Pyrasulfotole

Toxicity to reproduction

Administrative data

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 Oct 2003 to 07 Jul 2005

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:

2005

Report date:

2005

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Version / remarks:

adopted 22 Jan 2001

Deviations:

no

GLP compliance:

yes

Limit test:

no

Justification for study design:

SPECIFICATION OF STUDY DESIGN
- Premating exposure duration for parental (P0) animals: approximately 12 weeks
- Basis for dose level selection: Dose selection was based on results of a one-generation pilot study, where rats received 0, 100, 600 and 3600 ppm in the diet. In this study the NOEL was 100 ppm. From 600 ppm onwards P0 males exhibited reduced body weights. P0 females showed reduced body weights at 3600 ppm during gestation and lactation. There was an increase in food intake at 3600 ppm in P0 rats. The pup weights were significantly reduced at 3600 ppm.
- Termination time for F2: after a 4-week lactation period
- Route of administration: dietary admix

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 animals

Species:

rat

Strain:

Wistar

Remarks:

Crl: (WI) WU BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River GmbH, Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: (P) 6 - 7 wks; (F1) 4 wks
- Weight at study initiation: (P) 109 - 149 g (males), 101 -137 g (females); (F1) 68 - 119 g (males), 65 - 106 g (females)
- Fasting period before study: no
- Housing: singly (except when co-housed for matings) in Makrolon® cages Type Illh on low-dust soft-wood shavings or nesting material (day 20 GD to 7 LD)
- Diet: Kliba 3883.9.25 supplied by Provimi Kliba SA, Kaiseraugst, Switzerland, ad libitum
- Water: tap water, ad libitum
- Acclimation period: approximately one week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 - 25
- Humidity (%): 50 - 60
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 06 Oct 2003 To: 02 Aug 2004

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

PREPARATION OF DIETARY MIXTURES:
- Rate of preparation of diet (frequency): at least weekly
- Mixing appropriate amounts with: Kliba 3883.9.25
- Storage temperature of food: room temperature

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: 12 nights during the three-week mating period
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as Day 0 of pregnancy
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged: individually
- Any other deviations from standard protocol: no second mating period with different males was initiated if first mating period was unsuccessful

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis of dose formulations was performed using High Performance Liquid Chromatography (HPLC) with UV detection.

Homogeneity analysis: In samples taken from the top, middle and bottom of 5 and 5000 ppm mixtures the test substance was proven to be homogeneously distributed.
Stability analysis: The test substance concentration in 5 and 5000 ppm mixtures was within the range of ± 20% of their initially measured concentration after storage at animal room temperature for up to 15 days and after 9 weeks of freezer storage, which were 89 and 99% of nominal concentration.
Concentration analysis: The absence of the test substance in the control diet was confirmed. The test substance concentration (all concentrations including 0 ppm) measured at six time points were in the range of + 20% of the nominal concentrations.

Duration of treatment / exposure:

P0 females: During a 12-week premating period, through pairing, gestation and lactation (until LD 28) (in total approximately 22 weeks)
P0 males: During a 12-week premating period and through a 3-week pairing period (in total approximately 15 weeks)
F1/P1 females: Potentially in utero and through mother's milk, after onset of dietary consumption during lactation, during rearing (10 weeks), pairing (3 weeks) gestation (3 weeks) and lactation (4 weeks)
F1/P1 males: Potentially in utero and through mother's milk, after onset of dietary consumption during lactation, during rearing (10 weeks), pairing (3 weeks) gestation (3 weeks) and lactation (4 weeks)

Frequency of treatment:

Daily, 7 days/week

Details on study schedule:

- P1 parental animals not mated until 10 weeks after selected from the F1 litters
- Selection of parents from F1 generation when pups were 28 days of age
- Age at mating of the mated animals in the study: P0: 18 weeks, P1: 13 weeks

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

30 ppm

Remarks:

during premating:
equivalent to 2.50 and 3.09 mg/kg bw/day in P0 males and females, respectively
equivalent to 3.68 and 4.18 mg/kg bw/day in P1 males and females, respectively

Doses / concentrations 2

Dose / conc.:

300 ppm

Remarks:

during premating:
equivalent to 26 and 33 mg/kg bw/day in P0 males and females, respectively
equivalent to 34 and 39 mg/kg bw/day in P1 males and females, respectively

