diuron (ISO); 3-(3,4-dichlorophenyl)-1,1-dimethylurea

Carcinogenicity

Description of key information

Chronic toxicity/carcinogenicity studies are available to access the carcinogenic potential of Diuron.

The NOAEL for carcinogenicity was 1.0 mg/kg bw/day for males and 1.7 mg/kg bw/day for females (25 ppm), the lowest dose tested, based on increased incidences of urothelial hyperplasia in urinary bladder and renal pelvis of degree 2 at 10 or 17 mg/kg bw/day in males and females, respectively (250 ppm) (Schmidt, 1985, rat).

The LOAEL for chronic toxicity was 1.0 mg/kg bw/day for males and 1.7 mg/kg bw/day for females (25 ppm), based on haemosiderin deposition in the spleen of males and anaemia and increased spleen weights in females, respectively. No NOAEL could be established.

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:

01 Sep 1981 - 09 Sep 1983

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

other: Expert statement

Remarks:

Assessment of tumour responses

Reason / purpose for cross-reference:

other: Reference to amendment 1 and 2 of the final study report

Qualifier:

according to guideline

Guideline:

other: EPA 83-1

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

Version / remarks:

Not specified in study report.
Deviations according to current version adopted in 2018.

Deviations:

yes

Remarks:

Some currently required parameters (OECD TG 453, v2018) regarding clinical chemistry, urinalysis, and organ weights were not measured. These deviations do not affect the final outcome of the study.

GLP compliance:

yes

Species:

rat

Strain:

Wistar

Details on species / strain selection:

The study was conducted with rats, a species recommended for chronic toxicological/carcinogenicity studies in the respective test guidelines.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: BOR: WISW (SPF Cpb), SPF bred
- Source: Winkelmann, Borchen, Germany
- Age at study initiation: 6 - 7 weeks
- Mean body weight at study initiation: males: 85 g, female: 84 g
- Housing: conventionally in Makrolon cages type II, on dust-free wood granules, one animal per cage
- Diet: powdered feed, Altromin 1321 (Altromin GmbH, Lage, Germany), ad libitum
- Water: tap water in drinking water quality, ad libitum
- Acclimation period: one week

DETAILS OF FOOD AND WATER QUALITY: Diet and drinking water were analytically examined.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22
- Humidity (%): 50
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 01 Sep 1981 To: 09 Sep 1983

Route of administration:

oral: feed

Vehicle:

other: diet

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Mixing appropriate amounts with (Type of food): powdered feed, Altromin 1321 (Altromin GmbH, Lage, Germany)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The substance sample`s stability and homogeneity in the administration medium over the period of use was checked by analysis before start of the study. The content in the feed was regularly determined by analysis during the study.
The diet mixtures were analysed. After extracting the diet containing Diuron the concentration was measured by means of HPLC.
The feed mixtures were stable over the period of use and homogeneous mixtures were produced.
The test substance content determined by analysis did not differ from the nominal content to any great degree.

Duration of treatment / exposure:

12 months (interim sacrifice)
24 months (terminal sacrifice)

Frequency of treatment:

daily

Post exposure period:

none

Dose / conc.:

25 ppm (nominal)

Remarks:

actual test substance intake: males: 1.0 mg/kg bw/day, females: 1.7 mg/kg bw/day

Dose / conc.:

250 ppm (nominal)

Remarks:

actual test substance intake: males: 10 mg/kg bw/day, females: 17 mg/kg bw/day

Dose / conc.:

2 500 ppm (nominal)

Remarks:

actual test substance intake: males: 111 mg/kg bw/day, females: 203 mg/kg bw/day

No. of animals per sex per dose:

10 (interim sacrifice)
50 (terminal sacrifice)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The doses were based on a previous chronic feed study with rats.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily during the week, once daily on weekends and public holidays
- Cage side observations such as any alterations, clinicals signs and incidents were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once a week

BODY WEIGHT: Yes
- Time schedule for examinations: before study initiation, then weekly up to and including week 27, followed by 14-day intervals from week 28 to 104

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
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

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

OPHTHALMOSCOPIC EXAMINATION: No
- No detailed examinations performed.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: After 6, 12, and 18 months of dosing, and at the end of the dosing period after 24 months.
- Anaesthetic used for blood collection: Yes (diethyl ether). Except from blood samples for glucose concentration in plasma. These were obtained without anaesthesia.
- How many animals: 10 animals/sex/dose
- Parameters checked: Haematocrit, haemoglobin, red and white blood cell count, MCV, MCH, MCHC, platelets, differential white blood cell count
Reticulocytes from blood smears counted after 12 and 24 months, thromboplastin time measured after 24 months only.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: After 6, 12, and 18 months of dosing, and at the end of the dosing period after 24 months.
- How many animals: 10 animals/sex/dose
- Parameters checked: Total protein, glucose, cholesterol, urea, creatinine, total bilirubin, alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase

URINALYSIS: Yes
- Time schedule for collection of urine: After 6, 12, and 18 months of dosing, and at the end of the dosing period after 24 months.
- How many animals: 10 animals/sex/dose
- Animals fasted: Yes
- Parameters checked: semi-quantitative: glucose, blood, protein, bilirubin, ketone bodies, pH, urobilinogen, sediment; quantitative: protein

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- Organ weights from all animals at interim (12 months) and terminal (24 months) sacrifice: adrenals, testes, heart, kidneys, liver, lungs, spleen

HISTOPATHOLOGY: Yes
- organs preserved from all animals: adrenals, aorta, brain, epididymis, eyes, heart, kidneys, larynx, large intestine (caecum, colon, rectum), liver, lungs, lymph nodes (mesenteric and cervical), nervus ischiadicus, pancreas, pituitary, prostate, salivary glands, seminal vesicles, skeletal muscle, small intestine (duodenum, jejunum, ileum), spinal cord, spleen, stomach, testes, thymus, thyroids with parathyroid tissue, trachea, urinary bladder, uterus, femur, oesophagus, ovaries, sternum and all tissues showing gross changes

Histopathological findings of the urinary tract
Urothelial hyperplasias were graded by the following criteria:
Degree 1: approximately 4 - 6 layers of cells with normal structure
Degree 2: approximately 6 - 9 layers of cells and clear predominance of small cells
Degree 3: more than 9 layers of cells and barely apparent or absent covering layer, often nodular proliferation of urothelia with sinuate arrangement of basal membrane
Degree 1 hyperplasias were considered as physiological on the strength of the laboratory's experience with untreated animals of this strain and their occurence in control animals in the present study. Degree 2 and above was considered adverse.

