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REACH

Pyrasulfotole

EC number: 609-256-3 | CAS number: 365400-11-9

Life Cycle description
Uses advised against

Toxicological information

Endpoint summary

Administrative data

Description of key information

subacute toxicity:

OECD 410, dermal, rat, 28-days: NOAEL general toxicity 10 mg/kg bw/day

subchronic toxicity:

OECD 408, oral, rat, 90-days: NOAEL 30 ppm (equivalent to 1.96 mg/kg bw/day in males and 2.32 mg/kg bw/day in females)

chronic toxicity:

OECD 453, oral, rat, 6 months: NOAEL 25 ppm (eqivalent to 1.4 mg/kg bw/day in males and 1.8 mg/kg bw/day in females)

OECD 453, oral, rat, 12 months: NOAEL 25 ppm (equivalent to 1.1 mg/kg bw/day in males and 1.5 mg/kg bw/day in females)

OECD 453, oral rat, 24 months: NOAEL 25 ppm (equivalent to 1.0 mg/kg bw/day in males and 1.4 mg/kg bw/day in females)

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference 1

Endpoint:: sub-chronic toxicity: oral
Type of information:: experimental study
Adequacy of study:: key study
Study period:: 13 Feb - 29 Jul 2003
Reliability:: 1 (reliable without restriction)
Rationale for reliability incl. deficiencies:: guideline study
Reason / purpose for cross-reference:: reference to other study
Reason / purpose for cross-reference:: other: reference to review article
Qualifier:: according to guideline
Guideline:: OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:: adopted 21 Sep 1998
Deviations:: no
Qualifier:: according to guideline
Guideline:: OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:: adopted 25 Jun 2018
Deviations:: yes
Remarks:: Rats were housed individually instead of in groups per sex. Phytoestrogen levels were not determined in the diet. T4, T3 and TSH levels were not assessed. No vaginal smears taken for oestrus cycle assessment.
GLP compliance:: yes (incl. QA statement)
Remarks:: Groupe Interministeriel des Produits Chimiques, Secrétariat du GlPC, Paris Cedex, France
Limit test:: no
Species:: rat
Strain:: Wistar
Remarks:: 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
- Age at study initiation: 6 weeks
- Weight at study initiation: 191 - 215 g (males), 160 - 187 g (females)
- Fasting period before study: no
- Housing: individually in suspended stainless steel wire mesh cages
- Diet: rodent powdered and irradiated diet A04C-10P1 from U.A.R. (Usine d'Alimentation Rationnelle, Villemoisson-sur-Orge, France), ad libitum
- Water: filtered and softened water from the municipal water supply, ad libitum
- Acclimation period: 7 days

DETAILS OF FOOD AND WATER QUALITY: Routine analyses of feed and water indicated that there was no contamination which could have compromised the study.

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

IN-LIFE DATES: From: 13 Feb 2002 To: 24 May 2002
Route of administration:: oral: feed
Vehicle:: unchanged (no vehicle)
Details on oral exposure:: DIET PREPARATION
- Method of preparation: The test substance was ground to a fine powder before being incorporated into the diet by dry mixing.
- Rate of preparation of diet (frequency): The test substance formulations were prepared approximately every three weeks. There were four preparations during the study.
- Mixing appropriate amounts with: Certified rodent powdered and irradiated diet
- Storage temperature of food: - 15°C
Analytical verification of doses or concentrations:: yes
Details on analytical verification of doses or concentrations:: The stability of the test substance in the diet was demonstrated before the start of the study at concentrations of 2 and 15000 ppm for a time (95 days) which covered the period of storage and usage for the study. The homogeneity of the test substance in diet was verified on the first preparation at the lowest and highest concentrations to demonstrate adequate formulation procedures. The dietary levels of the test substance were verified at each concentration of the four preparations.

Using HPLC with UV detection the homogeneity and concentration of the test substance in the feed were demonstrated to be within target limits of 85 - 115% of the nominal concentration. The test substance was found to be stable in the rodent diet at concentrations of 2 and 15 000 ppm over a 82 days or 95 days period respectively at room temperature.
Duration of treatment / exposure:: 90 days
Frequency of treatment:: Daily, 7 days a week
Dose / conc.:: 2 ppm
Remarks:: equivalent to 0.13 and 0.15 mg/kg bw/day in males and females, respectively
Dose / conc.:: 30 ppm
Remarks:: equivalent to 1.96 and 2.32 mg/kg bw/day in males and females, respectively
Dose / conc.:: 1 000 ppm
Remarks:: equivalent to 66 and 77 mg/kg bw/day in males and females, respectively
Dose / conc.:: 7 000 ppm
Remarks:: equivalent to 454 and 537 mg/kg bw/day in males and females, respectively
Dose / conc.:: 12 000 ppm
Remarks:: equivalent to 830 and 956 mg/kg bw/day in males and females, respectively
No. of animals per sex per dose:: 10
Control animals:: yes, plain diet
Positive control:: no
Observations and examinations performed and frequency:: CAGE SIDE OBSERVATIONS: Yes
- Time schedule: check for mortality/moribundity: twice daily; recording of clinical signs: at least once daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once during acclimatization and at least weekly during the treatment period

BODY WEIGHT: Yes
- Time schedule for examinations: twice during the acclimatization period, on the first day of test substance administration, then at least weekly throughout the treatment period and before necropsy

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

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

WATER CONSUMPTION AND COMPOUND INTAKE: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: once during acclimatization and during weeks 2, 4, 8 and 12
- Dose groups that were examined: acclimatization period: all animals, during the dosing period: all surviving animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on study days 91, 92, 93 or 94, prior to sacrifice
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all
- Parameters examined: red blood cell count, haemoglobin, haematocrit, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, white blood cell count and differential count evaluation and platelet count, prothrombin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on study days 91, 92, 93 or 94, prior to sacrifice
- Animals fasted: Yes
- How many animals: all
- Parameters examined: any significant change in the general appearance of the plasma and the serum was recorded; 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 concentrations; globulin and albumin/globulin ratio values were calculated

URINALYSIS: Yes
- Time schedule for collection of urine: on study days 85, 86 or 87
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: Yes
- Parameters examined: any significant change in the general appearance of the urine was recorded; urinary volume, pH, urinary refractive index, glucose, bilirubin, ketone bodies, occult blood, protein, urobilinogen; microscopic examination of urinary sediment: red blood cells, white blood cells, epithelial cells, bacteria, casts and crystals

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during the acclimatization phase and during week 12
- Dose groups that were examined: all
- Battery of functions tested: grasping reflex (by pulling the animal across a textured surface); righting reflex (by putting the animal on its back); corneal reflex (following touching of the cornea with a fine nylon string); pupillary reflex (by covering the eyes of the animal for a few seconds and then pupillary reflex is observed by focusing a light in the eyes; auditory startle reflex (by observation of the animal response to a clap of the hands); head shaking reflex (head shaking response of the animal due to blowing on the ear)
Sacrifice and pathology:: GROSS PATHOLOGY: Yes, all animals alive at scheduled necropsy as well as those found dead or killed for humane reasons

HISTOPATHOLOGY: Yes
The following organs or tissues were sampled: adrenal gland, aorta, articular surface (femotibial), bone (sternum), bone marrow (sternum), brain, epididymides, esophagus, exorbital (lachrymal) gland, eye and optic nerve, harderian glands, heart, intestine (duodenum, jejunum, ileum, cecum, colon, rectum), kidney, larynx/pharynx, liver, lungs, lymph nodes (submaxillary and mesenteric), mammary gland, nasal cavities, ovary, pancreas, pituitary gland, prostate gland, sciatic nerve, seminal vesicle, skeletal muscle, skin, spinal cord (cervical, thoracic, lumbar), spleen, stomach, submaxillary (salivary) gland, testis, thymus, thyroid (with parathyroids), tongue, trachea, urinary bladder, uterus (including cervix), vagina, organs and tissues with macroscopic findings
The dose of 12000 ppm was above the Maximum Tolerated Dose in the rat and therefore histopathological examinations were not conducted at this dose. Additionally, as 30 ppm was observed to be free of treatment-related effects, histopathological examination was not conducted at 2 ppm.

ORGAN WEIGHTS: Yes
The following organs were weighed: adrenal gland, brain, epididymidis, heart, kidney, liver, ovary, pituitary gland, protstate gland, spleen, testis, thymus, thyroid gland (with parathyroid), and uterus (including cervix). Paired organs were weighed together.
Statistics:: Means and standard deviations were calculated for each group and per time period for body weight change and average food consumption. All calculations and statistical analyses were performed using a dedicated computer system (Path/Tox System, version 4.2.2).
In general, Bartlett test was performed to compare the homogeneity of group variances.
If the Bartlett test was not significant (α = 0.05), means were compared using the analysis of variance (ANOVA). If the ANOVA was not significant (α = 0.05), the statistical procedure was stopped and group means were considered to be homogeneous. If the ANOVA was significant, group means were compared using the Dunnett test (2-sided).
If the Bartlett test was significant, means were compared using the non-parametric analysis of variance of Kruskal-Wallis. If the Kruskal-Wallis test was not significant (α = 0.05), the statistical procedure was stopped and group means were considered to be homogeneous. When the Kruskal-Wallis test was significant, group means were compared using the Dunn test (2-sided).
If one or more group variance(s) equaled 0, means were compared using non-parametric procedures. The levels of significance for each statistical comparison were 0.05 and 0.01.
Clinical signs:: effects observed, treatment-related
Description (incidence and severity):: At 12000 ppm and 7000 ppm in both sexes, treatment-related clinical signs affecting a large number of animals and over an extended period were yellow (intense) coloured urine associated on a few occasions with soiled anogenital area. A few other clinical signs were also treatment-related but noted on less occasions: few or no faeces, wasted appearance,general pallor, cold to touch, piloerection, reduced motor activity, laboured respiration, hunched posture, increased salivation and soiling around the mouth. White area on eyes were noted in one male and four females at 12000 ppm and in two males at 7000 ppm.
At 1000 ppm, coloured urine was also noted for all males on a few days and one female presented a white area on eyes.
No treatment-related clinical signs were noted at 30 and 2 ppm.

See Attachment 1 for summary data of clinical signs.
Mortality:: mortality observed, treatment-related
Description (incidence):: At 12000 ppm, 6 males were found dead or killed for humane reasons between Days 15 and 72. One female was killed for humane reasons on Day 13. On Day 72, it was decided to sacrifice the surviving males of this group.
At 7000 ppm, 2 males were found dead or killed for humane reasons on Day 8 or 70.
Another male at 7000 ppm was killed for humane reasons on Day 83 showing laboured and noisy respiration, and ocular and nasal discharge. This death was not considered to be treatment-related as the autopsy revealed a haematoma within the oral cavity which was confirmed by histological examination, and probably due to an accidental trauma.
No other mortalities occurred during the study.
Body weight and weight changes:: effects observed, treatment-related
Description (incidence and severity):: At 12000 ppm, a reduction in body weight gain was recorded in males: -70% during the first week of exposure and ranging from -11.5 to -56% on all intervals from Days 22 to 70 when compared to control values. In females, a marked body weight loss (-73 and -59 g) was noted on 2 females and body weight gain of the remainder females of the group was reduced during the first week of exposure when compared to controls. The body weight gain was slightly lower during a few other intervals. At 12 000 ppm, the mean body weights were lower than the control values reaching statistically significance at all time points in males and on several occasions only in females (first two weeks of treatment and on Day 57).
At 7000 ppm, the mean body weight gain of males was decreased (-51%) during the first week of treatment when compared to controls and during most intervals from Day 50 onwards. The mean body weight gain of females was reduced between Days 1 to 8 (-69%) and on a few occasions during the treatment. Mean body weights were statistically significantly lower for most time points for females and only on several occasions for males (first few weeks).
At 1000 ppm, the body weight gains were slightly reduced during the first week of treatment only but with no statistical significance (-10%).
At 30 and 2 ppm, body weight and body weight changes were unaffected by treatment.

See Attachment 2 for summary data on body weight and body weight gains.
Food consumption and compound intake (if feeding study):: effects observed, treatment-related
Description (incidence and severity):: At 12000 ppm, food consumption in males was lower than control values throughout the study. The most important reduction (-29%) was on Week 1 and statistically significance was reached during several intervals from Days 1 to 70. In females, the mean food consumption was lower than control value on the first week of treatment only (reduction of -28%) without reaching statistical significance.
At 7000 ppm, a reduction of food consumption was noted during the first week in both males (-28%) and females (-15%), the difference with controls reaching statistical significance in males only. Very slight reductions thereafter were also observed in both sexes but were not statistically significant.
At 1000, 30 and 2 ppm, food consumption was unaffected by treatment.

See Attachment 3 for summary data of food consumption findings. See Table 1 for mean achieved test item intake per group.
Food efficiency:: not examined
Description (incidence and severity):: Not applicable.
Water consumption and compound intake (if drinking water study):: not examined
Description (incidence and severity):: Not applicable.
Ophthalmological findings:: effects observed, treatment-related
Description (incidence and severity):: Corneal opacity (snow flake appearance) was noted in 5, 3 and 1 animals (from both sexes) at 12000, 7000 and 1000 ppm, respectively, and was associated with neovascularisation of cornea in 4, 2 and 1 animals in each respective group.
Haematological findings:: no effects observed
Clinical biochemistry findings:: effects observed, treatment-related
Description (incidence and severity):: No male animals were available for clinical biochemistry examination at 12000 ppm.
For males at 7000 and 1000 ppm, statistically significantly increased cholesterol concentrations were noted (+45% and +51%, respectively). Also statistically significantly increased triglyceride concentrations were noted (+68% and +112%, respectively). In females at 12000 ppm, statistically significantly increased cholesterol concentration was noted (+30%).

The other statistically significant differences noted were considered not to be relevant in view of their occurrence and/or their low magnitude.

See Attachment 4 for summary data of clinical chemistry findings.
Endocrine findings:: not examined
Description (incidence and severity):: Not applicable.
Urinalysis findings:: effects observed, treatment-related
Description (incidence and severity):: A tendency towards lower pH values were observed in both sexes from 1000 ppm and higher. In males, less crystals were observed from 1000 ppm and higher, compared to the control group. A tendency towards higher ketone levels were observed in both sexes from 1000 ppm and higher.
At 12000 ppm, high numbers of erythrocytes (as well as occult blood) and leukocytes were seen in 2/9 females and a high number of epithelial cells was seen in 3/9 females.
At 7000 ppm, 3/7 males had high numbers of erythrocytes (as well as occult blood), leukocytes and epithelial cells. 2/10 females had high numbers of erythrocytes (as well as occult blood) and leukocytes and 1/10 female had a high number of epithelial cells.
In the control group, one female had a high number of leukocytes.

See Attachment 5 for summary data of urinalysis findings.
Behaviour (functional findings):: no effects observed
Description (incidence and severity):: No changes were noted during the neurotoxicity assessment. At 12000 ppm, one female had no pupillary reflex; this finding was probably consecutive to the corneal opacity and the absence of possible correct evaluation of this reflex.
Immunological findings:: not examined
Description (incidence and severity):: Not applicable.
Organ weight findings including organ / body weight ratios:: effects observed, treatment-related
Description (incidence and severity):: Mean absolute and relative kidney weights were statistically significantly higher in males at 7000 ppm. In females at 12000 and 7000 ppm, there was a tendency towards higher kidney weights which could be related to macroscopic and/or histological changes.
There was a tendency towards higher kidney weights in females at 1000 ppm, but as this change was slight, not statistically significant and not related to any histological change, it was considered of doubtful toxicological significance.

Mean liver weights were statistically significantly higher in males at 7000 and 1000 ppm. In females, mean relative liver weights were found slightly statistically significantly higher at 7000 and 1000 ppm. At 12000 ppm, mean liver to body weight ratio was found statistically significantly higher than the control value, this change was probably linked to the low body weights.

The mean thymus weight was statistically significantly lower in females at 12000 ppm. Although histological examination was not performed, this change could be related to the stress induced by the treatment.

Other statistically significant changes were considered as incidental.

See Tables 2 - 3 and Attachment 6 for summary data of organ weight findings.
Gross pathological findings:: effects observed, treatment-related
Description (incidence and severity):: Unscheduled Death:
At 12000 ppm, 6 males were found dead or killed for humane reasons between Day 15 and 72. On Day 72, it was decided to sacrifice the surviving males.
Almost all males had similar abnormalities in the urinary tract associated with the presence of yellow calculi. Yellow gritty content with calculi in the pelvis and/or in the urinary bladder were observed in 9/10 males. Associated findings were observed in the kidneys: pelvic dilatation (9/10), obviously large kidneys (3/10), pale (2/10), mottled or red or white foci (3/10). Urinary bladders were found distended (5/10). One male found dead on Day 15 had no macroscopic calculi but there was a red content in the urinary bladder and a bilateral pelvic dilatation. Obviously large liver were found in 2/10 males. One female was killed for humane reasons on Day 13 with soiled fur around the nose and forelegs and a general thin/emaciated appearance.
At 7000 ppm, 3 males were found dead or killed for humane reasons. The male killed for humane reasons on Day 70 had urinary tract findings similar to those observed in males at 12000 ppm: a unilateral pelvic dilatation, a distended urinary bladder with red urine and a gritty content with yellow calculi. Other major findings included black foci in the glandular region of the stomach associated with dark content in the intestines and a soiled fur in the anogenital region. The male killed for humane reasons on Day 83 had a hematoma within the oral cavity probably due to an accidental trauma. This finding was confirmed by histological examination. The male found dead on Day 8 had a dark content in the stomach and red content in the urinary bladder. Multifocal non glandular gastric ulcers were reported after histological examination but no clear cause of death was identified.

Scheduled Sacrifice:
Significant macroscopic findings were observed in the urinary tract (kidneys, ureters, urinary bladder and/or urethra). They were observed in 4/9 females at 12000 ppm, in 4/7 males and 5/10 females at 7000 ppm and consisted of the presence of yellow calculi, similar to those observed in decedent animals.
Gritty content with yellow calculi up to 0.7 cm in diameter was observed within the kidneys, in the ureters, in the urinary bladder or in urethra. Associated to the calculi, findings included: pelvic dilatation, abnormal shape of the kidneys, mottled kidneys, distended urinary bladder.
Livers were found obviously enlarged in 3/10 males at 1000 ppm. Prominent lobulation was noted in 1/7 male at 7000 ppm and in 2/10 males at 1000 ppm.
The thyroid gland was found obviously enlarged in one male at 1000 ppm.
All other gross pathology changes were considered as incidental and not treatment-related.

See Attachment 7 for summary data of gross pathological findings.
Neuropathological findings:: not examined
Description (incidence and severity):: Not applicable.
Histopathological findings: non-neoplastic:: effects observed, treatment-related
Description (incidence and severity):: Due to early mortality in males at 12000 ppm, histological examination was not conducted in this group. The top dose group at which histological examinations were performed was 7000 ppm.

Unscheduled Death:
The male from the 7000 ppm dose group had lesions in organs of the urinary system similar to those found in scheduled sacrificed animals. One kidney had dilated renal pelvis associated with a slight multifocal simple urothelial hyperplasia, dilated cortical and medullary tubules, hyaline tubular casts. Urinary bladder had a mild multifocal to diffuse simple urothelial hyperplasia, a moderate serosal mixed cellular infiltrate and mild focal submucosal hemorrhage. Although the macroscopic urolithiasis was not confirmed histologically (probably removed during technical processing), these changes were considered as the consequences of the calculi.

Scheduled Sacrifice:
Treatment-related changes were found within the organs of the urinary system, the liver and the thyroid gland.
Histological changes associated with the presence of calculi (urolithiasis) were found in the kidneys / urinary bladder / ureters in 4/8 males and 6/10 females at 7000 ppm. Macroscopically observed calculi were not systematically found at the histological examination (probably removed during the technical process).
Associated histological changes included: pelvic dilatation (uni- or bilateral), urinary epithelial hyperplasia (pelvis, urinary bladder and ureters), interstial fibrosis of the urinary tract, cystitis, ureteritis. Although some of these findings could be found incidentally in control animals and were not found systematically in each animal, their combination and their incidence make the change clearly treatment-related and consecutive to the calculi.
In the liver, a slight to moderate diffuse centrilobular hepatocellular hypertrophy was observed in 6/7 males at 7000 ppm and 9/10 males at 1000 ppm. This change was found in 1/10 female at 7000ppm. In females, a periportal vacuolation was found in 8/10 animals at 7000 ppm and 3/10 animals at 1000 ppm.
In the thyroid gland, there was a slight to mild diffuse follicular cell hypertrophy/hyperplasia in 2/7 males at 7000 ppm and in 5/10 males at 1000 ppm. This change was associated with a diffuse loss of colloid in 5/7 males at 7000 ppm and in 9/10 males at 1000 ppm. At 30 ppm, because the loss of colloid was observed in 1/10 males only and was not associated with follicular cell hypertrophy/hyperplasia, this change was considered not to be toxicologically relevant.

See Attachment 8 for summary data of histopathological findings (non-neoplastic).
Histopathological findings: neoplastic:: no effects observed
Other effects:: not examined
Description (incidence and severity):: Not applicable.
Details on results:: Corneal opacities, occasionally accompanied by neovascularization and their histopathological correlates (keratitis, reactive epithelial hyperplasia, and vascularization) are considered a rat-specific phenomenon. Corneal changes were not seen in other species chronically treated with the test substance (i.e. mice and dogs). The test substance is an inhibitor of the HPPDase enzyme and induces increased plasma tyrosine levels. This effect is more pronounced in rats than in mice and dogs. Experimentally induced hypertyrosinemia has been shown to induce snow flake-like corneal lesions in rats but not in mice (M-210983-01-2). In mice and humans, even under conditions of strong HPPD inhibition, tyrosine concentrations will not increase to levels high enough to induce ocular toxicity and hence, this toxicity observed in the rat is inappropriate for extrapolation to humans (ECETOC TR No. 99).

