2-{2-chloro-4-(methylsulfonyl)-3-[(tetrahydrofuran-2-ylmethoxy)methyl]benzoyl}cyclohexane-1,3-dione

Repeated dose toxicity: oral

Administrative data

Endpoint:

chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 Oct 2002 - 22 Dec 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

Version / remarks:

adopted in 1981

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.33 (Combined Chronic Toxicity / Carcinogenicity Test)

Version / remarks:

adopted 18 Nov 1987

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.4300 (Combined Chronic Toxicity / Carcinogenicity

Version / remarks:

adopted Aug 1998

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: MAFF in Japan, notification 12 Nousan n° 8147

Version / remarks:

adopted 24 Nov 2000

GLP compliance:

yes (incl. QA statement)

Remarks:

INTERMINISTERIAL GROUP FOR CHEMICAL PRODUCTS, Paris, France

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2-{2-chloro-4-(methylsulfonyl)-3-[(tetrahydrofuran-2-ylmethoxy)methyl]benzoyl}cyclohexane-1,3-dione

Cas Number:

473278-76-1

Molecular formula:

C20 H23 Cl O7 S

IUPAC Name:

2-{2-chloro-4-(methylsulfonyl)-3-[(tetrahydrofuran-2-ylmethoxy)methyl]benzoyl}cyclohexane-1,3-dione

Test material form:

solid

Test animals

Species:

rat

Strain:

other: Wistar Rj:WI (IOPS HAN)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: R. Janvier, Le Genest St Isle, France
- Females nulliparous and non-pregnant: not specified
- Age at study initiation: 6 weeks
- Weight at study initiation: 180 - 252 g (males) and 133 - 186 g (females)
- Housing: 5 animals of same sex per cage in suspended, stainless steel and wire mesh cages with grided bottoms
- Diet: ground and irradiated rodent diet A04C-10P1 from SAFE (Scientific Animal Food and Engineering, Epinary-sur-Orge, France), ad libitum except at designated time periods
- Water: filtered and softened tap water from the municipal water supply, ad libitum
- Acclimation period: at least 13 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 40 - 70
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): every 4 weeks
- Mixing appropriate amounts with (Type of food): A04C-10P1
- Storage temperature of food: room temperature

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability of the test substance (batch number R146) in the diet was demonstrated in a previous study (2002) at concentrations of 1.25 and 15000 ppm for at least 59 days under frozen storage followed by 11 days storage at room temperature. The stability of the test substance was also checked during the present study at concentrations of 2 and 5000 ppm for at least 77 days storage at room temperature.
The homogeneity of the test substance in the diet was verified for all first loads of the formulation F1, for the extreme concentrations on the first loads (2 and 5000 ppm) of the formulations F6, F13, F18 and F24, on the first load at 50 ppm of the formulation F6ter and on the first load at 2 ppm of the formulations F16bis and F17 to demonstrate adequate formulation procedures. Triplicate samples were collected of the lowest and the highest dose (900 and 18000 ppm) from the top, the middle and the bottom of the formulations. The results obtained from the homogeneity checks on the first loads were taken as measured concentration. Additionally, during the study, the concentration of all loads at each dietary level was verified (if not submitted for homogeneity check) prior to administration to the animals for the formulations F1, F2, F3, F6, F9, F13, F15, F16, F18, F21 and F24. The concentration of the preparations was also checked on the second loads of all supplemental preparations made for one week administration, on loads 2 to 4 at 50 ppm of the formulation F6ter, and on loads 2 to 4 at 2 ppm of the formulation F17.

Duration of treatment / exposure:

52 weeks (interim)
68 weeks (recovery group)
104 weeks (terminal sacrifice)

Frequency of treatment:

continuously (via diet)

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

2 ppm

Remarks:

equivalent to approximately 0.09 and 0.13 mg/kg bw/day over the 12-month period in males and females, respectively and equivalent to approximately 0.08 and 0.11 mg/kg bw/day over the 24-month period in males and females, respectively

Doses / concentrations 2

Dose / conc.:

50 ppm

Remarks:

equivalent to approximately 2.33 and 3.21 mg/kg bw/day over the 12-month period in males and females, respectively and equivalent to approximately 2.03 and 2.83 mg/kg bw/day over the 24-month period in males and females, respectively

Doses / concentrations 3

Dose / conc.:

1 500 ppm

Remarks:

equivalent to approximately 72.0 and 99.6 mg/kg bw/day over the 12-month period in males and females, respectively and equivalent to approximately 62.4 and 88.6 mg/kg bw/day over the 24-month period in males and females, respectively

Doses / concentrations 4

Dose / conc.:

5 000 ppm

Remarks:

equivalent to approximately 245 and 337 mg/kg bw/day over the 12-month period in males and females, respectively and equivalent to approximately 214 and 296 mg/kg bw/day over the 24-month period in males and females, respectively

No. of animals per sex per dose:

10 (12 month interim sacrifice)
15 (recovery group)
50 (24 month sacrifice)

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: The dose levels were selected based on the results from a previous 90-day dietary study in the rat (2003).

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily (including weekends and public holidays)
- Cage side observations included: mortality and moribundity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least daily (clinical signs), at least weekly (detailed physical examination including palpation for masses), except on Week 21 for females where physical examination was done twice for males in
error

BODY WEIGHT: Yes
- Time schedule for examinations: on Days 3, 7 and 10 during the acclimatisation period, weekly for the first 13 weeks of study, approximately every 4 weeks thereafter and prior to necropsy

FOOD CONSUMPTION AND COMPOUND INTAKE:
The weight of food supplied and of that remaining at the end of the food consumption period was recorded for each animal. Food consumption was recorded twice weekly during the first 6 weeks of treatment, then weekly up to Week 13, and once approximately every 4 weeks thereafter.The weekly mean achieved dosage intake in mg/kg bw/day for Weeks 1 to 13, then 1 week per month thereafter was calculated.

