAEL (= NOEL) for preneoplastic tubular hyperplasia in the kidney was 1.8 mg/kg/day (measured dose). In the forestomach, the NOAEL (= NOEL) for preneoplastic effects and for tumours was 1.8 mg/kg/day. At the NOAEL (= NOEL) for preneoplastic lesions in the kidney and stomach, no tumours attributable to the test substance administration were noted.

Executive summary:

This tumorigenicity study in Fischer 344 rats with the test substance was conducted to determine the no-effect levels (NOEL) for preneoplastic and neoplastic lesions which had been observed in the kidney and forestomach of rats in a previous tumorigenicity study with this material. In this study, four groups of 65 rats per sex received the test substance in the diet at constant dose levels of 1.8, 3.8, 15 and 175 mg/kg/day (measured doses for the low and low-mid dose groups, corrected for average binding of the test substance to the feed. Nominal doses for the mid-high and high dose group, because of minimal influence of binding at these dose levels). A fifth group of 65 animals per sex served as the control group. Parameters evaluated included observations for overt toxicity, moribundity and mortality, ophthalmology, body weight and food consumption. Haematology, clinical chemistry and urinalysis evaluations were conducted routinely during the study. An interim necropsy of 10 rats per sex per group was conducted at one year. Based on survival criteria, all surviving high dose males were terminated during month 23 of the study and all surviving males in the remaining dose groups were terminated during month 26. Surviving females in all groups were terminated during month 29. At the one year and terminal necropsies, selected organs were weighed. Histopathologic examination of the kidneys, stomach and renal and mesenteric lymph nodes from all animals was conducted. No treatment-related effects were observed during the ophthalmoscopic and hematologic evaluations conducted during this study. Evaluation of other parameters revealed treatment-related effects, primarily involving the kidney and forestomach, at dose levels of 3.8 mg/kg/day or greater and based on this information the report describes the No Effect Level (NOEL) for these effects. Following assessment of the study data and the observed effects, the following No Adverse Effect Levels (NOAELs) are derived.

The progression from preneoplastic lesions to tumours in the kidney and forestomach of the test substance-treated rats occurs through nongenetic mechanisms. For nongenetic mechanisms, no tumours attributable to the compound would be expected at or below the no-adverse-effect level (NOAEL) for preneoplastic toxicity. During the blinded histopathologic examination of kidneys in this tumorigenicity study, the NOAEL for renal tumour formation in males was 3.8 mg/kg bw/day based on the increase in combined incidence of renal adenomas and carcinomas in males at 15 mg/kg bw/day compared to controls. In females, the NOAEL for renal tumour formation was 15 mg/kg bw/day with an increased combined incidence of adenomas and carcinomas observed only at the top dose of 175 mg/kg bw/day. The NOAEL for preneoplastic findings in the kidney (hyperplasia) was 1.8 mg/kg/day based on statistically significant increases in kidney weights (absolute weight for females and relative weight for males) at the interim sacrifice and an increased incidence of tubular hyperplasia in males

In the forestomach of animals in this study, tumours and preneoplastic lesions (e.g. squamous hyperplasia and hyperkeratosis) were observed and were associated with the irritative properties of the test substance at dose levels of 3.8 and higher. The NOAEL for effects in the forestomach was 1.8 mg/kg bw/day based on an increased incidence of hyperplasia, chronic irritation and papillomas at 3.8 mg/kg/day and above when compared to controls. 

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

1.8 mg/kg bw/day

Study duration:

chronic

Species:

rat

Quality of whole database:

GLP compliant EPA OPP 83-2 study

System:

urinary

Organ:

kidney

Carcinogenicity: via inhalation route

Endpoint conclusion

Endpoint conclusion:

no study available

Carcinogenicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Mode of Action Analysis / Human Relevance Framework

The progression from preneoplastic lesions to tumors in the kidney and forestomach of the test substance-treated rats apparently occurs through nongenetic mechanisms. For nongenetic mechanisms, no tumours attributable to the compound would be expected at or below the no-effect level (NOAEL) for preneoplastic toxicity. During the blinded histopathologic examination of kidneys in this tumorigenicity study, renal tumours and preneoplastic tubular epithelial hyperplasia were observed at dose levels of 15 and 175 mg/kg/day. No treatment related renal tumors were observed at the NOEL for the preneoplastic hyperplasia, 1.8 mg/kg/day.