Thyroid findings (increased weight, histopathological changes comprising changes in colloid, follicular cell hypertrophy and pigment deposition in the follicular epithelium) are considered a non-adverse and rat specific phenomenon. No changes of the thyroid were noted in either mice or dogs, the other two species in which repeated-dose studies with histopathological examination of the thyroid were conducted with the test substance. As the test substance through inhibition of the HPPDase enzyme increases plasma tyrosine concentration in the rat, it is quite possible that some of this increased tyrosine is taken up by the thyroid and stored in the colloid, either as free tyrosine or through either increasing the synthesis of thyroglobulin or altering its composition in terms of number of tyrosine residues per thyroglobulin molecule.
In the absence of signs of altered thyroid functions (such as effects on body weights, fertility and gestation indices or effects on offspring performance in the developmental neurotoxicity study), the observed morphological changes are considered to be non-adverse.
: Key result
Dose descriptor:: NOAEL
Effect level:: 30 ppm
Based on:: test mat.
Sex:: male/female
Basis for effect level:: other: no adverse effects observed at 30 ppm
Remarks on result:: other: equivalent to 1.96 and 2.32 mg/kg bw/day in males and females, respectively
: Key result
Dose descriptor:: LOAEL
Effect level:: 1 000 ppm
Based on:: test mat.
Sex:: male/female
Basis for effect level:: gross pathology; histopathology: non-neoplastic; ophthalmological examination; organ weights and organ / body weight ratios; serum/plasma biochemistry; urinalysis
Remarks on result:: other: equivalent to 66 and 77 mg/kg bw/day in males and females, respectively
: Key result
Critical effects observed:: yes
Lowest effective dose / conc.:: 1 000 ppm
System:: urinary
Organ:: kidney
Treatment related:: yes
Dose response relationship:: yes
Relevant for humans:: not specified
: Key result
Critical effects observed:: yes
Lowest effective dose / conc.:: 1 000 ppm
System:: hepatobiliary
Organ:: liver
Treatment related:: yes
Dose response relationship:: yes
Relevant for humans:: not specified
Critical effects observed:: yes
Lowest effective dose / conc.:: 1 000 ppm
System:: eye
Organ:: cornea
Treatment related:: yes
Dose response relationship:: yes
Relevant for humans:: no
Critical effects observed:: yes
Lowest effective dose / conc.:: 1 000 ppm
System:: endocrine system
Organ:: thyroid gland
Treatment related:: yes
Dose response relationship:: yes
Relevant for humans:: no
Conclusions:: The study was performed under GLP conditions and according to OECD TG 408 (adopted 1998).The primary effects after sub-chronic dietary administration in the rat were limited to the eyes, liver, and urinary tract in both sexes and thyroid gland in males. Characteristic corneal “snowflake” lesions were observed in both males and females at doses of 1000 ppm and above. Increased liver weights were correlated with an increased incidence of centrilobular hypertrophy, with the greatest increase in incidence observed in males. Increased cholesterol and triglyceride concentrations were also observed in males at 1000 and 7000 ppm. Urinary tract stones (found in a separate analysis to be primarily composed of the test substance, which has been demonstrated to be excreted in the urine at high concentrations after oral dosing) were observed in both males and females and were related to the histopathological findings including urothelial hyperplasia. In the thyroid, males at 1000 ppm and above showed increased incidences of follicular cell hypertrophy / hyperplasia and diffuse loss of colloid.

Based on these findings, the NOAEL in the male and female rat was 30 ppm (1.96 mg/kg bw/day in males, 2.32 mg/kg bw/day in females).

Based on the effects observed at 1000 ppm (equivalent to 66 and 77 mg/kg bw/day in males and females, respectively), the substance is classified “STOT RE 2, H373, urinary system, liver”.

Reference 2

Endpoint:: chronic toxicity: oral
Type of information:: experimental study
Adequacy of study:: key study
Study period:: 29 Jan 2003 - 21 Oct 2005
Reliability:: 1 (reliable without restriction)
Rationale for reliability incl. deficiencies:: guideline study
Reason / purpose for cross-reference:: reference to same study
Reason / purpose for cross-reference:: reference to same study
Reason / purpose for cross-reference:: reference to other study
Reason / purpose for cross-reference:: other: reference to review article
Qualifier:: according to guideline
Guideline:: OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)
Version / remarks:: adopted 12 May 1981
Deviations:: no
Qualifier:: according to guideline
Guideline:: OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)
Version / remarks:: adopted 25 Jun 2018
Deviations:: yes
Remarks:: no details on whether feed and water were analysed for contaminants
GLP compliance:: yes (incl. QA statement)
Remarks:: Secrétariat du Groupe Interministeriel des Produits Chimiques, Paris, France
Limit test:: no
Specific details on test material used for the study:: STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Homogeneity and stability of the test substance in the diet: analytically confirmed
Species:: rat
Strain:: Wistar
Remarks:: Rj:WI (IOPS HAN)
Details on species / strain selection:: no data provided
Sex:: male/female
Details on test animals or test system and environmental conditions:: TEST ANIMALS
- Source: R. Janvier (Le Genest St Isle, France)
- Age at study initiation: 7 weeks
- Weight at study initiation: mean group weights: 286 - 289 g (males), 193 - 196 g (females)
- Fasting period before study: no
- Housing: individually in suspended, stainless steel and wire mesh cages
- Diet: A04CP1-10 from S.A.F.E. (Scientific Animal Food and Engineering, Augy, France), ad libitum
- Water: filtered and softened tap water from the municipal water supply, ad libitum
- Acclimation period: 22 days

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

IN-LIFE DATES: From: 29 Jan 2003 To: 20 Feb 2004
Route of administration:: oral: feed
Vehicle:: unchanged (no vehicle)
Details on oral exposure:: PREPARATION OF DIETARY MIXTURES:

DIET PREPARATION
- Rate of preparation of diet (frequency): The test substance formulations were prepared to cover the dietary requirements over 4-weekly periods.
- Mixing appropriate amounts with (Type of food): A04CP1-10 from S.A.F.E.
- Storage temperature of food: at ambient temperature

VEHICLE
- No vehicle was used
Analytical verification of doses or concentrations:: yes
Details on analytical verification of doses or concentrations:: The stability of the test substance in the diet has been demonstrated in a previous and recent 90-day rat study (M-102924-01-2). The stability of the test substance at 2 and 15000 ppm in the diet was verified for up to at least 82 days, when kept at ambient temperature which covered the period of storage and usage in this study.

The homogeneity of the test substance in diet was verified from the first loads at 25, 250, 1000 and 2500 ppm on the first formulation (F1) and on the first loads at 25 and 2500 ppm of formulations F6, F12, F14, F20 and F26, to demonstrate adequate formulation procedures.
The concentration was checked for all loads at all dose levels for formulations F1, F2, F3, F6, F9, F12, F14, F17, F20, F23 and F26.

Results:
- Homogeneity Analysis: 82* - 115% of nominal concentration
- Concentration Analysis: 85 - 112% of nominal concentration
* 3/266 samples were slightly outside the in-house target range of 85 - 115% of nominal concentration. This minor deviation was not considered to have affected the integrity of the study.
Duration of treatment / exposure:: 12 months
Frequency of treatment:: Daily, 7 days a week
Dose / conc.:: 25 ppm
Remarks:: equivalent to 1.1 and 1.5 mg/kg bw/day in males and females, respectively
Dose / conc.:: 250 ppm
Remarks:: equivalent to 11 and 15 mg/kg bw/day in males and females, respectively
Dose / conc.:: 1 000 ppm
Remarks:: equivalent to 45 and 63 mg/kg bw/day in males and females, respectively
Dose / conc.:: 2 500 ppm
Remarks:: equivalent to 114 and 155 mg/kg bw/day in males and females, respectively
No. of animals per sex per dose:: 10
Control animals:: yes, plain diet
Details on study design:: - Dose selection rationale: Dose levels were selected based on the results from a previous 90-day dietary study in the rat (M-102924-01-2) where dietary administration of up to 7000 ppm in males and 12000 ppm in females resulted in increased plasma cholesterol and triglycerides, increased liver and kidney weights, yellow calculi in the urinary tract, urothelial hyperplasia, centrilobular hepatocellular hypertrophy, and diffuse thyroid follicular cell hyperplasia. The NOAEL in the rat 90-day study was 30 ppm, with a LOAEL of 1000 ppm.

- Rationale for animal assignment (if not random): On the day of randomization, animals were allocated to dose groups using a computerized randomization procedure that ensured a similar body weight distribution among groups for each sex. Selected animals were in a weight range from 243 to 325 g for the males and 169 to 221 g for the females at the start of exposure to the test substance, i.e., within ± 20% of the mean body weight on the day of randomization. Any animal deemed unsuitable for selection based on weight, ophthalmological abnormalities or health status was not used for the study.
Observations and examinations performed and frequency:: CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were checked for moribundity and mortality twice daily (once daily on weekends or public holidays). Clinical signs were recorded at least once daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed physical examinations including palpation for masses were performed weekly from study Day 1.

BODY WEIGHT: Yes
- Time schedule for examinations: At least weekly during the acclimatization period then weekly for the first 13 weeks of study, approximately every 4 weeks thereafter and prior to necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

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

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations:
all animals: during acclimatization (indirect ophthalmoscopy)
all surviving animals after approximately 3, 6 and 12 months (indirect ophthalmoscopy and slit lamp)
- Dose groups that were examined: all

HAEMATOLOGY: Yes
- Time schedule for collection of blood:
Blood analyses were performed on all surviving animals in Weeks 28/29 and 50/51.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- Parameters examined: hematocrit, hemoglobin, leukocyte count, erythrocyte count, platelet count, prothrombin time, leukocyte differential count, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, mean corpuscular volume, reticulocyte count

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:
Blood analyses were performed on all surviving animals in Weeks 28/29 and 50/51.
- Animals fasted: Yes
- Parameters examined: calcium, chloride, magnesium, inorganic phosphorus, potassium, sodium, alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, gamma glutamyltransferase, albumin, creatinine, urea, total cholesterol, glucose, total bilirubin, total protein, triglycerides

URINALYSIS: Yes
- Time schedule and for collection of urine: Prior to allocation of the animals to treatment groups, urinalysis was performed on thirty males and thirty females. Urinalysis was performed on all surviving animals in Weeks 13/14, 26/27/28 and 48/49.
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: Yes (diet and water)
- Parameters examined: appearance, volume, specific gravity/osmolality/refractive index, pH, sediment (microscopic), protein, glucose, ketones, bilirubin, blood/red blood cells, urobilinogen, creatinine

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:: GROSS PATHOLOGY: Yes
Necropsy included the examination of external surfaces, all orifices, all major organs, tissues and body cavities. All significant macroscopic abnormalities (including masses and their regional lymph nodes when possible) were recorded, sampled and examined microscopically.

ORGAN WEIGHTS: Yes (see Table 1)

HISTOPATHOLOGY: Yes (see Table 1)
Histopathology examinations were performed as follows:
- all organs and tissue samples from animals sacrificed or dying during the treatment period,
- all organs and tissue samples from animals of control and high dose groups,
- liver, lung, kidney, thyroid gland, urinary bladder, pancreas and eye from animals of the intermediate dose groups,
- gross abnormalities from all animals.
For all unscheduled sacrificed or dead animals on study, the cause of death was determined when it was possible.
Initial examinations were performed by the Principal Investigator in Histopathology. Following the initial examination, a review pathologist undertook an independent « peer-review » of representative slides and diagnoses according to standardized operating procedures. The diagnoses presented in this report represent the consensus opinion of the two pathologists.
Statistics:: See "Any other information on materials and methods incl. tables"
Clinical signs:: effects observed, treatment-related
Description (incidence and severity):: Treatment-related clinical signs consisted of an increased incidence of white area on the eye at the three highest dose levels in both sexes. In addition, there was an increased incidence of soiled fur/soiling around the anogenital region at the two highest dose levels in both sexes (see Tables 3 and 4).
Mortality:: mortality observed, treatment-related
Description (incidence):: After 1 year of treatment the mortality rate in males at 2500 ppm was higher than in the control group (8/65 deceased animals versus 2/65 in the control). At 25 ppm in males, the mortality rate was also slightly higher than in the control group (6/65), but as the incidence at 1000 and 250 ppm was similar to the controls (2/65 in both groups), the higher incidence at 25 ppm was considered to be incidental. In females, the mortality rate was similar in the treated and the control groups. One female from the control group died due to accidental trauma.
Body weight and weight changes:: effects observed, treatment-related
Description (incidence and severity):: At 2500 and 1000 ppm, during the first week of treatment, mean body weight was reduced by 3% (p < 0.05) and 2%, respectively, in males, whilst mean cumulative body weight gain was reduced by 16% (p < 0.01) and 18% (p < 0.01), respectively, compared to controls. Thereafter, mean body weight gain was consistently lower than in controls by up to 56% and 60%, respectively, the effect being statistically significant on several occasions. At the end of the treatment period, mean body weight was reduced by 6% (p < 0.01) and 5% (p < 0.01), respectively, and mean cumulative body weight gain was reduced by 10% (p < 0.01) and 9% (p < 0.01), respectively, compared to controls. In females, there was no treatment-related effect on mean body weight, body weight gain and cumulative body weight gain. The few minor differences from controls which attained statistical significance were sporadic and were considered to be chance findings reflecting the number of comparisons made. There was no treatment-related effect on mean body weight gain in males during the second half of the first year of treatment. The few minor differences from controls which attained statistical significance were sporadic and were considered to be chance findings reflecting the number of comparisons made. At the end of the treatment period, mean body weight was reduced by 6% (p<0.01), and 5%, respectively, and mean cumulative body weight gain was reduced by 10% (p < 0.01) and 8% (p < 0.05), respectively, compared to controls. In females, mean body weight, body weight gain and cumulative body weight gain were comparable with the controls during the second half of the study.

At 250 ppm, during the first week of treatment, mean body weight was reduced by 2% in males, whilst mean cumulative body weight gain was reduced by 14% (p < 0.01), compared to controls. During the first six months of the study, mean body weight, body weight gain and cumulative body weight gain were comparable to controls. The few minor differences reaching statistical significance were considered to reflect inter-individual variation rather than a treatment-related effect. In females, there was no treatment-related effect on mean body weight, body weight gain and cumulative body weight gain during the first 6 months. The few minor differences from controls which attained statistical significance were sporadic and were considered to be chance findings reflecting the number of comparisons made. there was no treatment-related effect on mean body weight gain in males during the second half of the first year of treatment. The few minor differences from controls which attained statistical significance were sporadic and were considered to be chance findings reflecting the number of comparisons made. At the end of the treatment period, mean body weight was reduced by 3%, and mean cumulative body weight gain was reduced by 5%, respectively, compared to controls. In females, mean body weight, body weight gain and cumulative body weight gain were comparable with the controls during the second half of the study.

At 25 ppm, there was no treatment-related effect on mean body weight, body weight gain and cumulative body weight gain in either sex throughout the study.The few minor differences from controls which attained statistical significance were sporadic and were considered to be chance findings reflecting the number of comparisons made.

The few minor differences from controls which attained statistical significance were sporadic and were considered to be chance findings reflecting the number of comparisons made.

There was no relevant change in terminal body weights of treated animals when compared to controls.
Food consumption and compound intake (if feeding study):: no effects observed
Food efficiency:: not examined
Description (incidence and severity):: Not applicable.
Water consumption and compound intake (if drinking water study):: not examined
Description (incidence and severity):: Not applicable.
Ophthalmological findings:: effects observed, treatment-related
Description (incidence and severity):: Treatment-related findings noted in Weeks 23/24 consisted of corneal opacity, neovascularisation, oedema of the cornea, and "snow flake-like" corneal opacity at 2500 and 1000 ppm in both sexes and 250 ppm in males, as well as neovascularisation of the cornea and "snow flake-like" corneal opacity in females at 250 ppm. In Weeks 49/50/51 treatment-related findings consisted of corneal opacity, neovascularisation and oedema of the cornea, and "snow flake-like" corneal opacity at 2500, 1000 and 250 ppm in both sexes. See attachment 1 (attached background material) for tabulated ophthalmological results.
Haematological findings:: no effects observed
Clinical biochemistry findings:: effects observed, treatment-related
Description (incidence and severity):: In males, higher mean total cholesterol concentrations were observed at 2500, 1000 and 250 ppm. See attachment 1 (attached background material) for tabulated mean cholesterol data (males).
Endocrine findings:: not examined
Description (incidence and severity):: Not applicable.
Urinalysis findings:: effects observed, treatment-related
Description (incidence and severity):: When compared to the control groups, a tendency towards higher ketone levels was observed at 2500 and 1000 ppm in both sexes.
Lower mean pH values were noted in males at 2500, 1000 and 250 ppm. See attachment 1 (attached background material) for tabulated mean pH values (males).
In females lower mean pH values were noted only in Week 13/14, at 2500 ppm (-10%, p < 0.01), 1000 ppm (-6%, p < 0.01) and 250 ppm (-6%, p < 0.01). Correlated with the lower mean pH values, lower amounts of crystals were generally observed in the urine. Additionally, when compared to the controls, a tendency towards higher protein levels was seen in males at Week 26/27/28 at all dietary levels tested.
Other statistically significant differences were considered not to be relevant in view of their sporadic occurrence and/or the absence of a clear dose-effect relationship.
Behaviour (functional findings):: not examined
Description (incidence and severity):: Not applicable.
Immunological findings:: not examined
Description (incidence and severity):: Not applicable.
Organ weight findings including organ / body weight ratios:: effects observed, treatment-related
Description (incidence and severity):: In males only, mean liver weights were found to be statistically significantly higher at 2500, 1000 and 250 ppm (see Table 6). These differences were associated with microscopic hepatocellular hypertrophy and were considered to be treatment-related.
In males only, mean absolute and relative kidney weights were found to be higher and in most cases statistically different at 2500, 1000 and 250 ppm, when compared to controls (see Table 7). These differences were associated with relevant microscopic findings and were considered to be treatment-related.
Other organ weight changes were considered to be incidental in origin.
Gross pathological findings:: effects observed, treatment-related
Description (incidence and severity):: a/ Unscheduled deaths
Five animals (three males: one at 250 and two at 2500 ppm; and two females: one at 0 and one at 1000 ppm) scheduled for sacrifice after 12 months died / were sacrificed on Days 139, 169, 313 and 351 (two animals), respectively. The macroscopic findings observed on these five animals did not support a directly treatment-related death.
b/ Terminal sacrifice:
At 2500, 1000 and 250 ppm, enlarged liver was observed in males only (3/8, 3/10 and 3/9, respectively). This finding was correlated with microscopic centrilobular hypertrophy and was considered to be treatment-related.
At 2500 and 1000 ppm, presence of eye opacity was found in both sexes (5/8 and 5/10 in males and 7/10 and 5/9 in females, respectively). This finding was correlated with microscopic relevant findings and was considered to be treatment-related.
All others gross changes were considered to be incidental and not treatment-related.
Neuropathological findings:: not examined
Description (incidence and severity):: Not applicable.
Histopathological findings: non-neoplastic:: effects observed, treatment-related
Description (incidence and severity):: a/ Unscheduled deaths
Three males and two females died / were sacrificed prior to the scheduled 12 months sacrifice date. The pattern of mortality and the reasons for removal from the study did not indicate an effect of treatment and were considered to be incidental.

b/ Terminal sacrifice
Effects of treatment were seen in the eyes and pancreas of both sexes, and liver, kidneys and thyroid glands of males.
In the eyes, unilateral or bilateral inflammation, regenerative hyperplasia and neovascularisation of the cornea were seen in both sexes dosed at 2500 and 1000 ppm and in males at 250 ppm. Unilateral changes were generally more frequent than bilateral. These findings were considered to be treatment-related. The changes observed at 25 ppm were considered not to be toxicologically relevant as they occurred at a very low incidence and severity.
In the liver, centrilobular hepatocellular hypertrophy and centrilobular vacuolation were noted in males dosed at 2500, 1000 or 250 ppm. These were considered to reflect an adaptive response to treatment rather than an adverse effect.
In the thyroid gland, focal follicular cell hyperplasia was noted in 4/8 males dosed at 2500 ppm. Diffuse hypertrophy of follicular cells and increased follicular diameter were observed in males at 2500, 1000 or 250 ppm versus none in the controls. A higher incidence of altered basophilic colloid and brown pigment in follicular cells were noted in males at 2500, 1000 or 250 ppm. All these findings were considered to be treatment-related.
In the kidneys, a higher incidence and severity of chronic progressive nephropathy in males dosed at 2500, 1000 or 250 ppm was noted.
In the pancreas, diffuse degeneration/atrophy of acinar tissue and/or interstitial fibrosis or inflammation was seen in animals dosed at 2500 ppm and one male dosed at 1000 ppm.
A variety of other spontaneous changes was noted in control and treated animals with no indication of an effect of treatment. The spectrum of these findings is mainly consistent with changes commonly encountered in laboratory rats of this age kept under laboratory conditions.

See attachment 2 (attached background material) for tabulated histopathological findings in the eye, liver, thyroid and kidneys.
Histopathological findings: neoplastic:: no effects observed
Description (incidence and severity):: A small number of tumours was noted in animals from treated and control groups with no evidence of an effect of treatment.
Other effects:: not examined
Description (incidence and severity):: Not applicable.
Details on results:: Corneal opacities, occasionally accompanied by neovascularization and their histopathological correlates (keratitis, reactive epithelial hyperplasia, and vascularization) are considered a rat-specific phenomenon. Corneal changes were not seen in other species chronically treated with the test substance (i.e. mice and dogs). The test substance is an inhibitor of the HPPDase enzyme and induces increased plasma tyrosine levels. This effect is more pronounced in rats than in mice and dogs. Experimentally induced hypertyrosinemia has been shown to induce snow flake-like corneal lesions in rats but not in mice (M-210983-01-2). In mice and humans, even under conditions of strong HPPD inhibition, tyrosine concentrations will not increase to levels high enough to induce ocular toxicity and hence, this toxicity observed in the rat is inappropriate for extrapolation to humans (ECETOC TR No. 99).