Other examinations:

- Tissue sections of the spleen and bone marrow (femur) were also morphometrically examined.
- All animals that died or were sacrificed during the study were dissected and grossly examined as well.
- Additional examinations were performed after revision of guideline data requirements and addressed in subsequent amendments:
Rossberg, V.M. & Wirnitzer, U. (1994): Addendum to Bayer-Report 13962
includes new weight curves, body weight tables, recalculated feed intake data, neoplasia incidences
Rossberg, V.M. & Eiben, R. (1997): Second amendment to Bayer-Report 13962
includes mammary gland examination

Statistics:

Arithmetic group means and standard deviation were calculated. Statistical significance was assessed with U-test, Mann & Whitney and Wilcoxon.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

In some cases, reddish discoloured urine was noted, particularly in males dosed at 2500 ppm in the second half of the study. This finding might be attributed to the urothelial alterations which occurred in this group.
No further significant differences in behaviour or appearance were noted between the treated and control groups.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

An overall low mortality rate was observed. 22 of 200 males and 34 of 200 females died. Survival was not related to dose. Briefly, at the end of the study period 2, 8, 5, and 7 males and 11, 9, 3, and 11 females died or were sacrificed moribund in the control, 25, 250, and 2500 ppm group, respectively.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weight and body weight gain were not adversely affected in male and female rats dosed at 25 ppm.
The mean body weight of males at 250 and 2500 ppm was lowered compared to controls by 5 – 10% and 10 – 20%, respectively.
The mean body weight of females was 10 – 20% lower than controls in the 2500 ppm group. No effect on body weight was noted at 250 ppm.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption was not adversely affected in males and females of all dosed groups as compared to controls.
However, in relation to body weight, top dose animals of both sexes consumed approximately 20% more food, thus, actual doses were higher than planned by the nominal dose levels.

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

Animals of both sexes of all dose groups consumed about the same amount of food as the concurrent control group, i.e. 15 - 16 g food/animal/day.
Additional evaluation of the data, as reported in the first amendment to the study report, revealed a treatment-related decrease of food efficiency for male and female animals in the 2500 ppm dose group (males: 2.79, 2.81, 2.72, and 2.41; females: 1.45, 1.60, 1.56, and 1.10, with ascending dose, respectively) (see also Table 1 in "Any other information on results incl. tables").

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Statistically significant alterations in diverse blood parameters were noted from 6 months on until the end of the study. Briefly, higher leucocyte counts and changes in various red blood parameters were observed:
Mean leucocyte counts were increased in top dose animals of both sexes and male animals of the mid dose.
Red blood cell counts were decreased in males of the mid and top dose and in females at all dose levels.
Haemoglobin concentration was decreased in mid and top dose animals of both sexes.
Haematocrit values were decreased in top dose males and mid and top dose females.
Mean corpuscular volume as well as mean corpuscular haemoglobin were increased in male animals of the top dose and in female animals of all dose levels. Mean corpuscular haemoglobin concentration was decreased in mid and top dose animals of both sexes after 6 and 18 months of exposure, but not at study termination.
Animals in the mid and top dose groups exhibited higher quantities of pathological erythrocyte forms, apparent in the stainability of the cell contents or in the cell form (e.g. polychromasia, Jolly bodies, Heinz bodies, anisocytosis, anulocytosis).
After 12 months exposure, reticulocyte counts were increased in mid and top dose animals of both sexes. At study termination, reticulocyte counts were increased in male animals of the top dose only , but in female animals of all dose levels.
Thrombocytes, thromboplastin time, and differential cell count were not significantly affected by treatment, with the exception of decreased thromboplastin time in 25 ppm females after 24 months. Because the thromboplastin time was not dose-dependently changed, this finding is regarded incidental and not related to treatment.
In general, females seemed to be more susceptible to Diuron than males.
The findings indicate a non-progressive haemolytic (hyperchromic) anaemia. The primary target for these effects on the blood were considered to be the erythrocytes. The increase in leucocyte count might be caused by an increase in haematopoiesis due to increased breakdown of erythrocytes. These effects are considered treatment-related and adverse.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Increased bilirubin concentrations and blood urea concentrations in top dose (2500 ppm) animals of both sexes, accompanied by lowered cholesterol levels in males were considered to be treatment related. However, since the values for bilirubin and cholesterol were within the physiological range, these effects were not considered adverse. The mean urea concentrations were outside or at the limit of physiological reference values and might be attributed to the urothelial morphological changes or increased amino acid catabolism.
High dose males and females revealed temporary slightly lower alanine aminotransferase and aspartate aminotransferase activities.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Higher blood/erythrocyte contents were noted in animals of the 2500 ppm dose group (males after 12 and 24 months, females after 18 and 24 months). Glucose, urobilinogen, protein excretion, ketone body and bilirubin contents, and pH values were not altered in all treatment groups compared to control.
Quantitative determinations of protein excretion in the urine did not reveal any abnormalities.
Diuron did not affect urinary parameters in male and female animals dosed at 25 and 250 ppm.

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):

Absolute and relative spleen weights were dose-dependently increased at interim and terminal sacrifice in males and females.
At terminal sacrifice, absolute and relative spleen weights were statistically significantly increased in males of the mid and top dose groups and females of all treated groups.
Statistically significantly increased relative liver weights were noted in males of the mid and top dose levels and females of all dose groups at end of the study.
Male rats showed increased relative kidney weights at the top dose, reaching statistical significance at interim and terminal sacrifice, however no similar results were obtained for females.
Relative weights of the other organs were occasionally altered, but with regard to the lower body weights clinically and toxicologically not relevant.
Diuron did not affect organ weights in males dosed at the low dose (25 ppm).

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Gross pathological changes at interim sacrifice after 12 months in males and females at 250 and 2500 ppm consisted of swollen/enlarged and black coloured spleens, dark colouration of livers in females at 2500 ppm and one case of hardened ovaries at top dose was noted.
In animals that died spontaneously or were sacrificed because of moribundity, neoplastic lesions were noted that may have caused death.
Towards the end of the study swelling and enlargement of spleens, black colouration of spleens were found for both sexes at 250
and 2500 ppm dose levels and in females at 25 ppm. Males of the top dose showed increased incidences of hardened urinary bladder wall.
The same changes (swelling/enlargement and black colouration of spleen) were observed in mid and top dose animals of both sexes and in females of the low dose group at terminal sacrifice after 24 months exposure. Hardness or thickening of the wall of the urinary bladder were observed in approximately 50% of male and 25% of female animals in the top dose groups.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