In the forestomach of animals in this study, tumours and preneoplastic lesions (e.g. squamous hyperplasia and hyperkeratosis) were observed and were associated with the irritative properties of the test substance at dose levels of 3.8, 15 and 175 mg/kg/day. The NOAEL for irritation was 1.8 mg/kg/day. In the absence of the irritation-related, preneoplastic lesions, no treatment related tumours were observed in the forestomach at 1.8 mg/kg/day.

Justification for classification or non-classification

Based on the available information the substance is classified as carcinogenic category 2, H351: Suspected of causing cancer, in accordance with EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation No. 1272/2008.

Additional information

EPA OPP 83-2 - Wilson 1989

This tumorigenicity study in Fischer 344 rats with the test substance was conducted to determine the no-effect levels (NOEL) for preneoplastic and neoplastic lesions which had been observed in the kidney and forestomach of rats in a previous tumorigenicity study with this material. In this study, four groups of 65 rats per sex received the test substance in the diet at constant dose levels of 1.8, 3.8, 15 and 175 mg/kg/day (measured doses for the low and low-mid dose groups, corrected for average binding of the test substance to the feed. Nominal doses for the mid-high and high dose group, because of minimal influence of binding at these dose levels). A fifth group of 65 animals per sex served as the control group. Parameters evaluated included observations for overt toxicity, moribundity and mortality, ophthalmology, body weight and food consumption. Haematology, clinical chemistry and urinalysis evaluations were conducted routinely during the study. An interim necropsy of 10 rats per sex per group was conducted at one year. Based on survival criteria, all surviving high dose males were terminated during month 23 of the study and all surviving males in the remaining dose groups were terminated during month 26. Surviving females in all groups were terminated during month 29. At the one year and terminal necropsies, selected organs were weighed. Histopathologic examination of the kidneys, stomach and renal and mesenteric lymph nodes from all animals was conducted. No treatment-related effects were observed during the ophthalmoscopic and hematologic evaluations conducted during this study. Evaluation of other parameters revealed treatment-related effects, primarily involving the kidney and forestomach, at dose levels of 3.8 mg/kg/day or greater and based on this information the report describes the No Effect Level (NOEL) for these effects. Following assessment of the study data and the observed effects, the following No Adverse Effect Levels (NOAELs) are derived.

The progression from preneoplastic lesions to tumours in the kidney and forestomach of the test substance-treated rats occurs through nongenetic mechanisms. For nongenetic mechanisms, no tumours attributable to the compound would be expected at or below the no-adverse-effect level (NOAEL) for preneoplastic toxicity. During the blinded histopathologic examination of kidneys in this tumorigenicity study, the NOAEL for renal tumour formation in males was 3.8 mg/kg bw/day based on the increase in combined incidence of renal adenomas and carcinomas in males at 15 mg/kg bw/day compared to controls. In females, the NOAEL for renal tumour formation was 15 mg/kg bw/day with an increased combined incidence of adenomas and carcinomas observed only at the top dose of 175 mg/kg bw/day. The NOAEL for preneoplastic findings in the kidney (hyperplasia) was 1.8 mg/kg/day based on statistically significant increases in kidney weights (absolute weight for females and relative weight for males) at the interim sacrifice and an increased incidence of tubular hyperplasia in males

In the forestomach of animals in this study, tumours and preneoplastic lesions (e.g. squamous hyperplasia and hyperkeratosis) were observed and were associated with the irritative properties of the test substance at dose levels of 3.8 and higher. The NOAEL for effects in the forestomach was 1.8 mg/kg bw/day based on an increased incidence of hyperplasia, chronic irritation and papillomas at 3.8 mg/kg/day and above when compared to controls.