Reaction mass of 3-(difluoromethyl)-1-methyl-N-[(1RS,4SR,9RS)-1,2,3,4-tetrahydro-9-isopropyl-1,4-methanonaphthalen-5-yl]pyrazole-4-carboxamide and of 3-(difluoromethyl)-1-methyl-N-[(1RS,4SR,9SR)-1,2,3,4-tetrahydro-9-isopropyl-1,4-methanonaphthalen-5-yl]pyrazole-4-carboxamide; isopyrazam

Endpoint summary

Description of key information

- Oral: NOAEL = 5.5 and 6.9 mg/kg bw/day in males and females, respectively, rats, chronic toxicity, 2 years, dietary, according to OECD TG 453, Milburn 2008

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 Mar 2006 to 13 Jul 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

Version / remarks:

1981

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.4300 (Combined Chronic Toxicity / Carcinogenicity)

Version / remarks:

1998

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1988

Qualifier:

according to guideline

Guideline:

other: According to Japanese Ministry of Agriculture, Forestry and Fisheries, Test Data for Registration of Agrochemicals, 12 Nohsan No. 8147, Agricultural Production Bureau.

Version / remarks:

24 Nov 2000

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

HsdRCCHan

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 5-6 weeks old
- Weight at study initiation: Males 104 - 151 g and females 93 - 138 g
- Housing: Animals were housed, sexes separately, in multiple rat racks suitable for animals of this strain and the weight range expected during the course of this study. They were housed 5 per cage initially and in fours after they had been assigned to experimental groups.
- Acclimatisation period: approximately 1 week
- Diet Test and control diets based on CT1 diet, ad libitum
- Water Mains water, supplied by an automatic system, ad libitum
- Acclimation period: approximately 1 week prior to the start of the study.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity: 30 - 70 %
- Air changes (hr) : At least 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 21 Mar 2006 To: 13 Jul 2008

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
- Mixing appropriate amounts with feed: The test and control diets were prepared (using RM1 diet), and transferred to the animal rooms as required. The diets were prepared in 60 kg batches from 1000 g premixes prepared by mixing the appropriate amount of test substance with milled diet. The amount of test substance required was made up to 60 kg diet.
- Test diets were stored at room temperature for up to 6 weeks.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Samples from all dietary levels (including controls) were taken prior to the start of the study at approximately three-monthly intervals throughout the study and analysed quantitatively for test substance.
- Prior to the start of the study the homogeneity of the test substance in RM1 diet was determined by analysing samples from the low and high dose levels and the chemical stability of the test substance in diet was determined for these same diets over a period of up to 42 days at room temperature.
- Samples were extracted with acetonitrile and aliquots of supernatant diluted with acetonitrile, as appropriate, to give sample solution concentrations within the range of the calibration standards used. Samples and standards were analysed by High Performance Liquid Chromatography (HPLC).

Duration of treatment / exposure:

104 weeks, with animals designated for the interim kill were exposed for 52 weeks.

Frequency of treatment:

Continuously

Dose / conc.:

100 ppm

Remarks:

Group 2: Dietary equivalent to 5.5 and 6.9 mg/kg bw/day for males and females, respectively

Dose / conc.:

500 ppm

Remarks:

Group 3: Dietary equivalent to 27.6 and 34.9 mg/kg bw/day for males and females, respectively

Dose / conc.:

3 000 ppm

Remarks:

Group 4: Dietary equivalent to 173.5 and 232.8 mg/kg bw/day for males and females, respectively

No. of animals per sex per dose:

52 in main study 104 weeks, and in addition 12 for interim sacrifice after week 52.

Control animals:

yes, plain diet

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS:
- Time schedule: twice daily. Prior to the start of the study, all rats were examined to ensure that they were normal. Cage-side observations included recording any changes in clinical condition or behaviour.

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: weekly, generally at the same time that the body weights were recorded. Any rats requiring euthanasia were killed and examined post mortem. Any rats found dead were examined post mortem as soon as possible after death.

BODY WEIGHT:
- Time schedule for examinations: The body weight of each rat was recorded immediately before feeding of the experimental diets commenced and then on the same day, where practicable, every subsequent week for weeks 2 - 15 of the study and then every 2 weeks until termination. All rats were weighed at termination.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Time schedule: Weight of food given and food residues were recorded for the first 14 weeks of the study, week 16 and thereafter every fourth week.
- Food consumption was calculated as a mean value (g food/rat/day) for each cage.
- The food utilisation value per cage was calculated as the body weight gained by the rats in the cage per 100 g of food eaten for the first 12 weeks of the study.
- Dose rates (based on nominal dietary levels of test substance) were calculated in terms of mg substance/kg body weight.