At interim sacrifice after 12 months of exposure, spleens in all treatment groups showed a positive Prussian blue-reaction (indicative for iron deposition). Further, haemosiderin accumulation appeared increased in treated animals of all dose groups and both sexes compared to control. Quantitative evaluation by morphometric examination revealed a clear increase of the percentage of the surface area of deposited haemosiderin particles in mid and top dose animals. This finding was much more pronounced in female than in male animals.
In the liver, a slightly increased positive Prussian blue-reaction was observed in males of the top dose and females of the mid and top dose when compared to control.
An increased positive Prussian blue-reaction was also observed in kidneys of male top dose animals.
There was an increase in urothelial hyperplasia in the renal pelvis and urinary bladder of treated animals (Table 6 and 7 in "Any other information on results incl tables"). In the renal pelvis, a non-adverse degree 1 urothelial hyperplasia was noted in control animals (males: 5/10, females: 1/10), low dose (males: 3/10, females: 2/10), mid dose (males: 6/10, females: 5/10), as well as in top dose animals (males: 5/10, females: 4/10). Urothelial hyperplasia of degree 2 and above was considered adverse and was not observed in any control animal at this necropsy time point. Degree 2 hyperplasia was observed at the low dose in 1/10 male and 1/10 female rats, and in 5/10 top dose males.
In the urinary bladder, a non-adverse degree 1 urothelial hyperplasia was noted in some control animals (males: 3/10, females: 0/10), low dose (males: 5/10, females: 3/10), mid dose (males: 4/10, females: 5/10), as well as in some top dose animals (males: 0/10, females: 5/10). Urothelial hyperplasia of degree 2 and above was considered adverse and was not observed in any control animal at this necropsy time point. Degree 2 hyperplasia was observed in mid dose males (1/10) and high dose males and females (2/10 and 1/10, respectively). Degree 3 hyperplasia was found in high dose animals of both sexes (males: 5/10, females: 2/10). In some cases, a papillary urothelial hyperplasia was observed (degree 2 in 1/10 male control, degree 3 with deep growth in 3/10 males and 1/10 females in the top dose group).
A high incidence of C cell hyperplasia in the thyroid was present in all examined animals of the control and top dose group, in particular in males. However, this finding is known as a strain-specific spontaneous alteration.
In the trachea, occasional slight inflammatory alterations were noted in males (4/10) and females (2/10) of the top dose. In control animals such alterations were not observed.
All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered incidental or spontaneous in origin and were typical in respect to type, incidence and severity for rats of this strain and age without any relation to treatment.

No clear conclusion on non-neoplastic alterations could be drawn from animals that died spontaneously or were sacrificed moribund (due to the low number of affected animals). As the findings in those animals were of normal type, the pathological findings described below for terminal sacrifice at 24 months are summarized for all animals intended for 2-year treatment, including the animals which died prematurely or were sacrificed.
At terminal sacrifice after 24 months of exposure, haemosiderin accumulation in the spleen was observed in almost all animals of all study groups by positive Prussian blue-reaction. Quantitative evaluation by morphometric examination revealed an increase of the percentage of the surface area of deposited haemosiderin particles starting at the lowest dose tested. This finding was more pronounced in female than in male animals, however, less pronounced in animals after 24 months exposure compared to animals after 12 months exposure. Hyperaemia in the spleen was observed in males (15/49) in the 2500 ppm group and fibrosis in mid and high dose males (3/50 and 18/49, respectively) and top dose females (17/50). Based on the findings of the red blood cell parameters, the findings in the spleen are considered treatment-related.
Histopathological findings in the liver consisted mainly of round cell infiltrates, hyperaemia, in some cases substantial bile duct proliferation (males: 40/50, 37/50, 27/50, 34/49; females: 16/48, 10/50, 15/50, 25/50, with ascending dose), necrosis, and vacuolar degeneration of the liver cells. These alterations were randomly distributed throughout all study groups and represent largely the result of aging. The number of animals with tumour infiltrates from differing primary tumours in other organs showed a virtually even distribution throughout the groups.
In the kidneys, histopathological findings mainly comprised cortical scars, calcium deposits, protein cylinders in tubuli and round cell infiltrates (known to be slightly more common in male rats). Almost all animals of all study groups showed hyperaemia, equally distributed throughout the groups without dose-response correlation.
An increased incidence of hyperplasia of the urothelium was found in the renal pelvis and urinary bladder of treated animals (Table 6 and 7 in "Any other information on results incl tables"). In the renal pelvis (Table 7), a non-adverse degree 1 urothelial hyperplasia was noted in control animals (males: 31/50, females: 20/48), low dose (males: 30/50, females: 22/50), mid dose (males: 18/50, females: 12/50), as well as in top dose animals (males: 3/47, females: 5/47). Urothelial hyperplasia of degree 2 was observed in control animals (males: 5/50, females: 3/48), low dose (males: 7/50, females: 3/50), mid dose (males: 25/50, females: 30/50), as well as in top dose animals (males: 23/47, females: 33/47). Degree 3 hyperplasia was observed in 1/50 male control animals, mid dose (males: 2/50, females: 4/50), and top dose animals (males: 17/47, females: 4/47). Papilloma were observed in 1/47 top dose males and carcinoma in 2/47 top dose males.
In the urinary bladder (Table 6), a non-adverse degree 1 urothelial hyperplasia was noted in control animals (males: 11/50, females: 10/48), low dose (males: 5/50, females: 7/49), mid dose (males: 15/50, females: 9/50), as well as in some top dose animals (males: 1/49, females: 4/50). Urothelial hyperplasia of degree 2 was observed in single control animals (males: 2/50, females: 1/48), mid dose (males: 1/50, females: 3/50), and top dose animals (males: 3/49, females: 17/50). Degree 3 hyperplasia was found in mid dose females (5/50) and top dose animals of both sexes (males: 10/49, females: 9/50). These findings were considered treatment-related.
In the thyroid, high incidences of C cell hyperplasia - a strain-specific spontaneous alteration - were noted particularly in the males, as was the case for the interim autopsy. The severity of the hyperplasia was usually slight.
The histological changes noted in trachea, larynx, and oesophagus, such as occasional round cell infiltrates, blood and/or detritus in the lumen, were findings largely attributable to the terminal stage of the animals.
Morphometric examination of the bone marrow revealed a dose-dependent increase of the percentage of the surface area of the haematopoietic (activated) bone marrow in treated animals when compared to controls. In mid dose and top dose animals of both sexes an activation of the bone marrow was noted, with a correlating decrease in the fat marrow proportion. This finding was considered treatment-related. Further, males at the interim sacrifice revealed an activated haematopoetic bone marrow already at 25 ppm. However, as the differences to controls were slight and no correlating fall in fat marrow proportion was observed at this dose, the biological significance of bone marrow activation at 25 ppm remains unclear.
Hyperkeratosis was observed in the forestomach (males: 1/50, 3/50, 3/50, 1/49; females: 4/48, 0/50, 2/50, 1/50, with ascending dose) and ulcers appeared occasionally in the glandular stomach (males: 0/50, 1/50, 0/50, 0/49; females: 2/48, 2/50, 1/50, 1/50, with ascending dose). These alterations were evenly distributed throughout the groups. Secondary tumour infiltrates were found in the area of the serosa in some animals.
All other findings in the cerebrum, cerebellum, pituitary, eye, salivary glands, neck lymph nodes, larynx, oesophagus, heart muscle, aorta, parathyroid, lungs, fore- and glandular stomach, pancreas, mesenteric lymph nodes, small and large intestine, adrenals, testicles, epididymis, prostate, seminal vesicle, uterus, and ovaries occurred either individually or were biologically equally distributed over control and treatment groups. They were considered incidental or spontaneous in origin and were typically expected in respect to type, incidence and severity for conventionally housed rats of this strain and age without any relation to treatment.
The number of animals with tumour infiltrates from differing primary tumours in other organs showed a virtually even distribution throughout all study groups.