Thyroid findings (increased weight, histopathological changes comprising changes in colloid, follicular cell hypertrophy and pigment deposition in the follicular epithelium) are considered a non-adverse and rat specific phenomenon. No changes of the thyroid were noted in either mice or dogs, the other two species in which repeated-dose studies with histopathological examination of the thyroid were conducted with the test substance. As the test substance through inhibition of the HPPDase enzyme increases plasma tyrosine concentration in the rat, it is quite possible that some of this increased tyrosine is taken up by the thyroid and stored in the colloid, either as free tyrosine or through either increasing the synthesis of thyroglobulin or altering its composition in terms of number of tyrosine residues per thyroglobulin molecule.
In the absence of signs of altered thyroid functions (such as effects on body weights, fertility and gestation indices or effects on offspring performance in the developmental neurotoxicity study), the observed morphological changes are considered to be non-adverse.
: Key result
Dose descriptor:: NOAEL
Effect level:: 25 ppm
Based on:: test mat.
Sex:: male/female
Basis for effect level:: other: no adverse effects noted at 25 ppm
Remarks on result:: other: equivalent to 1.1 and 1.5 mg/kg bw/day in males and females, respectively
: Key result
Dose descriptor:: LOAEL
Effect level:: 250 ppm
Based on:: test mat.
Sex:: male/female
Basis for effect level:: other: findings at 250 ppm, decreased body weights, increased cholesterol, reduced urinary pH, increased liver and kidney weights, histopathological effects in cornea, liver, kidney and thyroid.
Remarks on result:: other: equivalent to 11 and 15 mg/kg bw/day in males and females, respectively
: Key result
Critical effects observed:: yes
Lowest effective dose / conc.:: 1 000 ppm
System:: gastrointestinal tract
Organ:: pancreas
Treatment related:: yes
Dose response relationship:: yes
Relevant for humans:: not specified
Critical effects observed:: yes
Lowest effective dose / conc.:: 250 ppm
System:: urinary
Organ:: kidney
Treatment related:: yes
Dose response relationship:: yes
Relevant for humans:: no
Critical effects observed:: yes
Lowest effective dose / conc.:: 250 ppm
System:: eye
Organ:: cornea
Treatment related:: yes
Dose response relationship:: yes
Relevant for humans:: no
Critical effects observed:: yes
Lowest effective dose / conc.:: 250 ppm
System:: endocrine system
Organ:: thyroid gland
Treatment related:: yes
Dose response relationship:: yes
Relevant for humans:: no
Conclusions:: The study was performed under GLP conditions and according to OECD TG 453 (adopted 1981). Based on increased mortality at 2500 ppm in males, this dose is considered to have exceeded the Maximum Tolerated Dose. Treatment-related findings were seen in the eyes, liver, kidney, thyroid, and pancreas. Findings in the eye were related to the biochemical mechanism of the test substance as an HPPDase inhibitor leading to increased tyrosinemia in rats, and were considered not to be relevant for humans. The observations in the liver, including centrilobular hepatocellular hypertrophy, were evaluated as being adaptive responses to treatment rather than adverse effects. The finding of chronic progressive nephropathy is considered to be a rodent-specific finding not relevant to man. The increased incidence of acinar degeneration / atrophy in the pancreas was only observed at 1000 and 2500 ppm, and may indicate a tyrosine-linked effect as well, as these findings were not observed in the mouse or dog, which are not as sensitive as the rat to the effects of tyrosinemia.

The NOAEL in this study was therefore considered to be 25 ppm (1.1 mg/kg bw/day in males and 1.5 mg/kg bw/day in females), while the LOAEL was 250 ppm (11 mg/kg bw/day in males and 15 mg/kg bw/day in females).

Reference 3

Endpoint:: chronic toxicity: oral
Type of information:: experimental study
Adequacy of study:: key study
Study period:: 29 Jan 2003 - 21 Oct 2005
Reliability:: 1 (reliable without restriction)
Rationale for reliability incl. deficiencies:: guideline study
Reason / purpose for cross-reference:: reference to same study
Reason / purpose for cross-reference:: reference to same study
Reason / purpose for cross-reference:: reference to other study
Reason / purpose for cross-reference:: other: reference to review article
Qualifier:: according to guideline
Guideline:: OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)
Version / remarks:: adopted 12 May 1981
Deviations:: no
Qualifier:: according to guideline
Guideline:: OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)
Version / remarks:: adopted 25 Jun 2018
Deviations:: yes
Remarks:: no details on whether feed and water were analysed for contaminants
GLP compliance:: yes (incl. QA statement)
Remarks:: Groupe Interministeriel des Produits Chimiques, Paris, France
Specific details on test material used for the study:: STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Homogeneity and stability of the test substance in the diet: analytically confirmed
Species:: rat
Strain:: Wistar
Remarks:: Rj:WI (IOPS HAN)
Details on species / strain selection:: no data provided
Sex:: male/female
Details on test animals or test system and environmental conditions:: TEST ANIMALS
- Source: R. Janvier (Le Genest St Isle, France)
- Age at study initiation: 7 weeks
- Weight at study initiation: mean group weights: 286 - 289 g (males), 193 - 196 g (females)
- Fasting period before study: no
- Housing: individually in suspended, stainless steel and wire mesh cages
- Diet: A04CP1-10 from S.A.F.E. (Scientific Animal Food and Engineering, Augy, France), ad libitum
- Water: filtered and softened tap water from the municipal water supply, ad libitum
- Acclimation period: 22 days

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

IN-LIFE DATES: From: 29 Jan 2003 To: 03 Mar 2005
Route of administration:: oral: feed
Vehicle:: unchanged (no vehicle)
Details on oral exposure:: PREPARATION OF DIETARY MIXTURES:
- Rate of preparation of diet (frequency): The test substance formulations were prepared to cover the dietary requirements over 4-weekly periods apart from the last formulation which covered the dietary needs until the end of the study. Twenty-six formulations (F1 to F26) were prepared during the study at each concentration.
Formulations F1 to F10 consisted of 4 loads of 45 kg at each concentration. In addition, an additional load (load 4 Bis) was prepared for formulation F2 to cover for accidental loss of diet. Formulations F11 to F13 consisted of 3 loads of 45 kg at each concentration. Formulations F14 to F25 consisted of 2 loads of 56 kg at each concentration. Formulation F26 consisted of 3 loads of 61 kg at each concentration.
- Mixing appropriate amounts with (Type of food): A04CP1-10 from S.A.F.E.
- Storage temperature of food: at ambient temperature

VEHICLE
- No vehicle was used.
Analytical verification of doses or concentrations:: yes
Details on analytical verification of doses or concentrations:: The stability of the test substance in the diet has been demonstrated in a previous and recent 90-day rat study (M-102924-01-2). The stability of the test substance at 2 and 15000 ppm in the diet was verified for up to at least 82 days, when kept at ambient temperature which covered the period of storage and usage on this study.
Twenty-six formulations (F1 to F26) were prepared during the study at each concentration. The homogeneity of the test substance in diet was verified from the first loads at 25, 250, 1000 and 2500 ppm on the first formulation (F1) and on the first loads at 25 and 2500 ppm of formulations F6, F12, F14, F20 and F26, to demonstrate adequate formulation procedures.
The concentration was checked for all loads at all dose levels for formulations F1, F2, F3, F6, F9, F12, F14, F17, F20, F23 and F26.

Results:
- Homogeneity Analysis: 82* - 115% of nominal concentration
- Concentration Analysis: 85 - 112% of nominal concentration
* 3/266 samples were slightly outside the in-house target range of 85 - 115% of nominal concentration. This minor deviation was not considered to have affected the integrity of the study.
Duration of treatment / exposure:: 24 months
Frequency of treatment:: Daily, 7 days a week
Dose / conc.:: 25 ppm
Remarks:: equivalent to 1.0 and 1.4 mg/kg bw/day in males and females, respectively
Dose / conc.:: 250 ppm
Remarks:: equivalent to 10 and 14 mg/kg bw/day in males and females, respectively
Dose / conc.:: 1 000 ppm
Remarks:: equivalent to 41 and 57 mg/kg bw/day in males and females, respectively
Dose / conc.:: 2 500 ppm
Remarks:: equivalent to 104 and 140 mg/kg bw/day in males and females, respectively
No. of animals per sex per dose:: 10 (for the 6-month subchronic phase)
10 (for the 12-month chronic phase)
55 (for the carcinogenicity phase)
Control animals:: yes, plain diet
Details on study design:: - Toxicokinetic data: no data provided
- Dose selection rationale: Dose levels were selected based on the results from a previous 90-day dietary study in the rat (M-102924-01-2) where dietary administration of up to 7000 ppm in males and 12000 ppm in females resulted in increased plasma cholesterol and triglycerides, increased liver and kidney weights, yellow calculi in the urinary tract, urothelial hyperplasia, centrilobular hepatocellular hypertrophy, and diffuse thyroid follicular cell hyperplasia. The NOAEL in the rat 90-day study was 30 ppm, with a LOAEL of 1000 ppm.
- Rationale for animal assignment: On the day of randomization, animals were allocated to dose groups using a computerized randomization procedure that ensured a similar body weight distribution among groups for each sex. Selected animals were in a weight range from 243 to 325 g for the males and 169 to 221 g for the females at the start of exposure to the test substance, i.e., within ± 20% of the mean body weight on the day of randomization. Any animal deemed unsuitable for selection based on weight, ophthalmological abnormalities or health status was not used for the study.
- Section schedule rationale: At termination of the carcinogenicity phase, males were sacrificed prior to females due to a 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.
Observations and examinations performed and frequency:: CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were checked for moribundity and mortality twice daily (once daily on weekends or public holidays). Clinical signs were recorded at least once daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed physical examinations including palpation for masses were performed weekly from study day 1.

BODY WEIGHT: Yes
- Time schedule for examinations: At least weekly during the acclimatization period then weekly for the first 13 weeks of study, approximately every 4 weeks thereafter and prior to necropsy. An additional body weight session was scheduled for study day 673 to enable achieved test material intake figures to be calculated over this period with corresponding additional food consumption session.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

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

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations:
all animals: during acclimatization (indirect ophthalmoscopy)
all surviving animals: after approximately 3, 6, 9, 12 and 24 months (indirect ophthalmoscopy and slit lamp)
- Dose groups that were examined: all

HAEMATOLOGY: Yes
- Time schedule for collection of blood:
Blood analyses were performed on the first ten surviving rats per group in weeks 28/29, 50/51, 78/79 and 104/105.
At scheduled necropsy, blood smears were prepared for all animals not sampled for hematology. The blood smears were stained with Wright stain for possible differential white blood cell determination (on the request of the Study Pathologist).
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- Parameters examined: hematocrit, hemoglobin, leukocyte count, erythrocyte count, platelet count, prothrombin time, leukocyte differential count, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, mean corpuscular volume, reticulocyte count

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:
Blood analyses were performed on the first ten surviving rats per group in weeks 28/29, 50/51, 78/79 and 104/105.
- Parameters examined: calcium, chloride, magnesium, inorganic phosphorus, potassium, sodium, alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, gamma glutamyltransferase, albumin, creatinine, urea, total cholesterol, glucose, total bilirubin, total protein, triglycerides

URINALYSIS: Yes
- Time schedule and for collection of urine: Prior to allocation of the animals to treatment groups, urinalysis was performed on thirty males and thirty females.
Urinalysis was performed on the first ten surviving rats per group in weeks 13/14, 26/27/28, 48/49, 80/81 and 101/102.
- Metabolism cages used for collection of urine: not specified
- Animals fasted: Yes (diet and water)
- Parameters examined: appearance, volume, specific gravity/osmolality/refractive index, pH, sediment (microscopic), protein, glucose, ketones, bilirubin, blood/red blood cells, urobilinogen, creatinine

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:: GROSS PATHOLOGY: Yes
Necropsy included the examination of external surfaces, all orifices, all major organs, tissues and body cavities. All significant macroscopic abnormalities (including masses and their regional lymph nodes when possible) were recorded, sampled and examined microscopically.

ORGAN WEIGHTS: Yes (see Table 1)

HISTOPATHOLOGY: Yes (see Table 2)
Histopathological examinations were 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 was determined when it was possible.
Initial examinations were performed by the Principal Investigator in Histopathology. Following the initial examination, a review pathologist, undertook an independent « peer-review » of representative slides and diagnoses according to standardized operating procedures. The diagnoses presented in this report represent the consensus opinion of the two pathologists. In addition, a Scientific Advisory Panel, at Experimental Pathology Laboratories, Inc. PO BOX 474, Herndon, VA 20172-0474, U.S.A., peer reviewed a selection of thyroid gland slides.
Statistics:: See "Any other information on materials and methods incl. tables"
Clinical signs:: effects observed, treatment-related
Description (incidence and severity):: During the first 6 and 12 months of treatment, treatment-related clinical signs consisted of an increased incidence of white area on the eye at the three highest dose levels in both sexes. In addition, there was an increased incidence of soiled fur/soiling around the anogenital region at the two highest dose levels in both sexes (see Tables 3 and 4).
During the second year, treatment-related clinical signs consisted of an increased incidence of white area on the eye in the three highest dose levels in both sexes. There was also an increased incidence of soiling around the anogenital region at the three highest dose levels in males and the two highest dose levels in females (see Table 5).
Mortality:: mortality observed, treatment-related
Description (incidence):: After 6 months of treatment, the mortality rate at 2500 ppm in males was slightly higher than in the control group (4/75 deceased animals versus 1/75 in the control). The mortality rate in the remaining male treated groups was very low and similar to the control group. In females there were no deaths during the first 6 months.

After 1 year of treatment the mortality rate in males at 2500 ppm was higher than in the control group (8/65 deceased animals versus 2/65 in the control). At 25 ppm in males, the mortality rate was also slightly higher than in the control group (6/65), but as the incidence at 1000 and 250 ppm was similar to the controls (2/65 in both groups), the higher incidence at 25 ppm was considered to be incidental. In females, the mortality rate was similar in the treated and the control groups.
One female from the control group (chronic phase) died due to accidental trauma.

After 2 years of treatment, the mortality rate in males at 2500 ppm was statistically significantly higher than in the control group (30/55 deceased animals versus 30/55 in the control; see Table 6). There was no statistically significant difference between the remaining male treated groups and the controls. The mortality rate in females was similar across the groups with no indication of a treatment-related effect. One male from the control group and one male at 250 ppm, together with one female at 25 ppm and 1000 ppm and two females at 2500 ppm died during anesthesia for blood sampling.
One male from the control group was sacrificed due to accidental trauma.
Body weight and weight changes:: effects observed, treatment-related
Description (incidence and severity):: (for mean body weight and body weight gain data see Table 7)

6-month phase
At 2500 and 1000 ppm, during the first week of treatment, mean body weight was reduced by 3% (p < 0.05) and 2%, respectively, in males, whilst mean cumulative body weight gain was reduced by 16% (p < 0.01) and 18% (p < 0.01), respectively, compared to controls. Thereafter, mean body weight gain was consistently lower than in controls by up to 56% and 60%, respectively, the effect being statistically significant on several occasions. At the end of the 6-month period (Day 176), mean body weight was reduced by 6% (p < 0.01) and 5% (p < 0.01), respectively, and mean cumulative body weight gain was reduced by 10% (p < 0.01) and 9% (p < 0.01), respectively, compared to controls.
In females, there was no treatment-related effect on mean body weight, body weight gain and cumulative body weight gain throughout the first 6 months of treatment. The few minor differences from controls which attained statistical significance were sporadic and were considered to be chance findings reflecting the number of comparisons made.
At 250 ppm, during the first week of treatment, mean body weight was reduced by 2% in males, whilst mean cumulative body weight gain was reduced by 14% (p < 0.01), compared to controls.Thereafter, mean body weight, body weight gain and cumulative body weight gain were comparable to controls throughout the first 6 months of treatment. The few minor differences reaching statistical significance were considered to reflect inter-individual variation rather than a treatment-related effect. In females, there was no treatment-related effect on mean body weight, body weight gain and cumulative body weight gain throughout the first 6 months of treatment. The few minor differences from controls which attained statistical significance were sporadic and were considered to be chance findings reflecting the number of comparisons made.
At 25 ppm, there was no treatment-related effect on mean body weight, body weight gain and cumulative body weight gain in either sex over the first 6 months of treatment. The few minor differences from controls which attained statistical significance were sporadic and were considered to be chance findings reflecting the number of comparisons made.
There was no relevant change in terminal body weights of treated animals when compared to controls.

Chronic phase
At 2500, 1000 and 250 ppm, there was no treatment-related effect on mean body weight gain in males during the second half of the first year of treatment. The few minor differences from controls which attained statistical significance were sporadic and were considered to be chance findings reflecting the number of comparisons made. At the end of the first year of treatment (Day 344), mean body weight was reduced by 6% (p < 0.01), 5% and 3%, respectively, and mean cumulative body weight gain was reduced by 10% (p < 0.01), 8% (p < 0.05) and 5%, respectively, compared to controls. In females, mean body weight, body weight gain and cumulative body weight gain were comparable with the
controls during the second half of the first year of treatment.
At 25 ppm, there was no treatment-related effect on mean body weight, body weight gain and cumulative body weight gain in either sex throughout the second half of the first year of treatment. The few minor differences from controls which attained statistical significance were sporadic and were considered to be chance findings reflecting the number of comparisons made.
There was no relevant change in terminal body weights of treated animals when compared to controls.

Carcinogenicity phase
At 2500 ppm, throughout the second year of treatment, mean body weight was reduced by up to 14 and 8% in males and females, respectively, whilst mean cumulative body weight gain was reduced by up to 23 and 12% in males and females, respectively, compared to controls. In males, mean body weight gain was consistently lower at most time points throughout the second year of treatment, compared to the controls, the effect being statistically significant on several occasions. In females, the effect on mean body weight gain was more pronounced during the first half of the second year of treatment and was statistically significant (p < 0.01) at a few time points.
At 1000 ppm, throughout the second year of treatment, mean body weight was reduced by up to 12% and 10% in males and females, respectively, whilst mean cumulative body weight gain was reduced by up to 20 and 15% in males and females, respectively, compared to controls. In males, lower mean body weight gains were observed at most time points throughout the first 6 months of the second year of treatment, when compared to the controls, the effect being statistically significant on most occasions. In females, mean body weight gain was consistently lower at most time points throughout the second year of treatment, compared to the controls, although the effect was not statistically significant.
At 250 ppm, throughout the second year of treatment, mean body weight was reduced by up to 8% and 6% in males and females, respectively, whilst mean cumulative body weight gain was reduced by up to 13 and 9% in males and females, respectively, compared to controls. In males, lower mean body weight gains were observed at most time points throughout the first 6 months of the second year of treatment, when compared to the controls, the effect being statistically significant on a few occasions. In females, mean body weight gain was comparable to or slightly lower than the controls throughout the second year of treatment, although the effect was not statistically significant.
At 25 ppm, there was no treatment-related effect on mean body weight, body weight gain and cumulative body weight gain in either sex throughout the second year of treatment. The few minor differences from controls which attained statistical significance were sporadic and were considered to be chance findings reflecting the number of comparisons made.
Mean terminal body weights were lower and statistically different in males (-12% at 2500 ppm, -8% at 1000 ppm, -7% at 250 ppm) when compared to controls.
Food consumption and compound intake (if feeding study):: no effects observed
Description (incidence and severity):: Mean food consumption was similar to controls throughout the chronic phase in both sexes and at all dose levels evaluated. The few minor differences from controls which attained statistical significance were sporadic and were considered to be chance findings reflecting the number of comparisons made.
Food efficiency:: not examined
Description (incidence and severity):: Not applicable.
Water consumption and compound intake (if drinking water study):: not examined
Description (incidence and severity):: Not applicable.
Ophthalmological findings:: effects observed, treatment-related
Description (incidence and severity):: At the 6 month (Weeks 23 to 24), ophthalmological examination treatment-related findings consisted of corneal opacity, neovascularisation and oedema of the cornea, and "snow flake-like" corneal opacity at 2500 and 1000 ppm in both sexes and 250 ppm in males, and neovascularisation of the cornea and "snow flake-like" corneal opacity in females at 250 ppm.

At the 1 year (Weeks 49/50/51) and 2 year (Weeks 102/103) treatment-related findings consisted of corneal opacity, neovascularisation and oedema of the cornea, and "snow flake-like" corneal opacity at 2500, 1000 and 250 ppm in both sexes.

At the two year examination the number of males at 25 ppm with corneal opacity and neovascularisation of the cornea was very slightly higher than in the control group. However, in the absence of treatment-related ocular findings at the histopathological examination, these non-specific changes were considered to be incidental.

Other ophthalmological changes were considered to be chance findings as they occurred in isolation, in a non dose-related manner or at a similar frequency in the control and treated group.

See attachment 1 (attached background material) for tabulated ophthalmological results.
Haematological findings:: no effects observed
Description (incidence and severity):: No toxicologically relevant variation was noted in the parameters assayed at any blood sampling for any dose and either sex.
The few statistically significant differences were considered not to be relevant in view of their low magnitude, the absence of a clear dose-effect relationship and/or their isolated occurrence.
Clinical biochemistry findings:: effects observed, treatment-related
Description (incidence and severity):: In males, higher mean total cholesterol concentrations were observed at 2500, 1000 and 250 ppm throughout the study. The increase at 25 ppm at 6 and 12 months was within historical control data (2.02 ± 0.47 at 7 months, 2.41 ± 0.57 at 12 months), and as cholesterol concentration at 25 ppm returns to normal levels for the remainder of the study, this increase was considered not to be treatment-related. See attachment 1 (attached background material) for tabulated mean cholesterol data (males).