FUNCTIONAL OBSERVATION BATTERY
- Time schedule for examinations: Detailed clinical assessments (during which each rat was removed from its cage and physically examined for changes in general health status) and quantitative assessments of landing foot splay, muscle weakness (fore- and hind-limb grip strength) and sensory perception (tail-flick test) were made in week 50 for all interim kill animals.
- The observations were made by one observer who was ‘blind’ with respect to the animal’s treatment, and recorded on a computer system by personnel not directly involved in the clinical observations. The presence and/or absence of all listed observations were recorded and the degree of condition noted (slight, moderate or extreme) where appropriate. The examination proceeded from the least to the most interactive test with observations recorded as follows:
- Assessment in the home cage: bizarre behaviour (circling, head flicking, head searching, walking backwards, rolling over sideways, paw flicking), vocalisation.
- Removal from the cage: approach response, response to touch (increased, decreased), vocalisation.
- In the standard arena: activity (increased, decreased), comatose, prostration, hunched posture, bizarre behaviour (circling, head flicking, head searching, walking backwards, rolling over sideways, paw flicking), convulsions (tonic, clonic), vocalisation, ataxia, tremors, reduced stability, abnormal gait, splayed gait, tiptoe gait, reduced limb function (fore, hind), upward curvature of the spine, downward curvature of the spine, piloerection, sides pinched in, ungroomed appearance, urinary incontinence, diarrhoea.
- Handling the animal: response to touch, convulsions, vocalisation, tremors, piloerection, skin colour (pale, hyperaemia, cyanosis), ungroomed appearance, hyperthermia, hypothermia, chromodacryorrhea, lachrymation, ptosis, enophthalmos, exophthalmos, miosis, mydriasis, stains around the mouth, stains around the nose, salivation, respiratory abnormalities (breathing rate, breathing depth, laboured breathing, gasping, irregular breathing, whistling, wheezing, croaking), thin appearance sides pinched in, dehydration, abdominal tone (increased, decreased), urinary incontinence, diarrhoea.
- Reflex tests: righting reflex (from supine position), response to sound (to finger click/clap), splay reflex (degree of splay when animal lifted by base of tail), visual placing response (animal is lifted by base of tail and slowly moved downwards towards the edge of arena), pupil response to light (after eye has been held closed for 10 seconds), palpebral membrane reflex (palpebral membrane touched with bristle and blink response observed), corneal reflex (hair is touched against cornea and blink reflex observed - only performed if palpebral reflex is absent), pinna reflex (bristle poked into ear canal), foot withdrawal reflex (to toe pinch).
- Quantitative measures: fore-limb and hind-limb grip strength, landing foot splay, time to tail flick.
- In addition, any other observations that may facilitate interpretation of the data were recorded.

OPHTHALMOSCOPIC EXAMINATION
- Time schedule for examinations: The eyes of all surviving main study animals from the control and high dose groups were examined during week 50 and the eyes of all surviving animals were examined in weeks 101 to 103 (prior to termination).
- The examination was carried out using an ophthalmoscope. The animals were examined after instillation of 0.5 % v/v tropicamide into the eyes to dilate the pupils. The eyes of animals designated for the main study were examined pre-experimentally, except for 4 control females, which were not examined in error. This error is considered not to have affected the integrity of the study.

HAEMATOLOGY AND CLINICAL CHEMISTRY: MICROSCOPY
- Time schedule for examinations: Blood was collected via the tail vein and samples were taken for haematology from pre-designated animals (n = 13 per group) in weeks 14, 27, 53 and 79. Blood was collected via the tail vein and samples were taken for clinical chemistry from a further designated thirteen male and thirteen female rats per group at weeks 14, 27, 53 and 79.
- Any designated rat which died or was killed before the scheduled time was replaced, as necessary, by an alternative animal where possible in order to maintain acceptable group sizes (minimum 10/sex/group). All surviving rats were bled by cardiac puncture at the interim kill in week 53 or at termination in week 105 for haematology and clinical chemistry.

HAEMATOLOGY
- The following measurements were made or determined on samples collected using EDTA as an anticoagulant: haemoglobin, mean cell haemoglobin concentration, haematocrit, platelet count, red blood cell count, total white cell count, mean cell volume, differential white cell count, mean cell haemoglobin and blood cell morphology.
- Clotting measurements consisting of prothrombin time and activated partial thromboplastin time were made on samples of blood collected in tubes containing sodium citrate as an anticoagulant. Blood cell morphology, including a differential white blood cell count was assessed by automated methods for all animals. Manual blood films were prepared and analysed as necessary.

CLINICAL CHEMISTRY (BLOOD)
- The following measurements were made on the plasma from blood samples collected into tubes containing lithium heparin as an anticoagulant: urea, alkaline phosphatase activity, creatinine aspartate, aminotransferase activity, glucose alanine aminotransferase activity, albumin, gamma-glutamyl transferase activity, total protein calcium, cholesterol, phosphorus (as phosphate), triglycerides, sodium, total bilirubin, potassium, creatine kinase activity and chloride.

CLINICAL CHEMISTRY (URINE)
- Time schedule for examinations: Individual urine samples were collected from a pre-designated thirteen males and thirteen females per group at weeks 13, 26, 52, 78 and 104. These were the same animals as those used for haematology analyses. Any designated rat which died or was killed before the scheduled time was replaced, as necessary, by an alternative animal where possible in order to maintain acceptable group sizes (minimum 10/sex/group).
- Samples were collected over a period of 16 - 18 hours during which the rats were housed individually in metabolism cages and denied access to food. The following parameters were measured or assessed and recorded on each urine sample: colour, appearance, volume, pH, specific gravity, protein, glucose, ketones, bilirubin and blood.

MOTOR ACTIVITY
- Locomotor activity was monitored by an automated activity recording apparatus. All interim kill animals were tested in week 50.
- Each observation period was divided into ten scans of five minutes duration. Treatment groups were counter balanced across test times and across devices. Motor activity was assessed in a separate room to minimise disturbances. During motor activity assessment, animals did not have access to food, water or items of environmental enrichment.

Sacrifice and pathology:

GROSS PATHOLOGY
- With the exception of animals found dead, all rats were killed by over exposure to halothane Ph. Eur. vapour followed by exsanguination by cardiac puncture.
- From all animals at interim kill and those from scheduled termination, the following organs were removed, trimmed free of extraneous tissue and weighed: adrenal, glands liver, brain, ovaries, epididymides, spleen, heart testes, kidneys and uterus (with cervix). Paired organs were weighed together.

