Pyrasulfotole

Repeated dose toxicity: oral

Administrative data

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

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to other study

Reference

Endpoint:

additional toxicological information

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

15 Mar - 28 Jul 1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Type of study / information:

This exploratory study was conducted primarily in order to determine the difference in development of corneal opacities between rats and mice after administration of diets containing dietary tyrosine. A secondary objective was to determine whether there was a difference in sensitivity between two strains of rats (Brown Norway and CD).

Qualifier:

no guideline available

Principles of method if other than guideline:

Groups of five male and five female CD rats, Brown Norway rats and CD 1 mice were administered either basal diet or diets supplemented with 2 or 5% tyrosine for 14 days. Animals were weighed weekly, observed for clinical signs daily and subjected to ophthalmological examinations on Days 2, 3, 7, 8 and 14. At study termination, eyes were taken from selected animals for histological examination and plasma taken for free plasma tyrosine analysis of selected groups.

GLP compliance:

yes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Aldrich, France, batch 68160-123
- Purity: 98%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: in an air-tight, light-resistant container at approximately 5°C
- Homogeneity and stability of the test substance in the diet were analytically verified. On all occasions results for homogeneity, concentrations and stability were within the acceptable ranges. The phenylalanine and tyrosine content of basal diet was analyzed (total amino acid levels of protein origin)
and were found to be 0.77 ± 0.04% and 0.55 ± 0.03% respectively.

No deaths occurred during the study. No treatment-related clinical signs were observed in CD rats of either sex at 2%, or in Brown Norway rats or CD 1 mice in either sex at 2 or 5% tyrosine. The observations in the 5% tyrosine male CD rat group were restricted to dark urine (all males during the second week of study and 3/5 females on Day 14) and one male which appeared thin and showed ptosis and piloerection from Days 10 and 13, respectively. In this animal, corneal opacity was severe and visible to the naked eye.

Body weights and food consumption were not affected by treatment.

No treatment-related corneal effects were observed in female groups of rats at 2% or 5% tyrosine. Male CD and Brown Norway rats were not affected at 2% tyrosine. No mice of either sex were affected at any dose. Slight corneal opacities were noted for 3 of 5 male CD rats at 5% tyrosine on Day 2. On the following day, these opacities had progressed to "moderate" and a fourth animal in the group showed a "slight" opacity. By Day 7, the opacities in these four animals had become "severe" and the fifth male had developed a "slight" opacity. By Day 14, two males developed a very severe opacity and showed signs of edema and vascularisation of the cornea. In three animals, congestion of the iris became evident. Only one Brown Norway rat at 5% tyrosine developed any corneal changes; in this animal the corneal opacity was slight and was only observed on Day 14.

Table 1: Individual ophthalmological findings

Group/Dietary tyrosine (%)	Animal No.	Sex	Study Day
			2	3	7	8	14
CD rats
0	all	-	-	-	-	-	-
2	all	-	-	-	-	-	-
5	ET3M 1380	M	Sl B	M B	SE B	Se B Im	Se B Im
	ET3M 1381	M	Sl U	M B If	Se B Is	Se B Is	VS B Is EV
	ET3M 1382	M	Sl B	M B	Se B	Se B Im	VS B Is EV
	ET3M 1383	M	-	Sl U	Se B	Se B	Se B
	ET3M 1384	M	-	-	Sl B	Sl B	Sl B
	all females	F	-	-	-	-	-
Brown Norway rats
0	all	-	-	-	-	-	-
2	all	-	-	-	-	-	-
5	ET6M 1410	M	-	-	-	-	-
	ET6M 1411	M	-	-	-	-	-
	ET6M 1412	M	-	-	-	-	Sl B
	ET6M 1413	M	-	-	-	-	-
	ET6M 1414	M	-	-	-	-	-
	all females	F	-	-	-	-	-
CD 1 mice
0	all	-	-	-	-	-	-
2	all	-	-	-	-	-	-
5	all	-	-	-	-	-	-

-             no finding

Sl           Slight opacity                B            Bilateral opacity

M          Moderate opacity          If            Iris failed to dilate

Se          Severe opacity              Im         Congestion of iris, mild

VS         Very severe opacity      Is           Congestion of iris, severe

U           Unilateral opacity           EV         Edema and vascularization of cormea

In male CD rats, the 2% and 5% tyrosine diets caused a 3-fold increase in plasma tyrosine to 59 mg/L and a 5-fold increase to 114 mg/L, respectively. Only the 5% group developed corneal opacity. In females the basal levels were lower than males, but the percentage increase in plasma tyrosine was similar in the 5% group for males and females.