In females at 2500 ppm, at Month 24, mean total cholesterol and triglyceride concentrations were higher (+63% and +135% respectively, not statistically significant) relative to the control group. In females at Month 7, when compared to the controls, statistically significant differences were noted at 2500, 1000 and 250 ppm in total cholesterol and triglyceride concentrations. However in the absence of a clear dose-effect relationship and in view of the variation of the individual values, these changes were considered not to be relevant.
Other statistically significant differences were considered not to be relevant in view of their low magnitude, the absence of a clear dose-effect relationship and/or their isolated occurrence.
Endocrine findings:: not examined
Description (incidence and severity):: Not applicable.
Urinalysis findings:: effects observed, treatment-related
Description (incidence and severity):: When compared to the control groups, a tendency towards higher ketone levels was observed at 2500 and 1000 ppm in both sexes at all collection periods and at 250 ppm in males at Month 19 and 24 only.
Throughout the study lower mean pH values were noted in males at 2500, 1000 and 250 ppm. See attachment 1 (attached background material) for tabulated mean pH values (males). In females lower mean pH values were noted only on Month 3 - 4, at 2500 ppm (-10%, p < 0.01), 1000 ppm (-6%, p < 0.01) and 250 ppm (-6%, p < 0.01). Correlated with the lower mean pH values, lower amounts of crystals were generally observed in the urine.
Additionally, when compared to the controls, a tendency towards higher protein levels was seen in males from Month 6-7 at all dietary levels tested in this study.
Other statistically significant differences were considered not to be relevant in view of their sporadic occurrence and/or the absence of a clear dose-effect relationship.
Behaviour (functional findings):: not examined
Description (incidence and severity):: Not applicable.
Immunological findings:: not examined
Description (incidence and severity):: Not applicable.
Organ weight findings including organ / body weight ratios:: effects observed, treatment-related
Description (incidence and severity):: Mean terminal body weights were lower and statistically different in males (-12% at 2500 ppm, -8% at 1000 ppm, -7% at 250 ppm) when compared to controls.
At 2500 ppm, 1000 ppm and at 250 ppm in males, mean liver weights were higher and statistically different for most parameters, when compared to controls (see Table 7). These differences were associated with microscopic hepatocellular hypertrophy and were considered to be treatment-related.
At 2500 ppm, 1000 ppm and at 250 ppm in males, mean kidney weights were higher and statistically different for all parameters when compared to controls (see Table 8). These differences were associated with relevant microscopic findings and were considered to be treatment-related.
At 2500 ppm and at 1000 ppm in males, mean spleen weights were higher and statistically different for all parameters when compared to controls. As these differences were not associated with relevant microscopic findings, they were considered not to be toxicologically significant.
All other organ weight differences were judged to be incidental in view of their individual variation.
Gross pathological findings:: effects observed, treatment-related
Description (incidence and severity):: a/ Unscheduled deaths:
289 animals died prematurely before the end of the study (see Table 4).
A higher incidence of eye opacity was found in treated groups at 2500, 1000 and 250 ppm when compared to controls. This change was dose-related, was correlated with relevant histopathological findings and was considered to be treatment-related.
A higher incidence of irregular surface and paleness of the kidney was found in treated males at 2500 and 1000 ppm than in controls. These changes were correlated with relevant histopathological findings and were considered to be treatment-related.
All other gross pathology changes were considered as incidental and not treatment-related.

b/ Terminal sacrifice:
A higher incidence of eye opacity was found in treated animals at 2500, 1000 and 250 ppm than in controls. This change was dose-related, was correlated with relevant histopathological findings and was considered to be treatment related.
A higher incidence of irregular surface of the kidney was found in treated males at 2500 and 1000 ppm than in controls. This change was correlated with relevant histopathological findings and was considered to be treatment-related.
All other gross pathology changes were considered as incidental and not treatment-related.

See attachment 2 (attached background material) for tabulated gross pathology findings in the eye and kidneys.
Neuropathological findings:: not examined
Description (incidence and severity):: Not applicable.
Histopathological findings: non-neoplastic:: effects observed, treatment-related
Description (incidence and severity):: Clear treatment-related effects were seen in the eyes, thyroid gland and pancreas in both sexes, and liver and kidney in males.
Treatment-related changes of little or no toxicological significance were seen at a higher incidence and/or severity in the sciatic nerve, skeletal muscle, pituitary gland, blood vessels, ovaries, uterus, vagina and brain.

In the eyes, the incidence of unilateral or bilateral inflammation of the cornea with regenerative hyperplasia was greater than controls in animals dosed at 2500, 1000 ppm and males at 250 ppm. Neovascularisation was greater than controls in animals dosed at 2500, 1000 or 250 ppm. In some animals there was mucous metaplasia, vacuolation or atrophy of the cornea or bilateral atrophy of the peripheral retina.

In the liver, minimal centrilobular hepatocellular hypertrophy was noted in animals dosed at 2500 and in males at 1000 or 250 ppm. This finding was considered to be an adaptive response to treatment rather than an adverse effect. The incidence of bile duct hyperplasia, fibrosis and peribiliary inflammatory cells was higher than controls in females at all dosage levels. Since these changes are a common finding in ageing rats and as there was no difference in severity and no dose-relationship, they were considered not to be treatment-related.

In the thyroid gland, colloid alteration showed a higher incidence and/or severity at 2500, 1000 or 250 ppm compared with controls. Brown pigment in follicular cells showed a higher incidence and/or severity at all dosage levels compared with controls. Alteration of colloid and brown (lipofuscin) pigment in the thyroid gland of rats were considered not to be an adverse finding, since these findings were seen in the controls and reflect a normal age-related physiological process associated with the rapid turnover of colloid which is unique to the rat. Diffuse follicular cell hypertrophy was only seen in a few females dosed at 2500, 1000 or 250 ppm but showed no effect of treatment in males. Focal follicular hyperplasia was seen in animals from all groups and the incidence was slightly greater than controls in males dosed at 1000 ppm.

In the pancreas, diffuse degeneration/atrophy of acinar tissue was recorded in a small number of animals dosed at 2500 and 1000 ppm. Focal degeneration/atrophy of acinar tissue was recorded in animals from all groups but the incidence was greater than controls in females dosed at 2500 ppm. Interstitial oedema was greater than controls in a small number of animals dosed at 1000 ppm.

In the kidneys, chronic progressive nephropathy (CPN) was seen in most animals on test but the severity was slightly greater than controls in males dosed at 2500, 1000 or 250 ppm. At 25 ppm, the difference was judged not to be relevant since there was no real difference in severity when compared to controls. A higher incidence and severity of hyperplasia of the collecting ducts were noted in males dosed at 2500 or 1000 ppm.

In the sciatic nerve, diffuse atrophy and degeneration of myelin/fibres was seen in animals from all groups but the incidence and severity of atrophy were greater than controls in animals dosed at 2500, 1000 or 250 ppm, whilst the incidence of degeneration was reduced in a similar fashion. Perivascular mineralisation was seen in animals from all groups but the incidence was greater than controls in males dosed at 250 ppm. Granuloma/cholesterol clefts were seen in animals from all groups but the incidence and severity were greater than controls in females dosed at 1000 ppm. These lesions were considered to be of minor toxicological importance as they occur spontaneously in ageing laboratory rats kept under laboratory conditions and the incidence and severity of diffuse atrophy and degeneration of myelin/fibres was within the in-house historical control range.

In the skeletal muscle, atrophy of myofibres was seen in animals from all groups but the incidence and severity were greater than controls in males dosed at 2500, 1000 or 250 ppm. However, this lesion was considered to be of minor toxicological importance as it occurs spontaneously in ageing laboratory rats kept under laboratory conditions and the incidence and severity of the lesion was within the in-house historical control range.

In the pituitary gland, focal hyperplasia of the pars distalis was noted in animals from all groups but the incidence was slightly higher than controls in animals dosed at 2500 ppm. It should be noted that there was no evidence of an effect of treatment on tumours of the pars distalis and this finding was considered to have occurred by chance.

In the blood vessels, a higher incidence of arterial inflammation with degeneration or intimal proliferation was seen in a number of tissues, mainly at 2500 or 1000 ppm in males. In other tissues the incidence was very low and there was no apparent difference from controls. This
change was considered to be of little toxicological importance.

In the ovaries, depletion of corpora lutea and follicles was recorded in all groups but the incidence was slightly greater than controls in animals dosed at 2500, 1000 or 250 ppm. In the uterus, squamous metaplasia of glands was higher in incidence than controls at 2500 ppm and a decrease in vaginal mucification with an increase in focal granular cell hyperplasia at the same dose level. When compared with controls, the changes were minor in severity and low in frequency, therefore they were considered to be of minor toxicological importance.

Focal mineralisation in the brain was seen in animals from all groups but the incidence was marginally higher than controls in males dosed at 2500 ppm but due to the low incidence and severity, it was considered to be of minor toxicologically importance as this finding occurs spontaneously in ageing laboratory rats kept under laboratory conditions and the incidence and severity was within the in-house historical control range.

See attachment 3 (attached background material) for tabulated histopathological findings in the eye, liver, thyroid and kidneys.
Histopathological findings: neoplastic:: effects observed, treatment-related
Description (incidence and severity):: In the eyes, squamous cell tumours were seen in the cornea of two males dosed at 2500 ppm (one papilloma and one carcinoma). Tumours of the cornea are not reported as spontaneous lesions and the occurrence of two in males dosed at 2500 ppm were considered to be an effect of treatment. In the light of persistent corneal inflammation and regenerative hyperplasia as an effect of treatment, these tumours were considered to have occurred as a result of a nongenotoxic proliferative mechanism.
In the thyroid gland, follicular cell adenoma was seen in five males dosed at 250 ppm (p<0.05). There were, however, no thyroid follicular cell adenomas at higher doses (1000 and 2500 ppm). In the absence of a dose relationship and in the face of very low tumor incidence, these follicular cell adenomas were considered not to be treatment-related.

A variety of spontaneous tumours was noted in control and treated animals with no indication of an effect of treatment. The spectrum of these findings is generally consistent with neoplastic changes commonly encountered in ageing laboratory rats kept under laboratory conditions.
Other effects:: not examined
Description (incidence and severity):: Not applicable.
Details on results:: Corneal opacities, occasionally accompanied by neovascularization and their histopathological correlates (keratitis, reactive epithelial hyperplasia, and vascularization) are considered a rat-specific phenomenon. Corneal changes were not seen in other species chronically treated with the test substance (i.e. mice and dogs). The test substance is an inhibitor of the HPPDase enzyme and induces increased plasma tyrosine levels. This effect is more pronounced in rats than in mice and dogs. Experimentally induced hypertyrosinemia has been shown to induce snow flake-like corneal lesions in rats but not in mice (M-210983-01-2). In mice and humans, even under conditions of strong HPPD inhibition, tyrosine concentrations will not increase to levels high enough to induce ocular toxicity and hence, this toxicity observed in the rat is inappropriate for extrapolation to humans (ECETOC TR No. 99).

Thyroid findings (increased weight, histopathological changes comprising changes in colloid, follicular cell hypertrophy and pigment deposition in the follicular epithelium) are considered a non-adverse and rat specific phenomenon. No changes of the thyroid were noted in either mice or dogs, the other two species in which repeated-dose studies with histopathological examination of the thyroid were conducted with the test substance. As the test substance through inhibition of the HPPDase enzyme increases plasma tyrosine concentration in the rat, it is quite possible that some of this increased tyrosine is taken up by the thyroid and stored in the colloid, either as free tyrosine or through either increasing the synthesis of thyroglobulin or altering its composition in terms of number of tyrosine residues per thyroglobulin molecule.
In the absence of signs of altered thyroid functions (such as effects on body weights, fertility and gestation indices or effects on offspring performance in the developmental neurotoxicity study), the observed morphological changes are considered to be non-adverse.
: Key result
Dose descriptor:: NOAEL
Remarks:: general toxicity
Effect level:: 25 ppm
Based on:: test mat.
Sex:: male/female
Basis for effect level:: other: no adverse effects noted at 25 ppm
Remarks on result:: other: equivalent to 1.0 and 1.4 mg/kg bw/day in males and females, respectively
: Key result
Dose descriptor:: LOAEL
Remarks:: general toxicity
Effect level:: 250 ppm
Based on:: test mat.
Sex:: male/female
Basis for effect level:: body weight and weight gain; gross pathology; histopathology: non-neoplastic; ophthalmological examination; organ weights and organ / body weight ratios; urinalysis
Remarks on result:: other: equivalent to 10 and 14 mg/kg bw/day in males and females, respectively
: Key result
Critical effects observed:: yes
Lowest effective dose / conc.:: 1 000 ppm
System:: gastrointestinal tract
Organ:: pancreas
Treatment related:: yes
Dose response relationship:: yes
Relevant for humans:: not specified
Critical effects observed:: yes
Lowest effective dose / conc.:: 250 ppm
System:: urinary
Organ:: kidney
Treatment related:: yes
Dose response relationship:: yes
Relevant for humans:: no
Critical effects observed:: yes
Lowest effective dose / conc.:: 250 ppm
System:: eye
Organ:: cornea
Treatment related:: yes
Dose response relationship:: yes
Relevant for humans:: no
Critical effects observed:: yes
Lowest effective dose / conc.:: 250 ppm
System:: endocrine system
Organ:: thyroid gland
Treatment related:: yes
Dose response relationship:: yes
Relevant for humans:: no
Conclusions:: The study was performed under GLP conditions and according to OECD TG 453 (adopted 1981). Based on increased mortality at 2500 ppm in males, this dose is considered to have exceeded the Maximum Tolerated Dose. Treatment-related findings were seen in the eyes, liver, kidney, thyroid, and pancreas. Findings in the eye were related to the biochemical mechanism of the test substance as an HPPDase inhibitor leading to increased tyrosinemia in rats, and were considered not to be relevant for humans. The observations in the liver, including centrilobular hepatocellular hypertrophy, were evaluated as being adaptive responses to treatment rather than adverse effects. The finding of chronic progressive nephropathy is considered to be a rodent-specific finding not relevant to man. Similarly, the findings of colloid alteration and pigment deposition were considered to be specific to the rat and not indicative of an adverse finding, but to be normal findings in ageing rats. The increased incidence of acinar degeneration / atrophy in the pancreas was only observed at 1000 and 2500 ppm, and may indicate a tyrosine-linked effect as well, as these findings were not observed in the mouse or dog, which are not as sensitive as the rat to the effects of tyrosinemia.

The NOAEL for general toxicity in this study was therefore considered to be 25 ppm (1.0 mg/kg bw/day in males and 1.4 mg/kg bw/day in females), while the LOAEL was 250 ppm (10 mg/kg bw/day in males and 14 mg/kg bw/day in females).

Reference 4

Endpoint:: chronic toxicity: oral
Type of information:: experimental study
Adequacy of study:: key study
Study period:: 29 Jan 2003 - 21 Oct 2005
Reliability:: 1 (reliable without restriction)
Rationale for reliability incl. deficiencies:: guideline study
Reason / purpose for cross-reference:: reference to same study
Reason / purpose for cross-reference:: reference to same study
Reason / purpose for cross-reference:: reference to other study
Reason / purpose for cross-reference:: other: reference to review article
Qualifier:: according to guideline
Guideline:: OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)
Version / remarks:: adopted 12 May 1981
Deviations:: no
Qualifier:: according to guideline
Guideline:: OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)
Version / remarks:: adopted 25 Jun 2018
Deviations:: yes
Remarks:: no details on whether feed and water were analysed for contaminants
GLP compliance:: yes (incl. QA statement)
Remarks:: Secrétariat du Groupe Interministeriel des Produits Chimiques, Paris, France
Limit test:: no
Species:: rat
Strain:: Wistar
Remarks:: 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)
- Age at study initiation: 7 weeks
- Weight at study initiation: mean group weights: 286 - 289 g (males), 193 - 196 g (females)
- Fasting period before study: no
- Housing: individually in suspended, stainless steel and wire mesh cages
- Diet: A04CP1-10 from S.A.F.E. (Scientific Animal Food and Engineering, Augy, France), ad libitum
- Water: filtered and softened tap water from the municipal water supply, ad libitum
- Acclimation period: 22 days

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

IN-LIFE DATES: From: 29 Jan 2003 To: 05 Sep 2003
Route of administration:: oral: feed
Vehicle:: unchanged (no vehicle)
Details on oral exposure:: PREPARATION OF DIETARY MIXTURES:

DIET PREPARATION
- Rate of preparation of diet (frequency): The test substance formulations were prepared to cover the dietary requirements over 4-weekly periods.
- Mixing appropriate amounts with (Type of food): A04CP1-10 from S.A.F.E.
- Storage temperature of food: at ambient temperature

VEHICLE
- No vehicle was used
Analytical verification of doses or concentrations:: yes
Details on analytical verification of doses or concentrations:: The stability of the test substance in the diet has been demonstrated in a previous and recent 90-day rat study (M-102924-01-2). The stability of the test substance at 2 and 15000 ppm in the diet was verified for up to at least 82 days, when kept at ambient temperature, which covered the period of storage and usage in this study.

The homogeneity of the test substance in diet was verified from the first loads at 25, 250, 1000 and 2500 ppm on the first formulation (F1) and on the first loads at 25 and 2500 ppm of formulations F6, F12, F14, F20 and F26, to demonstrate adequate formulation procedures.
The concentration was checked for all loads at all dose levels for formulations F1, F2, F3, F6, F9, F12, F14, F17, F20, F23 and F26.

Results:
- Homogeneity Analysis: 82* - 115% of nominal concentration
- Concentration Analysis: 85 - 112% of nominal concentration
* 3/266 samples were slightly outside the in-house target range of 85 - 115% of nominal concentration. This minor deviation was not considered to have affected the integrity of the study.
Duration of treatment / exposure:: 6 months
Frequency of treatment:: Daily, 7 days a week
Dose / conc.:: 25 ppm
Remarks:: equivalent to 1.4 and 1.8 mg/kg bw/day in males and females, respectively
Dose / conc.:: 250 ppm
Remarks:: equivalent to 14 and 19 mg/kg bw/day in males and females, respectively
Dose / conc.:: 1 000 ppm
Remarks:: equivalent to 58 and 77 mg/kg bw/day in males and females, respectively
Dose / conc.:: 2 500 ppm
Remarks:: equivalent to 143 and 191 mg/kg bw/day in males and females, respectively
No. of animals per sex per dose:: 10
Control animals:: yes, plain diet
Details on study design:: - Dose selection rationale: Dose levels were selected based on the results from a previous 90-day dietary study in the rat (M-102924-01-2) where dietary administration of up to 7000 ppm in males and 12000 ppm in females resulted in increased plasma cholesterol and triglycerides, increased liver and kidney weights, yellow calculi in the urinary tract, urothelial hyperplasia, centrilobular hepatocellular hypertrophy, and diffuse thyroid follicular cell hyperplasia. The NOAEL in the rat 90-day study was 30 ppm, with a LOAEL of 1000 ppm.

- Rationale for animal assignment (if not random): On the day of randomization, animals were allocated to dose groups using a computerized randomization procedure that ensured a similar body weight distribution among groups for each sex. Selected animals were in a weight range from
243 to 325 g for the males and 169 to 221 g for the females at the start of exposure to the test substance, i.e., within ±20% of the mean body weight on the day of randomization. Any animal deemed unsuitable for selection based on weight, ophthalmological abnormalities or health status was not used for the study.
Observations and examinations performed and frequency:: CAGE SIDE OBSERVATIONS: Yes
- Time schedule: animals were checked for moribundity and mortality twice daily (once daily on weekends or public holidays). Clinical signs were recorded at least once daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: detailed physical examinations including palpation for masses were performed weekly from study day 1.

BODY WEIGHT: Yes
- Time schedule for examinations: at least weekly during the acclimatization period then weekly for the first 13 weeks of study, approximately every 4 weeks thereafter and prior to necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

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

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations:
all animals: during acclimatization (indirect ophthalmoscopy)
all surviving animals: after approximately 3 and 6 months (indirect ophthalmoscopy and slit lamp)
- Dose groups that were examined: all

HAEMATOLOGY: Yes
- Time schedule for collection of blood: blood analyses were performed on all surviving animals in weeks 28/29.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- Parameters examined: hematocrit, hemoglobin, leukocyte count, erythrocyte count, platelet count, prothrombin time, leukocyte differential count, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, mean corpuscular volume, reticulocyte count

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: blood analyses were performed on all surviving animals in weeks 28/29.
- Animals fasted: Yes
- Parameters examined: calcium, chloride, magnesium, inorganic phosphorus, potassium, sodium, alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, gamma glutamyltransferase, albumin, creatinine, urea, total cholesterol, glucose, total bilirubin, total protein, triglycerides

URINALYSIS: Yes
- Time schedule and for collection of urine: prior to allocation of the animals to treatment groups, urinalysis was performed on thirty males and thirty females. Urinalysis was performed on all surviving animals in weeks 13/14 and 26/27/28.
- Metabolism cages used for collection of urine: not specified
- Animals fasted: Yes (diet and water)
- Parameters examined: appearance, volume, specific gravity/osmolality/refractive index, pH, sediment (microscopic), protein, glucose, ketones, bilirubin, blood/red blood cells, urobilinogen, creatinine

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:: GROSS PATHOLOGY: Yes
Necropsy included the examination of external surfaces, all orifices, all major organs, tissues and body cavities. All significant macroscopic abnormalities (including masses and their regional lymph nodes when possible) were recorded, sampled and examined microscopically.