FOOD EFFICIENCY: No

WATER CONSUMPTION AND COMPOUND INTAKE: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: during acclimatization phase and at Month 4, 5, 12, 24 and at the end of reversibility phase
- Dose groups that were examined: all animals (acclimatization phase), all surviving animals (4, 5, 12, 24 months and after reversibility phase)

HAEMATOLOGY: Yes
- Time schedule for collection of blood: all animals: Weeks 13 - 14, 22 - 24, 51 - 53; recovery group: on Week 69 (Day 91 of recovery phase); terminal scrifice: Weeks 78 and 105 - 107; puncture of the retro orbital venous plexus
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all surviving animals (12 month interim sacrifice), first ten surviving animals (recovery group and 24 month terminal sacrifice)
- Parameters examined: Red blood cell count, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, reticulocyte count, white blood cell total and differential counts and platelet count, blood smears, prothrombin time and activated partial thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: all groups: Weeks 13 - 14, 22 - 24, 51 - 53; recovery group: on Week 69 (Day 91 of recovery phase); terminal sacrifice group: Weeks 78 and 105 - 107; puncture of the retro orbital venous plexus
- Animals fasted: Yes
- How many animals: all surviving animals (12 month interim sacrifice), first ten surviving animals (recovery group and 24 month terminal sacrifice)
- Parameters examined: Total bilirubin, glucose, urea, creatinine, total cholesterol, triglycerides, chloride, sodium, potassium, calcium and inorganic phosphorus concentrations, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase and gamma-glutamyltransferase activities, total protein and albumin

URINALYSIS: Yes
- Time schedule for collection of urine: all groups: Weeks 11 - 12, 25 - 26 and 49 - 50; recovery groups: Week 68 (day 87 of recovery phase); terminal sacrifice group: Week 77 - 79 and 104
- Metabolism cages used for collection of urine: No data
- Animals fasted: Yes
- Parameters examined: appearance, volume, pH, urinary refractive index, glucose, bilirubin, ketone bodies, occult blood, protein, urobilinogen, urinary sediment (microscopic exmination for grading of: red blood cells, white blood cells, epithelial cells, bacteria, casts and crystals)

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table 1)
On Days 366, 367, 368 and 371 of the dosing phase for the interim sacrifice, on Days 94 and 97 of the recovery phase for the reversibility sacrifice and on Days 729 to 752 of the dosing phase for the terminal sacrifice, all surviving animals were sacrificed by exsanguination under deep anesthesia (pentobarbital, intraperitoneal injection of 60 mg/kg bw). An approximately equal number of animals randomly distributed amongst all groups were sampled on each day at the chronic and recovery sacrifice. At termination of the carcinogenicity phase, males were sacrificed prior to females due to the higher mortality rate in this sex. Consequently, an approximately equal number of males or females randomly distributed amongst the groups were sampled on each day taking into account the mortality. Animals were diet fasted overnight prior to sacrifices. All animals, including animals either found dead or killed for humane reasons, were necropsied. The necropsy included the examination of all major organs, tissues and body cavities. The organs or tissues checked in table 1 were sampled and/or weighed. All significant macroscopic abnormalities (including masses and their regional lymph nodes when possible) were recorded, sampled and examined microscopically. Samples were fixed by immersion in neutral buffered 10% formalin with the exception of the eye, optic nerve, Harderian gland, epididymis and testis that were fixed in Davidson's fixative. Two femoral bone marrow smears were prepared from sacrificed animals, one of which was stained with May-Grünwald Giemsa, but not examined as no relevant changes were observed in hematology or bone marrow histology. The second smear was stored unstained.


HISTOPATHOLOGY: Yes (see table 1)
Samples (except exorbital lachrymal gland, larynx/pharynx and nasal cavities) were embedded in paraffin wax. Histological sections, stained with hematoxylin and eosin, were prepared from all organs and tissue samples. Histopathology examination of the chronic (52-week) phase was performed as follows:
- all organs and tissue samples from animals sacrificed or found dead during the treatment period,
- all organs and tissue samples from animals of control and high dose groups,
- liver, lung, kidneys, pancreas, eyes and gross abnormalities from animals of the intermediate dose groups.
Histopathological examinations were performed only on the liver, kidney, pancreas and eyes for animals which survived to scheduled sacrifice and all organs for animals which died before scheduled sacrifice. Histopathology examination of the carcinogenicity phase was performed on all organs and tissues embedded including gross abnormalities in all animals from all groups including decedents. For all unscheduled sacrificed or dead animals on study, the cause of death (whether or not a tumor was the cause of death) was determined when it was possible.

Statistics:

In general Bartlett test was performed. If the Bartlett test was not significant (p>0.05), means were compared using the analysis of variance (ANOVA). If the ANOVA was not significant (p>0.05), the group means were considered homogeneous and no further analysis was performed. If the ANOVA was significant (p≤0.05), means of the exposed groups were compared to the mean of the control group using the Dunnett test (2-sided). If the Bartlett test was significant (p≤0.05), group means were compared using the Kruskal-Wallis test. If the Kruskal-Wallis test was not significant (p>0.05), group means were considered homogeneous and no further analysis was performed. If the Kruskal-Wallis test was significant (p≤0.05), means of the exposed groups were compared to the mean of the control group using the Dunn test (2-sided). If the Bartlett test was significant for body weight and average food consumption/day parameters and selected haematology parameters (red blood cell count, platelet count, white blood cell count, neutrophil count, lymphocyte count, reticulocyte count) data were transformed using the log transformation for body weight and food consumption and the square root transformation for haematology parameters before the same methods were applied as mentioned above. If the Bartlett test was significant (p≤0.05) even after log transformation, statistical analysis was proceeded as mentioned above.
Individual statistic methods used for specific parameters are descibed in `Any other information on materials and methods´.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