Doses / concentrations 3

Dose / conc.:

3 000 ppm

Remarks:

during premating:
equivalent to 272 and 346 mg/kg bw/day in P0 males and females, respectively
equivalent to 354 and 393 mg/kg bw/day in P1 males and females, respectively

No. of animals per sex per dose:

25 M / 25 F

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: Dose selection was based on results of a one-generation pilot study (M-129357-01-2), where rats received 0, 100, 600 and 3600 ppm in the diet. In this study the NOEL was 100 ppm. From 600 ppm onwards F0 males exhibited reduced body weights. F0 females showed reduced body weights at 3600 ppm during gestation and lactation. There was an increase in food intake at 3600 ppm in F0 rats. Pup weights were significantly reduced at 3600 ppm. Therefore, this 2-generation reproduction study was dosed at 0, 30, 300 and 3000 ppm.

Positive control:

no

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily for mortality and morbidity (once daily on weekends and public holidays)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to the first administration in the diet and then weekly as a rule and during the pregnancy and lactation periods as follows:
- Pregnancy on Day 0 (=day of a sperm positive smear or vaginal plug) 7,14 and 20, - lactation on Day 0 (day of birth), 4, 7,14, 21 and 28.

BODY WEIGHT: Yes
- Time schedule for examinations: prior to the first administration and thereafter weekly up to necropsy (males and females not pregnant*) and during the pregnancy and lactation periods as follows:
- Pregnancy on Day 0, 7, 14 and 20,
- Lactation on Day 0, 4, 7,14, 21 and 28,
- On the day of scheduled necropsy.

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

Oestrous cyclicity (parental animals):

Oestrus cycle length determination was done by evaluation of vaginal smears over 19 consecutive days prior to the mating period. Smears were examined microscopically for large serrated cells indicating estrus had occurred. This data was used to determine the estrus cycle length and whether females were cycling properly.

Sperm parameters (parental animals):

Parameters examined in P and F1 male parental generations at 0 and 3000 ppm:
testis weight, epididymis weight, sperm count in testes, sperm count in epididymides, enumeration of cauda epididymal sperm reserve, sperm motility, sperm morphology

Litter observations:

STANDARDISATION OF LITTERS
- Performed on Day 4 postpartum: yes
- Maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed, examined for external defects and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 / F2 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD, F2 pups only), presence of nipples/areolae in male pups.
In all F1 offspring selected for further treatment, the age and body weight when balano-preputial separation or vaginal opening had occured were recorded.

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was not determined for pups born or found dead.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: no

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: no

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals when no longer needed for mating
- Maternal animals: All surviving animals at weaning of 28 days old pups or up to 2 days later

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera. In females, implantation sites were counted after staining with 10% aqueous ammonium sulfide.

HISTOPATHOLOGY / ORGAN WEIGHTS
- The tissues indicated in Table 1 (any other information on material and methods incl. tables) were prepared for microscopic examination and weighed, respectively.
Staging of ovarian follicles was done in F1 females (high and low concentration) only. All reproductive organs of female rats suspect of reduced fertility were investigated.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at 28 - 30 days of age.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGTHS
The tissues indicated in Table 2 (any other information on material and methods incl. tables) were prepared for microscopic examination and weighed, respectively.

Statistics:

Statistical evaluation was performed on an Alpha 800 5/500 computer (TASC-system) using the following methods:
a) Analysis of Variance (ANOVA) and in case of significant results Dunnett's test as post hoc
test for: body weights and body weight gains, food consumption, number of implantation sites per female, number of viable pups per female, organ weights at necropsy, number of estruses, time to insemination, life birth, viability and lactation rate
b) 2 by N CHI2 test; in case of significant differences Fisher's exact test with Bonferroni correction for: number of viable pups per group based on the number of implantations, insemination, fertility, gestation and rearing rate
c) Kruskall-Wallis test and in case of significant differences Dunnett's test for: number of prenatal loss per litter
The sperm and spermatid count data were not evaluated statistically, because there were no meaningful differences occurred between the high dose and control groups.
Generally, differences between the control group and groups treated with the test substance groups were considered as statistically significant when p < 0.05. Significant differences from the control are indicated with * for p < 0.05 and **for p < 0.01.
Follicle counts were evaluated statistically using the Wilcoxon Mann-Whitney test.

