Pyrasulfotole

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Two-Generation Toxicity Study (OECD 416), rats:

NOAEL general toxicity = 30 ppm (equivalent to 2.50 and 3.09 mg/kg bw/day in P0 males and females, respectively and equivalent to 3.68 and 4.18 mg/kg bw/day in P1 males and females, respectively)

NOAEL reproductive toxicity >= 3000 ppm (highest dose tested) (equivalent to 272 and 346 mg/kg bw/day in P0 males and females, respectively and to 354 and 393 mg/kg bw/day in P1 males and females, respectively)

NOAEL offspring = 300 ppm (corresponding to 22.25 mg/kg bw/day)

Link to relevant study records

Reference

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

Reason / purpose for cross-reference:

reference to other study

Reason / purpose for cross-reference:

other: reference to review article

Qualifier:

according to guideline

Guideline:

OECD Guideline 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

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

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

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

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

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

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

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: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

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

Thyroid findings (increased weight, histopathological changes comprising changes in colloid, follicular cell hypertrophy and pigment deposition in the follicular epithelium) are considered a non-adverse and rat specific phenomenon. No changes of the thyroid were noted in either mice or dogs, the other two species in which repeated-dose studies with histopathological examination of the thyroid were conducted with the test substance. As the test substance through inhibition of the HPPDase enzyme increases plasma tyrosine concentration in the rat, it is quite possible that some of this increased tyrosine is taken up by the thyroid and stored in the colloid, either as free tyrosine or through either increasing the synthesis of thyroglobulin or altering its composition in terms of number of tyrosine residues per thyroglobulin molecule.
In the absence of signs of altered thyroid functions (such as effects on body weights, fertility and gestation indices or effects on offspring performance in the developmental neurotoxicity study), the observed morphological changes are considered to be non-adverse.

Key result

Dose descriptor:

NOAEL

Remarks:

general toxicity

Effect level:

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

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

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

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

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

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

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

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

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: 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.

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.

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

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

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

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

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

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

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

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

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.

Behaviour (functional findings):

not examined

Description (incidence and severity):

Not applicable.

Developmental immunotoxicity:

not examined

Description (incidence and severity):

Not applicable.

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).

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

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

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

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

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.

Behaviour (functional findings):

not examined

Description (incidence and severity):

Not applicable.

Developmental immunotoxicity:

not examined

Description (incidence and severity):

Not applicable.

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).

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

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:

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

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

Key result

Reproductive effects observed:

no

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).
 

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

272 mg/kg bw/day

Study duration:

chronic

Species:

rat

Quality of whole database:

The available information comprises an adequate and reliable study (Klimisch score 1), and is thus sufficient to fulfil the standard information requirements set out in Annex IX, 8.7, of Regulation (EC) No 1907/2006.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Reliable studies for toxicity to reproduction are available. As in other repeated dose toxicity studies with 5-hydroxy-1,3-dimethylpyrazol-4-yl)(α,α,α-trifluoro-2-mesyl-p-tolyl) methanone in rats, corneal opacities were noted in the 2-generation reproduction study described below. 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 HPPDase inhibition, tyrosine concentrations will not increase to levels high enough to induce ocular toxicity and hence, this toxicity observed in the rat is inappropriate for extrapolation to humans (ECETOC TR No. 99, 2006).

Furthermore, and also as in other repeated dose toxicity studies with 5-hydroxy-1,3-dimethylpyrazol-4-yl)(α,α,α-trifluoro-2-mesyl-p-tolyl) methanone in rats, thyroid findings were noted (increased weight, histopathological changes comprising changes in colloid, follicular cell hypertrophy and pigment deposition in the follicular epithelium) in the 2-generation reproduction study described below.

The toxicological significance of these thyroid findings were evaluated in an additional regulatory toxicology position paper (M-301557-01-1, 2007). The overall conclusion was that these findings were considered a non-adverse and rat specific phenomenon, details for this conclusion are provided below:

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

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

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

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

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

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

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

One-generation reproductive toxicity study in the rat (pilot-study)

A one-generation study is available which was performed under GLP and according to OECD 416 (M-12935701-2). The study was planned as a dose range finding study for the two-generation reproductive toxicity study (M-263320-01-2) and was therefore terminated after succession of the first generation. The test substance was incorporated into rodent diet at concentrations of 0, 100, 600, and 3600 ppm and fed to 10 male and female Wistar rats per dose group in the P0 generation. Animals were dosed throughout the premating, mating, gestation, and lactation periods. The F1 generation was maintained until Day 28 post-partum. The dietary concentrations used provided doses, during the pre-mating phase, of 0, 7.6, 43.5 and 305.5 mg/kg bw/day for male P0 animals and 8.9, 55.1 and 400.8 mg/kg bw/day for female P0 animals.

Survival, appearance and behaviour of P0 rats were not affected up to 3600 ppm. In male P0 rats, body weight depression was evident from 600 ppm onwards. In P0 females, the body weight gain was decreased at some time points of gestation and lactation at 3600 ppm. At 3600 ppm, P0 rats exhibited an increase in food intake per kg body weight.

 At necropsy, parental rats showed no macroscopic treatment-related findings up to 3600 ppm. Increased absolute and relative weights of the testes and epididymides in all treatment groups were not considered to reflect a toxic effect, as they were not observed in the subsequent two-generation reproductive toxicity study, nor were any macroscopic effects noted in the present study. From 600 ppm onwards, increased relative liver and kidney weights were calculated in males.

 The indices of insemination, fertility, gestation, rearing and life births as well as mating performance, duration of pregnancy, birth weights, number of pups born, percentages of males born and prenatal loss were not affected at levels of up to 3600 ppm. At 3600 ppm, the number of implantation sites and the mean litter size were reduced.

 At 3600 ppm, F1 pup weights were retarded from Day 21 post-partum onwards. No treatment related changes to the viability and lactation indices were observed up to 3600 ppm. Clinical observation of F1 pups revealed no remarkable changes up to 3600 ppm. At 3600 ppm, pups exhibiting renal pelvic dilation occurred. There was a tendency for reduced absolute and relative spleen weights in male and female F1 pups from 600 ppm onwards.