First year:
MALES
50 ppm: white area on eyes (45/60)
1500 ppm: white area on eyes (49/60)
5000 ppm: cold appearance (7/75), dental abnormality (13/75), general pallor (9/75), reduced motor activity (9/75), soiled anogenital region (7/75), localized soiled fur (7/75), wasted appearance (5/75) and white area on eyes (53/75; progressively disappearing within 3 month recovery, except of 1 animal)

FEMALES
2 ppm: white area on eyes (13/75)
50 ppm: white area on eyes (48/60)
1500 ppm: white area on eyes (55/60), soiled anogenital region (4/60)
5000 ppm: soiled anogenital region (12/75), white area on eyes (58/75; progressively disappearing within 3 month recovery) and hair loss (23/75)

Second year:
MALES
50 ppm: focal swelling (10/48; principally of hindlimb), reduced motor activity (12/48) and white area on eyes (41/48)
1500 ppm: focal swelling (21/45; principally of hindlimb), white area on eyes (45/45) and reduced motor activity (9/45; not statistically significant)
5000 ppm: focal swelling (16/42; principally of hindlimb), reduced motor activity (11/42), skin lesions (13/42) and white area on eyes (41/42)

FEMALES
50 ppm: white area on eyes (42/50)
1500 ppm: white area on eyes (45/49)
5000 ppm: white area on eyes (38/49), chromodacryorrhea (10/49) and soiled anogenital region (6/49)

For more details see table 2 in section "any other information on results incl. tables".

Mortality:

mortality observed, treatment-related

Description (incidence):

MALES
0 ppm: 1/75 animals after 1 year, 29/50 animals after 2 years
2 ppm: 4/60 animals after 1 year, 33/50 animals after 2 years
50 ppm: 2/60 animals after 1 year, 40/50 animals after 2 years
1500 ppm: 5/60 animals after 1 year, 34/50 animals after 2 years
5000 ppm: 16/75 animals after 1 year, 33/50 animals after 2 years

Only death of high dose group males where 16/75 were found dead or were sacrificed prematurely for humane reasons, were considered to be treatment-related. However, this effect was transient, as the overall mortality rate over two years was similar to the controls.

FEMALES
0 ppm: 0/75 animals after 1 year, 23/50 animals after 2 years
2 ppm: 1/60 animals after 1 year, 27/50 animals after 2 years
50 ppm: 0/60 animals after 1 year, 14/50 animals after 2 years
1500 ppm: 2/60 animals after 1 year, 17/50 animals after 2 years
5000 ppm: 1/75 animals after 1 year, 13/50 animals after 2 years

For more details see table 3 in section "any other information on results incl. tables".

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

First year:
MALES
2 and 50 and ppm: Mean body weight and body weight gain were comparable to the controls.
1500 ppm: Compared to control mean body weight and mean body weight gain were slightly lower by up to 6 and 11% (p<0.01 or p<0.05), respectively.
5000 ppm: Compared to the control during the first week of treatment, mean body weight and mean body weight gain were lower by 5 and 21% (p<0.01), respectively. Compared to the control group by the end of first year, mean body weight and mean body weight gain were lower by 4 and 6%(p<0.05), respectively.

FEMALES:
2 and 50 and ppm: Mean body weight and body weight gain were comparable to the controls.
1500 ppm: Compared to control mean body weight and mean body weight gain were slightly lower by up to 6 and 18% (p<0.01 or p<0.05), respectively.
5000 ppm: Compared to the control group during the first week of treatment, mean body weight and mean body weight gain were lower by 4 and 29% (p<0.01), respectively. Compared to the control group by the end of first year, mean body weight and mean body weight gain were lower by 6 and 11% (p<0.01), respectively.

Recovery period: During the 3-month recovery period, mean weekly body weight gain in both sexes previously treated at 5000 ppm was comparable to the controls or slightly reduced.

Second year:
MALES
2 ppm: Mean body weight and body weight gain were comparable to the controls.
50 ppm: Compared to control mean body weight and mean body weight gain were slightly lower by up to 11 and 17% (p<0.01 or p<0.05), respectively.
1500 ppm: Compared to control mean body weight and mean body weight gain were slightly lower by up to 12 and 17% (p<0.01 or p<0.05), respectively.
5000 ppm: Compared to the control mean body weight and mean body weight gain were lower by 12 and 17% (p<0.01 or p<0.05), respectively.

FEMALES:
2 ppm: Mean body weight and body weight gain were comparable to the controls.
50 ppm: Compared to control mean body weight and mean body weight gain were slightly lower by up to 8 and 12% (p<0.01 or p<0.05), respectively.
1500 ppm: Compared to control mean body weight and mean body weight gain were slightly lower by up to 16 and 23% (p<0.01 or p<0.05), respectively.
5000 ppm: Compared to the control mean body weight and mean body weight gain were lower by 16 and 23% (p<0.01 or p<0.05), respectively.

For more details see tables 4 and 5 in section "any other information on results incl. tables".

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

First year:
MALES
5000 ppm: decreased mean food consumption (6%; not statistically significant) in first weeks of treatment

FEMALES
5000 ppm: decreased mean food consumption (5%; p<0.05) in first weeks of treatment

The other few minor differences (reaching statistical significance) from controls were considered to reflect interindividual variation rather than a treatment-related effect.

Recovery phase:
During the 3-month recovery period, no relevant change was observed in the animals previously treated at 5000 ppm during one year.