HISTOPATHOLOGY
- All animals were examined post mortem. This involved an external observation and a detailed internal examination of all organs and structures.
- The following tissues were examined in situ, removed and examined and fixed in an appropriate fixative: gross lesions including masses, oesophagus, adrenal gland, ovary, aorta, oviduct, brain (cerebrum, cerebellum and brainstem), pancreas, bone - femur (including knee joint) parathyroid gland, bone marrow – femur, Peyer’s patches, caecum, pharynx, cervix, pituitary gland, colon, prostate gland, duodenum, rectum, epididymis, salivary gland eyes (retina, optic nerve), seminal vesicle, ex-orbital lachrymal, gland skin, harderian gland, spinal cord (cervical, thoracic, lumbar) heart, spleen, ileum, sternum, jejunum, stomach, kidney, testis, larynx, thymus, liver, thyroid gland, lung, trachea, lymph node – cervical, urinary bladder, lymph node – mesenteric, uterus, mammary gland - (females only), vagina, nerve – sciatic, voluntary muscle and nose.
All tissues from all animals were submitted for histology. Tissues for histology were routinely processed, embedded in paraffin wax, sectioned at 5 μm and stained with haematoxylin and eosin.

MICROSCOPIC EXAMINATION
- All submitted tissues were examined by light microscopy.

Statistics:

- All data were evaluated using the MIXED procedure in SAS (2004). Body weights were considered by analysis of covariance on initial (week 1) body weight,
separately for males and females. Food consumption and food utilisation during the periods weeks 1 - 4, 5 - 8, 9 - 12 and 1 - 12 were considered by analysis of variance, separately for males and females.
- Haematology data were considered by analysis of variance, separately for males and females.
- Motor activity measurements, time to tail-flick (in the sensory function test), landing foot splay and grip strengths were considered by analysis of variance, separately for males and females.
- Organ weights were considered by analysis of variance and analysis of covariance on final bodyweight, separately for males and females. Summary data are presented for organ to body weight ratios but these were not analysed statistically as the analysis of covariance provides a better method of allowing for differences in terminal body weights.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were more males in the 3000 ppm group with observations of scabs and subcutaneous masses than in the control group. There were more incidences of scabs in females in the 3000 ppm group than in the control group, although the number of animals affected was similar. All other observations affected a small number of animals or were recorded in similar numbers of control and treated rats and are considered not to be related to treatment.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Survival was good with approximately 75% of males and 67% of females on the study surviving to scheduled termination after two years. There were no treatment related effects on survival.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weights of animals receiving 3000 ppm test substance were reduced in comparison with control throughout the study. The maximum difference from control was 14 % in males and 30 % in females. Body weight gain in females at 3000 ppm was approximately 40% lower than control at the end of the study.
Body weights of males at 500 ppm groups were similar to control throughout the study. Body weights of females in the 500 ppm group were consistently lower than control. The effect was more pronounced during the second year of the study reaching a maximum of 12% below control for body weight and 17 % for body weight gain.
Body weights of males at 100 ppm groups were similar to control throughout the study. Body weights of females in the 100 ppm group were considered to be unaffected by treatment. During the second year of the study body weights in the 100 ppm females diverged from control attaining statistical significance towards the end of the study. However the body weights of the concurrent controls were consistently higher than historical control data, particularly during the second year of the study, whilst the growth curve of the 100 ppm group closely followed that typically seen in historical control curves. Therefore the body weights of females in the 100 ppm group were concluded to reflect the normal growth pattern of this strain of rat in this laboratory and the minor differences seen were considered to be unrelated to treatment.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Food consumption was reduced in rats in the 3000 ppm group from the start of the study to approximately week 11 in males and week 14 in females; thereafter values were similar to control. There were no consistent effects on food consumption in rats in the 100 or 500 ppm groups.
The efficiency of food utilisation from weeks 1 - 12 was reduced in both sexes in the 3000 ppm group. Food utilisation was slightly less efficient than control in females in the 500 ppm group and the difference was statistically significant overall for weeks 1 - 12. There were no effects on food utilisation in males in the 500 ppm group or either sex in the 100 ppm group.

Food efficiency:

effects observed, non-treatment-related

Description (incidence and severity):

Food utilisation for males fed 1000 ppm was statistically significantly less efficient than control between weeks 1 - 4, 5 - 8 and overall (weeks 1 - 13). The food utilisation for females fed 1000 ppm and both sexes at the lower dose levels was unaffected by treatment with test substance.

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

not examined

Ophthalmological findings:

effects observed, non-treatment-related

Description (incidence and severity):

In week 50 findings were similar in control rats and those receiving 3000 ppm. In weeks 101 to 103 there were more females with ophthalmoscopy findings in the 3000 ppm group than in the control group. For this reason the eyes of all animals from the 100 and 500 ppm groups were also examined. The number of males with findings was 21, 17, 13 and 23 in the control, 100, 500 and 3000 ppm groups respectively. The number of females with findings was 7, 5, 12 and 14 in the control, 100, 500 and 3000 ppm groups respectively. Although there were more females with findings in the 3000 ppm group than in the control group these comprised a variety of common findings and there was no evidence of any effect of treatment.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Treatment related effects were confined to differences in some parameters in females in the 3000 ppm group only. These comprised reduced haemoglobin, haematocrit and red blood cell count and increased platelet counts compared to control. White blood cell parameters were not affected by treatment. Other differences from control e.g. lower mean cell volume and mean cell haemoglobin in males in the 500 and 3000 ppm groups, were small and did not show a consistent pattern and were considered to be unrelated to treatment. There were no consistent effects on clotting times, small differences seen were inconsistent with time, sex and between the two measures of activated partial thromboplastin and prothrombin time.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Plasma triglycerides were reduced in the 3000 ppm group. This was evident throughout the study in males and from week 53 in females. Plasma triglycerides were also reduced in females in the 500 ppm group in the second year of the study. Differences in females at 100 ppm were intermittent, were not present at the end of the study and are considered to be unrelated to treatment.
Plasma cholesterol was increased in main study females in the 3000 ppm group at most time points. There was no effect in males or in the lower dose groups.
Plasma bilirubin levels were generally reduced in females in the 500 and 3000 ppm groups from week 27. There were some instances of lower plasma bilirubin levels in males in the 3000 ppm group. The isolated statistically significantly lower bilirubin value in females in the 100 ppm group at week 79 is considered not to be related to treatment, as the value is similar to the control value at week 53 and there is no difference from control at week 105.
Gamma glutamyl transferase activity was increased in males in the 500 and 3000 ppm groups from week 53. In females in the 3000 ppm group there were some high individual gamma glutamyl transferase values, but the difference from control was statistically significant in week 105 only.
Alanine transaminase activity was increased in males in the 3000 ppm group due to some high individual values and the difference from control was statistically significant in weeks 53 and 105. Activities of alkaline phosphatase and aspartate amino transferase were generally lower than control in males and females in the 3000 ppm group. There were a few instances of statistically significantly lower values in the 500 ppm group.
Sodium and chloride ions were marginally increased in females in the 3000 ppm group.
Chloride ions were occasionally higher than control in males in the 3000 ppm group but showed no consistent pattern and are considered to be unrelated to treatment. There was an isolated, higher calcium value at week 105 in females. There was no effect on plasma urea in males. In females in the 3000 ppm group values were statistically significantly increased in weeks 53 and 105.
There were a small number of high plasma creatinine values in the 3000 ppm group and values were statistically significantly higher than control in females in weeks 53 and 79, but in the absence of a difference at week 105, these differences are considered unrelated to treatment. Small differences from control in other parameters did not show a consistent pattern and were considered to be unrelated to treatment.