ORGAN WEIGHTS: Yes (see Table 1)

HISTOPATHOLOGY: Yes (see Table 1)
Histopathology examinations were performed as follows:
- all organs and tissue samples from animals sacrificed or dying during the treatment period
- all organs and tissue samples from animals of control and high dose groups
- liver, lung, kidney, thyroid gland, urinary bladder, pancreas and eye from animals of the intermediate dose groups
- gross abnormalities from all animals
For all unscheduled sacrificed or dead animals on study, the cause of death was determined when it was possible.
Initial examinations were performed by the Principal Investigator in Histopathology. Following the initial examination, a review pathologist undertook an independent « peer-review » of representative slides and diagnoses according to standardized operating procedures. The diagnoses presented in this report represent the consensus opinion of the two pathologists.
Statistics:: See "Any other information on materials and methods incl. tables"
Clinical signs:: effects observed, treatment-related
Description (incidence and severity):: Treatment-related clinical signs consisted of an increased incidence of white area on the eye at the three highest dose levels in both sexes. In addition, there was an increased incidence of soiled fur/soiling around the anogenital region at the two highest dose levels in both sexes (see Table 3).

The changes observed in the eye were considered to be as a consequence of the mode of action ofthe test substance, that in mammals inhibits a key enzyme involved in the catabolism of tyrosine and provokes a specific tyrosinaemia in the rat. The tyrosinemia and the resulting corneal lesions are not relevant to man.
Mortality:: mortality observed, treatment-related
Description (incidence):: The mortality rate at 2500 ppm in males was slightly higher than in the control group (4/75 deceased animals versus 1/75 in the control). The mortality rate in the remaining male treated groups was very low and similar to the control group. No deaths occurred in females.
Body weight and weight changes:: effects observed, treatment-related
Description (incidence and severity):: At 2500 and 1000 ppm, during the first week of treatment, mean body weight was reduced by 3% (p < 0.05) and 2%, respectively, in males, whilst mean cumulative body weight gain was reduced by 16% (p < 0.01) and 18% (p < 0.01), respectively, compared to controls (see Table 4). Thereafter, mean body weight gain was consistently lower than in controls by up to 56% and 60%, respectively, the effect being statistically significant on several occasions. At the end of the treatment period, mean body weight was reduced by 6% (p < 0.01) and 5% (p < 0.01), respectively, and mean cumulative body weight gain was reduced by 10% (p < 0.01) and 9% (p < 0.01), respectively, compared to controls. In females, there was no treatment-related effect on mean body weight, body weight gain and cumulative body weight gain. The few minor differences from controls which attained statistical significance were sporadic and were considered to be chance findings reflecting the number of comparisons made.

At 250 ppm, during the first week of treatment, mean body weight was reduced by 2% in males, whilst mean cumulative body weight gain was reduced by 14% (p < 0.01), compared to controls.Thereafter mean body weight, body weight gain and cumulative body weight gain were comparable to controls. The few minor differences reaching statistical significance were considered to reflect inter-individual variation rather than a treatment-related effect. In females, there was no treatment-related effect on mean body weight, body weight gain and cumulative body weight gain. The few minor differences from controls which attained statistical significance were sporadic and were considered to be chance findings reflecting the number of comparisons made.

At 25 ppm, there was no treatment-related effect on mean body weight, body weight gain and cumulative body weight gain in either sex over the first 6 months of treatment. The few minor differences from controls which attained statistical significance were sporadic and were considered to be chance findings reflecting the number of comparisons made.

There was no relevant change in terminal body weights of treated animals when compared to controls.
Food consumption and compound intake (if feeding study):: no effects observed
Food efficiency:: not examined
Description (incidence and severity):: Not applicable.
Water consumption and compound intake (if drinking water study):: not examined
Description (incidence and severity):: Not applicable.
Ophthalmological findings:: effects observed, treatment-related
Description (incidence and severity):: Treatment-related findings noted in Weeks 23/24 consisted of corneal opacity, neovascularisation, oedema of the cornea, and "snow flake-like" corneal opacity at 2500 and 1000 ppm in both sexes and 250 ppm in males, as well as neovascularisation of the cornea and "snow flake-like" corneal opacity in females at 250 ppm. See attachment 1 (attached background material) for tabulated ophthalmological results.
Haematological findings:: no effects observed
Clinical biochemistry findings:: effects observed, treatment-related
Description (incidence and severity):: In males, higher mean total cholesterol concentrations were observed at 2500, 1000 and 250 ppm. The higher values noted at 25 ppm after 7 months were within historical control data (2.02 ± 0.47), and as cholesterol concentration at 25 ppm returned to normal levels for the remainder of the study (12-month and carcinogenicity phase), this increase was considered not to be treatment-related. See attachment 1 (attached background material) for tabulated mean cholesterol data (males).

In females at Month 7, when compared to the controls, statistically significant differences were noted at 2500, 1000 and 250 ppm in total cholesterol and triglyceride concentrations. However in the absence of a clear dose-effect relationship and in view of the variation of the individual values, these changes were considered not to be relevant.
Endocrine findings:: not examined
Description (incidence and severity):: Not applicable.
Urinalysis findings:: effects observed, treatment-related
Description (incidence and severity):: When compared to the control groups, a tendency towards higher ketone levels was observed at 2500 and 1000 ppm in both sexes.

Lower mean pH values were noted in males at 2500, 1000 and 250 ppm throughout the study. See attachment 1 (attached background material) for tabulated mean pH values (males).

In females lower mean pH values were noted only in Week 13/14, at 2500 ppm (-10%, p<0.01), 1000 ppm (-6%, p < 0.01) and 250 ppm (-6%, p < 0.01). Correlated with the lower mean pH values, lower amounts of crystals were generally observed in the urine.

Additionally, when compared to the controls, a tendency towards higher protein levels was seen in males at Week 26/28 at all dietary levels tested. In the absence of an adverse kidney histopathology these findings were considered not adverse.

Other statistically significant differences were considered not to be relevant in view of their sporadic occurrence and/or the absence of a clear dose-effect relationship.
Behaviour (functional findings):: not examined
Description (incidence and severity):: Not applicable.
Immunological findings:: not examined
Description (incidence and severity):: Not applicable.
Organ weight findings including organ / body weight ratios:: effects observed, treatment-related
Description (incidence and severity):: In males only, mean liver weights were found to be statistically significantly increased at 2500, 1000 and 250 ppm (see Table 5) . These changes were correlated with histopathological changes and were thus regarded as treatment-related.
In males only, mean absolute and relative kidney weights were found statistically significantly higher at 2500 and at 1000 ppm (see Table 6). The change was not statistically significant at 250 ppm, but due to a dose-response, it was considered to be treatment-related at 2500, 1000 and 250 ppm.
Mean thyroid gland weights were found to be higher at 2500 ppm in both sexes and at 1000 and 250 ppm in males (see Table 7). At 2500 ppm in males, mean thyroid gland to body weight ratio was higher and statistically different when compared to controls and was considered to be treatment-related.
Other organ weight changes were considered to be incidental in origin.
Gross pathological findings:: effects observed, treatment-related
Description (incidence and severity):: a/ Unscheduled deaths
One control male rat was killed for humane reasons on Day 156. A palpable mass on the right kidney corresponding to a tubular cell carcinoma was found at necropsy.
All others gross changes were considered to be incidental and not treatment-related.

b/ Terminal sacrifice:
Enlarged liver was observed in a proportion of male animals in all treated groups (3/10 males at 2500 ppm, 1/10 males at 1000 ppm, 4/10 males at 250 ppm, and 2/10 males at 25 ppm). Prominent lobulation was observed at a higher incidence in treated males than in controls. These findings were correlated with centrilobular hypertrophy and were considered to be treatment-related.
A higher incidence of eye opacity was found in treated animals at 2500 ppm and in treated males at 1000 and 250 ppm. This finding was correlated with relevant microscopic findings and was considered to be treatment-related.
All others gross changes were considered to be incidental and not treatment-related.
Neuropathological findings:: not examined
Description (incidence and severity):: Not applicable.
Histopathological findings: non-neoplastic:: effects observed, treatment-related
Description (incidence and severity):: a/ Unscheduled deaths
One control male was killed for humane reasons and a malignant tubular cell carcinoma of the kidney was found at microscopy.

b/ Terminal sacrifice:
Effects of treatment were seen in the eyes, liver and pancreas of both sexes and thyroid glands and kidneys of males.

In the eyes, unilateral or bilateral inflammation of the cornea with regenerative hyperplasia and neovascularisation were seen in both sexes treated at 2500 and 1000 ppm and males at 250 ppm.

In the liver, centrilobular hepatocellular hypertrophy was noted in both sexes at 2500, 1000 or 250 ppm and in males at 25 ppm. The incidence of centrilobular vacuolation was higher than controls in males at 2500, 1000 or 250 ppm. These were considered to reflect an adaptive response to treatment rather than an adverse effect.

In the thyroid gland, a higher incidence of altered basophilic colloid was noted in males at 2500, 1000 and 250 ppm when compared with controls. Increased follicular diameter and brown pigment in follicular cells was observed with a higher incidence in males at 2500 ppm. The alteration of colloid and brown (lipofuscin) pigmentation were considered not to be adverse findings since these findings were observed in the controls and reflect a normal age-related physiological process associated with the rapid turnover of colloid which is unique to the rat.

In the kidney, there was a marginally higher incidence and severity of chronic progressive nephropathy in males dosed at 2500 ppm in comparison with controls. This finding was considered having no relevance for extrapolation in human risk assessment, as chronic nephropathy has been described to be a rodent-specify entity.

In the pancreas, diffuse degeneration/atrophy of acinar tissue was recorded in 7/10 males and 8/10 females dosed at 2500 ppm, and 1/9 females dosed at 1000 ppm.

A variety of spontaneous changes was noted in control and treated animals with no indication of an effect of treatment. The spectrum of these findings is mainly consistent with changes commonly encountered in laboratory rats of this age kept under laboratory conditions.

See attachment 2 (attached background material) for tabulated histopathological findings in the eye, liver, thyroid and kidneys.
Histopathological findings: neoplastic:: no effects observed
Description (incidence and severity):: The only tumor noted was a malignant tubular cell carcinoma of the kidney observed for a control animal.
Other effects:: not examined
Description (incidence and severity):: Not applicable.
Details on results:: Corneal opacities, occasionally accompanied by neovascularization and their histopathological correlates (keratitis, reactive epithelial hyperplasia, and vascularization) are considered a rat-specific phenomenon. Corneal changes were not seen in other species chronically treated with the test substance (i.e. mice and dogs). The test substance is an inhibitor of the HPPDase enzyme and induces increased plasma tyrosine levels. This effect is more pronounced in rats than in mice and dogs. Experimentally induced hypertyrosinemia has been shown to induce snow flake-like corneal lesions in rats but not in mice (M-210983-01-2). In mice and humans, even under conditions of strong HPPD inhibition, tyrosine concentrations will not increase to levels high enough to induce ocular toxicity and hence, this toxicity observed in the rat is inappropriate for extrapolation to humans (ECETOC TR No. 99).

Thyroid findings (increased weight, histopathological changes comprising changes in colloid, follicular cell hypertrophy and pigment deposition in the follicular epithelium) are considered a non-adverse and rat specific phenomenon. No changes of the thyroid were noted in either mice or dogs, the other two species in which repeated-dose studies with histopathological examination of the thyroid were conducted with the test substance. As the test substance through inhibition of the HPPDase enzyme increases plasma tyrosine concentration in the rat, it is quite possible that some of this increased tyrosine is taken up by the thyroid and stored in the colloid, either as free tyrosine or through either increasing the synthesis of thyroglobulin or altering its composition in terms of number of tyrosine residues per thyroglobulin molecule.
In the absence of signs of altered thyroid functions (such as effects on body weights, fertility and gestation indices or effects on offspring performance in the developmental neurotoxicity study), the observed morphological changes are considered to be non-adverse.
: Key result
Dose descriptor:: NOAEL
Effect level:: 25 ppm
Based on:: test mat.
Sex:: male/female
Basis for effect level:: other: no adverse effects observed at 25 ppm
Remarks on result:: other: equivalent to 1.4 and 1.8 mg/kg bw/day in males and females, respectively
: Key result
Dose descriptor:: LOAEL
Effect level:: 250 ppm
Based on:: test mat.
Sex:: male/female
Basis for effect level:: body weight and weight gain; clinical biochemistry; gross pathology; histopathology: non-neoplastic; ophthalmological examination; organ weights and organ / body weight ratios; urinalysis
Remarks on result:: other: equivalent to 14 and 19 mg/kg bw/day in males and females, respectively
: Key result
Critical effects observed:: yes
Lowest effective dose / conc.:: 1 000 ppm
System:: gastrointestinal tract
Organ:: pancreas
Treatment related:: yes
Dose response relationship:: yes
Relevant for humans:: not specified
Critical effects observed:: yes
Lowest effective dose / conc.:: 250 ppm
System:: eye
Organ:: cornea
Treatment related:: yes
Dose response relationship:: yes
Relevant for humans:: no
Critical effects observed:: yes
Lowest effective dose / conc.:: 2 500 ppm
System:: urinary
Organ:: kidney
Treatment related:: yes
Dose response relationship:: yes
Relevant for humans:: no
Critical effects observed:: yes
Lowest effective dose / conc.:: 250 ppm
System:: endocrine system
Organ:: thyroid gland
Treatment related:: yes
Dose response relationship:: yes
Relevant for humans:: no
Conclusions:: The study was performed under GLP conditions and according to OECD TG 453 (adopted 1981). Based on increased mortality at 2500 ppm in males, this dose is considered to have exceeded the Maximum Tolerated Dose. Treatment-related findings were seen in the eyes, liver, kidney, thyroid, and pancreas. Findings in the eye were related to the biochemical mechanism of the test substance as an HPPDase inhibitor leading to increased tyrosinemia in rats, and were considered not to be relevant for humans. The observations in the liver, including centrilobular hepatocellular hypertrophy, were evaluated as being adaptive responses to treatment rather than adverse effects. The finding of chronic progressive nephropathy is considered to be a rodent-specific finding not relevant to man. Similarly, the findings of thyroid colloid alteration and pigment deposition were considered to be specific to the rat and not indicative of an adverse finding, but to be normal findings in ageing rats. The increased incidence of acinar degeneration / atrophy in the pancreas was only observed at 1000 and 2500 ppm, and may indicate a tyrosine-linked effect as well, as these findings were not observed in the mouse or dog, which are not as sensitive as the rat to the effects of tyrosinemia.

The NOAEL in this study was therefore considered to be 25 ppm (1.4 mg/kg bw/day in males and 1.8 mg/kg bw/day in females), while the LOAEL was 250 ppm (14 mg/kg bw/day in males and 19 mg/kg bw/day in females).

Endpoint conclusion

Endpoint conclusion:: adverse effect observed
Dose descriptor:: NOAEL
: 1 mg/kg bw/day
Study duration:: chronic
Species:: rat
Quality of whole database:: The available information comprises adequate, reliable (Klimisch score 1) and consistent studies, and is thus sufficient to fulfil the standard information requirements set out in Annex VIII-IX, 8.6, of Regulation (EC) No 1907/2006.
System:: other: urinary and gastrointestinal (exocrine pancreas)
Organ:: kidney; pancreas

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

Link to relevant study records

Reference

Endpoint:: short-term repeated dose toxicity: dermal
Type of information:: experimental study
Adequacy of study:: key study
Study period:: 08 Feb - 12 Jul 2005
Reliability:: 1 (reliable without restriction)
Rationale for reliability incl. deficiencies:: guideline study
Qualifier:: according to guideline
Guideline:: OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)
Version / remarks:: adopted in 1981
Deviations:: no
GLP compliance:: yes
Limit test:: no
Species:: rat
Strain:: Wistar
Remarks:: HsdCpb:WU
Details on species / strain selection:: The rat is the recommended species for repeat-dose dermal toxicity studies. Animals of this strain have been used at the Bayer HealthCare AG for toxicological studies for many years. Historical data on their physiology and spontaneous alterations is available.
Sex:: male/female
Details on test animals or test system and environmental conditions:: TEST ANIMALS
- Source: Harlan Winkelmann GmbH Experimental Animal Breeders in Borchen, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: males: 9 - 10 weeks, females: 13 - 14 weeks
- Weight at study initiation: males: 222 - 262 g (mean 244 g), females: 202 - 230 g (mean 213 g)
- Fasting period before study: no
- Housing: individually in Type IIIh cages on wood shavings
- Diet: PROVIMI KLIBA® 3883.0.15 (Provimi Kliba SA, Kaiseraugst, Switzerland), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least seven days

DETAILS OF FOOD AND WATER QUALITY: analysis was performed

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 50 - 60
- Air changes (per hr): more than 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 08 Feb 2005 To: 09 Mar 2005
Type of coverage:: semiocclusive
Vehicle:: unchanged (no vehicle)
Remarks:: The test item was applied as undiluted solid onto a wet gauze pad.
Details on exposure:: TEST SITE
- Area of exposure: back and flank region, 6.0 x 5.0 cm = 30.0 cm²
- % coverage: greater than 10% of body surface area
- Type of wrap if used: the test item was applied to the moist gauze-layer of a "Cutiplast steril®" patch and secured in place with using Peha-Haft® placed on the rat's back; the gauze-layer was secured in place using "Peha-Haft®" cohesive stretch tape (approx. 8 x 23 cm).
- Time intervals for shavings: one day before the start of treatment and twice weekly afterwards

REMOVAL OF TEST SUBSTANCE
- Washing (if done): test item was removed with soap and water
- Time after start of exposure: 6 h

TEST MATERIAL
- Amount(s) applied: 10, 100 and 1000 mg/kg bw/day;
males reveived 9.6 - 12.8, 7.3 - 10.8 and 15.5 - 19.8 mg/cm²/day; females received 8.0 - 8.4, 5.7 - 9.5 and 15.0 - 16.1 mg/cm²/day
- For solids, paste formed: no

USE OF RESTRAINERS FOR PREVENTING INGESTION: yes ("Lomir Biomedical Inc." rat jacket)
Analytical verification of doses or concentrations:: no
Remarks:: (No dose formulation preparation or analysis was performed during the study, as the test item powder was applied to the rat skin without using any vehicle (only moistened with water)).
Details on analytical verification of doses or concentrations:: not applicable
Duration of treatment / exposure:: study period 28 days (males received 22 applications, females 23)
Frequency of treatment:: first three weeks: 5 days/week; thereafter: 7 days/week
Dose / conc.:: 10 mg/kg bw/day
Dose / conc.:: 100 mg/kg bw/day
Dose / conc.:: 1 000 mg/kg bw/day
No. of animals per sex per dose:: 10
Control animals:: yes, sham-exposed
Details on study design:: - Dose selection rationale: Dose levels were selected and determined by the sponsor according to results obtained in previous studies.
Observations and examinations performed and frequency:: CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Inspections on mortality and morbidity of the animals were done twice daily and on weekends / on public holidays once a day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: A careful clinical examination including observation outside the home cage in a standard arena (OFO) was made once prior to treatment and daily (careful clinical examination) or weekly (OFO) thereafter up to necropsies in all animals.

DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations: The shaved skin areas were examined before starting the study and each day of treatment during the study.

BODY WEIGHT: Yes
- Time schedule for examinations: The animals were weighed before the study was started and at the beginning of each study week.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

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

WATER CONSUMPTION: Yes
- Time schedule for examinations: Individual water intake was determined once weekly from the start of the study.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Ophthalmological examinations were performed before the start of the study and at the end of the treatment period.
- Dose groups that were examined: all (pre-treatment), groups 1 (control) and 4 (high dose) at the end of the treatment period

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood sampling was performed before the start of the study and towards the end of the treatment period (males day 25, females day 28).
- Anaesthetic used for blood collection: Yes (CO2/O2)
- Animals fasted: No
- How many animals: all
- Parameters examined: leucocyte count, differential blood count, erythrocytes, haemoglobin, haematocrit, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, mean corpuscular volume, thrombocytes, reticulocytes, hepatoquick

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood sampling was performed before the start of the study and towards the end of the treatment period (males day 25, females day 28).
- Animals fasted: No
- How many animals: all
- Parameters examined: alanine aminotransferse activity, albumin concentration, alkaline phosphatase activity, aspartate aminotransferase activity, total bilirubin concentration, cholesterol concentration, creatinine concentration, calcium concentration, chloride concentration, glucose concentration, potassium concentration, sodium concentration, inorganic phosphate concentration, total protein concentration, triglycerides, urea concentration

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:: GROSS PATHOLOGY: Yes, all animals alive at scheduled necropsy as well as those killed in moribund state

HISTOPATHOLOGY: Yes
- adrenal glands, aorta, brain (cerebrum, cerebellum, brain stem), epididymides, esophagus, eyes, eyelids, exorbital lacrimal glands, femur, harderian glands, head: nasal cavity, heart, intestine (incl. Peyer's patches): duodenum, jejunum, iileum, caecum, colon, rectum, remaining intestine, kidneys, larynx, liver, lungs, lymph nodes: mandibular and mesenteric, optic nerves, ovaries, oviducts, physical identifier, pancreas, pharynx, pituitary gland, prostate, salivary glands (parotid, submandibular, sublingual), sciatic nerve, seminal vesicles (incl. coagulation glands), skeletal muscle (tigh), skin (mammary region), spinal cord (cervical, thoracic, lumbar), spleen, sternum, stomach (forestomach and glandular stomach), sestes, thymus, thyroid glands (with parathyroids), tongue, trachea, ureters, urethra, urinary bladder, uterus (with cervix), vagina, Zymbal's glands, organs and tissues with macroscopic findings, skin - treated, skin - untreated

ORGAN WEIGHTS: Yes
- adrenal glands, kidneys, liver, brain, testes, epididymides, heart, ovaries, spleen, thyroid gland (in fixed state), thymus, uterus
Statistics:: As basis for statistical evaluation of numerical data SAS 6.12 was used. The results for the groups that received the test compound were compared with those for the control group. The statistical evaluation of data related to laboratory investigations, body and organ weights as well as food and water intake is performed using SAS® routines.
Clinical signs:: no effects observed
Dermal irritation:: no effects observed
Mortality:: no mortality observed
Body weight and weight changes:: no effects observed
Food consumption and compound intake (if feeding study):: no effects observed
Food efficiency:: not examined
Description (incidence and severity):: Not applicable.
Water consumption and compound intake (if drinking water study):: no effects observed
Ophthalmological findings:: no effects observed
Haematological findings:: effects observed, non-treatment-related
Description (incidence and severity):: In mid-dose males a statistically significantly higher MCHC value was noted (320 g/L Ery vs. 314 in the control). In mid-dose females a statistically significantly higher MCH value was noted (17.4 pg vs. 16.9 in the control). Both differences were only mimimal in degree and showed no dependency. Therefore these changes were considered to be not treatment-related.