Reproductive indices:

Insemination index (%) = (number of sperm positive females / number of females co-housed with a male) x 100
Fertility index (%) = (number of pregnant females / number ofsperm positive females) x 100
Gestation index (%) = (number of females completing delivery / number of pregnancies) x 100
Live birth index (%) = (number of live pups at birth / total number of pups born) x 100

Offspring viability indices:

Rearing index = (number of females rearing a litter to lactation day 21 / number of females delivering a litter) x 100
Viability index = (number of pups alive on lactation day 4 pre-culling / number of live pups born) x 100
Lactation index = (number of pups alive after 3 weeks / number of pups after four days (after culling)) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Eye opacities were noted in 15 males and 4 females at 3000 ppm (for details see Attachment 2 "attached background material").

Dermal irritation (if dermal study):

not examined

Description (incidence and severity):

Not applicable.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

At the end of the premating periods no adverse effect on body weights and body weight gain was noted up to 3000 ppm.
In the absence of a dose dependency the reduced (p < 0.05) body weight mean at termination in 300 ppm males is considered not to reflect a treatment-effect.
During gestation no effects on body weights were noted up to 3000 ppm. During lactation (lactation Days 0 - 4) reduced body weight gain was noted at 3000 ppm.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food intake during the premating and gestation period was not influenced by the treatment up to 3000 ppm.
Food consumption was slightly reduced at 3000 ppm during lactation Days 0 - 4.

For compound intake see Attachment 1 (attached background material).

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):

Diffuse or reticulate cornea opacities and/or corneal neovascularisation were noted, each with dose-dependently increasing incidences from 300 ppm onwards (for details see Attachment 2 "attached background material"). These effects correlated with histopathological findings.

Haematological findings:

not examined

Description (incidence and severity):

Not applicable.

Clinical biochemistry findings:

not examined

Description (incidence and severity):

Not applicable.

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

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Not applicable.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

At 300 and 3000 ppm, degenerative processes of the cornea (keratitis, reactive epithelial hyperplasia, vascularisation) were noted correlating in clinical observations, ophthalmoscopically and/or at necropsy.
At all dose levels changes in thyroid glands were evident. They were characterized by colloidal alteration, pigment deposition in the follicular epithelium from 30 ppm onwards as well as a follicular cell hypertrophy at 300 ppm and above. In low dose females, the thyroid gland was only marginally affected. In the absence of functional changes to the thyroid or progression to further histopathological changes at this dose, these findings are regarded to be non-adverse.

Further findings in the pituitary gland, kidneys and liver were only noticed in males:
In the anterior part of the pituitary gland of males treated at 300 and 3000 ppm there was an increase in the number of eosinophilic inclusions.
The liver of 300 and 3000 ppm males showed hepatocellular hypertrophy and cytoplasmic change indicating an increased metabolic activity induced by the test compound. In males treated at 300 ppm and above signs of an enhanced aging process ("early progressive nephropathy") were evident. This was concluded, because incidence and/or grade of basophilic tubules, tubular dilation with hyaline casts and mononuclear cell infiltrates increased by dose.
In most cases (thyroid, liver and kidneys) these findings were correlated with changes in organ weights in males.
Histopathology of reproduction organs inclusive ovarian follicle stages revealed no treatment effect.

See Attachment 9 for histopathological findings in F0 parental animals (attached background material).

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Description (incidence and severity):

Not applicable.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Details on results (P0)

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 (P0)

1

Key result

Dose descriptor:

NOAEL

Remarks:

general toxicity

Effect level:

30 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects noted at 30 ppm

Remarks on result:

other: equivalent to 2.50 and 3.09 mg/kg bw/day in P0 males and females, respectively

2

Key result

Dose descriptor:

LOAEL

Remarks:

general toxicity

Effect level:

300 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

ophthalmological examination

organ weights and organ / body weight ratios

histopathology: non-neoplastic

Remarks on result:

other: equivalent to 26 and 33 mg/kg bw/day in P0 males and females, respectively

3

Key result

Dose descriptor:

NOAEL

Remarks:

for reproductive function

Effect level:

3 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects on reproductive parameters noted up to 3000 ppm.