 The NOAELs for male and female P0 animals were concluded to be 100 and 600 ppm, respectively (equivalent to 7.6 and 55.1 mg/kg bw/day in males and females, respectively). The NOAEL for reproductive toxicity was concluded to be 600 ppm (equivalent to 43.5 and 55.1 mg/kg bw/day in males and females, respectively). The NOAEL for the F1 offspring was concluded to be 100 ppm.

Two-generation reproductive toxicity study in the rat

In this study - that was performed under GLP and according to OECD 416 (M-263320-01-2) - the test substance was incorporated into rodent diet at concentrations of 0, 30, 300, and 3000 ppm and fed to male and female Wistar rats in the P0 generation throughout premating, mating, gestation, and lactation periods. Following weaning of the F1 generation, the weanlings were maintained in their same dietary groups through adolescence, mating, gestation, and lactation. The dietary concentrations used provided doses, during the pre-mating phase, of 0, 2.5, 26, and 272 mg/kg bw/day for male P0, 0, 3.7, 34, and 354 mg/kg bw/day for male F1, 0, 3.1, 33, and 346 mg/kg bw/day for female P0, and 0, 4.2, 39, and 393 mg/kg bw/day for female F1 animals.

Parental Findings

There was no treatment-related increase in mortality in either the P0 or the P1 males or females at any dose level. Two P1 females at 3000 ppm were sacrificed in a moribund condition, but their deaths were not considered to be related to treatment. The only treatment-related clinical sign was increased incidence of eye opacities in P0 and P1 animals at 3000 ppm during pre-mating, gestation, and lactation phases. During ophthalmological examinations the incidence of either diffuse or reticulate corneal opacities and of corneal neovascularization was increased in both P0 and P1 males and females at 300 and 3000 ppm. There was no treatment-related effect on body weight in the P0 males or females. In the P1 adults, body weight and body weight gain were statistically significantly reduced in males at 300 and 3000 ppm, and body weight was statistically significantly reduced in females at 3000 ppm, for a final reduction in body weight at 3000 ppm at the end of the pre-mating period of 12% in males and 7% in females. There was no statistically significant effect on body weight at any dose during gestation in either the P0 or the P1 dams. During lactation Days 0-4, body weight gain of P0 dams at 3000 ppm and P1 dams at 300 and 3000 ppm was statistically significantly reduced compared to controls. These effects on body weight were considered to be treatment-related. There was no effect on food consumption during the pre-mating period in either male or female rats in the P0 or the P1 generation. Food consumption during lactation Days 0-4 was slightly but statistically non-significantly reduced at 3000 ppm in P0 dams and at 300 and 3000 ppm in P1 dams. This effect on food consumption was evaluated as related to treatment.

There was no treatment-related effect on either oestrus cyclicity or on any sperm analysis parameters in either the P0 or the P1 females or males, respectively. There were no treatment-related effects on any index of reproductive performance in either the P0 or the P1 adults. Both gestation and rearing indices were slightly decreased at 3000 ppm in the P1 adults, but these decreases were due to deaths not related to administration of the test substance, and therefore were themselves not related to treatment.

In P0 males, absolute kidney and liver weight were increased at 3000 ppm, and absolute thyroid weight was increased at 300 and 3000 ppm. Relative kidney, liver, and thyroid weights were increased in P0 males at 300 and 3000 ppm. There were slight increases in P0 females at 300 and 3000 ppm in absolute and relative kidney and liver weights. In P1 males, there were increases in absolute and relative liver, kidney, and thyroid weights at all doses which showed no relationship to dose. In P1 females, a dose-related increase in the absolute weight of liver and adrenals was seen only at 3000 ppm. Relative kidney, liver, and adrenal weight was increased in females at 3000 ppm. There were no treatment-related macroscopic findings in P0 male or female rats at any dose. In P1 males, one rat at 300 ppm and 14 males at 3000 ppm showed eye opacities. Two P1 females at 3000 ppm also showed eye opacities. There were no other treatment-related findings in P1 male or female rats.

There were no treatment-related effects on organs of the reproductive tract.

Treatment-related effects were observed in eyes and thyroid of both males and females of the P0 and P1 generations, and in pituitary, kidneys, and liver of P0 and P1 males only. Findings in the eyes, at 300 and 3000 ppm in both males and females of the P0 and P1 generations, included keratitis and vascularization, correlating with in-life observations. Thyroid findings included colloid alteration at all doses in male P0 and P1 animals and females of the P0 generations and from 300 ppm in P1 females, pigment deposition at all doses in P0 and P1 males and from 300 ppm in P0 and P1 females, and increased follicular cell hypertrophy in P0 males at 3000 ppm and P1 males from 3000 ppm and P0 females at 3000 ppm. Eosinophilic inclusions were increased in the anterior pituitary of males at 300 and 3000 ppm in both the P0 and P1 generations. Findings in the liver included hepatocellular hypertrophy, cellular alteration, and slight increases in periportal fat accumulation in P0 and P1 males at 300 and 3000 ppm. The kidneys of males in the P0 and P1 generation had increased incidence and / or severity of basophilic tubules and tubular dilation, in a dose-related manner from 300 ppm onwards.

Offspring findings

Dietary administration of the test substance did not affect number of implantations or prenatal loss. There was no effect on live birth index or on the ratio of males to females at any dose level. Viability indices were slightly, statistically non-significantly reduced in F2 pups at all doses. However, these decreases were considered not to be treatment-related through 300 ppm, as they were generally within the historical control range and at 30 ppm due to total litter loss of two dams before Day 4 of lactation. There was no effect on ano-genital distance in either males or females in the F2 generation. The only treatment-related clinical sign in pups was the finding of animals cold to the touch, which was increased in F1 pups at 3000 ppm only (12 pups) compared to control (5 pups). There were no treatment-related findings in F2 pups. Ophthalmological examination of pups at the time of weaning on postnatal Day 28 showed an increased incidence of corneal opacity and / or corneal neovascularization at 300 and 3000 ppm in both F1 and F2 pups, with a dose-related increase. Both litter (mean body weight for males and females taken together) and pup (mean body weight for males or females, taken separately) body weights were statistically significantly decreased in the F1 and F2 pups at 3000 ppm at weaning on Day 28. In F2 females at 3000 ppm, body weight was also statistically significantly decreased at lactation Day 21. These effects on pup and litter body weight were considered to be treatment-related. Treatment-related, statistically significant delays of preputial separation at 300 and 3000 ppm, and marginal delays of vaginal opening at 3000 ppm, were observed in the F1 pups.