In male Brown Norway rats, the basal plasma tyrosine levels were similar to female CD rats, as were the levels with 5% diets (68 mg/L). The levels for the 2% and 5% dietary male groups were about 3 fold and 5 fold the basal level, respectively. One male in the 5% group had a very high plasma tyrosine level (nearly 10 times that of the other four rats in the group), and was the only Brown Norway rat with a corneal lesion.

In CD 1 mice, the basal levels were similar to female CD rats and male Brown Norway rats but with a 5% tyrosine diet, there was no significant increase in plasma tyrosine levels.

Table 2: Mean plasma tyrosine concentrations

Animal strain	Dietary tyrosine (%)	Mean plasma tyrosine (mg/L)
		Males		Females
		Mean	S.D.	Mean	S.D.
CD rat	0	21	4.2	13	2.0
	2	59	4.8	-	-
	5	114	39.9	62	29.8
Brown Norway rat	0	12	0.6	-	-
	2	32	15.0	-	-
	5	68*	13.0	-	-
CD 1 mouse	0	13	1.8	-	-
	5	18	7.1	-	-

- not determined                   * excluding one high outlier

At necropsy, no significant gross changes attributable to treatment were observed except for the corneal opacities described below.

Histopathology

No treatment-related changes were seen in female CD rats at any dose level or in males at 2% tyrosine. The two male rats examined at 5% tyrosine presented qualitatively and quantitatively similar lesions. They were characterized by the following changes:

- diffuse corneal epithelial intracytoplasmic vacuolation

- severe interstitial edema of the basal epithelial layer of the cornea

- edematous and/or swollen nuclear changes in the corneal epithelial cells

- inflammatory reaction involving the whole cornea: diffuse polymorphonuclear (PMNs) cell infiltration of the corneal stroma and epithelium, focal infiltration of the corneal epithelium and ciliary processes.

These changes were correlated to the corneal opacities (superficial keratitis) observed for almost two weeks at the ophthalmologic examinations.

No treatment-related changes were seen in male Brown Norway rats at 2% tyrosine. One of the examined eyes from the 5% male group was from the only animal in the group of five to show changes visible at ophthalmoscopy. The histological changes in this eye were characterized by:

- focal accumulation of PMNs in the anterior chamber

- mild diffuse infiltration of the corneal stroma by PMNs lining up at the base of the epithelium

- solitary focus of superficial epithelial desquamation of non-keratinized cells in central portion of cornea.

These changes were correlated with the observation of a slight corneal opacity which was first detected shortly before necropsy.

No treatment-related changes were observed for any group of male mice.

Conclusions:

This study demonstrates that a high dietary intake of free L-tyrosine leads to increased plasma tyrosine in rats, but not in mice. Furthermore, there was a strong correlation between the presence of corneal opacities and high plasma tyrosine. The same dietary treatment in mice did not result in corneal opacities.
CD rats were more susceptible to the corneal effects of dietary tyrosine than the other strain of rat tested. The higher basal plasma tyrosine levels in male CD rats in comparison with female CD rats or male Brown Norway rats, may be important in explaining different responses to elevated tyrosine intake between rat strains. However, this is not true for the inter species-comparison. Mice appear to be resistant to high tyrosine intake with no significant increase in plasma tyrosine and no corneal lesions.
The typical corneal lesion seen in the different strains of rat treated with tyrosine and those observed in regulatory studies have common characteristics. The lesions all have a "snow-flake" appearance and are variously described as keratitis or focal or multifocal opacities of the cornea. Following high tyrosine diets, the lesion appears rapidly (often 2-3 days) and is multifocal. The lesion in the single Brown Norway rat appeared late in the study (Day 14) but had the same appearance as lesions in the CD rat at Day 2 at ophthalmoscopy.
Most histological findings indicate an inflammatory reaction suggestive of a keratitis, in various corneal layers, sometimes with involvement of associated structures (eg. ciliary processes).