Endocrine findings:

not examined

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no effects on the urine parameters measured or assessed. Urine pH was lower than control in females in the 3000 ppm group in week 13, however as there were no differences from control at other time points, this is considered to be unrelated to treatment.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Functional observation battery: There were no treatment-related effects on landing foot splay, on fore limb grip strength or time to tail-flick. Hind limb grip strength was reduced compared to control in females in the 3000 ppm group. There was no effect in males in the 3000 ppm group or either sex in the 100 or 500 ppm groups.
All groups showed a typical pattern of reducing activity over the monitoring period with no evidence of an effect of treatment. A higher level of activity was recorded for 36-40 minutes in females in the 3000 ppm group than in the control group and this higher activity was also reflected in the overall activity of these animals. Activity of males in the 3000 ppm group and both sexes in the 100 or 500 ppm groups was similar to control.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Interim kill:
Liver weights were increased in the 3000 ppm group, by 17% in males and by 14% in females. There were no significant differences from control in the 100 or 500 ppm groups.
Adrenal weights and heart weights were lower than control in females in the 3000 ppm group.
Brain weights were higher than control in males in the 3000 ppm group.
At interim kill no treatment related differences were evident on the weights of epididymides, kidneys, ovaries, spleen, testes or uterus with cervix.

Terminal kill:
Liver weights were increased in the 3000 ppm group by 18% in males and by 27% in females. Weights were also increased by 12% in females in the 500 ppm group. There were no significant differences from control in either sex in the 100 ppm group.
Adrenal weights were lower than control in females in the 3000 ppm group when abnormally high adrenal weights were excluded.
Brain weights were higher than control in females in the 3000 ppm group.
At terminal kill no treatment related differences were evident on the weights of epididymides, heart, kidneys, ovaries, spleen, testes or uterus with cervix.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were a higher number of females in the 3000 ppm group with watery fluid in the abdominal cavity. These were all decedents (1, 0, 2, 5 in the control, 100, 500 and 3000 ppm groups respectively). At terminal kill there were also a higher number of females in the 3000 ppm group with pale spots or areas on the liver and with a liver mass. There were no treatment related macroscopic findings in males in the 3000 ppm group or either sex in the 100 or 500 ppm groups.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

12 month kill:
Treatment related findings were confined to the liver. There was an increased incidence of hepatocyte vacuolation, hepatocyte hypertrophy and increased hepatocyte/Kupffer cell pigmentation in the livers of all males and females in the 3000 ppm group. These findings were also present, although the severity was less, in the majority of males and females in the 500 ppm group. Liver findings in males and females in the 100 ppm group were similar to control indicating that there was no treatment related effect at this dose level.

Main study:
Treatment related findings were seen in the liver, kidneys (females) and mesenteric lymph nodes. There were treatment-related increases in the incidence of several changes in the liver which were sometimes accompanied by an increase in severity.

At 100 ppm, liver effects were centrilobular minimal hepatocyte hypertrophy (both sexes) and minimal centrilobular hepatocyte pigmentation (females). In males there was also a slightly higher incidence of findings typically seen in aging rats which were minimal hyperplasia of the bile ducts, accompanied in some animals by minimal fibrosis. The findings at 100 ppm were predominantly observed in animals at week 105. The earliest incidence of bile duct hyperplasia was observed at week 80 and the earliest incidence of pigmentation in females was seen at week 72.

Brown pigment was seen in the kidney tubules of animals from treated and control groups but the incidence was significantly greater than controls in females in the 500 and 3000 ppm groups. There was an increased incidence of sinus erythrocytosis in the mesenteric lymph nodes of males in the 3000 ppm group.

An increased incidence of eosinophilic droplets was seen in the nasal epithelium of both sexes in
the 500 ppm group but in the absence of a similar increase in the 3000 ppm group, this is considered not to be related to treatment.

A few spontaneous findings showed a slightly lower incidence in some treated groups in comparison with controls. These findings included indentation of the ventral brain, chronic cardiomyopathy, progressive chronic nephropathy, epithelial mineralisation of the renal pelvis, dilation /secretion of the mammary gland ducts and diffuse hyperplasia of the thyroid C-cells. A lower incidence and/or severity of spontaneous changes are often associated with decreased weight gain and are not usually regarded as an adverse effect of treatment.