There were no treatment-related effects on organ weights in either F1 or F2 weanlings. Absolute brain weight was statistically significantly decreased in male and female F1 and F2 weanlings. However, relative brain weight was unaffected, and thus the decrease in absolute brain weight was considered to be due to decreased body weight. Absolute spleen weights were statistically significantly decreased in F1 male and female weanlings at 3000 ppm, and in males relative spleen weight was also slightly but statistically significantly decreased. However, this effect was not observed in the F2 weanlings and thus was considered not to be treatment-related. Macroscopic findings: Corresponding with the increased mortality during lactation at 3000 ppm in the F2 pups compared to controls, treatment-related findings in this group at gross necropsy included increased incidence of autolysis and no milk in the stomach. The incidence of dilated and / or enlarged kidneys was increased at 3000 ppm in F1 and F2 pups.

Conclusions

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).

 

Effects on developmental toxicity

Description of key information

Developmental toxicity study (OECD 414), rats:

NOAEL maternal animals: 10 mg/kg bw/day

NOAEL fetuses: 10 mg/kg bw/day

NOAEL fetal abnormalities: 300 mg/kg bw/day (highest dose tested)

Developmental toxicity study (OECD 414), rabbits:

NOAEL maternal animals: 75 mg/kg bw/day

NOAEL fetuses: 10 mg/kg bw/day

NOAEL fetal abnormalities: 250 mg/kg bw/day (highest dose tested)

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02 Sep 2003 to 01 Jan 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

adopted 22 Jan 2001

Deviations:

yes

Remarks:

animals were housed with a16-hour light period

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

adopted 25 Jun 2018

Deviations:

yes

Remarks:

see "principles of method if other than guideline"

Principles of method if other than guideline:

Deviations to OECD guideline 414 (2018): Diet not analysed (phytoestrogens); no investigations on thyroid weight and histopathology; reproductive tract of fetuses or cryptorchidism not examined; no comparison between external vs. internal (gonadal) sex morphology; animals were housed with a16-hour light period.

GLP compliance:

yes (incl. QA statement)

Remarks:

Groupe Interministeriel des Produits Chimiques, Paris, France

Limit test:

no

Species:

rabbit

Strain:

New Zealand White

Remarks:

KBL (NZW) IOPS/SPF

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Elevage Scientifique des Dombes (ESD), Chatillon sur Chalaronne, France
- Age at study initiation: approximately 18 weeks
- Fasting period before study: no
- Housing: individually in polycarbonate cages on a perforated cage floor
- Diet: laboratory animal pellets 110C-10 from S.A.F.E. (Scientific Animal Food and Engineering, Augy, France), ad libitum
- Water: municipal tap water, ad libitum
- Acclimation period: 4 - 5 days prior to first treatment

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 - 21
- Humidity (%): 40 - 70
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 8/16

IN-LIFE DATES: From: 02 Sep 2003 To: 22 Oct 2003

Route of administration:

oral: gavage

Vehicle:

other: 0.5% aqueous solution of methylcellulose 400 (Fluka, Mulhouse, France)

Details on exposure:

PREPARATION OF DOSE FORMULATIONS:
Formulations were prepared six times during the study and stored at approximately 5°C (± 3°C).
The suspensions were mixed continuously before and during treatment with an electromagnetic stirrer.

VEHICLE
- Amount of vehicle: 4 mL/kg bw

Details on analytical verification of doses or concentrations:

Analysis of dose formulations was performed using High Performance Liquid Chromatography (HPLC) with UV detection.
Homogeneity of the suspensions was checked during the first formulation for the lowest and highest concentrations. All concentrations were checked for all formulations. Stability of the compound in suspension in the vehicle was determined before the start of the study at 0.1 and 250 g/L in 0.5% aqueous methylcellulose in a previous range-finding study, where the test substance was found to be stable over a 29-day period under the conditions of study utilization.

Homogeneity and concentration of dosing suspensions of the test substance were between 91 and 106% of nominal values and within the in-house target range of 90 to 110% of nominal concentration.

Details on mating procedure:

- Impregnation procedure: purchased timed pregnant
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: observed copulation referred to as day 0 of pregnancy

Duration of treatment / exposure:

Gestation day (GD) 6 to 28

Frequency of treatment:

once daily, 7 days a week

Duration of test:

23 days of treatment

Dose / conc.:

10 mg/kg bw/day

Dose / conc.:

75 mg/kg bw/day

Dose / conc.:

250 mg/kg bw/day

No. of animals per sex per dose:

25 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The range of dosages was selected in agreement with the Sponsor Representative and based on the results obtained in a previous range finding study in pregnant rabbits (Wason, 2002), where animals received doses of 0, 75, 250, 500, 750 and 1000 mg/kg bw/day from GD 6 to GD 28.
- Rationale for animal assignment: On each day of mating, the females were allocated to control and treated groups using a computerized randomization procedure. Body weight means were checked to ensure similar means among all groups.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were examined from GD 0 through GD 21. All cages were checked for dead or moribund animals twice daily, once in the morning and again in the afternoon (except at weekends and public holidays when checking was carried out once daily). Clinical signs were recorded once daily.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: GD 3, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26 and 29.