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

After 12 months of exposure, one neoplastic alteration, namely a benign tumour of the ovary, was found in one top dose female (1/10).

In the following, observations from all animals intended for 2-year treatment are shown, including animals that died or were sacrificed moribund prior to study termination.

At terminal sacrifice after 24 months of exposure, increased incidences of malignant neoplasias, mainly of the urothelium (renal pelvis and urinary bladder), were observed at the top dose of 2500 ppm (Table 6 and 7 in "Any other information on results incl tables"): Urothelial neoplastic alterations in the renal pelvis (Table 7) were found in 2/47 top dose males, including one papilloma and two transitional epithelial carcinomas. No neoplasias in the renal pelvis were observed in females of any dose group.
In the urinary bladder (Table 6), transitional epithelial carcinoma was noted in one male of the control and mid dose group each (1/50 and 1/49, respectively). Further, one carcinoma was found in situ in one female animal of the mid dose group (1/50). The incidence for neoplasias was increased in top dose animals, where 67% of male and 22% of female animals (males: 33/49, females: 11/50) exhibited transitional epithelial carcinoma sometimes with partial hornification, compared to 2% of controls. In four cases of the top dose males, transitional epithelial carcinomas were present with portions revealing a differentiation from squamous epithelial carcinoma. Benign transitional epithelial papillomas were found in the urinary bladder in one female animal of the control group (1/48), two female animals of the mid dose group (2/50), and in some animals of the top dose group (male: 3/49, female 2/50). The distribution of the number of malignant and/or benign neoplastic alterations in the urinary bladder in males and females of the control group and 25 ppm and 250 ppm dose groups was inconspicuous. The other findings in the urinary bladder (largely round cell infiltrates (males and females), concrement, and detritus in the lumen (males)) were normal in type and distribution throughout the groups.
A re-evaluation (first addendum) of the malignant neoplasias of kidney and urinary organs in male rats, confirmed the high incidence of transitional epithelial carcinoma of the urinary bladder in animals treated with 2500 ppm. 33 cases were found in the top dose group as compared to 1 for the controls and other treatment groups.
The incidence and distribution of urothelial neoplasias (renal pelvis and urinary bladder) in the 2500 ppm dose group together with the increase in degree 2 and 3 hyperplasia in the mid and top dose groups was considered test-substance related and adverse.

The type and group distribution of the neoplasias of the sexual organs were inconspicuous for male animals of all dose groups (Leydig cell tumours with an incidence of 2% to 10% without apparent dose correlation, one mesothelioma).
In the ovaries, isolated benign and malignant neoplasias were found, in all cases one-sided. The incidence was maximum 2% in control, low dose, as well as top dose animals.
In the uterus (Table 8 in "Any other information on results incl. tables"), adenocarcinoma were found in 5/48 control, 5/50 low, and 5/50 mid dose females, which corresponds to an incidence of 10%. In high dose females adenocarcinomas were observed in 9/50 animals (18%). One little differentiated uterus carcinoma was observed in one top dose animal. Benign mucosal polyps were found in all study groups (control: 7/48, low dose: 7/50, mid dose: 6/50, top dose: 3/50), with the highest incidence of 15% in controls. Moreover, malignant neoplasias of various types (sarcomas and carcinomas) were found in single animals of the low, mid and high dose group at an incidence maximum of 4% apart from adenocarcinomas (fibromyoma: 1/50 low dose females; leiomyosarcoma: 1/50 low dose females; endometrium sarcoma: 2/50 high dose females and squamous epithelial carcinoma in 1/50 mid and 1/50 high dose females; please refer to Table 8).
In summary, the incidence of rat uterus adenocarcinomas was slightly increased in the top dose group compared to the control, low, and mid dose groups, while the number of benign neoplasias (polyps) was simultaneously lower in high dose females than in control, low, and mid dose females. The sum of polyps and adenocarcinoma was comparable among the groups (12, 12, 11 and 13 adenocarcinoma plus polyps in control, low, mid, and top dose females, respectively), and thus, no difference in the overall incidence of neoplasias in the rat uterus caused by Diuron was observed. Further, comparably high incidences of both adenocarcinomas and of other malignant neoplasias were found in the historical control data (please refer to Stropp, 2018, expert statement). Specifically adenocarcinomas of the uterus are subject to a high rate of spontaneous variation (spontaneous incidence of uterus’ adenocarcinoma 2% - 20%, mean approx. 8%). As the incidence of adenocarcinomas of the present study does not exceed the historical control data range, and the sum of polyps and adenocarcinoma in the rat uterus is comparable among the control and all treatment groups, a treatment related increase in rat uterus adenocarcinoma is not considered to be present.
A re-assessment of the historical control data (Stropp, 2018, expert statement) confirmed the high variation of incidences of adenocarcinomas in the uterus in this rat strain. 21 long-term/carcinogenicity studies, which were performed between Jan 1979 and Feb 1984, were re-evaluated. The incidence of uterus adenocarcinomas was 0 - 20% in the historical control database. In one long-term/carcinogenicity study of the historical control database, which was performed adjacent to the present study, 10 out of 50 control animals showed adenocarcinomas in the uterus. Therefore, the incidence of adenocarcinomas of all treatment groups in the Diuron study was within the range of the appropriate historical control data.
In addition, there is no evidence of any pre-neoplastic, neoplastic, or non-neoplastic uterus effect at the 12 months interim sacrifice as well as at the end of the study after 24 months.