A variety of other spontaneous changes was noted with no evidence of an effect of treatment. The incidence and the spectrum of these findings are consistent with changes commonly encountered in rats of this age kept under laboratory conditions

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

In the 3000 ppm group there were increased incidences of uterine endometrial adenocarcinoma and liver hepatocellular adenoma in females. A reduced incidence of mammary fibroadenoma was also seen in females in the 3000 ppm group. All findings were statistically significant using the Peto-trend test. Endometrial adenocarcinoma of the uterus was considered to have led to the death or early termination of five females in the 3000 ppm group.
There was a higher incidence of thyroid follicular cell adenoma observed in the 3000 ppm males (7/64 – 10.9 % compared to controls 1/64 – 1.56 %) that was statistically significant when analysed with the Peto-trend test, but was not significant using Fisher’s exact test.
The historical control incidence from 3 previous studies in this laboratory (PR1248, PR1321 and PR1324) ranged from 1.6 to 9.6 %. While the incidence in 3000 ppm males was marginally outside the laboratories historical control range, an incidence of 10.9 % is within the range of control values held in the RITA data base (Wistar rats), which shows a wide variation from 0 to 28 % (104 studies carried out between 1983 and 2004). In the period 1997 – 2004 there were 32 recorded studies. In seven of these studies the number of thyroid adenomas was greater than 10.9 %, demonstrating that 10.9% is within the previously recorded range for the wider control population (1997 – 12 %; 1998 (x2) – 12%; 1998 – 14 %; 1999 – 28.0 %; 2000 – 14.7 %, 2002 – 11.8 %). No treatment related neoplastic findings were seen at 100 or 500 ppm.

Key result

Dose descriptor:

NOAEL

Effect level:

100 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

histopathology: non-neoplastic

Remarks on result:

other: Dietary equivalent to 5.5 and 6.9 mg/kg bw/day in males and females, respectively.

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

3 000 ppm

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

3 000 ppm

System:

female reproductive system

Organ:

uterus

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

Verification of the Test Diets

The mean concentrations for all batches of diet preparations analysed were within 10 % of the nominal concentration. The homogeneity of the test substance in diet preparations at concentrations of 100 ppm and 3000 ppm, (for a batch size of 60 kg), was determined and considered satisfactory, percentage deviations from the overall mean were within 2 %. The reanalysis of the test substance in diet preparations at concentrations of 100 ppm and 3000 ppm when stored at room temperature was shown to be satisfactory for 42 days, covering the period of dosing.

Table 1. Intergroup comparison of hind limb grip strength (g) in females.

Dietary concentration of test substance (ppm)	0	100	500	3000
Week 50	686	578	558	489**

** Statistically significant difference from control group mean, p<0.01 (Student’s t-test, 2-sided)

Table 2. Intergroup comparison of adjusted body weight (g): selected weeks.

Week	Dietary Concentration of test substance (ppm)
	Males				Females
	0	100	500	3000	0	100	500	3000
1	125.8	125.7	125.6	126.8	113.1	112.6	111.1	112.0
2	166.4	166.2	165.4	159.1**	134.3	134.4	133.7	129.3**
13	373.3	367.9	370.7	344.7**	230.1	224.7*	222.9**	206.9**
27	440.6	433.6	438.9	405.4**	256.5	251.0	249.3*	227.2**
39	483.2	474.9	481.5	439.9**	277.1	273.2	270.5	238.2**
53	523.3	513.1	521.8	469.4**	305.4	297.6	294.7*	248.0**
79	571.5	565.1	573.2	504.2**	363.5	346.4*	332.8**	267.5**
105	610.1	603.3	623.3	530.2**	403.5	382.0*	359.2**	296.2**

* Statistically significant difference from control group mean, p<0.05 (Student’s t-test, 2-sided)

** Statistically significant difference from control group mean, p<0.01 (Student’s t-test, 2-sided

Table 3. Intergroup comparison of food utilisation (g body weight gained/100 g food consumed).

Week	Dietary Concentration of test substance (ppm)
	Males				Females
	0	100	500	3000	0	100	500	3000
1-4	22.88	22 72	22.92	22.07**	14.86	14.50	14.19	12.53**
5-8	10.24	10.21	10.42	937**	6.34	6.07	6.05	5.64**
9-12	6.26	6.25	6.20	5.47**	3.23	3.04	321	2.71**
Overall (1-12)	13.20	13.10	13.20	12.30**	8.03	7.80	7.73*	6.86**

* Statistically significant difference from control group mean, p<0.05 (Student’s t-test, 2-sided)

** Statistically significant difference from control group mean, p<0.01 (Student’s t-test, 2-sided)

Table 4. Intergroup comparison of liver weights adjusted for body weight (g).

Organ/week	Dietary Concentration of test substance (ppm)
	Males				Females
	0	100	500	3000	0	100	500	3000
Liver, 53	15.1	14.3	15.2	17.7**	9.0	8.9	9.6	10.3**
Liver , 105	16.6	16.5	17.5*	19.5**	10.6	11.2	11.9**	13.4**

* Statistically significant difference from control group mean, p<0.05 (Student’s t-test, 2-sided)

** Statistically significant difference from control group mean, p<0.01 (Student’s t-test, 2-sided)

Table 5. Incidence of selected microscopic non-neoplastic findings in the liver at week.

Week	Dietary Concentration of test substance (ppm)
	Males				Females
	0	100	500	3000	0	100	500	3000
Total number examined (decedents)	52 (15)	52 (14)	52 (11)	52 (12)	52 (14)	52 (17)	52 (22)	52 (19)
Hypertrophy, hepatocellular, centrilobular	0 (0)	12 (0)**	45 (6)**	49 (11)**	0 (0)	22 (5)**	49 (19)**	50 (17)**
Vacuolation, hepatocellular, centrilobular	3 (2)	7 (1)	32 (6)**	39 (6)**	1 (1)	0 (0)	18 (4)**	3 (0)
Pigment, hepatocellular, centrilobular	0 (0)	0 (0)	1   (0)	32 (6)**	3 (0)	34 (12)**	49 (19)**	46 (16)**
Hyperplasia, bile ducts	3 (1)	11 (2)*	19 (2)**	12 (1)**	15 (4)	12 (5)	17  (9)	23 (6)
Fibrosis, bile ducts	1 (1)	5 (1)	10 (0)**	12 (1)**	6 (1)	6 (2)	12 (5)	11 (3)
Altered hepatocytes, eosinophilic	7 (1)	1  (2)	23 (3)**	32 (3)**	9 (2)	15 (3)	26 (11)**	29 (7)**

Table 6. Incidence of selected microscopic non-neoplastic findings in the female kidney at week 105.