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

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29
- Organs examined: visceral organs, the number of ribs recorded and the kidney and urinary bladder examined for the presence of gritty material

OTHER:
- Liver weights were recorded and the liver was preserved in 10% neutral buffered formalin, but no histopathological examination was conducted

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: Uterine horn(s) without visible implantations were immersed in a 20% solution of ammonium sulfide according to the SALEWSKI method in order to visualize any sites which were not apparent

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: half per litter

Statistics:

Bartlett test was performed to compare the homogeneity of group variances. If the Bartlett test was not significant, means were compared using the analysis of variance (ANOVA). If the ANOVA was not significant, the group means were considered homogeneous and no further analysis was performed. If the ANOVA was significant, a Dunnett test (2-sided) was performed. If the Bartlett test was significant for body weight change and corrected body weight change, a Kruskal-Wallis test was performed. If the Kruskal-Wallis test was not significant, group means were considered homogeneous and no further analysis was performed. If the Kruskal-Wallis test was significant, means of exposed groups were compared to the mean of the control group using the Dunn test (2-sided).
If the Bartlett test was significant for food consumption or for liver weight, a log transformation of the data was performed.
-If the Bartlett test on log transformed data was not significant, means were compared using the ANOVA on log transformed data. If the ANOVA was not significant, the group means were considered homogeneous and no further analysis was performed. If the ANOVA was significant, a Dunnett test (2-sided) on log transformed data was performed.
- If the Bartlett test on log transformed data was significant even after transformation, group means were compared using the non-parametric Kruskal-Wallis test. If the Kruskal-Wallis test was not significant, the group means were considered homogeneous and no further analysis was performed. If the Kruskal-Wallis test was significant, means of exposed groups were compared to the mean of the control group using the Dunn test (2-sided).
For fetal sex and fetal death status, groups were compared using the Chi-square test for fetal sex parameter, using the Fisher Exact test (2-sided) for fetal death status parameter.

Indices:

- Pre-implantation loss
- Post-implantation loss
- Number of live fetuses
- Number of dead fetuses
- Percentage of dead fetuses per litter
- Percentage of male fetuses per litter
- Mean fetal body weight per litter
- Mean fetal body weight per group
- Mean fetal body weight per sex
- Percentage of fetuses affected per litter
- Mean percentage of fetuses affected per group

Historical control data:

Historical control data on fetal morphology data for six studies conducted in the laboratory in New Zealand White rabbits are available and presented in the report (see Attachment 7). Additional historical control data on fetal body weights from seven studies were provided in a separate document (M-284605-01-1, see Attachment 8).

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

All 25 females at 250 mg/kg bw/day were observed with intense yellow sediment in the urine on one or more occasion. In addition, at this dosage five females had few faeces on a number of occasions, even through this finding is very common in this strain, the incidence at the high dose is slightly higher than would be expected by chance. At 75 mg/kg bw/day, one female had intense yellow sediment in the urine on two occasions and one female beige sediment in the urine on four occasions.
No treatment-related signs were noted at 10 mg/kg bw/day.

See Attachment 1 for a summary of clinical findings (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, treatment-related

Description (incidence and severity):

At 250 mg/kg bw/day, overall body weight change was reduced by 31% between GD 6 to 29. The effect was most pronounced between GD 8 to 10 when body weight remained static compared with a 0.03 kg body weight gain in the controls, the difference was statistically significant (p ≤ 0.01). Body weight change was unaffected by treatment at 75 or 10 mg/kg bw/day.
Corrected maternal body weight change (maternal body weight change independent of the uterine weight) was more pronounced at 250 mg/kg bw/day (-0.21 kg) compared with the controls (-0.11 kg), though not statistically significant. Corrected maternal body weight change at 75 or 10 mg/kg bw/day was comparable with the control group.

See Attachment 2 for a summary of body weight and body weight gain data (attached background material).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

At 250 mg/kg bw/day, food consumption was reduced between each interval phase from GD 6 to 29 by between 9 and 24%, the effect being statistically significant (p ≤ 0.01) on several occasions, when compared with the controls. Food consumption was unaffected by treatment at 75 or 10 mg/kg bw/day.

See Attachment 3 for a summary of food consumption data (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:

not examined

Description (incidence and severity):

Not applicable.

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):

Liver weight was increased by 22% (125.3 g, p<0.01) at 250 mg/kg bw/day, in comparison with the control group (102.6 g).
There was no effect on liver weight at 75 or 10 mg/kg bw/day.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Prominent lobulation of the liver was recorded in 2/25 females at 250 mg/kg bw/day.
There was an isolated incidence of prominent lobulation in the liver at both 75 and 10 mg/kg bw/day. However, in isolation, with no corresponding liver weight effect or dose relationship, this finding is considered to be fortuitous at these two dose levels. Accordingly, no treatment-related macroscopic findings were noted at 75 or 10 mg/kg bw/day.

Neuropathological findings:

not examined

Description (incidence and severity):

Not applicable.

Histopathological findings: non-neoplastic:

not examined

Description (incidence and severity):

Not applicable.

Histopathological findings: neoplastic:

not examined

Description (incidence and severity):

Not applicable.

Other effects:

not examined

Description (incidence and severity):

Not applicable.

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

no effects observed

Other effects:

not examined

Description (incidence and severity):

Not applicable.

Details on maternal toxic effects:

Adverse maternal effects were limited to few faeces, reduced food consumption, reduced body weight gain and increased liver weights at 250 mg/kg bw/day.

Key result

Dose descriptor:

NOAEL

Effect level:

75 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: no adverse effects noted at 75 mg/kg bw/day

Key result

Dose descriptor:

LOAEL

Effect level:

250 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

organ weights and organ / body weight ratios

Key result

Abnormalities:

no effects observed

Description (incidence and severity):

No organ changes were noted.

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 250 mg/kg bw/day male fetal weights were reduced by 14%, female fetal weights by 16% and body weight for the combined sexes by 15%. In each case the effect was statistically significant (p ≤ 0.01).

Slightly reduced fetal weights noted at 75 and 10 mg/kg bw/day were considered to be chance findings in the light of the slight effect, lack of dose response and the fact that values were within the in-house historical control range. In additional position papers (M-284605-01-1 and M-301557 01-1) on this topic it is further mentioned that control fetal weight in this study is slightly above the historical control studies in terms of mean body weights and percentile ranges, and thus the slight difference between this control group and the 10 and 75 mg/kg bw/day groups are not due to an effect of treatment on fetal body weights. Instead, they are due to concurrent control values which are slightly higher than normal.

See Attachment 4 for a summary of fetal body weights (attached background material).