Remarks on result:

other: equivalent to 272 and 346 mg/kg bw/day in P0 males and females, respectively

Target system / organ toxicity (P0)

1

Critical effects observed:

yes

Lowest effective dose / conc.:

300 ppm

System:

endocrine system

Organ:

pituitary gland

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

2

Critical effects observed:

yes

Lowest effective dose / conc.:

300 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.:

300 ppm

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

4

Critical effects observed:

yes

Lowest effective dose / conc.:

300 ppm

System:

eye

Organ:

cornea

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

5

Critical effects observed:

yes

Lowest effective dose / conc.:

300 ppm

System:

endocrine system

Organ:

thyroid gland

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Eye opacities were noted in 15 males and 7 females at 3000 ppm (for details see Attachment 2 "attached background material").

Dermal irritation (if dermal study):

not examined

Description (incidence and severity):

Not applicable.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Two females at 3000 ppm were euthanized in moribund condition (for details see Attachment 2 "attached background material"). Post-mortem examination including histopathology revealed no test substance specific organ lesions. Therefore, the clinical symptoms of these animals are considered incidental.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

During gestation no effects on body weights were noted up to 3000 ppm. During lactation (lactation Days 0 - 4) statistically significantly reduced body weight gain was noted at 300 and 3000 ppm.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food intake during the premating and gestation period was not influenced by the treatment up to 3000 ppm. During lactation (lactation Days 0 - 4) a slight reduction in food intake in dams ingesting 300 and 3000 ppm was noted.

For compound intake see Attachment 1 (attached background material).

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):

Diffuse or reticulate cornea opacities and/or corneal neovascularisation were noted, each with dose-dependently increasing incidences from 300 ppm onwards (for details see Attachment 2 "attached background material"). These effects correlated with histopathological findings.

Haematological findings:

not examined

Description (incidence and severity):

Not applicable.

Clinical biochemistry findings:

not examined

Description (incidence and severity):

Not applicable.

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):

In treated males, higher absolute and relative kidney and thyroid weights were found at 30 ppm and above. These deviations were mostly statistically significant and nearly in all cases dose-dependently distributed. In the absence of a histopathological correlate increased kidney weights at 30 ppm were considered not adverse. 3000 ppm females showed increased relative (p < 0.01) kidney weights.
There were significantly increased relative liver weights in males receiving 300 or 3000 ppm.
In 3000 ppm females slightly increased absolute and relative weights of the adrenals were observed (each p < 0.01).
Statistically significantly decreased absolute weights of the epididymis, testis, prostate at 3000 ppm and of the brain in male and female rats at 3000 ppm are attributed to differences in body weights.
There were decreased absolute spleen weights in 300 and 3000 ppm males, most likely due to decreased body weights as relative spleen weights were unaffected.

See Attachment 4 for mean absolute and relative organ weights of parental F1 parental animals (attached background material).

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Eye opacities were noted in one 300 ppm male and fourteen 3000 ppm males as well as in two 3000 ppm females.

Neuropathological findings:

not examined

Description (incidence and severity):

Not applicable.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

The eyes of parental rats showed degenerative processes of the cornea (keratitis, reactive epithelial hyperplasia, vascularisation) beginning at 300 ppm and above correlating with findings seen at clinical observations, ophthalmoscopically and/or at necropsy.
In the thyroid glands treatment-related findings were evident beginning at 30 ppm. They were characterized by colloidal alteration, pigment deposition in the follicular epithelium from 30 ppm onwards as well as a follicular cell hypertrophy at 300 ppm and above. In low dose females the thyroid gland was only marginally affected. In the absence of functional changes to the thyroid or progression to further histopathological changes at this dose, these findings are regarded to be non-adverse.

Further findings in the pituitary gland, kidneys and liver were only noticed in males:
In the anterior part of the pituitary gland of males treated at 300 ppm there was an increase in the number of eosinophilic inclusions.
The liver of 300 and 3000 ppm males was showing hepatocellular hypertrophy and cytoplasmic change indicating an increased metabolic activity induced by the test compound. However, there was also a slightly increased periportal fat accumulation in high dose males.
In males treated at 300 ppm and above signs of an enhanced aging process ("early progressive nephropathy") were evident. This was concluded, because incidence and/or grade of basophilic tubules, tubular dilation with hyaline casts increased by dose. In most cases (thyroid, liver and kidneys) these findings were correlated with changes in organ weights in males.