Cross-reference 2

Reason / purpose for cross-reference:

other: reference to review article

Reference

Endpoint:

additional toxicological information

Type of information:

other: ECETOC TR No. 99: Toxicological Modes of Action: Relevance for Human Risk Assessment

Adequacy of study:

other information

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Publication prepared by experts of the European Centre for Ecotoxicology and Toxicology of Chemicals

Qualifier:

no guideline required

Executive summary:

Triketone-mediated tyrosinaemia and corneal opacity

Various triketones, including 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC) and mesotrione, have been reported to cause corneal opacity in rats but not in mice. Investigative toxicology studies have revealed that this species-specific ocular toxicity is caused by the action of high concentrations of tyrosine at the target site, rather than by the direct action of xenobiotics, or metabolites thereof, on the eye.

The basis for the species difference in expression of toxicity is due to differences in the disposition of tyrosine, the ultimate toxiphore, between rats and mice. The rate-limiting step in the removal of excess tyrosine is its conversion to 4-hydroxyphenylpyruvate by tyrosine aminotransferase (TAT). Rats have much lower activity of hepatic TAT than mice and are therefore unable to prevent the build up of high and toxic tyrosine levels in plasma. In contrast, mouse hepatic TAT activity is of sufficient magnitude to prevent tyrosine concentrations of reaching toxic levels.

Data indicate the activity of hepatic TAT in humans to be similar to mice, suggesting that the mouse is a more appropriate model than the rat to investigate the potential hazard of mesotrione-induced ocular toxicity in humans.

NTBC is a very potent inhibitor of 4-hydroxyphenyl pyruvate dioxygenase (HPPD). For NTBC, which is used internationally in the treatment of children suffering from the rare hereditary disease Tyrosinaemia Type I, there is currently over 1000 patient-years clinical experience without evidence for adverse ocular effects. Furthermore studies have been conducted with NTBC and mesotrione in healthy male volunteers. With mesotrione, only marginally increased plasma tyrosine concentrations were observed which quickly returned to control levels. Steady state plasma tyrosine concentrations with NTBC were also much lower than those observed in the rat. No ocular toxicity was associated on human exposure to either NTBC or mesotrione.

The corn herbicide 2-(4-methylsulfonyl-2-nitrobenzoyl-1,3-cyclohexanedione (mesotrione) and other triketones, such as NTBC are inhibitors of HPPD. In mammals, HPPD is the second enzyme of the catabolic cascade for removal of excess dietary tyrosine. The biological consequence of HPPD inhibition in mammals has been shown to be a rise in plasma tyrosine.

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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Test guideline 1

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

Test guideline 2

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

Test material

Test material information

Constituent 1

Reference substance name:

4-[2-methanesulfonyl-4-(trifluoromethyl)benzoyl]-1,3-dimethyl-1H-pyrazol-5-ol

EC Number:

609-256-3

Cas Number:

365400-11-9

Molecular formula:

C14H13F3N2O4S

IUPAC Name:

4-[2-methanesulfonyl-4-(trifluoromethyl)benzoyl]-1,3-dimethyl-1H-pyrazol-5-ol

Test material form:

solid: crystalline

Test animals

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

Administration / exposure

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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

2 ppm

Remarks:

equivalent to 0.13 and 0.15 mg/kg bw/day in males and females, respectively

Doses / concentrations 2

Dose / conc.:

30 ppm

Remarks:

equivalent to 1.96 and 2.32 mg/kg bw/day in males and females, respectively

Doses / concentrations 3

Dose / conc.:

1 000 ppm

Remarks:

equivalent to 66 and 77 mg/kg bw/day in males and females, respectively

Doses / concentrations 4

Dose / conc.:

7 000 ppm

Remarks:

equivalent to 454 and 537 mg/kg bw/day in males and females, respectively

Doses / concentrations 5

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

Examinations

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.

Results and discussion

Results of examinations

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.

Effect levels

1

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

2

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

Target system / organ toxicity

1

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

2

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

3

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

4

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

Any other information on results incl. tables

Table 1: Mean achieved test item intake per group

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

* from week 1 to 10 only

Table 2: Kidney weight changes (% change when compared to controls)

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

NS: Not statistically significant

NC: No relevant change

*:    no surviving animals

Table 3: Liver weight changes (% change when compared to controls)

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

NS: Not statistically significant

NC: No relevant change

*:    no surviving animals

Table 4: Incidence and severity of treatment-related liver changes at terminal sacrifice

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

#:   2 ppm not examined

Applicant's summary and conclusion

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”.