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Anogenital distance of all rodent fetuses:

not examined

Description (incidence and severity):

Not applicable.

Changes in postnatal survival:

not examined

Description (incidence and severity):

Not applicable.

External malformations:

effects observed, treatment-related

Description (incidence and severity):

The number of runt fetuses (bw < 28.0 g) was clearly increased at 250 mg/kg bw/day, where the mean percentage of fetuses classified as runts was 20.1% compared with 5.2% in the control group. At 75 mg/kg bw/day, the mean percentage of runt fetuses was very similar to the control value, whereas at 10 mg/kg bw/day the mean percentage was higher. However, in the absence of a dose-related response, the apparent effect at 10 mg/kg bw/day is considered to be incidental.

The following anomalities were considered incidental due to their isolated occurrence and/or coverage by historical control data: one incidence of umbilical hernia at 250 mg/kg bw/day, two incidences of fetuses (from one litter) with malrotated forepaws at 250 mg/kg bw/day.

See attachment 5 for a summary of external fetal observations (attached background material).

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

A small number of malformations were recorded and were confined to the 10 mg/kg bw/day dose level and the control group. In the absence of findings at the two higher dose levels, these malformations are regarded as incidental.

A few treatment-related altered ossification patterns and increased incidence of minor skeletal variants were noted at 75 and 250 mg/kg bw/day:

At 250 mg/kg bw/day, the following anomaly; 13th thoracic rib (unilateral/bilateral) with the presence of 27 pre-sacral vertebrae, was increased compared with the control group. At this dosage, the incidence of the following variants was higher than in the controls: atlas centrum unossified, extra ossification site between the atlas and axis, 13th thoracic rib (unilateral/bilateral) or 13th thoracic rib (bilateral, short unilaterally), 1st metacarpal incomplete ossification or unossified, insertion point(s) of pelvic girdle on 2nd sacral vertebra, and bilateral incomplete ossification of the pubis.

At 75 mg/kg bw/day, the following anomaly was slightly increased; 13th thoracic rib (unilateral/bilateral) with the presence of 27 pre-sacral vertebrae. In addition, a number of variants were increased including; extra ossification site between the atlas and axis, 13th thoracic rib (unilateral/bilateral) or 13th thoracic rib (bilateral, short unilaterally), and insertion point(s) of pelvic girdle on 2nd sacral vertebra.

At 10 mg/kg bw/day, there was slight increase in the variant finding 13th thoracic rib (unilateral/bilateral) or 13th thoracic rib (bilateral, short unilaterally), which was marginally outside the in-house historical control range. However, the percentage of dams having 13/13 ribs or 12/13 ribs was 79%, 83%, 96% and 77% at 0, 10, 75 and 250 mg/kg bw/day, respectively, confirming that this finding is a common variant at both the maternal and fetal level.

No other variants or anomalies are considered to be related to treatment as they occurred at a similar frequency in the control group, did not occur in a dose-related manner or were within in-house historical control ranges.

See attachment 6 for a summary of fetal skeletal observations (attached background material).

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

The few malformations noted were distributed across the groups including the control group, and in the absence of a dose-related increase, none of the malformations observed are considered to be treatment-related. A small number of variants and anomalies appeared across the groups, including controls, none of the findings occurred in a dose-related manner.

Other effects:

not examined

Description (incidence and severity):

Not applicable.

Details on embryotoxic / teratogenic effects:

No treatment related findings indicative of teratogenicity were seen in th study. Adverse fetal effects included reduced fetal weight and increased incidence of runt fetuses at 250 mg/kg bw/day, as well as treatment-related altered ossification patterns and increased incidence of minor skeletal variants at 75 and 250 mg/kg bw/day.

Key result

Dose descriptor:

NOAEL

Effect level:

10 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects were noted at 10 mg/kg bw/day

Key result

Dose descriptor:

LOAEL

Effect level:

75 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

skeletal malformations

Key result

Abnormalities:

no effects observed

Description (incidence and severity):

No treatment-related fetal malformations were noted.

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

250 mg/kg bw/day

Treatment related:

yes

Relation to maternal toxicity:

developmental effects as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

yes

Conclusions:

The study was performed under GLP conditions and according to OECD TG 414 (adopted 2001). Clinical signs were limited to yellow or beige sediments in the urine of several animals at 75 and 250 mg/kg bw/day. Maternal body weight change was decreased at 250 mg/kg bw/day, as well as a decrease in corrected maternal body weight change at this dose. Food consumption was reduced at 250 mg/kg bw/day throughout the study. Maternal liver weight was increased at 250 mg/kg bw/day. Per group, the percentage of dams with more than 24 ribs ranged from 77% to 96%, indicating that the majority of healthy, successfully reproducing adults commonly have either 25 or 26 ribs instead of the 24 ribs which are commonly accepted as the "correct" number of ribs. Fetal body weight was decreased at 250 mg/kg bw/day, and the incidence of runt fetuses was increased at 250 mg/kg bw/day. The only treatment-related findings in the pups which were increased by administration of the test substance were decreases or delays in ossification and increased incidence of 13th thoracic ribs. These findings were considered to be related - as was demonstrated in the rat - to increased maternal plasma tyrosine concentration after administration of an HPPDase inhibitor. Based on decreased maternal body weight and food consumption, the maternal LOAEL was 250 mg/kg bw/day. The maternal NOAEL in the rabbit was 75 mg/kg bw/day. Due to decreased ossification and other minor skeletal findings, the fetal LOAEL was 75 mg/kg bw/day, while the NOAEL was 10 mg/kg bw/day

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 Oct 2003 - 30 Jan 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

adopted 22 Jan 2001

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

adopted 25 Jun 2018

Deviations:

yes

Remarks:

see "principles of method if other than guideline"

Principles of method if other than guideline:

Deviations to OECD guideline 414 (2018): Diet not analysed (phytoestrogens); no investigations on the thyroid (weight, histopathology, no T4, T3/TSH levels measured); anogenital distance of pups not measured; reproductive tract of fetuses or cryptorchidism not examined; no comparison between external vs. internal (gonadal) sex morphology.