The relatively low rate of fibroadenomas of the mammary gland observed in the present study was re-evaluated. A systematically examination (second addendum) revealed no treatment-related effects at any dose level.

All other neoplastic findings occurred either sporadically or were biologically equally distributed over control and treated animals. They were considered incidental and spontaneous in origin and were typically expected in respect to type, incidence and severity for conventionally housed rats of this strain and age without any relation to treatment.

The earliest time at which a neoplasia was detected by histology in one of the animals was after about 16 months of the study in the case of the males (carcinoma of pituitary, dose group 250 ppm), and in the case of the females after about 12 months (adenocarcinoma of mamma, 25 ppm dose group). The majority of the neoplasias were found in the animals sacrificed after 24 months (end of study) and in the animals which died or were sacrificed towards the end of the study.

Key result

Dose descriptor:

NOAEL

Remarks:

carcinogenicity

Effect level:

25 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: neoplastic

Remarks on result:

other: actual test substance intake: males: 1.0 mg/kg bw/day, females: 1.7 mg/kg bw/day

Key result

Dose descriptor:

LOAEL

Remarks:

carcinogenicity

Effect level:

250 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: neoplastic

Remarks on result:

other: actual test substance intake: males: 10 mg/kg bw/day, females: 17 mg/kg bw/day

Key result

Dose descriptor:

LOAEL

Remarks:

chronic toxicity

Effect level:

25 ppm

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other: no NOAEL could be established

Remarks:

actual test substance intake: males: 1.0 mg/kg bw/day

Key result

Dose descriptor:

LOAEL

Remarks:

chronic toxicity

Effect level:

25 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

haematology

organ weights and organ / body weight ratios

Remarks on result:

other: no NOAEL could be established

Remarks:

actual test substance intake: females: 1.7 mg/kg bw/day

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

25 ppm

System:

other: urinary tract and erythrocytes

Organ:

other: spleen/erythrocytes and urinary tract

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Table 1: Summary table of a combined chronic dietary toxicity and carcinogenicity study in rats with Diuron compared to controls (after 24 months)

	Males				Females
Dose level (ppm)	0	25	250	2500	0	25	250	2500
No. of animals examined	50	50	50	50	50	50	50	50
No. of mortalities	2	8	5	7	11	9	3	11
Clinical signs (“bloody urine”)	1	0	2	28	7	6	2	7
Body weight (g) at termination	418	415	401*	357**	249	260	257	215**
Body weight gain (0-103 weeks) (g)	332	330	316	272	165	178	173	131
Food consumption (g/d)	16	16	16	16	16	15	15	16
Food efficiency	2.79	2.81	2.72	2.41	1.45	1.60	1.56	1.10
Compound intake (mg/kg bw/d)	0	1.02	10.5	111	0	1.69	16.9	203

* p < 0.05, ** p < 0.01

Table 2: Incidences of neoplasia after 24 months (end of the study) and from animals dying spontaneously or moribund and sacrificed

Dose (ppm)	Males				Females
	0	25	250	2500	0	25	250	2500
Animals examined	50	50	50	49	48	50	50	50
Total neoplasias	21	16	19	57	31	31	31	39
incl.benign	18	12	11	18	23	20	19	19
malignant	3	4	8	39	8	11	12	29
Number animals with neoplasias	19	14	16	41	26	27	22	29
Number animals with multiple neoplasias	2	2	3	13	4	4	7	9
Number animals with only benign neoplasias	17	10	8	4	18	16	10	4
Number animals with only malignant neoplasias	2	2	6	25	6	10	6	19
Number animals with benign and malignant neoplasias	0	2	2	12	2	1	6	6

Table 3: Absolute and relative spleen weights, autopsy after 12 and 24 months

Diuron concentration ppm	12 months		24 months
	male / female (mg), abs	male / female (mg/100 g)	male / female (mg), abs	male / female (mg/100 g)
0	598/407	155/192	791/493	188/200
25	724**/460	165**/212	841/717**	201/277**
250	707**/634**	190**/300	877**/797**	218**/311**
2500	1329**/1162**	375**/615**	1514**/1265**	427**/570**

** p < 0.01

Table 4: Results of haematological examination (group means after 24 months)

Dose ppm	LEU giga/L	ERY tera/L	HGB g/L	MCV fL	THROM giga/L	RETI 0/00	TP SEC	HCT L/L	MCH pg	MCHC g/L
Male
0	6.7	8.24	155	57	736	17	27.8	0.47	18.8	332
25	6.1	8.14	158	58	774	22	27.1	0.47	19.5	336
250	8.6**	7.90	151	59	893	21	26.8	0.46	19.1	326
2500	11.3	6.82**	143*	65**	896	72**	29.0	0.44	20.9**	325
Female
0	7.3	7.69	155	59	836	17	25.5	0.45	20.2	344
25	7.3	6.86*	144	63*	884	23*	23.8*	0.43	21.2*	336*
250	6.1	6.40**	141**	65**	828	30**	26.1	0.42**	22.1*	338
2500	13.4**	5.95**	138**	67**	901	62**	25.6	0.40**	23.2**	346

LEU - leucoytes, ERY - erythrocytes, HGB - haemoglobin, MCV - mean corpuscular volume, THROM - thrombocytes (platelets)

RETI - reticulocytes, TP - prothrombin time, HCT - haematocrit, MCH - mean corpuscular haemoglobin, MCHC - mean corpuscular haemoglobin concentration; * p < 0.05, ** p < 0.01

Table 5: Clinical chemical parameters in blood and protein concentration in urine means after 24 months

Dose/ppm	AP U/L	ASAT U/L	ALAT U/L	BILI µmol/L	PROT g/L	UREA mmol/L	CREA µmol/L	PROT/U g/L	CHOL mmol/L	GLUC mmol/L
Male
0	187	36.0	46.1	2.8	69.0	5.68	55	2.42	3.83	5.62
25	170	37.3	45.0	2.6	68.9	5.88	57*	3.62	3.49	5.67
250	154	35.4	41.7	3.0	69.3	6.44	64*	3.43	3.97	5.59
2500	188	37.8	39.4	4.2**	65.0	7.68**	60	1.95	2.66**	4.75*
Female
0	165	75.3	61.3	3.3	71.1	6.86	62	0.64	2.38	5.21
25	138	49.6*	48.1	3.2	69.7	7.07	62	0.66	2.32	4.84
250	140	58.2	48.9	2.6*	71.7	7.65*	55	0.66	2.20	4.65
2500	166	43.2**	52.1	4.6**	71.0	8.49**	57	0.84	2.69	5.03