Second year:
Mean food consumption was similar to controls during the second year of the study in both sexes and at all dose levels. The few minor increases (reaching statistical significance) in males at 5000 ppm were considered to reflect inter-individual variation rather than a treatment-related effect.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

effects observed, treatment-related

Description (incidence and severity):

First year:
At 5000, 1500 and 50 ppm, the following treatment-related ophthalmological findings were observed after 4 months of exposure: corneal opacity, neovascularization of the cornea and snow flake-like corneal opacity in both sexes, in association with oedema of the cornea in males only. The incidence of these findings was already marked by Month 4 and increased throughout the duration of the study. The type of corneal lesions observed at 5000, 1500 and 50 ppm in the rat (together with the eye keratitis noted at the microscopic examination), are based on characteristics of the compound that inhibits 4-hydroxyphenylpyruvic acid
dioxygenase (4-HPPDase), an enzyme of the tyrosine catabolic pathway. These lesions are related to an increase in plasma tyrosine level caused by a blockade of the 4-HPPDase enzyme in the rat. However, the rat is a species particularly sensitive to inhibition of the 4-HPPDase enzyme and is atypical in its susceptibility to develop tyrosine-related eye lesions. Therefore, although these lesions (corneal lesions and eye keratitis) were treatment-related, they were considered not to be toxicologically relevant to man. At 2 ppm, no treatment-related ophthalmological change was noted, as the incidences of ophthalmological findings were similar to the control groups at all time points.Other ophthalmological findings recorded were those commonly recorded or were recorded in one
or two animals only and were thus considered not to be related to test substance administration.

Recovery phase:
After 13 weeks of recovery, corneal opacity, oedema of the cornea and snow flake-like corneal opacity were totally reversible whilst neovascularization of the cornea persisted in some high dose group animals affected during the chronic phase.

Second year:
At the end of 2 years of exposure, the following treatment-related ophthalmological findings were observed:
At 5000 ppm, the incidence of corneal opacity, neovascularization and oedema of the cornea and snow flake-like corneal opacity was statistically significantly elevated in both sexes. At 1500 and 50 ppm, the incidence of corneal opacity, neovascularization and oedema of the cornea was statistically significantly elevated in both sexes, in association with a statistically significant increase in the incidence of snow flake-like corneal opacity in females. At 2 ppm, ophthalmological examination did not reveal any statistically significant changes. Other ophthalmological findings recorded were those commonly recorded or were recorded in one or two animals only and were thus considered not to be related to the test substance administration.

For more details see table 6 in section "any other information on results incl. tables".

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

First year:
MALES: Increased total cholesterol levels (up to 57%; p<0.01) and/or triglyceride levels (up to 72%; p<0.01) in males and femles, respectively, at concentrations of 50, 1500 and 5000 ppm; decreased mean alkaline phosphatase activity (up to -27%; p<0.05) at 5000 and 1500 ppm at Months 3 and 6; slight variations (not toxicologically relevant) in total protein (up to 13%; p<0.01) and albumin concentrations (up to 12%; p<0.01) and consequently in globulin concentrations and albumin/globulin ratio (calculated parameters) at 5000, 1500 and 50 ppm at Month 3

FEMALES: Increased mean total cholesterol (up to 32%; p<0.01) and/or triglycerides concentrations (up to 20%; p<0.01) at 50, 1500 and 5000 ppm

Recovery period: All changes detected during first year were found to be reversible, as after the 3-month recovery period.

Second year:
MALES: Increased mean total cholesterol concentrations (+36%; p<0.05) at 5000 ppm after 18 month but reversible within 24 month

FEMALES: Increased mean total cholesterol concentrations (+27%; p<0.05) at 5000 ppm after 18 month but reversible within 24 month

For more details see table 7 in section "any other information on results incl. tables".

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

First year:
MALES: Higher ketone level at Month 3 (5000 and 1500 ppm), Month 6 (5000 ppm) and Month 12 (5000, 1500 and 50 ppm); lower mean pH (50, 1500 and 5000 ppm); higher protein level Month 3 (5000 ppm) and at Months 6 and 12 (5000, 1500 and 50 ppm); lower amount of crystals (50, 1500 and 5000 ppm)

FEMALES: Higher ketone level at all sampling periods at 50, 1500 and 5000 ppm; lower mean pH (50, 1500 and 5000 ppm)

Recovery period: All changes detected during the chronic phase were found to be reversible after the 3-month recovery period, with the exception of the mean pH value that remained lower in the females previously treated at 5000 ppm for one year.

Second year:
MALES: Higher ketone level at all sampling periods at 50, 1500 and 5000 ppm; lower mean pH values at Months 18 and 24 (50, 1500 and 5000 ppm); higher protein levels and lower amounts of crystals at Month 18 only (50, 1500 and 5000 ppm)

FEMALES: Higher ketone level at all sampling periods at 50, 1500 and 5000 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):

First year:
MALES: higher mean absolute and/or relative liver and kidney weights at 50, 1500 and 5000 ppm compared to control; increased brain weight by 7 and 6% at 1500 and 5000 ppm, respectively

Recovery phase:
Mean terminal body weight was lower in both sexes at 5000 ppm (-10 and -12% in males and females, respectively, p<0.05), when compared to controls. All organ weight changes detected at the end of the chronic (first year) phase were found to be reversible and therefore not toxicologically relevant. The few changes noted after the 3-month recovery period were considered to be incidental in view of their individual variation.