GLP compliance:

yes (incl. QA statement)

Remarks:

Groupe Interministeriel des Produits Chimiques, Paris, France

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl: CD (SD)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, St Germain-sur-l'Arbresle, France
- Weight at study initiation: 226 - 308 g
- Fasting period before study: no
- Housing: individually in suspended, stainless steel, wire mesh cages
- Diet: certified rodent pelleted and irradiated diet A04C-10 from S.A.F.E. (Scientific Animal Food and Engineering, Epinay-sur-Orge, France, ad libitum
- Water: municipal water, ad libitum
- Acclimation period: 19 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 40 - 70
- Air changes (per hr): target of 15 (not monitored)
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 28 Oct 2003 To: 18 Nov 2003

Route of administration:

oral: gavage

Vehicle:

other: 0.5% aqueous solution of methylcellulose 400 (Fluka, Mulhouse, France)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulations were prepared twice during the study and stored at approximately 5°C (± 3°C).
The suspensions were mixed continuously before and during treatment with an electromagnetic stirrer.

VEHICLE
- Amount of vehicle (if gavage): 10 mL/kg bw

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 of the suspensions was checked on the first formulation (F1) for the lowest and the highest concentrations (1 and 30 g/L). In addition, the intermediate concentration of the first formulation (F1) and all concentrations of the second formulation (F2) were checked. Stability of the test substance in suspension in the vehicle at concentrations of 0.1 and 250 g/L was determined in a previous range-finding study, and was found to be stable for 29 days under similar conditions to those of the current study.

Homogeneity and concentration checks of dosing suspensions were between 91 and 104% of nominal values, which is within the in-house target range of 90 to 110% of nominal.

Details on mating procedure:

- Impregnation procedure: cohoused with stock males of the same strain and same supplier
- M/F ratio per cage: 1/1
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy

Duration of treatment / exposure:

Gestation day (GD) 6 to 20

Frequency of treatment:

once daily, 7 days a week

Duration of test:

15 days of treatment

Dose / conc.:

10 mg/kg bw/day

Dose / conc.:

100 mg/kg bw/day

Dose / conc.:

300 mg/kg bw/day

No. of animals per sex per dose:

25 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The range of doses was selected in agreement with the Sponsor Representative and based on results obtained in a range-finding study (Langard-Lerche, 2002), where pregnant rats received 0, 25, 75, 250, 500 or 800 mg/kg bw/day. The high dose of 300 mg/kg bw/day was expected to cause slight maternal toxicity and possibly slight developmental toxicity, but not exceed a Maximum Tolerated Dose (MTD). The mid dose of 100 mg/kg bw/day was considered to be a suitable intermediate dose, should the high dose prove to be too high. The low dose of 10 mg/kg bw/day was expected to be a clear No Observed Effect Level (NOEL) for both maternal and developmental toxicity.

- Rationale for animal assignment (if not random): The females were assigned to control and treated groups using a body weight procedure for each day of pairing. Body weight means were checked after the mating period to ensure similar means among all groups.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were examined from GD 0 through GD 21. All cages were checked for dead or moribund animals twice daily, once in the morning and again in the afternoon (except at weekends and public holidays when checking was carried out once daily). Clinical signs were recorded once daily.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: GD 0, 6, 8, 10, 12, 14, 16, 18 and 21.

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

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21
- Organs examined: visceral organs, the number of ribs recorded and the kidney and urinary bladder examined for the presence of gritty material

OTHER:
- Liver weights were recorded and the liver was preserved in 10% neutral buffered formalin, but no histopathological examination was conducted

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: Uterine horn(s) without visible implantations were immersed in a 10% solution of ammonium sulfide according to the SALEWSKI method in order to visualize any sites which were not apparent

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter

Statistics:

Bartlett test was performed to compare the homogeneity of group variances. If the Bartlett test was not significant (p > 0.05), means were compared using the analysis of variance (ANOVA). If the ANOVA was not significant (p > 0.05), the group means were considered homogeneous and no further analysis was performed. If the ANOVA was significant (p ≤ 0.05), a Dunnett test (2-sided) was performed. If the Bartlett test was significant (p ≤ 0.05) for body weight change and corrected body weight change, a Kruskal-Wallis test was performed. If the Kruskal-Wallis test was not significant (p > 0.05), group means were considered homogeneous and no further analysis was performed. If the Kruskal-Wallis test was significant (p ≤ 0.05), means of exposed groups were compared to the mean of the control group using the Dunn test (2-sided).
If the Bartlett test was significant (p ≤ 0.05) for food consumption or for liver weight, a log transformation of the data was performed.
-If the Bartlett test on log transformed data was not significant (p > 0.05), means were compared using the ANOVA on log transformed data. If the ANOVA was not significant (p > 0.05), the group means were considered homogeneous and no further analysis was performed. If the ANOVA was significant (p ≤ 0.05), a Dunnett test (2-sided) on log transformed data was performed.
- If the Bartlett test on log transformed data was significant even after transformation (p ≤ 0.05), group means were compared using the non-parametric Kruskal-Wallis test. If the Kruskal-Wallis test was not significant (p > 0.05), the group means were considered homogeneous and no further analysis was performed. If the Kruskal-Wallis test was significant (p ≤ 0.05), means of exposed groups were compared to the mean of the control group using the Dunn test (2-sided).
For fetal sex and fetal death status, groups were compared using the Chi-square test for fetal sex parameter, using the Fisher Exact test (2-sided) for fetal death status parameter.

Indices:

- Pre-implantation loss
- Post-implantation loss
- Number of live fetuses
- Number of dead fetuses
- Percentage of dead fetuses per litter
- Percentage of male fetuses per litter
- Mean fetal body weight per litter
- Mean fetal body weight per group
- Mean fetal body weight per sex
- Percentage of fetuses affected per litter
- Mean percentage of fetuses affected per group

Historical control data:

Historical control data for six studies conducted in the laboratory in Sprague-Dawley CD rats are available and presented in the report (see Attachment 7).

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

In the high dose group, all females had increased salivation, 7/25 had intense yellow urine, 5/25 had vaginal discharge and 8/25 anogenital soiling, on one or more occasions. In the mid dose group, 7/25 females had increased salivation and 1/25 females had brown soiling around the anogenital region, on at least one occasion. No treatment-related signs were observed at the low dose.
All other clinical signs were considered to be incidental and not related to treatment.