AP - alkaline phosphatase, ASAT - aspartate aminotransferase, ALAT - alanine aminotransferase, BILI - bilirubin, PROT - protein, UREA - urea, CREA - creatinine, PROT/U - protein urine quantitative, CHOL - cholesterol, GLUC - glucose

* p < 0.05, ** p < 0.01

Table 6: Histopathological findings of the urinary bladder in rats administered Diuron for 12 or 24 months

Sex		Males				Females
Dose                                        [ppm]		0	25	250	2500	0	25	250	2500
[mg/kg bw/day]		0	1.0	10	111	0	1.7	17	203
Necropsy after 12 months exposure
Urinary bladder	[N]	10	10	10	10	10	10	10	10
Urothelial hyperplasia	[n]
Degree 1		3	5	4	0	0	3	5	5
Degree 2		0	0	1	2	0	0	0	1
Degree 3		0	0	0	5	0	0	0	2
Degree 3 + atypia		0	0	0	0	0	0	0	0
Papillary urothelial hyperplasia	[n]
Degree 1		0	0	0	0	0	0	0	0
Degree 2		1	0	0	0	0	0	0	0
Degree 3 + deep growth		0	0	0	3	0	0	0	1
Neoplasias	[n]	0	0	0	0	0	0	0	0
Concrement in lumen	[n]	4	7	0	0	0	0	0	0
Detritus in lumen	[n]	4	1	5	2	0	1	1	0
Round cell infiltrates	[n]	0	2	0	0	0	0	0	0
Necropsy after 24 months exposure (including dead/moribund sacrificed animals)
Urinary bladder	[N]	50	50	49	49	48	49	50	50
Urothelial hyperplasia	[n]
Degree 1		11	5	15	1	10	7	9	4
Degree 2		2	0	1	3	1	0	3	17
Degree 3		0	0	0	10	0	0	5	9
Degree 3 + atypia		0	0	0	1	0	0	0	0
Papillary urothelial hyperplasia	[n]	0	0	0	0	0	0	0	0
Papilloma	[n]	0	0	0	3	1	0	2	2
Carcinoma in situ	[n]	0	0	0	0	0	0	1	0
Carcinoma	[n]	1	0	1	33	0	0	0	11
I*		1	0	1	29	0	0	0	11
II**		0	0	0	4	0	0	0	0
Concrement in lumen	[n]	4	7	8	5	1	0	0	0
Blood in lumen	[n]	2	0	0	0	0	0	0	0
Detritus in lumen	[n]	9	4	8	1	2	0	1	2
Round cell infiltrates	[n]	17	16	13	11	7	9	12	3
Tumor infiltrates	[n]	0	0	2	0	0	0	0	0

[N] = animals evaluated; [n] = animals affected

* transitional epithelial carcinoma (partly with hornification)

** transitional epithelial carcinoma with portions revealing differentiation from squamous epithelial carcinoma

Table 7: Histopathological findings of the renal pelvis in rats administered Diuron for 12 or 24 months

Sex		Males				Females
Dose                                        [ppm]		0	25	250	2500	0	25	250	2500
[mg/kg bw/day]		0	1.0	10	111	0	1.7	17	203
Necropsy after 12 months exposure
Renal pelvis	[N]	10	10	10	10	10	10	10	10
Urothelial hyperplasia	[n]
Degree 1		5	3	6	5	1	2	5	4
Degree 2		0	1	0	5	0	1	0	0
Degree 3		0	0	0	0	0	0	0	0
Neoplasias	[n]	0	0	0	0	0	0	0	0
Detritus	[n]	0	0	0	1	1	0	0	0
Blood	[n]	0	0	0	0	1	0	0	0
Necropsy after 24 months exposure (including dead/moribund sacrificed animals)
Renal pelvis	[N]	50	50	50	47	48	50	50	47
Urothelial hyperplasia	[n]
Degree 1		31	30	18	3	20	22	12	5
Degree 2		5	7	25	23	3	3	30	33
Degree 3		1	0	2	17	0	0	4	4
Papilloma	[n]	0	0	0	1	0	0	0	0
Carcinoma	[n]	0	0	0	2	0	0	0	0
Detritus	[n]	4	2	1	0	1	2	3	1
Haemorrhage	[n]	2	0	0	0	0	0	0	0
Round cell infiltrates	[n]	10	4	3	3	1	4	2	1
Bacteria	[n]	0	0	0	1	0	0	0	0
Calcium deposits	[n]	0	1	1	2	4	7	7	3
Expanded	[n]	1	0	2	2	2	4	0	2
Hydronephrosis	[n]	0	0	0	0	0	0	0	1
Tumour infiltrates	[n]	0	0	1	1	0	0	0	0

[N] = animals evaluated; [n] = animals affected

Table 8: Histopathological findings of the uterus in female rats administered Diuron for 12 or 24 months

Dose                            [ppm]	0	25	250	2500
[mg/kg bw/day]	0	1.7	17	203
Necropsy after 24 months exposure (including dead/moribund sacrificed animals)
Animals examined	48	50	50	50
Neoplastic observations in the uterus (all)
Uterus adenomacarcinoma	5/48 (10.4%)	5/50 (10%)	5/50 (10%)	10*/50 (20%)
Rat uterus polyps	7/48 (15%)	7/50 (14%)	6/50 (12%)	3/50 (6%)
Sum of adenocarcinoma and uterus polyps	12	12	11	13
Fibromyoma	0	1	0	0
Leiomvosarcoma	0	1	0	0
Endometrium sarcoma	0	0	0	2
Squamous epithelial carcinoma	0	0	1	1
Non-neoplastic observations in the uterus (all)
Animals examined	48	50	50	50
Glandular-cystic hyperplasia	5	5	2	7
Dilated	0	1	0	0
Hyperemia	1	0	0	2
Hemorrhage	0	1	0	0
Hemosiderin storage	0	1	0	0
Haematometra	0	1	0	0
Endometritis	0	0	0	1
Proliferated mucosa	0	1	1	0
Necropsy after 12 months exposure
Animals examined	10	1	2	10
Neoplastic changes in uterus	0	0	0	0
Non-neoplastic observations after 12 months of exposure (all)
Animals examined	10	1	2	10
Endometritis	1	0	0	1
Cavity dilated	0	1	1	0
Atrophic mucosa	0	1	1	0
Glandular cystic hyperplasia	0	0	0	1
Detritus in lumen	0	0	1	0
Hyperemia	0	0	2	0
Hemosiderin storage	0	1	0	0

*9 adeno-carcinoma + 1 little differentiated carcinoma

Executive summary:

In the present chronic toxicity/carcinogenicity study according to EPA 83-1 guidelines (comparable to OECD 453) rats were administered 25, 250, or 2500 ppm of Diuron in the feed for 24 months. The mortality rates were low overall and did not correlate with doses. The intake of 2500 ppm resulted in an increased incidence of neoplasia of the urothelium in both sexes. Considering severe hyperplasia as a possible early stage of neoplastic alterations, the increased incidence of urothelial hyperplasia in urinary bladder and renal pelvis of degree 2 at 250 ppm was considered as LOAEL in regard to carcinogenicity. Thus, a NOAEL of 25 ppm is derived for carcinogenicity, corresponding to an actual test substance intake of 1.0 mg/kg bw/day for males and 1.7 mg/kg bw/day for females. As the incidence of adenocarcinoma in the rat uterus did not exceed the historical control data range, and the sum of polyps and adenocarcinoma in the rat uterus was comparable among the control and all treatment groups, the occurrence of uterus adenocarcinoma is not considered as treatment related to Diuron.