Second year:
MALES: higher mean abosolute and/or relative liver and kidney weights (statistically significant for most parameters) at 50,1500 and 5000 ppm compared to control; decreased mean absolute brain weight at 50, 1500 and 5000 ppm (not toxicological relevantas since they were observed for one parameter only, were opposite in both sexes and were not associated with any change at the macroscopic and microscopic examinations)

FEMALES: higher mean abosolute and relative liver weights at 1500 and 5000 ppm compared to control; lower absolute and mean kidney to brain weights at 1500 ppm (non-treatment related since no dose-relationship); decresed ovary weights at 5000 ppm (this increase was attributable to a bilateral mass noted on ovary of this female, which contributed by itself to the increase in ovary weights in this group) and 50 and 1500 ppm (not toxicological relevant since it was observed for this parameter only and was not associated with any change at the macroscopic and microscopic examinations); increased brain to body weight at 1500 and 5000 ppm (not toxicological relevantas since they were observed for one parameter only, were
opposite in both sexes and were not associated with any change at the macroscopic and microscopic examinations)

For more details see table 8 in section "any other information on results incl. tables".

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

First year:
- Unscheduled death:
MALES
5000 ppm: hemorrhagic sydrome in 4 animals found dead at the beginning of the study: multiple heamorrages in the subcutis, brain and testis in 2 animals found dead on Day 9 and 25; subcutaneous hematoma in 2 animals found dead on Day 24 and 25; The hemorrhagic lesions were the cause of death and were considered to be treatment-related in these 4 animals; non treatment-related findings: irregular surface of the kidney and bilateral eye opacity in one animal killed on Day 24

- Terminal sacrifice:
MALE/FEMALE: eye opacity at 50, 1500 and 5000 ppm

Recovery period:
- Unschedules death:
MALES: dark contents in the stomach and intestines in two animals found dead on Day 29, red foci in the epidymidis, brain hematoma, uniliteral eye opacity in one animal found dead on Day 11
FEMALES: irregular surface on the kidney in one animal found dead on Day 55

Second year:
- Unscheduled death:
MALES:
50 ppm: eye opacity
1500 ppm: eye opacity
5000 ppm: eye opacity, pale appearance of animals

FEMALES
1500 ppm: eye opacity
5000 ppm: irregular surface of kidney, skin allopecia (not toxicological relevant since no relevant microscopic changes occured)

- Terminal sacrifice:
MALES
50 ppm: eye opacity, irregular surface on kidney
1500 ppm: eye opacity, irregular surface on kidney (not statistically significant)
5000 ppm: eye opacity, irregular surface on kidney

FEMALES
50 ppm: eye opacity, enlarged pituitary gland (not toxocilogically relevant since no dose-relationsship)
1500 ppm: eye opacity
5000 ppm: eye opacity, enlarged pituitary gland (not toxocilogically relevant since no dose-relationsship)

For more details see tables 9 and 10 in section "any other information on results incl. tables".

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

First year:
- Unscheduled death: hemorrhagic syndrome in 4/9 animals found dead; progressive nephropathy and keratitis in one male killed on Day 241
- Terminal sacrifice:
MALES:
Kidney: chronic progressive nephropathy at 50, 1500 and 5000 ppm (including: tubular cell regeneration, thickened basement membranes (glomerular and tubular), interstitial fibrosis, inflammation, dilated/cystic tubules, protein casts, debris, mesangial proliferation, glomerular sclerosis, and hypertrophy/hyperplasia of tubular epithelium)

Pancreas: minimal to moderate acinar atrophy/fibrosis (focal/multifocal) at all dose levels (non-treatment-related since no dose-relationship)

Eye: minimal to marked keratitis (including following changes in the cornea: acute to chronic active inflammation, keratinization, epithelial vacuolization, neovascularization) at 50, 1500 and 5000 ppm (not relevant for human since inhibition of key enzyme provoking systemic tyrosinaemia is relevant in the rat only)

Liver: centrilobular hepatocellular hypertrophy (diffuse) in 2/10 animals at 1500 ppm and in 3/5 animals at 5000 ppm (adaptive response)

FEMALES:
Pancreas: minimal to moderate interstitial inflammation (focal/multifocal) at 5000 ppm (not toxicological relevant since no dose-effect); minimal to moderate acinar atrophy/fibrosis (focal/multifocal) at all dose levels (non-treatment-related since no dose-relationship)

Eye: minimal to marked keratitis (including: acute to chronic active inflammation, keratinization, epithelial vacuolization, neovascularization) at 50, 1500 and 5000 ppm (not relevant for human since inhibition of key enzyme provoking systemic tyrosinaemia is relevant in the rat only)

Urinary bladder: transitional cell hyperplasia in 5/10 animals at 5000 ppm (reversible since not observed after 24 months)
Adrenal gland: zona glomerulosa hypertrophy in 7/10 animals at 5000 ppm (not toxicological relevant since low severity of findings and reversible since not observed after 24 months)

Recovery period:
- Unscheduled death: hemorrhagic syndrome in 3 males found dead at beginning of recovery period; Kidney: progressive nephropathy in one female found dead on Day 55

- Terminal sacrifice:
MALES
5000 ppm:
Kidney: minimal to severe chronic progressive nephropathy and minimal to slight interstitial inflammation (focal/multifocal)
Eye: keratitis (not relvant for human; see comment at chronic phase)

FEMALES
5000 ppm:
Pancreas: interstitial inflammation iin 7/14 animals; minimal to slight acinar atrophy/fibrosis (non-treatment-related); basophilic changes in 4/14 animals (non-treatment related)
Eye: keratitis (not relvant for human; see comment at chronic phase)

Second year:

MALES:
Kidney: minimal to severe chronic progressive nephropathy at 50, 1500 and 5000 ppm (no relevance for human since age related, spontaneously occuring in rats and a high incidence in control gorup)

Pancreas: minimal to moderate interstitial inflammation (focal/multifocal) in the pancreas at 1500 and 5000 ppm (not toxicologically relevant since no real difference in severity to controls); minimal to severe acinar atrophy/fibrosis (focal/multifocal) at 1500 and 5000 ppm (statistically significant)