See Attachment 1 for a summary of clinical signs (attached background material).

Dermal irritation (if dermal study):

not examined

Description (incidence and severity):

Not applicable.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One control female was killed in a moribund condition on GD 17 due to accidental trauma.
There were no other mortalities during the course of the study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weight gain was reduced by 84% (p ≤ 0.01) at 300 mg/kg bw/day and by 53% (p ≤ 0.05) at 100 mg/kg bw/day between GD 6 to 8, when compared with the controls. Thereafter, body weight gain between each interval phase was essentially comparable between the groups, though the initially observed reduction in absolute body weight observed at the two highest dose levels was maintained throughout treatment.
No effect on body weighs was observed at 10 mg/kg bw/day.

Maternal corrected body weight gain was 16% less (p ≤ 0.05) at 300 mg/kg bw/day than in the control group, but was unaffected by treatment at the lower two dose levels.

See Attachment 2 for body weight and body weight gain for pregnant animals (attached background information).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption was reduced between GD 6 and 16 at 300 mg/kg bw/day, compared with controls. The effect was most pronounced between GD 6 to 8, when there was a 15% reduction (p ≤ 0.01) and GD 8 to 10 when the reduction was 7% (p ≤ 0.05).
At 100 and 10 mg/kg bw/day, food consumption was comparable with the controls throughout treatment.

See Attachment 3 for summary data of maternal food consumption (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:

not examined

Description (incidence and severity):

Not applicable.

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):

There was a minimal increase in liver weight of 6% (p ≤ 0.05) at 300 mg/kg bw/day and a trend towards increased liver weight (+5%, not statistically significant) at 100 mg/kg bw/day.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

One high dose female had yellow sediment in the kidney with a gritty content found in the urinary bladder.
The few other macroscopic findings were considered to be incidental as they did not occur in a dose-related manner and are common findings in this age and strain of rat.

Neuropathological findings:

not examined

Description (incidence and severity):

Not applicable.

Histopathological findings: non-neoplastic:

not examined

Description (incidence and severity):

Not applicable.

Histopathological findings: neoplastic:

not examined

Description (incidence and severity):

Not applicable.

Other effects:

not examined

Description (incidence and severity):

Not applicable.

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

not examined

Description (incidence and severity):

Not applicable.

Changes in number of pregnant:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Grossly enlarged placentas were observed for two and four fetuses from the same litter in the mid and high dose, respectively.

Details on maternal toxic effects:

Adverse maternal effects were limited to clinical signs and reduced body weight gain (days 6 and 8) at 100 and 300 mg/kg bw/day, as well reduced food consumption between gestation days 6 and 16 at 300 mg/kg bw/day.

Key result

Dose descriptor:

NOAEL

Effect level:

10 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: no adverse effects observed at 10 mg/kg bw/day

Key result

Dose descriptor:

LOAEL

Effect level:

100 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

Key result

Abnormalities:

no effects observed

Description (incidence and severity):

No organ changes were noted.

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean fetal body weights (calculated by sex and combined sex) were reduced by 5 - 6% at 300 mg/kg bw/day. The change was statistically significant (p ≤ 0.01) for each parameter. At 100 mg/kg bw/day, there was 2% reduction for each parameter compared with controls, the effect being statistically significant (p ≤ 0.05) for male body weights.
Mean fetal body weights were unaffected by treatment at 10 mg/kg bw/day.

See Attachment 4 for summary data of fetal body weights (attached background material).

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Anogenital distance of all rodent fetuses:

not examined

Description (incidence and severity):

Not applicable.

Changes in postnatal survival:

not examined

Description (incidence and severity):

Not applicable.

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

At 300 mg/kg bw/day, there were four fetuses from two different litters (3 fetuses from one litter and 1 fetus from a second litter), with the malformation hindpaw polydactyly. The three fetuses from the same litter at 300 mg/kg/day with hindpaw polydactyly also had an anomaly consisting of a combination of forelimb hyperflexion, digits on forepaws misshapen and digits on both forepaws and hindpaws malpositioned. The low incidence of this finding, the lack of other related findings in either forepaws or hindpaws in further fetuses, and the lack of ossification of the extra phalanges in the 2 fetuses of these 4 which were examined by skeletal staining suggest that this malformation was due to chance and was not an indication of a teratogenic potential of the test substance.

The only other malformation observed was a single fetus with an umbilical hernia at the low dose, which was therefore considered to be incidental.

See Attachment 5 for a summary of external fetal observations (attached background material).

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

The only malformations observed were in two fetuses from the same litter at 300 mg/kg bw/day, which consisted of a combination of short tibia and femur, supernumerary cartilaginous phalanges, fused cartilage for a number of metatarsals and phalanges, and unossified 5th metatarsal. These two fetuses were two of the four fetuses which had the external observations hindpaw polydactyly. At such a low incidence these findings are considered to be incidental.
The number of other anomalies noted was few and either occurred in a non dose-related manner, in isolation or were inside the in-house historical control range and are therefore considered to be chance findings.

At 300 mg/kg bw/day, the following variants occurred at a higher incidence than in the controls: enlarged fontanelle, 5th or 6th sternebra unossified, extra ossification point on the 14th thoracic vertebra, and incomplete ossification of the 5th metacarpal. At both 300 and 100 mg/kg bw/day, the following variants occurred with a higher frequency than in controls and in a dose-related manner: incompletely ossified or bipartite 5th and/or 6th sternebra, 3rd and/or 4th proximal phalanges of the forepaw unossified, and short 14th thoracic rib. At all three dose levels tested the following variants were increased in a dose-related manner, though the effects at 10 mg/kg bw/day were slight: 7th cervical centrum unossified, 1st metatarsal unossified, and less than 9 sacrocaudal vertebrae ossified.
No other variants were considered to be related to treatment as they occurred at a similar frequency in the control group, did not occur in a dose-related manner or were within in-house historical control ranges.

See Attachment 6 for a summary of skeletal fetal observations (attached background material).