Histopathology of reproduction organs inclusive ovarian follicle stages revealed no treatment effect.

See Attachment 9 for histopathological findings in F1 parental animals (attached background material).

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Description (incidence and severity):

Not applicable.

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related effects on reproduction performance were noted at any dose.
Insemination and fertility indices as well as gestation length and number of litters born were not changed by the treatment up to 3000 ppm.

At 3000 ppm the number of F2 pups born was slightly lower (p > 0.05) than at 0 ppm due to two pre-scheduled deaths during pregnancy in this group. Subsequently, also the rearing and gestation indices were lower (p > 0.05) . As these deaths are considered incidental, the reduced gestation and rearing index at 3000 ppm is not indicative for a treatment effect on reproductive performance.

Details on results (P1)

Corneal opacities
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 (P1)

1

Key result

Dose descriptor:

NOAEL

Remarks:

general toxicity

Effect level:

30 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects were noted at 30 ppm.

Remarks on result:

other: equivalent to 3.68 and 4.18 mg/kg bw/day in P1 males and females, respectively

2

Key result

Dose descriptor:

LOAEL

Remarks:

general toxicity

Effect level:

300 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

ophthalmological examination

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

Remarks on result:

other: equivalent to 34 and 39 mg/kg bw/day in P1 males and females, respectively

3

Key result

Dose descriptor:

NOAEL

Remarks:

for reproduction

Effect level:

3 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effcts on reproductive parameters noted up to 3000 ppm.

Remarks on result:

other: equivalent to 272 and 346 mg/kg bw/day in P1 males and females, respectively

Target system / organ toxicity (P1)

1

Critical effects observed:

yes

Lowest effective dose / conc.:

300 ppm

System:

endocrine system

Organ:

pituitary gland

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

2

Critical effects observed:

yes

Lowest effective dose / conc.:

300 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.:

300 ppm

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

4

Critical effects observed:

yes

Lowest effective dose / conc.:

300 ppm

System:

eye

Organ:

cornea

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

5

Critical effects observed:

yes

Lowest effective dose / conc.:

300 ppm

System:

endocrine system

Organ:

thyroid gland

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

At 3000 ppm more F1 pups (n = 12, in two litters) were found to be cold to touch than in the other groups (n = 5, 5 and 0 at 0, 30 and 300 ppm, respectively)

Dermal irritation (if dermal study):

not examined

Description (incidence and severity):

Not applicable.

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

Viability indices were not affected in F1 pups.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 3000 ppm decreased pup (males and females) and litter weights were evident on Day 21 p.p. (p > 0.05) and Day 28 p.p. (p ≤ 0.01). Offspring body weights and litter weights were not changed toxicologically relevantly up to 300 ppm.

At the end of the premating period no adverse effect on body weights and body weight gain was noted at 30 ppm (males) and up to 300 ppm (females). From 300 ppm onwards (males), statistically significantly reduced body weights and body weight gain were evident. At 3000 ppm, females showed statistically significantly reduced body weights. At the end of the premating period at 3000 ppm the body weight depression was 12% in males and 7% in females.

See Attachment 8 for mean F1 litter and pup weights (attached background material).

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

The food intake during the premating period was not influenced by the treatment up to 3000 ppm.

For compound intake see Attachment 1 (attached background material).

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 300 and 3000 ppm diffuse or reticulate cornea opacity and/or cornea neo-vascularisation increased dose-dependently. No changes were obvious in pups at 30 ppm.

See Attachment 5 for ophthalmological findings in F1 weanlings (attached background material).

Haematological findings:

not examined

Description (incidence and severity):

Not applicable.

Clinical biochemistry findings:

not examined

Description (incidence and severity):

Not applicable.

Urinalysis findings:

not examined

Description (incidence and severity):

Not applicable.

Sexual maturation:

effects observed, treatment-related

Description (incidence and severity):

Balano-preputial separation in post weanlings was statistically significantly delayed at 300 ppm (marginally, mean age 44.2 days) and at 3000 ppm (more pronounced, mean age 46.3 days) compared to the control (mean age 41.0 days). The corresponding body weights were higher (p < 0.01) at 300 and 3000 ppm compared to controls. Balano-preputial separation was unaffected at 30 ppm (mean age 41.8 days).