	Dietary Concentration of test substance (ppm)
Finding	0	100	500	3000
Pigment, brown, tubules	18/52	24/52	30/52*	42/52**

* Statistically significant difference from control incidence, p<0.05 (Fisher’s exact test)

** Statistically significant difference from control incidence, p<0.01 (Fisher’s exact test)

Table 7. Incidence of selected microscopic non-neoplastic findings in the male mesenteric lymph node at week 105.

	Dietary Concentration of test substance (ppm)
Finding	0	100	500	3000
Erythrocytosis, sinus	3/52	8/52	8/51	14/52**

** Statistically significant difference from control incidence, p<0.01 (Fisher’s exact test)

Table 8. Incidence of selected microscopic neoplastic findings at 52 & 105 weeks.

	Dietary Concentration of test substance (ppm)
	0	100	500	3000
Total number examined (12 at interim kill, 52 up to 105 weeks)	64	64	64	64
Males: thyroid follicular cell adenoma Total incidence Incidence at interim kill	1   0	4   0	2   0	7   1
Females: thyroid follicular cell adenoma Total incidence Incidence at interim kill	5   0	1   0	3   0	5   2
Females: hepatocellulair adenoma Females: uterine endometrial adenocarcinoma	1 1	1 2	1 3	11 15

Conclusions:

Oral administration of test substance in the diet at dose levels of 100 ppm (5.5 and 6.9 mg/kg/day in males and females) there were minimal treatment related non-neoplastic changes in the liver that were considered not to be adverse. 5.5 mg/kg bw/day was considered to be the no observed adverse effect level (NOAEL).

Executive summary:

In an OECD TG 453 study in compliance with GLP, groups of 64 male and 64 female HsdRCCHan: WIST rats were fed diets containing 0 (control), 100, 500 or 3000 ppm test substance for up to 2 years (mean dietary equivalent to 5.5, 27.6 and 173.5 mg/kg bw/day for males and 6.9, 34.9 and 232.8 mg/kg bw/day for females, respectively). Twelve males and twelve females from each group were designated for interim kill after 52 weeks. The surviving animals continued to termination after 104 weeks on test. Clinical observations, body weights and food consumption were measured and blood and urine samples were taken for clinical pathology. A functional observation battery of tests and locomotor activity monitoring were performed during week 50 on the interim kill animals. An ophthalmoscopic examination was performed on all main study animals pre study, during weeks 50 and during weeks 101 to 103. All animals, including any found dead or killed prematurely were examined post mortem and tissues were taken for subsequent histopathology examination. At the scheduled 53 or 105 week examinations post mortem, cardiac blood samples were taken for clinical pathology and selected organs were weighed.

There were no treatment related effects on survival. There were higher numbers of males in the 3000 ppm group with scabs and subcutaneous masses than in the control group. There was no evidence of any effect of treatment on ophthalmoscopy findings. Body weights of animals receiving 3000 ppm test substance were reduced throughout the study. The maximum difference from control was 14 % in males and 30 % in females for body weight and 17 % and 40 % respectively for body weight gain. Body weights of females in the 500 ppm group were consistently lower than control. The effect was more pronounced during the second year of the study reaching a maximum of 12 % below control for body weight and 17 % for body weight gain. Body weights of females in the 100 ppm group were considered to be unaffected by treatment. Food consumption and the efficiency of food utilisation were reduced in rats in the 3000 ppm group in the early stages of the study only. There were no treatment related effects on the functional observation clinical assessments, landing foot splay, motor activity, time to tail flick or fore limb grip strength. Hind limb grip strength was lower than control in females in the 3000 ppm group reflecting their lower body weights. There were some indications of a small effect on haemoglobin and related parameters in rats in the 3000 ppm group. Platelet counts were increased in females in the 3000 ppm group at most time points. White blood cell parameters were not affected by treatment and there were no consistent effects on clotting times. There were a number of blood clinical chemistry findings in the 3000 ppm group. Plasma triglycerides were reduced in both sexes, plasma cholesterol was increased and plasma bilirubin levels were generally reduced in females. There was some evidence of increases in gamma glutamyl transferase and alanine transaminase activities. Activities of alkaline phosphatase and aspartate amino transferase were generally lower than control. Sodium and chloride ions were generally increased in females. There were a few small changes in blood clinical chemistry findings in the 500 ppm group but none in the 100 ppm group. There were no treatment-related effects on the urine parameters measured or assessed. Liver weights were increased in the 3000 ppm group at interim and terminal kill and in females in the 500 ppm group at terminal kill only. There were changes in the liver of rats in the 500 and 3000 ppm groups (hepatocellular hypertrophy, pigment in centrilobular hepatocytes, eosinophilic foci of altered hepatocytes, vacuolation of centrilobular hepatocytes and hyperplasia and fibrosis of the bile ducts). Minimal treatment related changes at 100 ppm were hepatocellular hypertrophy in both sexes, bile duct hyperplasia in males and pigment in centrilobular hepatocytes in females. An increased incidence of brown pigment was seen in the kidney of females in the 500 and 3000 ppm groups. There was an increased incidence of sinus erythrocytosis in the mesenteric lymph nodes of males in the 3000 ppm group. There were increased incidences of uterine endometrial adenocarcinoma and liver hepatocellular adenoma and a reduced incidence of mammary fibroadenoma in females at 3000 ppm. No effect on tumour incidence was seen in males or at the 100 or 500 ppm dose levels. Uterine endometrial adenocarcinoma was considered to have led to the death or early termination of five females in the 3000 ppm group.