See Attachment 2 for summary data of haematological findings.
Clinical biochemistry findings:: effects observed, non-treatment-related
Description (incidence and severity):: The activities of aspartate aminotransferase were decreased at 10 mg/kg bw/day and above and of alkaline phosphatase at 100 mg/kg bw/day and above in both sexes.
The concentrations of cholesterol, triglycerides, total protein and albumin were higher, and the concentrations of creatinine lower in males at 1000 mg/kg bw/day. In females, the concentration of bilirubin was higher at 100 and 1000 mg/kg bw/day.
The concentration of sodium was decreased at 100 mg/kg bw/day and above in both sexes.
Calcium was increased at 1000 mg/kg in females, and inorganic phosphate decreased in males at 10 mg/kg bw/day and above.
All these partly statistically significant differences were either of a minimal degree, and/or not dose-related, and/or were observed only in one sex, and/or were all in the range of historical values. Therefore, they were considered as not treatment-related.

See Attachment 3 for summary data of clinical chemistry findings and Attachment 5 for laboratory historical control data.
Endocrine findings:: not examined
Description (incidence and severity):: Not applicable.
Urinalysis findings:: not examined
Description (incidence and severity):: Not applicable.
Behaviour (functional findings):: not examined
Description (incidence and severity):: Not applicable.
Immunological findings:: not examined
Description (incidence and severity):: Not applicable.
Organ weight findings including organ / body weight ratios:: effects observed, treatment-related
Description (incidence and severity):: Absolute and relative liver weights were statistically significantly increased at 1000 mg/kg bw/day in both sexes.

Absolute mean liver weight was statistically significantly increased in females at 10 mg/kg bw/day. However, relative mean liver weight of these animals was similar to the control group. In the absence of liver weight effects at 100 mg/kg bw/day this finding was considered incidental in nature.

See Table 2 for mean liver weight data on Day 28. See Attachment 4 for summary data of organ weight findings.
Gross pathological findings:: no effects observed
Neuropathological findings:: not examined
Description (incidence and severity):: Not applicable.
Histopathological findings: non-neoplastic:: effects observed, treatment-related
Description (incidence and severity):: Locally, no evidence of any findings was detected at the application site of the skin.
In the pancreas, focal degeneration was detected at 100 mg/kg bw/day and above in both sexes.
In the thyroid gland, follicular cell hypertrophy was detected in both sexes in all dose groups, including controls. However, an increased incidence occurred in males at 1000 mg/kg bw/day (5-5-4-9 affected animals with increasing doses). Furthermore, colloid alteration of the thyroid was detected with an increased incidence in males (1-4-6-10 affected animals with increasing doses). The incidence occurred together with an increasing grading in the upper dose groups.
Slight hepatocellular hypertrophy was observed at 1000 mg/kg bw/day, in males.
Hypertrophy of the pars distalis of pituitary was observed at 1000 mg/kg bw/day in males.

See Attachment 1 for summary data of histopathological (non-neoplastic) findings.
Histopathological findings: neoplastic:: no effects observed
Other effects:: not examined
Description (incidence and severity):: Not applicable.
Details on results:: Thyroid findings (increased weight, histopathological changes comprising changes in colloid, follicular cell hypertrophy and pigment deposition in the follicular epithelium) are considered a non-adverse and rat specific phenomenon. No changes of the thyroid were noted in either mice or dogs, the other two species in which repeated-dose studies with histopathological examination of the thyroid were conducted with the test substance. As the test substance through inhibition of the HPPDase enzyme increases plasma tyrosine concentration in the rat, it is quite possible that some of this increased tyrosine is taken up by the thyroid and stored in the colloid, either as free tyrosine or through either increasing the synthesis of thyroglobulin or altering its composition in terms of number of tyrosine residues per thyroglobulin molecule.
In the absence of signs of altered thyroid functions (such as effects on body weights, fertility and gestation indices or effects on offspring performance in the developmental neurotoxicity study), the observed morphological changes are considered to be non-adverse.
: Key result
Dose descriptor:: NOAEL
Remarks:: local
Effect level:: 1 000 mg/kg bw/day
Based on:: test mat.
Remarks:: equivalent to 15.0 mg/m2
Sex:: male/female
Basis for effect level:: other: No dermal irritation observed up to the highest dose tested
: Key result
Dose descriptor:: NOAEL
Effect level:: 10 mg/kg bw/day
Based on:: test mat.
Sex:: male/female
Basis for effect level:: other: No adverse effects seen at 10 mg/kg bw/day
: Key result
Dose descriptor:: LOAEL
Effect level:: 100 mg/kg bw/day
Based on:: test mat.
Sex:: male/female
Basis for effect level:: other: histopathological effects in the pancreas at 100 mg/kg bw/day
: Key result
Critical effects observed:: yes
Lowest effective dose / conc.:: 100 mg/kg bw/day
System:: gastrointestinal tract
Organ:: pancreas
Treatment related:: yes
Dose response relationship:: yes
Relevant for humans:: not specified
Critical effects observed:: yes
Lowest effective dose / conc.:: 100 mg/kg bw/day
System:: endocrine system
Organ:: thyroid gland
Treatment related:: yes
Dose response relationship:: yes
Relevant for humans:: no
Critical effects observed:: yes
Lowest effective dose / conc.:: 1 000 mg/kg bw/day
System:: hepatobiliary
Organ:: liver
Treatment related:: yes
Dose response relationship:: yes
Relevant for humans:: not specified
Critical effects observed:: yes
Lowest effective dose / conc.:: 1 000 mg/kg bw/day
System:: endocrine system
Organ:: pituitary gland
Treatment related:: yes
Dose response relationship:: yes
Relevant for humans:: not specified
Conclusions:: The study was performed under GLP conditions and according to OECD TG 410 (adopted 1981).There were few effects of repeated dermal application of the test substance to either male or female rats. There were no local effects on either skinfold thickness or on signs of irritation. The local NOAEL was determined to be 1000 mg/kg bw/day (equivalent to 15 mg/m2 skin), while the systemic NOAEL was 10 mg/kg bw/day, based on histopathological effects observed at 100 and 1000 mg/kg bw/day in both males and females.

Based on the effects observed in the pancreas at 100 mg/kg bw/day, the substance is classified "STOT RE 2, H373, gastro-intestinal".

Endpoint conclusion

Endpoint conclusion:: adverse effect observed
Dose descriptor:: NOAEL
: 10 mg/kg bw/day
Study duration:: subacute
Species:: rat
Quality of whole database:: The available information comprises an adequate and reliable study (Klimisch score 1), and is thus sufficient to fulfil the standard information requirements set out in Annex VIII - IX, 8.6, of Regulation (EC) No 1907/2006.
System:: other: hepatobiliary, gastrointestinal and endocrine
Organ:: liver; pancreas; pituitary gland

Repeated dose toxicity: dermal - local effects

Link to relevant study records

Reference

Endpoint:: short-term repeated dose toxicity: dermal
Type of information:: experimental study
Adequacy of study:: key study
Study period:: 08 Feb - 12 Jul 2005
Reliability:: 1 (reliable without restriction)
Rationale for reliability incl. deficiencies:: guideline study
Qualifier:: according to guideline
Guideline:: OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)
Version / remarks:: adopted in 1981
Deviations:: no
GLP compliance:: yes
Limit test:: no
Species:: rat
Strain:: Wistar
Remarks:: HsdCpb:WU
Details on species / strain selection:: The rat is the recommended species for repeat-dose dermal toxicity studies. Animals of this strain have been used at the Bayer HealthCare AG for toxicological studies for many years. Historical data on their physiology and spontaneous alterations is available.
Sex:: male/female
Details on test animals or test system and environmental conditions:: TEST ANIMALS
- Source: Harlan Winkelmann GmbH Experimental Animal Breeders in Borchen, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: males: 9 - 10 weeks, females: 13 - 14 weeks
- Weight at study initiation: males: 222 - 262 g (mean 244 g), females: 202 - 230 g (mean 213 g)
- Fasting period before study: no
- Housing: individually in Type IIIh cages on wood shavings
- Diet: PROVIMI KLIBA® 3883.0.15 (Provimi Kliba SA, Kaiseraugst, Switzerland), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least seven days

DETAILS OF FOOD AND WATER QUALITY: analysis was performed

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 50 - 60
- Air changes (per hr): more than 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 08 Feb 2005 To: 09 Mar 2005
Type of coverage:: semiocclusive
Vehicle:: unchanged (no vehicle)
Remarks:: The test item was applied as undiluted solid onto a wet gauze pad.
Details on exposure:: TEST SITE
- Area of exposure: back and flank region, 6.0 x 5.0 cm = 30.0 cm²
- % coverage: greater than 10% of body surface area
- Type of wrap if used: the test item was applied to the moist gauze-layer of a "Cutiplast steril®" patch and secured in place with using Peha-Haft® placed on the rat's back; the gauze-layer was secured in place using "Peha-Haft®" cohesive stretch tape (approx. 8 x 23 cm).
- Time intervals for shavings: one day before the start of treatment and twice weekly afterwards

REMOVAL OF TEST SUBSTANCE
- Washing (if done): test item was removed with soap and water
- Time after start of exposure: 6 h

TEST MATERIAL
- Amount(s) applied: 10, 100 and 1000 mg/kg bw/day;
males reveived 9.6 - 12.8, 7.3 - 10.8 and 15.5 - 19.8 mg/cm²/day; females received 8.0 - 8.4, 5.7 - 9.5 and 15.0 - 16.1 mg/cm²/day
- For solids, paste formed: no

USE OF RESTRAINERS FOR PREVENTING INGESTION: yes ("Lomir Biomedical Inc." rat jacket)
Analytical verification of doses or concentrations:: no
Remarks:: (No dose formulation preparation or analysis was performed during the study, as the test item powder was applied to the rat skin without using any vehicle (only moistened with water)).
Details on analytical verification of doses or concentrations:: not applicable
Duration of treatment / exposure:: study period 28 days (males received 22 applications, females 23)
Frequency of treatment:: first three weeks: 5 days/week; thereafter: 7 days/week
Dose / conc.:: 10 mg/kg bw/day
Dose / conc.:: 100 mg/kg bw/day
Dose / conc.:: 1 000 mg/kg bw/day
No. of animals per sex per dose:: 10
Control animals:: yes, sham-exposed
Details on study design:: - Dose selection rationale: Dose levels were selected and determined by the sponsor according to results obtained in previous studies.
Observations and examinations performed and frequency:: CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Inspections on mortality and morbidity of the animals were done twice daily and on weekends / on public holidays once a day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: A careful clinical examination including observation outside the home cage in a standard arena (OFO) was made once prior to treatment and daily (careful clinical examination) or weekly (OFO) thereafter up to necropsies in all animals.

DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations: The shaved skin areas were examined before starting the study and each day of treatment during the study.

BODY WEIGHT: Yes
- Time schedule for examinations: The animals were weighed before the study was started and at the beginning of each study week.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

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

WATER CONSUMPTION: Yes
- Time schedule for examinations: Individual water intake was determined once weekly from the start of the study.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Ophthalmological examinations were performed before the start of the study and at the end of the treatment period.
- Dose groups that were examined: all (pre-treatment), groups 1 (control) and 4 (high dose) at the end of the treatment period

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood sampling was performed before the start of the study and towards the end of the treatment period (males day 25, females day 28).
- Anaesthetic used for blood collection: Yes (CO2/O2)
- Animals fasted: No
- How many animals: all
- Parameters examined: leucocyte count, differential blood count, erythrocytes, haemoglobin, haematocrit, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, mean corpuscular volume, thrombocytes, reticulocytes, hepatoquick

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood sampling was performed before the start of the study and towards the end of the treatment period (males day 25, females day 28).
- Animals fasted: No
- How many animals: all
- Parameters examined: alanine aminotransferse activity, albumin concentration, alkaline phosphatase activity, aspartate aminotransferase activity, total bilirubin concentration, cholesterol concentration, creatinine concentration, calcium concentration, chloride concentration, glucose concentration, potassium concentration, sodium concentration, inorganic phosphate concentration, total protein concentration, triglycerides, urea concentration

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:: GROSS PATHOLOGY: Yes, all animals alive at scheduled necropsy as well as those killed in moribund state

HISTOPATHOLOGY: Yes
- adrenal glands, aorta, brain (cerebrum, cerebellum, brain stem), epididymides, esophagus, eyes, eyelids, exorbital lacrimal glands, femur, harderian glands, head: nasal cavity, heart, intestine (incl. Peyer's patches): duodenum, jejunum, iileum, caecum, colon, rectum, remaining intestine, kidneys, larynx, liver, lungs, lymph nodes: mandibular and mesenteric, optic nerves, ovaries, oviducts, physical identifier, pancreas, pharynx, pituitary gland, prostate, salivary glands (parotid, submandibular, sublingual), sciatic nerve, seminal vesicles (incl. coagulation glands), skeletal muscle (tigh), skin (mammary region), spinal cord (cervical, thoracic, lumbar), spleen, sternum, stomach (forestomach and glandular stomach), sestes, thymus, thyroid glands (with parathyroids), tongue, trachea, ureters, urethra, urinary bladder, uterus (with cervix), vagina, Zymbal's glands, organs and tissues with macroscopic findings, skin - treated, skin - untreated

ORGAN WEIGHTS: Yes
- adrenal glands, kidneys, liver, brain, testes, epididymides, heart, ovaries, spleen, thyroid gland (in fixed state), thymus, uterus
Statistics:: As basis for statistical evaluation of numerical data SAS 6.12 was used. The results for the groups that received the test compound were compared with those for the control group. The statistical evaluation of data related to laboratory investigations, body and organ weights as well as food and water intake is performed using SAS® routines.
Clinical signs:: no effects observed
Dermal irritation:: no effects observed
Mortality:: no mortality observed
Body weight and weight changes:: no effects observed
Food consumption and compound intake (if feeding study):: no effects observed
Food efficiency:: not examined
Description (incidence and severity):: Not applicable.
Water consumption and compound intake (if drinking water study):: no effects observed
Ophthalmological findings:: no effects observed
Haematological findings:: effects observed, non-treatment-related
Description (incidence and severity):: In mid-dose males a statistically significantly higher MCHC value was noted (320 g/L Ery vs. 314 in the control). In mid-dose females a statistically significantly higher MCH value was noted (17.4 pg vs. 16.9 in the control). Both differences were only mimimal in degree and showed no dependency. Therefore these changes were considered to be not treatment-related.

See Attachment 2 for summary data of haematological findings.
Clinical biochemistry findings:: effects observed, non-treatment-related
Description (incidence and severity):: The activities of aspartate aminotransferase were decreased at 10 mg/kg bw/day and above and of alkaline phosphatase at 100 mg/kg bw/day and above in both sexes.
The concentrations of cholesterol, triglycerides, total protein and albumin were higher, and the concentrations of creatinine lower in males at 1000 mg/kg bw/day. In females, the concentration of bilirubin was higher at 100 and 1000 mg/kg bw/day.
The concentration of sodium was decreased at 100 mg/kg bw/day and above in both sexes.
Calcium was increased at 1000 mg/kg in females, and inorganic phosphate decreased in males at 10 mg/kg bw/day and above.
All these partly statistically significant differences were either of a minimal degree, and/or not dose-related, and/or were observed only in one sex, and/or were all in the range of historical values. Therefore, they were considered as not treatment-related.

See Attachment 3 for summary data of clinical chemistry findings and Attachment 5 for laboratory historical control data.
Endocrine findings:: not examined
Description (incidence and severity):: Not applicable.
Urinalysis findings:: not examined
Description (incidence and severity):: Not applicable.
Behaviour (functional findings):: not examined
Description (incidence and severity):: Not applicable.
Immunological findings:: not examined
Description (incidence and severity):: Not applicable.
Organ weight findings including organ / body weight ratios:: effects observed, treatment-related
Description (incidence and severity):: Absolute and relative liver weights were statistically significantly increased at 1000 mg/kg bw/day in both sexes.

Absolute mean liver weight was statistically significantly increased in females at 10 mg/kg bw/day. However, relative mean liver weight of these animals was similar to the control group. In the absence of liver weight effects at 100 mg/kg bw/day this finding was considered incidental in nature.

See Table 2 for mean liver weight data on Day 28. See Attachment 4 for summary data of organ weight findings.
Gross pathological findings:: no effects observed
Neuropathological findings:: not examined
Description (incidence and severity):: Not applicable.
Histopathological findings: non-neoplastic:: effects observed, treatment-related
Description (incidence and severity):: Locally, no evidence of any findings was detected at the application site of the skin.
In the pancreas, focal degeneration was detected at 100 mg/kg bw/day and above in both sexes.
In the thyroid gland, follicular cell hypertrophy was detected in both sexes in all dose groups, including controls. However, an increased incidence occurred in males at 1000 mg/kg bw/day (5-5-4-9 affected animals with increasing doses). Furthermore, colloid alteration of the thyroid was detected with an increased incidence in males (1-4-6-10 affected animals with increasing doses). The incidence occurred together with an increasing grading in the upper dose groups.
Slight hepatocellular hypertrophy was observed at 1000 mg/kg bw/day, in males.
Hypertrophy of the pars distalis of pituitary was observed at 1000 mg/kg bw/day in males.

See Attachment 1 for summary data of histopathological (non-neoplastic) findings.
Histopathological findings: neoplastic:: no effects observed
Other effects:: not examined
Description (incidence and severity):: Not applicable.
Details on results:: Thyroid findings (increased weight, histopathological changes comprising changes in colloid, follicular cell hypertrophy and pigment deposition in the follicular epithelium) are considered a non-adverse and rat specific phenomenon. No changes of the thyroid were noted in either mice or dogs, the other two species in which repeated-dose studies with histopathological examination of the thyroid were conducted with the test substance. As the test substance through inhibition of the HPPDase enzyme increases plasma tyrosine concentration in the rat, it is quite possible that some of this increased tyrosine is taken up by the thyroid and stored in the colloid, either as free tyrosine or through either increasing the synthesis of thyroglobulin or altering its composition in terms of number of tyrosine residues per thyroglobulin molecule.
In the absence of signs of altered thyroid functions (such as effects on body weights, fertility and gestation indices or effects on offspring performance in the developmental neurotoxicity study), the observed morphological changes are considered to be non-adverse.
: Key result
Dose descriptor:: NOAEL
Remarks:: local
Effect level:: 1 000 mg/kg bw/day
Based on:: test mat.
Remarks:: equivalent to 15.0 mg/m2
Sex:: male/female
Basis for effect level:: other: No dermal irritation observed up to the highest dose tested
: Key result
Dose descriptor:: NOAEL
Effect level:: 10 mg/kg bw/day
Based on:: test mat.
Sex:: male/female
Basis for effect level:: other: No adverse effects seen at 10 mg/kg bw/day
: Key result
Dose descriptor:: LOAEL
Effect level:: 100 mg/kg bw/day
Based on:: test mat.
Sex:: male/female
Basis for effect level:: other: histopathological effects in the pancreas at 100 mg/kg bw/day
: Key result
Critical effects observed:: yes
Lowest effective dose / conc.:: 100 mg/kg bw/day
System:: gastrointestinal tract
Organ:: pancreas
Treatment related:: yes
Dose response relationship:: yes
Relevant for humans:: not specified
Critical effects observed:: yes
Lowest effective dose / conc.:: 100 mg/kg bw/day
System:: endocrine system
Organ:: thyroid gland
Treatment related:: yes
Dose response relationship:: yes
Relevant for humans:: no
Critical effects observed:: yes
Lowest effective dose / conc.:: 1 000 mg/kg bw/day
System:: hepatobiliary
Organ:: liver
Treatment related:: yes
Dose response relationship:: yes
Relevant for humans:: not specified
Critical effects observed:: yes
Lowest effective dose / conc.:: 1 000 mg/kg bw/day
System:: endocrine system
Organ:: pituitary gland
Treatment related:: yes
Dose response relationship:: yes
Relevant for humans:: not specified
Conclusions:: The study was performed under GLP conditions and according to OECD TG 410 (adopted 1981).There were few effects of repeated dermal application of the test substance to either male or female rats. There were no local effects on either skinfold thickness or on signs of irritation. The local NOAEL was determined to be 1000 mg/kg bw/day (equivalent to 15 mg/m2 skin), while the systemic NOAEL was 10 mg/kg bw/day, based on histopathological effects observed at 100 and 1000 mg/kg bw/day in both males and females.

Based on the effects observed in the pancreas at 100 mg/kg bw/day, the substance is classified "STOT RE 2, H373, gastro-intestinal".

Endpoint conclusion

Endpoint conclusion:: no adverse effect observed
Dose descriptor:: NOAEL
: 15 mg/cm²
Study duration:: subacute
Species:: rat
Quality of whole database:: The available information comprises an adequate and reliable study (Klimisch score 1), and is thus sufficient to fulfil the standard information requirements set out in Annex VIII-IX, 8.6, of Regulation (EC) No 1907/2006.

Mode of Action Analysis / Human Relevance Framework

Data on the mode of action and human relevance for HPPDase inhibitors, specifically in relation to effects seen in the eye and thyroid gland are discussed in detail within the endpoint summary for Repeated Dose Toxicity (Additional Information field).

Additional information

Reliable studies on the repeated dose toxicity of (5-hydroxy-1,3-dimethylpyrazol-4-yl)(α,α,α-trifluoro-2-mesyl-p-tolyl) methanone are available. The data comprises a dermal short-term repeated dose toxicity study as well as oral short-term, subchronic and chronic repeated dose toxicity studies. All studies were performed according to GLP. Table 1 summarizes the repeated-dose toxicity studies performed with the test substance.