Chronic Diuron-treatment further resulted in non-progressive haemolytic (hyperchromic) anaemia in male and female rats. In females, anaemia and increased spleen weights were observed starting at the lowest dose applied of 25 ppm. In males, marginally increased haemosiderin deposits were observed in the spleen at the same concentration, which indicated destruction of erythrocytes. Thus, in both sexes, no NOAEL was established for chronic toxicity. The lowest dose applied of 25 ppm was considered as LOAEL in both sexes. The main target organs for chronic toxicity following chronic exposure to Diuron were the urothelium of the urinary tract and erythrocytes/spleen.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

1 mg/kg bw/day

Study duration:

chronic

Species:

rat

System:

urinary

Organ:

bladder

Carcinogenicity: via inhalation route

Endpoint conclusion

Endpoint conclusion:

no study available

Carcinogenicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the available data on carcinogenicity (neoplasias of the urothelium) Diuron needs to be classified:

DSD: Cat 3, R40

CLP: Carc. Cat. 2, H351

Additional information

The carcinogenic potential of Diuron was tested in a reliable 2-year chronic toxicity/carcinogenicity study in rats conducted according to EPA 83-1 guideline (comparable to OECD 453) (Schmidt, 1985).

Wistar rats were randomly assigned to 4 groups of 50 males and 50 females each. Ten additional males and females per group were included for interim sacrifice at 12 months. Three dose groups were administered 25, 250, or 2500 ppm of Diuron in the feed for 12 or 24 months. This corresponded to an actual test substance intake of 1.0, 10, and 111 mg/kg bw/day in males, and 1.7, 17, and 203 mg/kg bw/day in females.

A low mortality rate was observed among all test groups, which was unaffected by treatment. Reddish discoloured urine was noted particularly in males dosed at 2500 ppm, which might be attributed to urothelial alterations. Body weight and body weight gain were decreased in males starting at 250 ppm and in females of the high dose group. Further, food efficiency was decreased in male and female animals in the 2500 ppm dose group. Haematological examinations revealed that chronic Diuron treatment resulted in non-progressive haemolytic (hyperchromic) anaemia in male and female rats. In females, anaemia and increased spleen weights were observed starting at the lowest dose applied of 25 ppm. In males, marginally increased haemosiderin deposits were observed in the spleen at the same concentration, which is indicative for destruction of erythrocytes. Based on the available data no NOAEL was established for chronic toxicity. The lowest dose applied of 25 ppm was considered as LOAEL for chronic toxicity in both sexes based on haemosiderin deposition in the spleen of males and anaemia/increased spleen weights in females.

Non-neoplastic findings included fibrosis of the spleen in males at 250 and 2500 ppm and in females at 2500 ppm, which might have occurred secondary to haematotoxicity.

Histological examinations of the uterus revealed that the incidence of rat uterus adenocarcinomas was slightly increased in the high dose group compared to the control, low, and mid dose groups, while the number of benign neoplasias (polyps) was simultaneously lower in high dose females than in control, low, and mid dose females. The sum of polyps and adenocarcinoma was comparable among the groups (12/48, 12/50, 11/50 and 13/50 adenocarcinoma plus polyps in control, low, mid, and high dose females, respectively), and thus, no difference in the overall incidence of neoplasias in the rat uterus caused by Diuron was observed. Further, comparably high incidences of adenocarcinomas were found in the historical control data. Adenocarcinomas of the uterus were subject to a high rate of spontaneous variation (spontaneous incidence of uterus’ adenocarcinoma 2% - 20%). As the incidence of adenocarcinomas in the rat uterus did not exceed the historical control data range (10.4, 10 , 10, and 20% for control and Diuron treatment groups with ascending dose), and the sum of polyps and adenocarcinoma in the rat uterus was comparable among the control and all treatment groups, the occurrence of uterus adenocarcinoma is rather considered as spontaneous and not related to treatment with Diuron.

A re-assessment of the historical control data (expert statement by Stropp, 2018 provided in the technical dossier) confirmed the high variation of incidences of adenocarcinomas in the uterus in this rat strain. 21 long-term/carcinogenicity studies, which were performed between Jan 1979 and Feb 1984, were re-evaluated. The incidence of uterus adenocarcinomas was 0 - 20% in the historical control database. In one long-term/carcinogenicity study of the historical control database, which was performed adjacent to the present study, 10 out of 50 control animals showed adenocarcinomas in the uterus. Therefore, the incidence of adenocarcinomas of all treatment groups in the Diuron study (10 - 20%) was within the range of the appropriate historical control data.

Moreover, there is no evidence of any pre-neoplastic, neoplastic, or non-neoplastic uterus effect at the 12 months interim sacrifice as well as at the end of the study after 24 months.

The intake of 2500 ppm Diuron resulted in an increased incidence of neoplasia of the urothelium in both sexes (67% and 22% for males and females, respectively). In comparison, maximum incidences of 2% for these lesions were found at lower dose levels. Considering severe hyperplasia as a possible early stage of neoplastic alterations, the increased incidence of urothelial hyperplasia in urinary bladder and renal pelvis of degree 2 at 250 ppm was considered as LOAEL in regard to carcinogenicity. Thus, a NOAEL of 25 ppm is derived for carcinogenicity, corresponding to an actual test substance intake of 1.0 mg/kg bw/day for males and 1.7 mg/kg bw/day for females.

In conclusion, the main target organs for carcinogenicity and chronic toxicity following chronic exposure to Diuron were the urothelium of the urinary tract and the erythrocytes together with the spleen, respectively. Based on the increased incidence of urothelial hyperplasia at the mid dose and the increased incidence of malignant neoplasias of the urothelium of the urinary bladder at the top dose, Diuron requires classification and labelling according to the criteria of 1272/2008/EC (CLP) as Carc. Cat 2, H351.