Vaginal opening was slightly but statistically significantly delayed at 3000 ppm (34.2 compared to 32.6 days of age in the control). The corresponding body weights were similar to those of the control.

See Attachment 6 for sexual maturation data of F1 post weanlings (attached background material).

Anogenital distance (AGD):

not examined

Description (incidence and severity):

Not applicable.

Nipple retention in male pups:

not examined

Description (incidence and severity):

Not applicable.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

At 3000 ppm decreased absolute brain weights (p ≤ 0.0.5 or p ≤ 0.01) were noted for male and female F1 and F2 weanlings. Because corresponding relative weights were not affected, this finding is considered to be secondary to body weight depression in this group.
Additionally, there were reduced absolute spleen weights (F1 weanlings) in this group (p ≤ 0.01), which correlated only in male F1 weanlings with a slight reduction in relative spleen weights (p ≤ 0.01). As this finding was not noted in F2 weanlings a treatment effect is not assumed.

No statistically significantly changes in absolute or relative organ weights were observed in male and female F1 or F2 weanlings up to 300 ppm.

See Attachment 7 for mean absolute organ weights of F1 weanlings (attached background material).

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

At 3000 ppm more weanlings exhibited dilated and/or enlarged kidneys than in the control (3 pups vs. 1 pup in the control).
Up to 300 ppm no remarkable incidences of macroscopical findings were found at pup or weanling necropsies.

See Attachment 10 for summary of necropsy findings in F1 pups / weanlings (attached background material).

Histopathological findings:

effects observed, treatment-related

Other effects:

not examined

Description (incidence and severity):

Not applicable.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Description (incidence and severity):

Not applicable.

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Description (incidence and severity):

Not applicable.

Details on results (F1)

Corneal opacities
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).

Effect levels (F1)

1

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

300 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects were noted at 300 ppm.

Remarks on result:

other: equivalent to 22.25 mg/kg bw/day

2

Key result

Dose descriptor:

LOAEL

Generation:

F1

Effect level:

3 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

sexual maturation

clinical signs

body weight and weight gain

ophthalmological examination

gross pathology

Remarks on result:

not measured/tested

Remarks:

equivalent to 228.59 mg/kg bw/day

Target system / organ toxicity (F1)

1

Critical effects observed:

yes

Lowest effective dose / conc.:

300 ppm

System:

eye

Organ:

cornea

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

2

Critical effects observed:

yes

Lowest effective dose / conc.:

3 000 ppm

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Results: F2 generation

General toxicity (F2)

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Description (incidence and severity):

Not applicable.

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

The viability indices were below control values from 30 ppm onwards. Viability indices were 92.45, 83.79, 78.89 and 71.60 at 0, 30, 300 and 3000 ppm, respectively. However, the differences from control were not statistically significant. Most viability indices were within the range of historical controls (78.74 to 100%) and were therefore considered to be incidentally lower rather than reflecting an adverse effect.
This assumption is supported by the following facts:
- At 30 ppm only two of 25 dams completely lost their litters up to day LD 4 whereas in other litters of this group no remarkable pup mortality was evident.
- As shown in other two-generation studies performed recently in that laboratory a broad variation range in viability indices can occur within different unaffected study groups indicating that a relative low viability can occur incidentally.
- A very slight (<10%) difference between the viability at 30 and 0 ppm exists. The viability index at 300 ppm is most probably also not indicative for a test substance effect as the lower limit of the historical control range is just met.
At 3000 ppm a slight reduction by the treatment must be stated causing a reduced litter size on LD 4.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 3000 ppm, decreased pup (males and females) and litter weights were evident on Day 21 p.p. (mostly p > 0.05) and Day 28 p.p. (p ≤ 0.01).
Offspring body weights and litter weights were not changed toxicologically relevantly up to 300 ppm.

See Attachment 8 for mean F2 litter and pup weights (attached background material).

Food consumption and compound intake (if feeding study):

not examined

Description (incidence and severity):

Not applicable.

Food efficiency:

not examined

Description (incidence and severity):

Not applicable.