In conclusion, oral administration of test substance in the diet at dose levels up to 3000 ppm for up to two years led to a marked effect on body weight/body weight gain, particularly in females. There were increases in uterine endometrial adenocarcinoma and liver hepatocellular adenoma in females at this dose level. Treatment-related non-neoplastic findings were seen in the liver, kidney (females) and mesenteric lymph nodes (males). A dose of 500 ppm was the no effect level for neoplastic changes. At this dose the main effects were non-neoplastic changes in the liver and kidney (females) and a reduction in body weight/body weight gain in females. At a dose of 100 ppm (5.5 and 6.9 mg/kg/day in males and females) there were minimal treatment related non-neoplastic changes in the liver that were considered not to be adverse. This dose was considered to be the no observed adverse effect level (NOAEL).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

5.5 mg/kg bw/day

Study duration:

chronic

Species:

rat

Quality of whole database:

This study was conducted in accordance with OECD TG 453 following GLP principles.

System:

hepatobiliary

Organ:

liver

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

All available data was assessed and the studies representing the worst-case effects were included as key studies. Other studies are included as supporting information. Sub-chronic and chronic oral toxicity studies in the rat, mouse and dog demonstrate that the primary target organ for the test substance is the liver (increased organ weight and centrilobular hepatocyte hypertrophy). Liver toxicity is usually accompanied by reductions in body weight and food consumption.

 

Chronic Toxicity

Two chronic toxicity studies are available. In the key study (Milburn 2008), performed according to OECD TG 453 and GLP, groups of 64 male and 64 female HsdRCCHan: WIST rats were fed diets containing 0 (control), 100, 500 or 3000 ppm test substance for up to 2 years (mean dietary equivalent to 5.5, 27.6 and 173.5 mg/kg bw/day for males and 6.9, 34.9 and 232.8 mg/kg bw/day for females, respectively). Twelve males and twelve females from each group were designated for interim kill after 52 weeks. The surviving animals continued to termination after 104 weeks on test. Clinical observations, body weights and food consumption were measured and blood and urine samples were taken for clinical pathology. A functional observation battery of tests and locomotor activity monitoring were performed during week 50 on the interim kill animals. An ophthalmoscopic examination was performed on all main study animals pre study, during weeks 50 and during weeks 101 to 103. All animals, including any found dead or killed prematurely were examined post mortem and tissues were taken for subsequent histopathology examination. At the scheduled 53 or 105 week examinations post mortem, cardiac blood samples were taken for clinical pathology and selected organs were weighed.

There were no treatment related effects on survival. There were higher numbers of males in the 3000 ppm group with scabs and subcutaneous masses than in the control group. There was no evidence of any effect of treatment on ophthalmoscopy findings. Body weights of animals receiving 3000 ppm test substance were reduced throughout the study. The maximum difference from control was 14 % in males and 30 % in females for body weight and 17 % and 40 % respectively for body weight gain. Body weights of females in the 500 ppm group were consistently lower than control. The effect was more pronounced during the second year of the study reaching a maximum of 12 % below control for body weight and 17 % for body weight gain. Body weights of females in the 100 ppm group were considered to be unaffected by treatment. Food consumption and the efficiency of food utilisation were reduced in rats in the 3000 ppm group in the early stages of the study only. There were no treatment related effects on the functional observation clinical assessments, landing foot splay, motor activity, time to tail flick or fore limb grip strength. Hind limb grip strength was lower than control in females in the 3000 ppm group reflecting their lower body weights. There were some indications of a small effect on haemoglobin and related parameters in rats in the 3000 ppm group. Platelet counts were increased in females in the 3000 ppm group at most time points. White blood cell parameters were not affected by treatment and there were no consistent effects on clotting times. There were a number of blood clinical chemistry findings in the 3000 ppm group. Plasma triglycerides were reduced in both sexes, plasma cholesterol was increased and plasma bilirubin levels were generally reduced in females. There was some evidence of increases in gamma glutamyl transferase and alanine transaminase activities. Activities of alkaline phosphatase and aspartate amino transferase were generally lower than control. Sodium and chloride ions were generally increased in females. There were a few small changes in blood clinical chemistry findings in the 500 ppm group but none in the 100 ppm group. There were no treatment-related effects on the urine parameters measured or assessed. Liver weights were increased in the 3000 ppm group at interim and terminal kill and in females in the 500 ppm group at terminal kill only. There were changes in the liver of rats in the 500 and 3000 ppm groups (hepatocellular hypertrophy, pigment in centrilobular hepatocytes, eosinophilic foci of altered hepatocytes, vacuolation of centrilobular hepatocytes and hyperplasia and fibrosis of the bile ducts). Minimal treatment related changes at 100 ppm were hepatocellular hypertrophy in both sexes, bile duct hyperplasia in males and pigment in centrilobular hepatocytes in females. An increased incidence of brown pigment was seen in the kidney of females in the 500 and 3000 ppm groups. There was an increased incidence of sinus erythrocytosis in the mesenteric lymph nodes of males in the 3000 ppm group. There were increased incidences of uterine endometrial adenocarcinoma and liver hepatocellular adenoma and a reduced incidence of mammary fibroadenoma in females at 3000 ppm. No effect on tumour incidence was seen in males or at the 100 or 500 ppm dose levels. Uterine endometrial adenocarcinoma was considered to have led to the death or early termination of five females in the 3000 ppm group.