Thyroid gland: colloid alteration and brown pigment in the cytoplasm of the thyroid follicular cells (not toxicologically relevant since similar alterations are present in control rats, indicating that these changes likely reflect a normal physiologic process associated with the unique rapid turnover of colloid) and follicular cell hypertrophy at 50, 1500 and 5000 ppm

Eye: marked keratitis at 50, 1500 and 5000 ppm (not toxicological relevant to human, see comment at chronic phase); atrophy/degeneration (multifocal/diffuse) in sciatic nerve at 50, 1500 and 5000 ppm (non-treatment related since high incidence in control groups and no dose-dependency)

Skeletal muscle: minimal to marked myofiber atrophy/degeneration at 50, 1500 and 5000 ppm (non-treatment related since secondary effect from sciatic nerve atrophy),

Hindleg/hindpawn: ulcerative necrotising inflammation at 1500 and 5000 ppm (indirect effect with minor toxicological relevance)

Liver: angiectasis (focal/multifocal) and extramedullary hematopoiesis (multifocal) at 1500 and 5000 ppm (not toxicologically relevant since age related and no dose-dependency); minimal to slight centrilobular hepatocellular hypertrophy (diffuse) at 1500 ppm (adaptive response)

Brain: minimal to slight mineralization (focal/multifocal) at 5000 ppm (non-treatment-related since also observed in control groups and within range of historical control data)

Pituitary gland: cyst(s)/pseudocyst(s) in pars distalis at 5000 ppm (not toxicologically relevant since it was a spontaneous finding)

Urinary bladder: proteinaceous material at 5000 ppm of animals found dead (not toxicologically relevant since it is a common finding and was obtained in isolation for this parameter)

FEMALES:
Pancreas: minimal to moderate interstitial inflammation (focal/multifocal) at 50, 1500 (not significant) and 5000 ppm (not toxicologically relevant since no real difference in severity to controls); minimal to severe acinar atrophy/fibrosis (focal/multifocal) at 2 (within range of historical data), 50, 1500 and 5000 ppm

Thyroid gland: colloid alteration, brown pigment in the cytoplasm of the thyroid follicular cells (not toxicologically relevant since similar alterations are present in control rats, indicating that these changes likely reflect a normal physiologic process associated with the unique rapid turnover of colloid) and follicular cell hypertrophy at 50, 1500 and 5000 ppm

Eye: marked keratitis at 50, 1500 and 5000 ppm (not toxicological relevant to human, see comment at chronic pahse); atrophy/degeneration (multifocal/diffuse) in sciatic nerve at 1500 ppm (non-treatment related since high incidence in control groups and no dose-dependency)

Skeletal muscle: minimal to marked myofiber atrophy/degeneration at 1500 and 5000 ppm (non-treatment related since secondary effect from sciatic nerve atrophy)

Liver: bile duct hyperplasia (focal/multifocal) at 50, 1500 and 5000 ppm (not toxicologically relevant since age related and no dose-dependency)

Ovaries: minimal to marked diffuse atrophy at 5000 ppm (non-treatment-related since within the range of historical control data)

For more details see table 11 in section "any other information on results incl. tables".

Histopathological findings: neoplastic:

no effects observed

Effect levels

Dose descriptor:

NOAEL

Remarks:

24 month

Effect level:

2 ppm

Based on:

test mat.

Remarks:

equivalent to 0.08 and 0.11 mg/kg bw/day in males and females, respectively

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Target system / organ toxicity

Critical effects observed:

yes

Lowest effective dose / conc.:

50 ppm

System:

endocrine system

Organ:

pancreas

thyroid gland

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Any other information on results incl. tables

Table 2. Treatment-related clinical signs

	Males					Females
Dose level (ppm)	0	2	50	1500	5000	0	2	50	1500	5000
First year (chronic phase)
Number of animals examined	75	60	60	60	75	75	60	60	60	75
Cold to touch	0	0	0	0	7*	nc	nc	nc	nc	nc
Dental abnormality	5	6	6	2	13*	nc	nc	nc	nc	nc
General pallor	0	0	0	1	9**	nc	nc	nc	nc	nc
Reduced moto activity	0	0	0	0	9**	nc	nc	nc	nc	nc
Soiled anogenital region	0	0	0	1	7**	0	0	2	4*	12**
Soiled fur, localized	0	0	1	1	7**	nc	nc	nc	nc	nc
Wasted	0	1	0	1	5*	nc	nc	nc	nc	nc
White area in eyes	1	1	45**	49**	53**	2	13**	48**	55**	58**
Hair loss	nc	nc	nc	nc	nc
Recovery phase
Number of animals examined on Week 1 of recovery	15	-	-	-	12	14	-	-	-	15
White area on eyes	0	-	-	-	9**	0	-	-	-	9**
Number of animals examined on Week 13 of recovery	15	-	-	-	11	14	-	-	-	14
White area on eyes	0	-	-	-	1	0	-	-	-	1
Second year (carcinogenicity phase)
Number of animals examined	21	17	10	16	17	27	22	36	33	36
Focal swelling	3	3	10*	21**	16**	nc	nc	nc	nc	nc
Reduced motor activity	5	6	12*	9	11*	nc	nc	nc	nc	nc
Skin lesions	7	10	6	9	13*	nc	nc	nc	nc	nc
White area on eye	3	3	41**	45**	41**	2	6	42**	45**	38**
Chromodacryorrhea	nc	nc	nc	nc	nc	1	1	3	4	10**
Soiled anogenital region	nc	nc	nc	nc	nc	0	0	0	3	6*

*•: p<0.05; **: p<0.01; nc: not concerned or no treatment-related change.