 

In mice, the carcinogenic potential of Diuron was tested in a 2-year combined chronic toxicity and carcinogenicity study conducted according to OECD 453 (Eiben, 1990).

NMRI mice (4 groups of 60 males and 60 females) were used in the study. Three dose groups received 25, 250 or 2500 ppm Diuron in the feed. The concurrent control group received standard, un-treated feed. The overall study period was 24 months with an interim sacrifice after 12 months on 10 animals per sex and per group.

Clinical symptoms, mortality and food consumption were not affected by treatment. A slight reduction in body weight was noted at the highest tested dose. Further, higher leucocyte counts and signs of increased erythrocyte decomposition were observed in the liver, spleen and kidneys indicating the presence of treatment-related anaemia. Liver toxicity was further indicated by altered enzyme activity of alanine-aminotransferase and histopathological findings in the liver. Based on these findings, a NOAEL for chronic toxicity of 250 ppm was established in male and female mice, corresponding to 50.8 and 77.5 mg/kg bw/day in males and females, respectively.

Concerning carcinogenicity, no indications for an increased number of benign and malignant tumours were observed. A slight but statistically significant increase in the incidence of mammary gland adenocarcinoma and a slight increase in the incidence of luteoma in the ovary was noted in high dose females. However, simultaneously to the increased incidence of luteoma in the ovary, a decrease in the number of granulosa-theca-cell tumours ("sex cord stromal tumours") was observed. In consequence, the total number of sex cord stromal tumours (i.e. luteomas and granulosa-theca-cell tumours) was not increased dose dependently (i.e. 11/45, 7/37, 15/46, 14/44 sex cord stromal tumours in total, with ascending dose). Of note, luteoma was also observed in one control animal at interim sacrifice after 12 months treatment. Thus, no obvious treatment related effect on the sex cord stromal tumours combined incidence was present. Further, the study duration of the present study in mice was 24 months and it is frequently observed that tumour incidences increase mainly at the end of life. This is especially relevant for the assessment of ovary tumours, as it was reported in the literature that there was a sharp increase in ovarian tumours in earlier studies with NMRI mice exceeding 18 months and up to 27 months (please refer to the expert statement provided in the technical dossier, Stropp, 2018). Thus, considering that both sex cord stromal tumours derive from the same cell origin, the granulosa cell, an age-related shift in the incidence of sex cord stromal tumours from granulosa-theca-cell tumours towards luteomas cannot be excluded. Hence, the increased incidence of luteoma was not considered as indicative for a carcinogenic potential of the test substance. Moreover, there is no evidence of any pre- or non-neoplastic ovary effect at the 12 months interim sacrifice as well as at the end of the study after 24 months in the present long-term toxicity/carcinogenicity study in mice. In addition, there were no ovary effects reported in repeated dose toxicity studies after oral, dermal or inhalation exposure in rats, rabbits and dogs (see above; rat uterus) (please refer to the expert statement provided in the technical dossier, Stropp, 2018). Thus, based on all available data, it is concluded that the observed ovary luteoma are sporadic findings of spontaneously occurring tumours without biological relevance for the assessment of Diuron.

Microscopic examinations of the mammary gland indicated a statistically significant increase in the incidence of adenocarcinoma at 2500 ppm compared to control (2/39, 1/32, 1/44, 6/39, with ascending dose; 5.1, 3.1, 2.3 and 15.4%). The neoplasias of the mammary gland occurred late towards the end of the study in both top dose (starting from day 574) and control animals for the first time. The available historical control data base points to a high biological variability of spontaneously occurring mammary gland tumours (incidence: 0 - 12.8%) (please refer to the expert statement provided in the technical dossier, Stropp, 2018). There is one study in the historical control database that showed a clearly higher incidence (5/39 (12.8%)) than in the Diuron control group (2/39 (5.1%)). Thus, although the incidence of mammary tumours was outside the range of the historical control data, a possible treatment-related increase in the tumour incidences of mammary tumours is not considered sufficiently proven by the obtained experimental data. Further, there is no evidence of any pre- or non-neoplastic mammary effect at the 12 months interim sacrifice as well as at the end of the study after 24 months in the long-term toxicity/carcinogenicity study in female mice. In addition, there were no mammary gland effects reported in repeated dose toxicity studies after oral, dermal or inhalation exposure in rats, rabbits and dogs (please refer to the expert statement provided in the technical dossier, Stropp, 2018).

Overall, based on all available data it was concluded that the tumour observations on ovary luteoma and mammary gland adenocarcinoma represent rather sporadic findings of spontaneously occurring tumours without biological relevance indicating carcinogenic properties of Diuron. Thus, a NOAEL of 2500 ppm was derived for carcinogenicity.

 

The experimental data (Schmidt, 1985 and Eiben, 1990) was retrospectively discussed in an expert statement, which assessed the tumour responses in the chronic toxicity/carcinogenicity studies in rats and mice with Diuron (Stropp, 2018). It is concluded that the available toxicological database shows a very consistent picture, with urinary tract, and spleen/erythrocyte damage as consistent target organs of systemic toxicity.

In contrast, there is no consistent picture related to effects in the rat uterus, mouse ovary, mouse mammary gland.

Where tumour observations were reported in other organs than the urinary bladder, these tumours were either within the range of the historical control data (rat, adenocarcinoma uterus), or where the historical control database is limited (mouse ovary luteoma, mouse mammary gland adenocarcinoma), there is for the ovar no obvious treatment related effect on the "sex cord stromal tumours" combined incidence and for the mammary gland there is one study in the limited historical control database that showed an incidence clearly higher than in the concurrent control group (5/39 (12.8%)), pointing to a variability that is of importance for the assessment of the Diuron study.

In addition, there is a lack of consistency over species, a lack of pre- or non-neoplastic effects of the respective organs in rats and mice in all available repeated dose toxicity studies, and a lack of mechanistic evidence (no genotoxic potential, no respective response in a mammary two-stage carcinogenesis studies in rats and mice, and no pre- or non-neoplastic findings in repeated dose toxicity studies. These aspects put the biological relevance of the observations further in question.

Overall, based on all available data it is concluded that the tumour observations rat uterus adenocarcinoma, mouse ovary luteoma and mouse mammary gland adenocarcinoma are sporadic findings of spontaneously occurring tumours without biological relevance for the assessment of Diuron.

Carcinogenicity of Diuron is concluded on the basis of bladder tumours resulting in classification as Carc. 2, H351. This classification is valid, because for the tumour observations outside of the urinary bladder there is no evidence that the observations are biologically relevant and therefore there is no "two-species and multi-site response".