Table 1: Repeated-dose toxicity studies performed with (5-hydroxy-1,3-dimethylpyrazol-4-yl)(α,α,α-trifluoro-2-mesyl-p-tolyl) methanone

Species and study type	NOAEL	LOAEL	Findings at LOAEL	Study ID
Mouse
90-day dietary 100, 750, 1500, 3000 ppm	3000 ppm (M: 617 mg/kg bw/day, F: 500 mg/kg bw/day,)	> 3000 ppm (M: > 500 mg/kg bw/day, F: > 617 mg/kg bw/day)	No relevant effects observed	M-103284-01-4
Rat
28-day dermal 10, 100, 1000 mg/kg bw/day	10 mg/kg bw/day	100 mg/kg bw/day	Increased liver weight, slight histopathological effects in pancreas and thyroid	M-263216-01-2
90-day dietary 2, 30, 1000, 7000 ppm	30 ppm (M: 1.96 mg/kg bw/day, F: 2.32 mg/kg bw/day)	1000 ppm (M: 66 mg/kg bw/day, F: 77 mg/kg bw/day)	Increased liver and kidney weights, histopathological effects in the kidney, cholesterol, triglycerides	M-102924-01-2
6-month rat dietary 25, 250, 1000, 2500 ppm	25 ppm (M: 1.4 mg/kg bw/day, F: 1.8 mg/kg bw/day)	250 ppm (M: 14 mg/kg bw/day, F: 19 mg/kg bw/day)	Decreased body weights, increased cholesterol, reduced urinary pH, increased liver, kidney and thyroid weights, histopathological effects in cornea, liver and thyroid	M-267037-02-1
12-month rat dietary 25, 250, 1000, 2500 ppm	25 ppm (M: 1. mg/kg bw/day, F: 1.5 mg/kg bw/day)	250 ppm (M: 11 mg/kg bw/day, F: 15 mg/kg bw/day)	Decreased body weights, increased cholesterol, reduced urinary pH, increased liver and kidney weights, histopathological effects in cornea, liver, kidney and thyroid	M-267037-02-1
24-month rat dietary 25, 250, 1000, 2500 ppm	25 ppm (M: 1.0 mg/kg bw/day, F: 1.4 mg/kg bw/day)	250 ppm (M: 10 mg/kg bw/day, F: 14 mg/kg bw/day)	Decreased body weights, reduced urinary pH, increased liver and kidney weights, histopathological effects in cornea, kidney and thyroid	M-267037-02-1
Dog
90-day dietary 100, 500, 1000 ppm	1000 ppm (M: 40 mg/kg bw/day, F: 33 mg/kgbw/day)	> 1000 ppm (M: > 40 mg/kg bw/day, F: > 33 mg/kg bw/day)	No relevant effects observed	M-257679-01-1
12-month dietary 250, 1000, 3000 ppm	1000 ppm (M: 34 mg/kg bw/day, F: 33 mg/kg bw/day)	3000 ppm (M: 101 mg/kg bw/day, F: 93 mg/kg bw/day)	M: increased liver, kidney weights, increased hepatocytomegaly F: increased liver, thyroid weights, increased hepatocytomegaly	M-267321-01-1

In studies with (5-hydroxy-1,3-dimethylpyrazol-4-yl)(α,α,α-trifluoro-2-mesyl-p-tolyl)methanone in rats that required repeated dosing, corneal opacities were noted. Corneal opacities, occasionally accompanied by neovascularization and their histopathological correlates (keratitis, reactive epithelial hyperplasia, and vascularization) are considered a rat-specific phenomenon. Corneal changes were not seen in other species chronically treated with the test substance (i.e. mice and dogs). The test substance is an inhibitor of the HPPDase enzyme and induces increased plasma tyrosine levels. This effect is more pronounced in rats than in mice and dogs. Experimentally induced hypertyrosinemia has been shown to induce snow flake-like corneal lesions in rats but not in mice (M-210983-01-2). In mice and humans, even under conditions of strong HPPD inhibition, tyrosine concentrations will not increase to levels high enough to induce ocular toxicity and hence, this toxicity observed in the rat is inappropriate for extrapolation to humans (ECETOC TR No. 99, 2006).

Furthermore, and also as in other toxicity studies with 5-hydroxy-1,3-dimethylpyrazol-4-yl)(α,α,α-trifluoro-2-mesyl-p-tolyl)methanone in rats, thyroid findings were noted (increased weight, histopathological changes comprising changes in colloid, follicular cell hypertrophy and pigment deposition in the follicular epithelium) in the rat repeated dose studies described below. The toxicological significance of these thyroid findings was evaluated and discussed in an additional regulatory toxicology position paper (M-301557-01-1, 2007). The overall conclusion was that these findings were considered a non-adverse and a rat specific phenomenon:

No changes of the thyroid were noted in either mice or dogs, the other two species in which repeated-dose studies with histopathological examination of the thyroid were conducted with the test substance. This suggests that potential thyroid effects of the test substance have only a limited toxicological significance, as the rat is known to be much more sensitive than either mice or dogs to alterations in thyroid homeostasis. The main function of the thyroid is to regulate overall metabolic processes through synthesis, storage, and release of thyroid hormones. These hormones, thyroxine and triiodothyronine (T4 and T3 respectively) are produced by the action of thyroid peroxidase which iodinates tyrosine residues available in the thyreoglobulin complexes in the colloid of the thyroid follicles.

As the test substance through inhibition of the HPPDase enzyme increases plasma tyrosine concentration in the rat, it is quite possible that some of this increased tyrosine is taken up by the thyroid and stored in the colloid, either as free tyrosine or through either increasing the synthesis of thyreoglobulin or altering its composition in terms of number of tyrosine residues per thyreoglobulin molecule. As plasma tyrosine concentrations increase to a much lesser extent after HPPDase inhibition in the mouse than in the rat, this would explain why colloid alteration is not observed in the mouse even after administration of doses of up to approximately 600 mg/kg bw/day.

If alteration in the appearance of the thyroid colloid had an effect on the function of the thyroid, such an effect might be manifested in altered maintenance of body weight through alteration of metabolic rate, through alteration in the ability of the dams to maintain gestation, or through effects on offspring performance in the developmental neurotoxicity study.

In the rat 90-day, multigeneration, and chronic / carcinogenicity studies, the alteration in thyroid colloid is seen at a lower dietary concentration of the test substance than is any effect on body weight or body weight gain. Additionally, the effect on offspring viability and body weight at parturition and during lactation is seen a greater dose than that in which thyroid colloid alteration is seen in the parental animals. These findings suggest that the altered colloid has no effect on thyroid function with regard to maintenance of normal body weight and development.

In the developmental neurotoxicity study, there was no biologically significant effect on any facet of learning or memory in the offspring at any dose. The ability of the offspring to master the water maze was unaffected. Although the latency to crossing in the passive avoidance test was slightly decreased at the high dose in both males and females, in the absence of other effects on learning and memory such a slight effect does not indicate that there was any mental impairment of the pups.

Histopathological sections of the thyroid from male and female rats from the rat chronic / oncogenicity study were examined by an independent panel of senior pathologists (M-266373-01-2, 2005) to determine whether the colloid alteration in the thyroid represented an adverse effect of the test substance. This expert group noted that the colloid alterations were present in all groups including controls, and that the morphology was similar between treated and control groups, with the primary difference being an increase in the number of follicles affected in treated groups. Additionally, colloid alterations were observed in the absence of follicular cell hypertrophy, and were not considered to indicate a persistent alteration in thyroid function.

From the observed and implied lack of functional consequences of the thyroid colloid alteration, as observed in multiple studies conducted with the test substance in the rat, it is clear that the finding of colloid alteration is of no toxicological significance and can be expected to be non-adverse.

Summary of Repeated Dose Toxicity Studies

Oral

90-day subchronic toxicity feeding study in the mouse:

In this study, which was conducted according to OECD 408, the test substance was incorporated into rodent diet and administered to 10 male and 10 female C57BL/6J mice per dose group at concentrations of 0, 100, 750, 1500, and 3000 ppm for 90 days (M-103284-01-4). These concentrations provided doses of 0, 16.5, 124, 259, and 500 mg/kg bw/day for males and 0, 19.7, 152, 326, and 617 mg/kg bw/day for females. Observations and examinations included clinical signs, body weights, and food consumption, clinical chemistry, urinalysis, gross necropsy, selected organ weights and histopathological examination.

No treatment-related mortalities occurred during the study and no treatment-related clinical signs were noted. There were no effects on body weight, body weight gain or food consumption. There were no treatment-related changes in any clinical chemistry parameters. Urine pH was statistically significantly increased at 3000 ppm in females, but was not analyzed statistically in males due to the small number of values obtained. There were no treatment-related effects on organ weights, gross necropsy or histopathological findings.

Conclusions

The only treatment-related finding was an increase in urinary pH in females at 3000 ppm. The NOAEL for the study is therefore considered to be 3000 ppm (500 mg/kg bw/day in males and 617 mg/kg bw/day in females).

90-day subchronic toxicity feeding study in the rat:

In this study that was performed according to OECD 408 (M-102924-01-2), the test substance was mixed into powdered rodent diet at dietary concentrations of 0, 2, 30, 1000, 7000, or 12000 ppm, to provide doses of 0.13, 1.96, 66, 454, and 830 mg/kg bw/day for males and 0.15, 2.32, 77, 537, and 956 mg/kg bw/day in females. Observations and examinations included clinical signs, detailed clinical examinations, neurological (reflex) examinations, body weights, and food consumption, ophthalmological examinations, clinical chemistry and haematology, urinalysis, gross necropsy, selected organ weights and histopathological examination.

There were no mortalities in the groups receiving 0, 2, 30, or 1000 ppm. At 7000 ppm, one male was found dead on Day 8, one male was sacrificed for humane reasons on Day 70, and one male was killed for humane reasons on Day 83. At 12000 ppm, one female was sacrificed on Day 13 for humane reasons. Four males in this treatment group were found dead (Days 15, 41, 45, and 72), and two males were killed for humane reasons (Days 43 and 64), it was decided to sacrifice the surviving males of this group on Day 72. No clinical signs were noted at either 2 or 30 ppm. At 1000 ppm and above, the significant clinical findings were yellow-coloured urine and white areas on the cornea. There were no effects noted at examination of reflexes in Week 12. Body weight was decreased in both males and females at higher doses, with both 7000 ppm and 12000 ppm decreasing body weight gain by more than 10% in females. There were occasional slight decreases in food consumption by both males and females at 7000 and 12000 ppm, but there was no alteration in food consumption at concentrations of 1000 ppm or below. Neovascularization of the cornea and characteristic "snowflake" corneal opacities were noted at 1000 and 12000 ppm in males, and 1000 ppm, 7000, and 12000 ppm in females. There were no biologically significant effects on haematological parameters at any dose in either males or females. In males, bilirubin, aspartate and alanine aminotransferases, urea, and creatinine were biologically and or statistically significantly increased at the dietary concentration of 7000 ppm, while cholesterol and triglycerides were increased at 1000 and 7000 ppm. Alkaline phosphatase was decreased at 1000 ppm and above when compared to controls. Very few of these parameters showed any response in females. At urinalysis, ketone levels were increased from 1000 ppm in both males and females. This is likely due to detection of the triketone structure of the test substance itself, as the vast majority of the parent molecule is excreted in the urine unchanged. There was an increased incidence of occult blood, erythrocytes, leukocytes, and epithelial cells in the urine in both males and females at 7000 ppm and in females at 12000 ppm (males in the 12000 ppm group did not survive until the end of the study and urine was therefore not collected). Mean absolute and relative liver and kidney weights were biologically and statistically significantly increased in both males and females at 1000 ppm and above. In male rats either found dead or sacrificed prior to the end of the study (3 at 7000 ppm and 10 at 12000 ppm), the principal cause of death was considered to be related to calculi in the urinary tract. Clearly treatment-related macroscopic findings seen in these animals included gritty content in and dilation of the renal pelvis, obviously large kidneys, pale or mottled colour of the kidneys, and foci on the kidneys, and red or gritty content in the urinary bladder, distension of the urinary bladder, and red foci in the bladder, and obviously large liver. Other findings which are less clearly related to treatment included dilation, dark content, and black foci of the stomach, soiled fur, and dark content in the intestines. In animals which survived to the end of the study, treatment-related macroscopic findings were seen at 1000 ppm and above. In both males and females from 1000 ppm, these findings included corneal opacities, abnormal shape of the kidneys, mottled kidneys, dilation of and gritty content in the renal pelvis, gritty content and distension of the urinary bladder, and gritty content of the ureters. A few females at 7000 and 12000 ppm were noted with obviously small kidneys, and one female at 12000 ppm showed gritty content in the urethra. A few males had obviously large liver at 1000 ppm, with prominent lobulation at 1000 and 7000 ppm. One male had obviously large thyroid at 1000 ppm. Microscopic pathology: The dose of 12000 ppm was above the Maximum Tolerated Dose in the rat and therefore histopathological examinations were not conducted at this dose. Additionally, as 30 ppm was observed to be free of treatment-related effects, histopathological examination was not conducted at 2 ppm. Treatment-related changes observed in final-sacrifice animals were within the urinary tract and the liver in both sexes, and the eyes and thyroid gland in males.

Conclusions

The primary effects of the test substance after subchronic dietary administration in the rat were limited to the eyes, liver, and urinary tract in both sexes and thyroid gland in males. Characteristic corneal "snowflake" lesions were observed in both males and females at doses of 1000 ppm and above (not relevant to humans). Increased liver weights were correlated with an increased incidence of centrilobular hypertrophy, with the greatest increase in incidence observed in males. Increased cholesterol and triglyceride concentrations were also observed in males at 1000 and 7000 ppm. Urinary tract stones (found to be primarily composed of the test substance, which has been demonstrated to be excreted in the urine at high concentrations after oral dosing) were observed in both males and females and were related to the histopathological findings including urothelial hyperplasia. In the thyroid, males at 1000 ppm and above showed increased incidences of follicular cell hypertrophy / hyperplasia and diffuse loss of colloid (not relevant to human).

Based on these findings, the NOAEL for dietary administration of the test substance in the male and female rat was 30 ppm (1.96 mg/kg bw/day in males, 2.32 mg/kg bw/day in females).

6-month chronic toxicity feeding study in the rat:

In the 6-month toxicity phase of this combined chronic and carcinogenicity study (M-267037-02-1), that was performed according to OECD 453, the test substance was administered at dietary concentrations of 0, 25, 250, 1000, and 2500 ppm to groups of 10 male and 10 female Wistar rats for 6 months. These dietary concentrations provided doses of 1.4, 14, 58 and 143 mg/kg bw/day for males and 1.8, 19, 77 and 191 mg/kg bw/day for females. Observations and examinations included clinical signs, detailed clinical examinations, body weights, and food consumption, ophthalmological examinations, clinical chemistry and haematology, urinalysis, gross necropsy, selected organ weights and histopathological examination.

A treatment-related increase in mortality was observed in males at 2500 ppm (4/75 deceased animals versus 1/75 in the control), there was no treatment-related effect on mortality in females at any dose level. Treatment-related clinical signs of toxicity included white area on the eye and soiled fur in one or more areas. An increased incidence of these findings was observed at 250 ppm and above in both males and females. Body weight and body weight gain were reduced in males and females at the higher doses, with a greater effect observed in males than in females. There was no effect on food consumption at any dose in either males or females.

During urinalysis, higher ketone concentrations were noted in both males and females at 1000 ppm and above. These were due to the triketone form of the molecule, which is largely excreted unchanged in the urine. Urine pH was decreased at 250 ppm and above in males at all time points, while in females at 250 ppm and above urine pH was decreased only at the 3-month time point. Urinary protein was increased in males at all doses.

Liver and kidney weight was statistically significantly increased in males at 250 ppm and above, and thyroid weights was slightly increased at 250 ppm and above. There were no biologically significant effects on organ weights in females at any group or time point.

At necropsy, treatment-related findings were observed in the eyes, liver, and kidney. Eye opacities seen in both males and females were related to increased tyrosinemia and are considered not to be relevant for human risk assessment. Enlarged liver was observed in males at all doses, with prominent lobulation at 25, 250, and 1000 ppm.

Non-neoplastic microscopic findings: Treatment-related findings were observed in the eyes, liver, pancreas, thyroid gland, and kidneys. In the eyes, the incidence of corneal inflammation was increased in males at dietary concentrations of 250 ppm and above, and in females at 1000 and 2500 ppm. Regenerative hyperplasia of the cornea was increased in males at 250 ppm and above, and in females at 1000 and 2500 ppm only. Neovascularization of the cornea was increased in males at 250 ppm and above, and in females at 1000 and 2500 ppm. Centrilobular hepatocellular hypertrophy was increased in males at all doses and in females from 250 ppm. Centrilobular hepatocellular vacuolation increased in incidence in males from 250 ppm, but was not observed in females. In the pancreas, an increased incidence of diffuse acinar degeneration / atrophy was reported in both males and females at 2500 ppm. In the thyroid, the incidence of altered colloid was increased in males in all treatment groups. The incidence of increased follicular diameter was increased in males from 250 ppm, but this finding was not observed in females. Pigment deposition in the follicular cells was increased in males from 250 ppm. The incidence of chronic progressive nephropathy was slightly increased in males at 2500 ppm.

Conclusions

Based on increased mortality at 2500 ppm in males, this dose is considered to have exceeded the Maximum Tolerated Dose. Treatment-related findings were seen in the eyes, liver, kidney, thyroid, and pancreas. Findings in the eye were related to the biochemical mechanism of the test substance as an HPPDase inhibitor leading to increased tyrosinemia in rats, and were considered not to be relevant for humans. The observations in the liver, including centrilobular hepatocellular hypertrophy, were evaluated as being adaptive responses to treatment rather than adverse effects. The finding of chronic progressive nephropathy is considered to be a rodent-specific finding not relevant to man. Similarly, the findings of thyroid colloid alteration and pigment deposition were considered to be specific to the rat and not indicative of an adverse finding, but to be normal findings in ageing rats. The increased incidence of acinar degeneration / atrophy in the pancreas was only observed at 1000 and 2500 ppm, and may indicate a tyrosine-linked effect as well, as these findings were not observed in the mouse or dog, which are not as sensitive as the rat to the effects of tyrosinemia.

The NOAEL in this study was therefore considered to be 25 ppm (1.4 mg/kg bw/day in males and 1.8 mg/kg bw/day in females), while the LOAEL was 250 ppm (14 mg/kg bw/day in males and 19 mg/kg bw/day in females).

12-month chronic toxicity feeding study in the rat:

In the 12-month toxicity phase of this combined chronic and carcinogenicity study (M-267037-02-1), that was performed according to OECD 453, the test substance was administered at dietary concentrations of 0, 25, 250, 1000, and 2500 ppm to groups of 10 male and 10 female Wistar rats for 12 months. These dietary concentrations provided doses of 1.1, 11, 45 and 114 mg/kg bw/day for males and 1.5, 15, 63 and 155 mg/kg bw/day for females. Observations and examinations included clinical signs, detailed clinical examinations, body weights, and food consumption, ophthalmological examinations, clinical chemistry and haematology, urinalysis, gross necropsy, selected organ weights and histopathological examination.

A treatment-related increase in mortality was observed in males at 2500 ppm (8/65 deceased animals versus 2/65 in the control). There was no treatment-related effect on mortality in other male treatment groups or in females at any dose level. Treatment-related clinical signs of toxicity included white area on the eye and soiled fur in one or more areas. An increased incidence of these findings was observed at 250 ppm and above in both males and females. Body weight and body weight gain were reduced in males and females at the higher doses, with a greater effect observed in males than in females. There was no effect on food consumption at any dose in either males or females.

There was no treatment-related effect on haematology. Plasma cholesterol was biologically and statistically significantly increased at 250 ppm and above. This effect on plasma cholesterol was considered to be the only treatment-related clinical chemistry finding. During urinalysis, higher ketone concentrations were noted in both males and females at 1000 ppm and 2500 ppm. These were due to the triketone form of the test substance, which is largely excreted unchanged in the urine. Urine pH was decreased at 250 ppm and above in males at all time points, while in females at 250 ppm and above urine pH was decreased only at the 3-month time point. Urinary protein was increased in males at all doses from Month 7 onwards, however, at 25 ppm there was little difference when compared to the historical control data, and only at a few time points was the percentage of animals with a given multistix reading for urinary protein greater than the historical controls at this does level, and this exceedance was generally only by a small margin. It is concluded that the apparent increase in urinary protein excretion at 25 ppm does not represent a specific effect of the test substance on the kidney of toxicological significance (M-301557-01-1).

Liver and kidney weight was statistically significantly increased in males at 250 ppm and above. There were no effects on organ weights in females. At necropsy, treatment-related findings were observed in the eyes, liver, and kidney. Eye opacities seen in both males and females were related to increased tyrosinemia and are considered not to be relevant for human risk assessment. Enlarged liver was observed in males at 250 ppm and above.

Non-neoplastic microscopic findings: Treatment-related findings were observed in the eyes, liver, pancreas, thyroid gland, and kidneys. Findings observed in other organs were considered to be of little to no toxicological relevance or not to be treatment-related. In the eyes, the incidence of corneal inflammation was increased in males at dietary concentrations of 250 ppm and above, and in females at 1000 and 2500 ppm. Regenerative hyperplasia of the cornea was increased in males at 250 ppm and above, and in females at 1000 and 2500 ppm. Neovascularization of the cornea was increased in males at 250 ppm and above, and in females at 1000 and 2500 ppm. Centrilobular hepatocellular hypertrophy was increased in males only at 250 ppm and above. Centrilobular hepatocellular vacuolation increased in incidence in males from 250 ppm, but was not observed in females.