Table 3. Mortality incidence after 1 and 2 years of treatment (unscheduled deaths of animals)

	First year of treatment (chronic phase)
Dose level (ppm)	0	2	50	1500	5000
Males
Number of animals	75	60	60	60	75
Mortality	1	4	2	5	16
Females
Number of animals	75	60	60	60	75
Mortality	0	1	0	2	1
	Second year of treatment (carcinogenicity phase)
Males
Number of animals	50	50	50	50	50
Mortality	29	33	40	34	33
Females
Number of animals	50	50	50	50	50
Mortality	23	27	14	17	13

 

Table 4. Body weights and body weight gains in males

Dose level [ppm]	0	2		50			1500		5000
	g	g	% of control		g	% of control	g	% of control	g	% of control
BODY WEIGHTS
Initial (Day 1)	218	218	100		217	100	216	99	215	99
Week 1 (Day 8)	276	278	101		278	101	270	98	261**	95
Week 13 (Day 92)	535	547	102		538	101	504**	94	500**	93
Week 25 (Day 176)	630	639	101		631	100	602*	96	585**	93
Week 49 (Day 344)	708	709	100		706	100	682	96	677*	96
Week 77(Day 372)	737	742	101		685*	93	664**	90	671**	91
Final (Day 708)	660	679	103		606	92	600*	91	630	95
BODY WEIGHT GAINS
Week 1 (Days 1 – 8)	58	61	105		61	105	54**	93	46**	79
Weeks 1 – 13 (Days 1 – 92)	318	329	103		320	101	288**	91	284**	89
Weeks 13 – 25 (Days 92 – 176)	95	91	96		94	99	96	101	86	91
Weeks 25 - 49 (Days 176 – 344)	79	73	92		75	95	81	103	92	116
Weeks 49 – 77 (Days 344 – 540)	38	36	95		-17**	nc	-11	nc	-11**	nc
Weeks 77 – 101 (Days 540 – 708)	-47	-54	115		-64	136	-67	143	-44	94
Overall (Days 1 – 708)	446	463	104		392	88	383	86	418	94

*: p≤0.05; **: p≤0.01; nc: not calculated

 

Table 5. Body weights and body weight gains in females

Dose level [ppm]	0	2		50			1500		5000
	g	g	% of control		g	% of control	g	% of control	g	% of control
BODY WEIGHTS
Initial (Day 1)	162	162	100		162	100	162	100	163	101
Week 1 (Day 8)	190	190	100		189	99	185*	97	182**	96
Week 13 (Day 92)	291	291	100		289	99	280**	96	282*	97
Week 25 (Day 176)	327	324	99		325	99	317*	97	318*	97
Week 49 (Day 344)	363	356	98		359	99	345*	95	341**	94
Week 77(Day 372)	433	428	99		412	99	385**	89	393**	91
Final (Day 708)	474	483	102		438	92	405**	85	401**	85
BODY WEIGHT GAINS
Week 1 (Days 1 – 8)	28	28	100		26	93	23**	82	20**	71
Weeks 1 – 13 (Days 1 – 92)	129	128	99		127	98	118**	91	119**	92
Weeks 13 – 25 (Days 92 – 176)	37	33	89		36	97	37	100	36	97
Weeks 25 - 49 (Days 176 – 344)	36	33	92		34	94	27	75	23*	64
Weeks 49 – 77 (Days 344 – 540)	71	69	97		52	73	43**	61	45**	63
Weeks 77 – 101 (Days 540 – 708)	42	52	124		32	76	28	67	13**	31
Overall (Days 1 – 708)	309	321	104		276	89	245**	79	239**	77

*: p≤0.05; **: p≤0.01; nc: not calculated

Table 6. Ophthalmological findings

	Males					Females
Dose level (ppm)	0	2	50	1500	5000	0	2	50	1500	5000
Month 4 (Weeks 14 - 15)
Number of animals examined	75	60	60	59	63	75	60	60	59	75
Corneal opacity	0	0	12**	14**	8**	0	0	5*	7**	4
Neovascularization of the cornea	0	0	30**	38**	34**	1	1	30**	47**	23**
Oedema of the cornea	0	0	12**	12**	6**	1	2	1	1	2
Corneal opacity « snow flake »	0	0	22**	27**	29**	0	0	29**	41**	22**
Month 5 (Weeks 21 – 22)
Number of animals examined	75	60	59	59	63	75	60	60	59	75
Corneal opacity	0	0	23**	21**	22**	0	0	7**	25**	9**
Neovascularization of the cornea	0	0	33**	40**	39**	1	0	40**	50**	37**
Oedema of the cornea	0	0	23**	22**	22**	1	1	4	24**	9**
Corneal opacity « snow flake »	0	0	13**	22**	18**	0	0	35**	28**	32**
Month 12 (Weeks 50 – 51)
Number of animals examined	75	60	59	59	63	75	59	60	58	74
Corneal opacity	0	0	36**	37**	36**	1	1	21**	41**	28**
Neovascularization of the cornea	0	0	44**	45**	49**	1	1	46**	53**	50**
Oedema of the cornea	0	0	36**	39**	45**	1	2	26**	45**	37**
Corneal opacity « snow flake »	0	0	8**	8**	17**	0	0	27**	16**	24**
Week 13 of recovery phase
Number of animals examined	15	-	-	-	11	14	-	-	-	14
Neovascularization of the cornea	0	-	-	-	2	0	-	-	-	6**
Month 24 – 25 (Weeks 104 – 106)
Number of animals examined	21	17	11	17	17	29	25	37	33	39
Corneal opacity	0	0	10**	16**	11**	0	3	15**	26**	19**
Neovascularization of the cornea	0	0	10**	17**	17**	0	3	30**	32**	30**
Oedema of the cornea	0	0	10**	17**	17**	0	3	20**	31**	25**
Corneal opacity « snow flake »	0	0	0	1	7**	0	0	16**	12**	12**