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

not examined

Ophthalmological findings:

effects observed, treatment-related

Description (incidence and severity):

At 300 and 3000 ppm diffuse or reticulate cornea opacity and/or cornea neo-vascularisation increased dose-dependently. No changes were obvious in pups at 30 ppm.

See Attachment 5 for ophthalmological findings in F2 weanlings (attached background material).

Haematological findings:

not examined

Description (incidence and severity):

Not applicable.

Clinical biochemistry findings:

not examined

Description (incidence and severity):

Not applicable.

Urinalysis findings:

not examined

Description (incidence and severity):

Not applicable.

Sexual maturation:

not examined

Description (incidence and severity):

Not applicable.

Anogenital distance (AGD):

no effects observed

Nipple retention in male pups:

not examined

Description (incidence and severity):

Not applicable.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

At 3000 ppm reduced absolute brain weights (p ≤ 0.05 or p ≤ 0.01) were noted for male and female weanlings. Because corresponding relative weights were not affected, this finding is considered to be secondary to reduced body weights in this group.
No statistically significantly changes in absolute or relative organ weights were observed in weanlings up to 300 ppm.

See Attachment 7 for mean absolute organ weights of F2 weanlings (attached background material).

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

At 3000 ppm more autolytic weanlings (5 pup vs. 1 pup in the control) or pups without milk in the stomach (4 vs. 1 in the control) were noted.
At 3000 ppm more weanlings exhibited dilated and/or enlarged kidneys than in the control (3 vs.1 in the control).

See Attachment 10 for summary of necropsy findings in F2 pups / weanlings (attached background material).

Histopathological findings:

not examined

Description (incidence and severity):

Not applicable.

Other effects:

not examined

Description (incidence and severity):

Not applicable.

Developmental neurotoxicity (F2)

Behaviour (functional findings):

not examined

Description (incidence and severity):

Not applicable.

Developmental immunotoxicity (F2)

Developmental immunotoxicity:

not examined

Description (incidence and severity):

Not applicable.

Details on results (F2)

Corneal opacities
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).

Effect levels (F2)

1

Key result

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

300 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects were noted at 300 ppm.

Remarks on result:

other: equivalent to 22.25 mg/kg bw/day

2

Key result

Dose descriptor:

LOAEL

Generation:

F2

Effect level:

3 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

body weight and weight gain

gross pathology

Remarks on result:

other:

Target system / organ toxicity (F2)

1

Critical effects observed:

yes

Lowest effective dose / conc.:

300 ppm

System:

eye

Organ:

cornea

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

2

Critical effects observed:

yes

Lowest effective dose / conc.:

3 000 ppm

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Applicant's summary and conclusion

Conclusions:

The study was performed under GLP conditions and according to OECD TG 416 (adopted 2001). Parental body weight was statistically and biologically significantly decreased by administration of high doses of the test substance by dietary incorporation. Treatment-related clinical signs in adult animals were limited to an increased incidence of eye opacity in both males and females. Liver, kidney and thyroid weights were increased, as well as the incidence of microscopic findings. In the liver, the microscopic findings were indicative of an adaptive response and of increased metabolic activity. Kidney findings were generally characterized as the result of an enhanced aging progress, and may be due to either excretion of the test substance in the urine, or to increased excretion of tyrosine in the urine due to HPPDase inhibition. The findings in the thyroid at 30 ppm were considered to be non-adverse in the absence of either functional changes or increase in other microscopic findings at this dose. The NOAEL for parental systemic toxicity was therefore 30 ppm (2.50 mg/kg bw/day in males, 3.09 mg/kg bw/day in females).
There was no effect on either sperm parameters or on oestrus cyclicity at any dose, nor were there any effects on fertility, gestation index or length, or rearing indices. The reproductive NOAEL was therefore 3000 ppm (272.42 mg/kg bw/day in males, 345.67 mg/kg bw/day in females).
In pups, viability was reduced at 3000 ppm, and clinical signs were also increased in this dietary group. Pup and litter weights were reduced at 3000 ppm near the end of lactation, but there was no effect on pup body weight at birth. Preputial separation and vaginal opening were delayed with a greater effect on preputial separation. At necropsy, the incidence of dilated and/or enlarged kidneys was increased at 3000 ppm. The offspring NOAEL was therefore 300 ppm (22.25 mg/kg bw/day).