In the pancreas, diffuse acinar degeneration / atrophy was reported in males at 1000 and 2500 ppm, and in females at 2500 ppm. In the thyroid, the incidence of altered colloid and increased follicular diameter was increased in males from 250 ppm. Pigment deposition in the follicular cells was increased in males from 250 ppm. The incidence of focal follicular cell hyperplasia was slightly increased in males at 2500 ppm. Diffuse follicular cell hypertrophy was noted in a few males and females from 250 ppm.

The incidence of chronic progressive nephropathy was slightly increased in males from 250 ppm and in females at 250 and 1000 ppm (one animal each). The relevance of chronic progression nephropathy at 25 ppm was discussed in detail in regulatory position papers M-301557-01-1 and M-284620-01-1 (2007). On review of the concurrent and historical control data, and considering the absence of long-term damage to the kidneys similar to chronic progressive nephropathy in the other species tested (mouse and dog), the chronic progressive nephropathy observed at 25 ppm was not considered to be biologically significantly increased.

Neoplastic microscopic findings: A small number of tumors was noted in animals from treated and control groups with no evidence of an effect of treatment.

Conclusions

Based on increased mortality at 2500 ppm in males, this dose is considered to have exceeded the Maximum Tolerated Dose. Treatment-related findings were seen in the eyes, liver, kidney, thyroid, and pancreas. Findings in the eye were related to the biochemical mechanism of the test substance as an HPPDase inhibitor leading to increased tyrosinemia in rats, and were considered not to be relevant for humans. The observations in the liver, including centrilobular hepatocellular hypertrophy, were evaluated as being adaptive responses to treatment rather than adverse effects. The finding of chronic progressive nephropathy is considered to be a rodent-specific finding not relevant to man. The increased incidence of acinar degeneration / atrophy in the pancreas was only observed at 1000 and 2500 ppm, and may indicate a tyrosine-linked effect as well, as these findings were not observed in the mouse or dog, which are not as sensitive as the rat to the effects of tyrosinemia.

The NOAEL in this study was therefore considered to be 25 ppm (1.1 mg/kg bw/day in males and 1.5 mg/kg bw/day in females), while the LOAEL was 250 ppm (11 mg/kg bw/day in males and 15 mg/kg bw/day in females).

24-month chronic toxicity feeding study in the rat:

In this study conducted according to OECD 453, (5-hydroxy-1,3-dimethylpyrazol-4-yl)(α,α,α-trifluoro-2-mesyl-p-tolyl)methanone was incorporated into rodent diet and administered at concentrations of 0, 25, 250, 1000, and 2500 ppm to groups of 75 male and female Wistar rats (M-267037-01-2). In addition to the 24 -month phase, the study also included a 6-month and 1-year repeated-dose toxicity phase.

The administered concentrations provided final doses of 0, 1.0, 10, 41, and 105 mg/kg bw/day for males and 0, 1.4, 14, 57, and 141 mg/kg bw/day for females. Body weight and food consumption were measured weekly for the first 13 weeks of the study, then every 4 weeks through the remainder of the study until necropsy. Ophthalmological examinations were conducted on all animals during acclimatization and at Months 3, 6, 12, 18, and 24 of treatment. Blood for haematological and clinical chemical examination was collected after 6, 12, and 24 months from overnight-fasted rats. Urine was collected overnight at 3, 6, 12, 18, and 24 months from animals fasted overnight of food and water.

A treatment-related increase in mortality was observed in males at 2500 ppm. There was no treatment-related effect on mortality in other male treatment groups or in females at any dose level. Treatment-related clinical signs of toxicity included white area on the eye and soiled fur in one or more areas. An increased incidence of these findings was observed at 250, 1000, and 2500 ppm in both males and females. Body weight and body weight gain were reduced in males and females at the higher doses, with a greater effect observed in males than in females. There was no effect on food consumption at any dose in either males or females.

Treatment-related findings (corneal opacity, neovascularization of the cornea, oedema of the cornea, and "snow flake" corneal opacities) were seen during ophthalmoscopy in both males and females. At 6 and 12 months, increased incidence of these findings was seen only at 250, 1000, and 2500 ppm. At 24 months, these findings were increased compared to controls at all doses in males. However, at 25 ppm there was only a slight increase in corneal opacity, neovascularization of the cornea, and oedema of the cornea in males in neovascularization of the cornea in females. The findings at the dose level of 25 ppm were discussed in additional regulatory position papers M-284620-01-1 and M-301557-01-1 (2007). A comparison was made with the laboratory historical control data for rat chronic studies, from this it was evident that the incidence of the findings at 25 ppm fell within the historical control range for the majority of effects. For males at 25 ppm, the incidence of corneal opacity and neovascularization were marginally outside the historical control range, as was eye opacity in females at this dose level. Due to the low incidence of these findings, especially in comparison to their incidence at the higher dose levels, they were not considered indicative of a major adverse effect. The findings are related to tyrosinemia resulting from inhibition of 4-hydroxyphenylpyruvate dioxygenase (HPPDase), a key enzyme in the tyrosine catabolic pathway. As humans are capable of metabolizing and excreting excess tyrosine while rats are much less capable of this metabolism, the corneal effects of HPPDase inhibitors are considered to be relevant for rats but not for man.

There was no treatment-related effect on haematology at any dose or time point in either male or female rats. Plasma cholesterol was biologically and statistically significantly increased at 250 ppm and above at Months 7 and 12, and was biologically and/or statistically significantly increased at 1000 and 2500 ppm at 18 and 24 months. This effect on plasma cholesterol was considered to be the only treatment-related clinical chemistry finding. Although plasma cholesterol was statistically significantly increased in males at 25 ppm at the 7-month time point, this value was within historical control data and cholesterol concentrations in this dose group returned to control levels by the 12-month time point; therefore, this increase was considered not to be treatment-related.

During urinalysis, higher ketone concentrations were noted in both males and females at 1000 ppm and 2500 ppm at all collection periods, and in males at 250 ppm at Months 19 and 24 only. These were due to the triketone structure of the molecule, which is largely excreted unchanged in the urine. Urine pH was decreased at 250 ppm and above in males at all time points, while in females at 250 ppm and above urine pH was decreased only at the 3-month time point. Urinary protein was increased in males at all doses from Month 7 onwards, however, at 25 ppm there was little difference when compared to the historical control data, and only at a few time points was the percentage of animals with a given multistix reading for urinary protein greater than the historical controls at this does level, and this exceedance was generally only by a small margin. It is concluded that the apparent increase in urinary protein excretion at 25 ppm does not represent a specific effect of the test substance on the kidney of toxicological significance (M-301557-01-1).

Liver and kidney weight was statistically significantly increased in males only at the higher doses in this study. There were no biologically significant effects on organ weights in females at any group. At necropsy, treatment-related findings were observed in the eyes, liver, and kidney. Eye opacities seen in both males and females were related to increased tyrosinemia and are considered not to be relevant for human risk assessment. An increased incidence of pale kidneys or irregular surface of the kidney was noted in males at 1000 and 2500 ppm.

In the eyes, the incidence of corneal inflammation was increased in males at 250 ppm and above, and in females at 1000 and 2500 ppm. At 24 months, there was a very slight increase in corneal inflammation in males at 25 ppm and in females at 250 ppm. Regenerative hyperplasia of the cornea was increased in males at 250 ppm and above, and in females at 1000 and 2500 ppm, only. Neovascularization of the cornea was increased in males 250 ppm and above, and in females at 250 ppm and above. There was an increase in males in the incidence of mucous metaplasia of the cornea at 250 ppm and above. Corneal atrophy was increased in males at 250 ppm and above and in females at 1000 and 2500 ppm, and peripheral retinal atrophy was increased in both males and females at 250 ppm and above. The significance of the retinal atrophy was discussed in an additional regulatory toxicology position paper (M-286627-01-1, 2007). This finding is considered to be subsequent to and a result of excessive corneal inflammation resulting from the marked corneal opacities developing in these same groups. Of the animals reported with peripheral retinal atrophy of any sort, the majority (approximately 80% in each group) were also reported with corneal atrophy, lending support to this hypothesis. It is concluded that the finding of peripheral retinal atrophy, as observed in the 1000 and 2500 ppm groups of this rat carcinogenicity study, is related to corneal inflammation resulting from increased plasma tyrosine concentration. These increased plasma tyrosine concentrations are likewise the result of the test substance administration, through its mode of action as an HPPDase inhibitor. As the rat is more sensitive than man to the downstream effects of HPPDase inhibition, including corneal opacities and inflammation, these findings are not relevant to man.

Centrilobular hepatocellular hypertrophy was increased in males from 250 ppm. In females centrilobular hepatocellular hypertrophy was only reported in one animal at 2500 ppm. In the pancreas, diffuse acinar degeneration / atrophy was reported in both males and females; the incidence of diffuse acinar degeneration / atrophy was increased in males and females at 1000 and 2500 ppm. The incidence of focal acinar degeneration / atrophy was increased in females at 2500 ppm.

In the thyroid, the incidence of altered colloid was increased in males and females from 250 ppm and above. Pigment deposition in the follicular cells was increased in both males and females in all dietary groups. The incidence of focal follicular cell hyperplasia was slightly increased in males from 250 ppm. In females, this finding was only at a very low incidence. No clear relationship of follicular cell hyperplasia to dose was evident. Diffuse follicular cell hypertrophy was noted in a few males and females from 250 ppm, again in the absence of a clear dose-relationship. The observed thyroid findings are considered a rat-specific phenomenon of HPPDase inhibitors without relevance for humans (a detailed justification is included above).

There was no substantive dose-related increase in the incidence of chronic progressive nephropathy in males at 24 months, and the incidence in control animals at 24 months of 80% indicates that this is a very common finding in the aging male rat. Chronic progressive nephropathy was observed in females, with no dose relationship. Hyperplasia of the collecting ducts was increased in males at 1000 and 2500 ppm. Collecting duct hyperplasia was also noted as slightly increased at 25 ppm, however, the severity of this finding was very low and was comparable to or even less severe than that in historical and concurrent controls. Therefore, this finding at 25 ppm was not considered indicative of an adverse effect of the test substance (discussed in detail in regulatory position paper M-301557-01-1, 2007).

Conclusions

Based on increased mortality at 2500 ppm in males, this dose is considered to have exceeded the Maximum Tolerated Dose. Treatment-related findings were seen in the eyes, liver, kidney, thyroid, and pancreas. Findings in the eye were related to the biochemical mechanism of the test substance as an HPPDase inhibitor leading to increased tyrosinemia in rats, and were considered not to be relevant for humans. The observations in the liver, including centrilobular hepatocellular hypertrophy, were evaluated as being adaptive responses to treatment rather than adverse effects. The finding of chronic progressive nephropathy is considered to be a rodent-specific finding not relevant to man. Similarly, the findings of colloid alteration and pigment deposition were considered to be specific to the rat and not indicative of an adverse finding, but to be normal findings in ageing rats. The increased incidence of acinar degeneration / atrophy in the pancreas was only observed at 1000 and 2500 ppm, and may indicate a tyrosine-linked effect as well, as these findings were not observed in the mouse or dog, which are not as sensitive as the rat to the effects of tyrosinemia.

The NOAEL for general toxicity in this study was therefore considered to be 25 ppm (1.0 mg/kg bw/day in males and 1.4 mg/kg bw/day in females), while the LOAEL was 250 ppm (10 mg/kg bw/day in males and 14 mg/kg bw/day in females).

90-day subchronic toxicity feeding study in the Beagle dog:

In this study, that was conducted according to OECD 409, the test substance was administered to groups of 4 male and 4 female beagle dogs by dietary admix at doses of 0, 100, 500 and 1000 ppm (M- 257679-01-1). These concentrations provided doses of 0, 3, 17, and 40 mg/kg bw/day for males and 0, 3, 17, and 33 mg/kg bw/day for females. Observations and examinations included clinical signs, body weights, and food consumption, ophthalmological examinations, clinical chemistry and haematology, urinalysis, gross necropsy, selected organ weights and histopathological examination.

All animals survived until scheduled necropsy and there were no treatment-related clinical signs in either males or females. Body weight and food consumption were unaffected. There were no ophthalmological findings at any dose level which were ascribed to treatment. There were no treatment-related haematological, clinical chemistry or urinalysis findings at any dose level. Gross necropsy finding, organ weights and histopathology revealed no treatment-related findings in either males or females at any dose.

Conclusions

In the absence of any findings, the NOAEL for this study was 1000 ppm (40 mg/kg bw/day in males and 33 mg/kg bw/day in females).

12-month chronic toxicity feeding study in the Beagle dog:

In this study, which was performed according to OECD 452, the test substance was incorporated into diet and administered to groups of 4 male and 4 female beagle dogs at doses of 0, 250, 1000, and 3000 ppm for one year (M-267321-01-1). These concentrations provided doses of 0, 7, 34, and 101 mg/kg bw/day for males and 0, 9, 33, and 93 mg/kg bw/day for females. Observations and examinations included clinical signs, body weights, and food consumption, ophthalmological examinations, clinical chemistry and haematology, urinalysis, gross necropsy, selected organ weights and histopathological examination.

All animals survived until scheduled necropsy and there were no treatment-related clinical signs in either males or females. There were no biologically relevant effects on body weights and food consumption. There were no abnormalities noted at ophthalmological examination. There were no treatment-related haematological, clinical chemistry or urinalysis findings. Treatment-related changes in organ weights were noted for liver, thyroid, and kidney at 3000 ppm only, specifically increased absolute and relative liver weight in males and females, increased absolute and relative kidney weight in males, and increased absolute and relative thyroid weight in females. There were no observations at gross necropsy which were considered to be related to treatment. The only treatment-related histopathology findings were seen in the liver in male and female dogs at 3000 ppm, in which the incidence of centrilobular and/or midzonal hepatocytomegaly was increased (2 in males, 3 in females) compared to controls (0 in both males and females).

Conclusions

The toxicity of the test substance by dietary administration in the dog was limited. In this one-year study, administration of 3000 ppm caused increases in absolute and relative liver weights in males and females, absolute and relative kidney weights in males, and absolute and relative thyroid weights in females. The treatment-related histopathological lesions were limited to increased incidence of hepatocytomegaly in both males and females at 3000 ppm. The NOAEL for this study was 1000 ppm (34 mg/kg bw/day for males and 33 mg/kg bw/day for females).

Dermal:

In a 28-day subacute dermal toxicity study conducted according to OECD 410 the test substance was administered to rats (M-263216-01-2). The test substance was moistened with tap water and applied under a semiocclusive bandage to the shaved skin of 10 male and 10 female Wistar rats per treatment group, at dose levels of 0, 10, 100, and 1000 mg/kg bw/day. The substance was applied five days per week for the first three weeks of the study, and seven days per week for the final week, so that all animals were dosed daily for at least the last 8 days of the study. On each treatment day, the application period was 6 hours, after which remaining test substance was washed off with water. Observations and examinations included clinical signs, ophthalmological examinations in all high-dose and control animals, body weights, food and water consumption, evaluation eventual skin reactions, haematology, clinical chemistry, gross necropsy, selected organ weights and histopathology.

There were no mortalities or clinical signs, nor were treatment-related effects observed at ophthalmological investigation of either male or female rats at 1000 mg/kg bw/day. There were no treatment-related effects on body weights, food or water consumption, haematological and clinical chemistry parameters. Absolute and relative liver weight was statistically significantly increased in both males and females at 1000 mg/kg bw/day. Absolute liver weight was statistically significantly increased in females at 10 mg/kg bw/day, but as there was no dose-relationship and as body weight was also increased at this dose, this was considered not to be treatment-related but to be due to increased body weight. There were no observations at necropsy which were treatment-related.

There was no evidence of any reaction at the application sites to dermal application. Focal degeneration of the pancreas was observed in both males and females at 100 and 1000 mg/kg bw/day. Thyroid follicular cell hypertrophy was seen in both males and females in both control and treated animals. Alteration of thyroid colloid was seen at an increased incidence and severity in 100 and 1000 mg/kg bw/day in males. Hepatocellular hypertrophy and hypertrophy of the pars distalis of pituitary were observed in males at 1000 mg/kg bw/day. These findings in the pancreas, thyroid, pituitary and liver are considered to be treatment-related.

Conclusions

There were few effects of repeated dermal application of the test substance to either male or female rats. There were no local effects on either skinfold thickness or on signs of irritation. The local NOAEL was determined to be 1000 mg/kg bw/day (equivalent to 15 mg/m2 skin), while the systemic NOAEL was 10 mg/kg bw/day, based on histopathological effects observed at 100 and 1000 mg/kg bw/day in both males and females.

Justification for classification or non-classification

Based on the effects observed in the oral and dermal repeated dose toxicity study at 1000 ppm (M: 66 mg/kg bw/day; F: 77 mg/kg bw/day) and 100 mg/kg bw/day, respectively, the substance is classified “STOT RE 2, H373” liver, urinary system, exocrine pancreas.

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Sex	Males			Females
Dose group (ppm)	1000	7000	12000	1000	7000	12000
Mean absolute kidney weight	NC	+27% p<0.01	*	+8% NS	+19% NS	+23% NS
Mean kidney to body weight ratio	NC	+37% p<0.01	*	+9% NS	+27% p<0.05	+31% NS
Mean kidney to brain weight ratio	+8% NS	+31% p<0.01	*	+10% NS	+26% p<0.05	+26% NS

Sex	Males			Females
Dose group (ppm)	1000	7000	12000	1000	7000	12000
Mean absolute liver weight	+22% p<0.01	+16% p<0.05	*	+9% NS	NC	NC
Mean liver to body weight ratio	+21% p<0.01	+22% p<0.01	*	+10% p<0.01	+13% p<0.01	+10% p<0.05
Mean liver to brain weight ratio	+26% p<0.01	+20% p<0.05	*	+12% p<0.05	+12% p<0.05	NC

Incidence of treatment-related clinical signs after 6 months of treatment (first day of appearance)
Sex	Male					Female
Dose level (ppm)	0	25	250	1000	2500	0	25	250	1000	2500
Group size	75	75	75	75	75	75	75	75	75	75
White area on eye	0	1 (40)	38 (13)	41 (13)	48 (6)	0	1 (14)	5 (69)	36 (41)	52 (27)
Soiled fur generalized	0	0	2 (138)	4 (138)	13 (131)	0	0	2 (159)	4 (159)	19 (159)
Soiled fur localized	0	0	0	0	6 (34)0	0	0	0	1 (167)	1 (167)
Soiled anogenital region	0	0	0	1 (68)	2 (111)	0	0	1 (104)	1 (139)	25 (35)

Incidence of treatment-related clinical signs after 6 months of treatment (first day of appearance)
Sex	Male					Female
Dose level (ppm)	0	25	250	1000	2500	0	25	250	1000	2500
Group size	75	75	75	75	75	75	75	75	75	75
White area on eye	0	1 (40)	38 (13)	41 (13)	48 (6)	0	1 (14)	5 (69)	36 (41)	52 (27)
Soiled fur generalized	0	0	2 (138)	4 (138)	13 (131)	0	0	2 (159)	4 (159)	19 (159)
Soiled fur localized	0	0	0	0	6 (34)0	0	0	0	1 (167)	1 (167)
Soiled anogenital region	0	0	0	1 (68)	2 (111)	0	0	1 (104)	1 (139)	25 (35)

Incidence of treatment-related clinical signs after 6 months of treatment (first day of appearance)
Sex	Male					Female
Dose level (ppm)	0	25	250	1000	2500	0	25	250	1000	2500
Group size	75	75	75	75	75	75	75	75	75	75
White area on eye	0	1 (40)	38 (13)	41 (13)	48 (6)	0	1 (14)	5 (69)	36 (41)	52 (27)
Soiled fur generalized	0	0	2 (138)	4 (138)	13 (131)	0	0	2 (159)	4 (159)	19 (159)
Soiled fur localized	0	0	0	0	6 (34)0	0	0	0	1 (167)	1 (167)
Soiled anogenital region	0	0	0	1 (68)	2 (111)	0	0	1 (104)	1 (139)	25 (35)

Mean achieved test item intake (weeks 1-13)
Diet concentration [ppm]	Males [mg/kg bw/d]	Females [mg/kg bw/d]
2	0.13	0.15
30	1.96	2.32
1000	66	77
7000	454	535
12000	830*	956

Sex	Male					Female
Dose (ppm)	0	2#	30	1000	7000	0	2#	30	1000	7000
Number of animals	10	-	10	10	7	10	-	10	10	10
Hepatocellular hypertrophy, centrilobular, diffuse
- slight	0	-	0	8	1	0	-	0	0	1
- mild	0	-	0	1	4	0	-	0	0	0
- moderate	0	-	0	0	1	0	-	0	0	0
Total	0	-	0	9	6	0	-	0	0	1
Periportal vacuolation, hepatocellular, diffuse
- slight	0	-	0	0	0	0	-	0	1	5
- mild	0	-	0	0	0	0	-	0	2	3
Total	0	-	0	0	0	0	-	0	3	8

Mean achieved test item intake (mg/kg bw/day)
Sex	Males				Females
Dose levels (ppm)	25	250	1000	2500	25	250	1000	2500
Weeks 1 - 13	1.4	14	58	143	1.8	19	77	191
Weeks 1 - 52	1.1	11	45	114	1.5	15	63	155

Mortality incidence after 2 years of treatment
Sex	Males					Females
Dose level (ppm)	0	25	250	1000	2500	0	25	250	1000	2500
Group size	55	55	55	55	55	55	55	55	55	55
Mortality [% mortality)	30 (54.5)	38 (69.1)	25 (45.5)	29 (52.3)	40* (72.7)	28 (50.9)	19 (34.5)	25 (45.5)	22 (40.0)	26 (47.3)

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Pyrasulfotole

Endpoint summary

Administrative data

Description of key information

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Repeated dose toxicity: via oral route - systemic effects

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Reference 3

Reference 4

Endpoint conclusion

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Endpoint conclusion

Repeated dose toxicity: dermal - local effects

Link to relevant study records

Reference

Endpoint conclusion

Mode of Action Analysis / Human Relevance Framework

Additional information

Justification for classification or non-classification

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