*: p≤0.05; **: p≤0.01

Table 7. Changes in clinical chemistry parameters compared to controls [%]

Dose level (ppm)	50			1500			5000
Month	3	6	12	3	6	12	3	6	12
Cholesterol concentration
Males	+20%*	+20%	+38%**	+21% *	+24%**	+29**	+46%**	+44%**	+57%**
Females	-4%	+9%	-1%	+6%	+13%*	+12%	+24%**	+26%**	+32%**
Triglyceride concentration
Males	+44%*	+15%	+42%*	+48%*	+50%	+44%	+51%**	+34%*	+72%**
Females	+40%**	+9%	+11%	+49%**	+21%*	+6%	+62%**	+18%	+20%
Alkaline phosphatase activity
Males	-23%	-11%	-2%	-27%*	-23%**	-20%	-32%**	-20%**	-17%

*: p≤0.05; **: p≤0.01

Table 8. Changes in Organ weights

	Males					Females
Dose level (ppm)	0	2	50	1500	5000	0	2	50	1500	5000
First year (chronic phase)
Number of animals examined	10	9	9	10	5	10	10	10	8	10
Mean absolute liver weight (g)	13.8	13.0	16.5	16.3	16.7	7.5	7.9	8.0	8.5	7.8
Mean liver to bw ratio (%)	2.00	1.92	2.38**	2.44**	2.67**	2.30	2.33	2.34	2.56	2.45
Mean absolute kidney weight (g)	3.45	3.47	4.04*	3.94	3.63	2.32	2.47	2.39	2.36	2.26
Mean kidney to bw ratio (%)	0.501	0.510	0.586*	0.593**	0.589*	0.710	0.730	0.695	0.709	0.715
Second year (carcinogenicity phase)
Number of animals examined	21	17	10	16	17	27	22	36	33	36
Mean absolute liver weight (g)	12.6	13.5	14.0	15.7**	15.7**	10.5	11.0	10.6	10.1	10.7
Mean liver to bw ratio (%)	2.09	2.09	2.51**	2.84**	2.74**	2.40	2.52	2.63	2.69*	2.81**
Mean absolute kidney weight (g)	0.665	0.673	0.801	0.921**	0.812*	0.709	0.706	0.725	0.736	0.766
Mean kidney to bw ratio (%)	168.4	182.4	196.7*	225.7**	208.3**	143.1	139.2	137.1	128.8**	137.9

*: p≤0.05; **: p≤0.01

Table 9. Macroscopic changes at terminal sacrifice of first year and recovery phase

	Males					Females
Dose level (ppm)	0	2	50	1500	5000	0	2	50	1500	5000
First year (chronic phase)
Eye opacity	0/10	0/9	6/9**	4/10*	2/5	1/10	0/10	6/10*	4/8	7/10**
Second year (carcinogenicity phase)
Eye opacity	1/21	1/17	7/10**	9/16**	14/17**	1/27	3/23	22/36**	30/33**	23/37**
Irregular surface on kidney	2/21	2/17	7/10**	6/16	7/17*	nc	nc	nc	nc	nc

*: p≤0.05; **: p≤0.01

Table 10. Macroscopic changes at unscheduled sacrifice (incidence) in second year (carcinogenicity phase)

	Males					Females
Dose level (ppm)	0	2	50	1500	5000	0	2	50	1500	5000
Eye opacity	1/29	3/33	10/40*	13/34**	11/33**	1/23	1/27	1/14	6/17	3/13
Irregular surface on kidney	nc	nc	nc	nc	nc	0/23	0/27	1/14	1/17	3/13*

*: p≤0.05; **: p≤0.01

 

Table 11. Microscopic changes at terminal sacrifice

	Males					Females
Dose level (ppm)	0	2	50	1500	5000	0	2	50	1500	5000
First year (chronic phase)
Bilateral keratitis	0/10	0/9	6/9**	2/10	1/5	0/10	0/10	8/10**	6/8**	5/10*
Unilateral keratitis	0/10	0/9	3/9	3/10	2/5	0/10	0/10	1/10	1/8	5/10*
Recovery phase
Chronic progressive nephropathy	5/15	-	-	-	9/11	0/14	-	-	-	2/14
Interstitial inflammation of pancreas	2/15	-	-	-	7/11	1/14	-	-	-	3/14
Unilateral or bilateral keratitis in the eye	1/15	-	-	-	6/11	0/14	-	-	-	7/14
Second year (carcinogenicity phase)
Follicular cell hypertrophy in thyroid gland	3/50	6/47	16/48**	19/50**	27/48**	1/50	3/50	22/50**	20/49**	19/50**
Chronic progressive nephropathy	30/50	35/50	43/50**	42/50**	38/50**	16/50	17/50	25/50	20/50	24/50
Acinar atrophy/fibrosis in the pancreas	18/50	24/48	23/49	33/49**	36/48**	13/50	22/50*	28/50**	32/48**	31/50**
Unilateral or bilateral keratitis in the eye	1/49	1/50	41/49**	47/49**	42/50**	1/50	2/50	36/50**	44/49**	38/50**
Ulcerative necrotising inflammation in hindleg and/or hindpawn of samples	3/7	3/7	7/13	13/13	18/21	1/1	0/0	3/3	3/5	2/4

*: p≤0.05; **: p≤0.01

Applicant's summary and conclusion

Conclusions:

The NOAEL over a 24-month period of dietary administration with the test substance for males and females was 2 ppm for both (equivalent to 0.08 and 0.11 mg/kg bw/day in males and females, respectively).