In conclusion, oral administration of test substance in the diet at dose levels up to 3000 ppm for up to two years led to a marked effect on body weight/body weight gain, particularly in females. There were increases in uterine endometrial adenocarcinoma and liver hepatocellular adenoma in females at this dose level. Treatment-related non-neoplastic findings were seen in the liver, kidney (females) and mesenteric lymph nodes (males). A dose of 500 ppm was the no effect level for neoplastic changes. At this dose the main effects were non-neoplastic changes in the liver and kidney (females) and a reduction in body weight/body weight gain in females. At a dose of 100 ppm (5.5 and 6.9 mg/kg/day in males and females) there were minimal treatment related non-neoplastic changes in the liver that were considered not to be adverse. This dose was considered to be the no observed adverse effect level (NOAEL).

 

The other chronic toxicity study, performed according to OECD TG 452 under GLP, is a supporting study (Jackson 2008). In this study, dogs were exposed via capsules to 25, 100 and 250 mg/kg bw/day for 12 months. The main observed effects were related to adaptive liver responses in males, such as increased liver weights (up to 52%) and changes in related blood parameters (increased alkaline phosphatase, alanine aminotransferase and glutamate dehydrogenase levels, in combination with decreased bilirubin and albumin). No liver hypertrophy was observed. The NOAEL was set at 25 mg/kg bw/day.

 

Sub-chronic toxicity

Five sub-chronic toxicity studies are available, two in rats, one in mice and two in dogs. In the key study (Shearer & Wood 2009), groups of 10 male and 10 female Han Wistar rats fed diets containing 0, 100, 250 or 2000 ppm of two different syn:anti ratios of the test material for a period of at least 90 days. This study was conducted in accordance with OECD TG 408 following GLP principles.

Daily administration of the test material for at least 90 consecutive days was generally associated with a significant reduction in body weight change in females treated with either test material at 2000 ppm and to a lesser extent in the males. Food utilisation was reduced in females receiving 2000 ppm. There were no treatment related effects on ophthalmoscopy observations or in the functional observation battery. There were no treatment related effects on haematology or urine parameters. Alkaline phosphatase activity was noted to be statistically significantly lower in both sexes at 2000 ppm compared to control. Alkaline phosphatase activity was slightly lower than control in females at 250 ppm. Liver weights were increased in both sexes in the 2000 ppm groups for both test materials. Thyroid weights were higher than control in males in the 2000 ppm groups for both test materials. Centrilobular hypertrophy and midzonal hepatocyte vacuolation were observed in both sexes and with both test materials at 2000 ppm. There were no treatment related findings in the liver at 100 or 250 ppm with either test material.

The no observed adverse effect level (NOAEL) was considered to be 250 ppm with either test material. This was equivalent to 20.29 and 24.14 mg/kg bw/day for males and females, respectively, for the test material with a syn:anti ratio of 89.5:6.9 and 20.75 and 24.15 mg/kg bw/day for males and females, respectively, for the test material with a syn:anti ratio of 63.3:27.5.

 

The other sub-chronic toxicity studies, either performed according to OECD TG 408 and GLP or OECD TG 409 and GLP, are supporting studies (Milburn 2007, Pinto 2008, Brammer 2007, Jackson 2008). Findings related to adaptive responses of the liver were consistent for the different test species. Unlike the studies in rats and mice, liver hypertrophy was not reported in dog.

 

Short-term toxicity

Five short-term toxicity studies are available, four in rat and one in mice. In the key study (Milburn 2007), groups of 5 male and 5 female HsdRccHan: WIST rats were given 0, 300, 4000, 8000 ppm of the test material in diet for 28 consecutive days in a study according to OECD TG 407 following GLP principles. Clinical observations, body weights and food consumption were measured throughout the study.

All animals survived to the end of the study and no clinical abnormalities were detected. The animals in the 8000 ppm group lost weight initially and by day 29 adjusted weights were 14 and 18 % below control in males and females, respectively. There was a reduction in growth in the 4000 ppm group; body weights at the end of the study were 12 and 10 % below control in males and females, respectively. There were no effects on body weight in males and females in the 300 ppm group. Food consumption of males and females receiving 4000 or 8000 ppm was lower than that of the controls. Prothrombin time was increased in males in the 8000 ppm group. Neutrophils and eosinophils were lower than control in females in the 8000 ppm group and neutrophils were lower in female in the 4000 ppm group. In the 4000 and 8000 ppm groups urea, cholesterol, and phosphorus levels were increased in females and triglycerides were reduced in males. In the 8000 ppm group only creatinine levels were lower and chloride levels were higher than control in males and potassium levels and gamma glutamyl transferase activity were higher than control in females. Liver weights were increased in males and females in the 4000 and 8000 ppm treatment groups. Testes weights in the 8000 ppm were higher than control, but only after adjustment for final body weight, which was significantly decreased. All animals in the 4000 and 8000 ppm groups showed minor histopathological changes in the liver in the form of minimal or slight centrilobular hepatocyte hypertrophy.

The no observed adverse effect level (NOAEL) was considered to be 300 ppm of the test material, equivalent to 29.4 and 28.1 mg/kg bw/day in males and females, respectively.

 

The other short-term toxicity studies, either performed according or similar to OECD TG 407 and following GLP, are supporting studies (Milburn 2007a & 2007b & 2008, Rattray and Milburn 2007). The three studies yielded a consistent array of effects consisting in increases of relative liver weight and slight alterations in certain haematological and biochemical parameters. These alterations were in general inconsistent among different studies except for the increases in cholesterol concentrations and decreases in triglyceride concentrations. There were no treatment-related macroscopic necropsy findings. However, treatment-related microscopic changes (hepatocellular hypertrophy) was observed for all three test material ratios. Thus, these changes are likely to be an indication of an adaptive response rather than of adversity.

Justification for classification or non-classification

Based on the available information, the effects on the liver are adaptive and therefore classification upon repeated exposure is not warranted according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation No. (EC) 1272/2008.