Visceral malformations:

no effects observed

Other effects:

no effects observed

Details on embryotoxic / teratogenic effects:

No treatment related findings indicative of teratogenicity were seen in th study. Adverse fetal effects included decreased fetal body weight and delays in ossification at 100 and 300 mg/kg bw/day. The sekeletal variations were characterized by enlarged fontanelle, 5th or 6th sternebra unossified, extra ossification point on the 14th thoracic vertebra, and incomplete ossification of the 5th metacarpal at 300 mg/kg bw/day. At both 100 and 300 mg/kg bw/day, incompletely ossified or bipartite 5th and/or 6th sternebra, 3rd and/or 4th proximal phalanges of the forepaw unossified, and short 14th thoracic rib. At all three dose levels tested the following variants were increased in a dose-related manner, though the effects at 10 mg/kg bw/day were slight: 7th cervical centrum unossified, 1st metatarsal unossified, and less than 9 sacrocaudal vertebrae ossified.

Key result

Dose descriptor:

NOAEL

Effect level:

10 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects were noted at 10 mg/kg bw/day

Key result

Dose descriptor:

LOAEL

Effect level:

100 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

skeletal malformations

Key result

Abnormalities:

no effects observed

Description (incidence and severity):

No treatment-related fetal malformations were noted.

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

100 mg/kg bw/day

Treatment related:

yes

Relation to maternal toxicity:

developmental effects as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

yes

Conclusions:

The study was performed under GLP conditions and according to OECD TG 414 (adopted 2001). Based on decreased maternal body weight gain between gestation days 6 and 8, as well as clinical signs observed at 100 and 300 mg/kg bw/day, the maternal NOAEL in this study was considered to be 10 mg/kg bw/day. The fetal effects observed included decreased fetal body weight and delays in ossification, and therefore the NOAEL was 10 mg/kg bw/day.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

10 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The available information comprises adequate and reliable studies (Klimisch score 1), and are thus sufficient to fulfil the standard information requirements set out in Annex IX, 8.7, of Regulation (EC) No 1907/2006.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Toxicity to reproduction: other studies

Additional information

Reliable studies on developmental toxicity / teratogenicity are available in rats and rabbits.

Developmental toxicity study in the rat

In this study - that was performed under GLP and according to OECD 414 (M-267036-01-2) - the test substance was administered to groups of 25 female Sprague Dawley rats at doses of 10, 100, and 300 mg/kg bw/day on gestation Days 6 through 20. On gestation Day 21, the rats were sacrificed and subjected to macroscopic examination. The number of ribs was counted and liver weight was measured. Gravid uterine weight was measured, and the number of corpora lutea, implantations, resorptions, and live and dead fetuses, and the sex and individual body weights of live fetuses were determined. Live fetuses were killed by subcutaneous injection of sodium pentobarbital and examined for external observations. Approximately half of the fetuses from each litter were preserved for free-hand sectioning, while the other half were skinned, eviscerated, and stained in alizarin red S and alcian blue. Fetal observations are classified as common variants (changes occurring in more than approximately 5% of the control population), minor anomalies (slight, relatively rare structural changes that are not obviously detrimental), and malformations (very rare or obviously lethal changes).

There were no treatment-related mortalities during the study. Treatment-related clinical signs were observed at 100 and 300 mg/kg bw/day, and included increased salivation, anogenital soiling, intense yellow urine, and vaginal discharge. There was no effect of treatment on pregnancy rate in any group. At 300 mg/kg bw/day, maternal body weight was slightly, and statistically not significantly, decreased during the latter part of gestation. Body weight change was statistically significantly decreased at 100 and 300 mg/kg bw/day in the interval of gestation Days 6-8. Thereafter, body weight change was similar across all groups. Maternal corrected body weight change was statistically significantly reduced at 300 mg/kg bw/day. There was a treatment-related, biologically and/or statistically significant decrease in food consumption at 300 mg/kg bw/day between gestation Days 6 and 16, compared to controls. The only treatment-related finding at gross necropsy was observed in one female at 300 mg/kg bw/day, which showed yellow sediment in the kidney and gritty content in the urinary bladder. Liver weight was statistically significantly increased in 300 mg/kg bw/day (15.6 g, p<0.05) and slightly increased at 100 mg/kg bw/day (15.4 g, not statistically significant) versus liver weight in control rats (14.7 g).

There were no treatment-related effects on the number of corpora lutea, implantation sites, either early or late resorptions, number of live fetuses, male/female ratio, or percent pre- or post-implantation loss per litter. Fetal body weight was biologically and/or statistically significantly decreased at 100 and 300 mg/kg bw/day.

Fetal external observations: A total of four fetuses at 300 mg/kg bw/day, from 2 different litters, presented the malformation of hindpaw polydactyly. The low incidence of this finding, the lack of other related findings in either forepaws or hindpaws, and the lack of ossification of the extra phalanges in the 2 fetuses of these 4 which were examined by skeletal staining suggest that this malformation was due to chance and was not an indication of a teratogenic potential of the test substance. One fetus at 10 mg/kg bw/day had the malformation of umbilical hernia, which was evaluated as not related to treatment. Three of the fetuses with hindpaw polydactyly (300 mg/kg bw/day), all of which were in the same litter, also showed an anomaly of forelimb hyperflexion, misshapen digits on the forepaws, and malpositioning of the digits on both the forepaws and the hindpaws. There were no other treatment-related anomalies or variants in any dose group. Examination of the fetuses for visceral findings revealed no malformations, and no treatment-related variants or anomalies, in any group. Fetal skeletal observations: Two of the four fetuses with the external observation of hindpaw polydactyly had malformations of short tibia and femur, supernumerary cartilaginous phalanges, fused cartilage for several metatarsals and phalanges, and unossified 5th metatarsal. These findings were considered not to be related to treatment, because of their low incidence. The only anomaly which was evaluated as related to treatment was an increase at 300 mg/kg bw/day of 27 pre-sacral vertebrae.

Treatment-related variants observed in this study were primarily linked to altered ossification.

Conclusions

Based on decreased maternal body weight gain between gestation Days 6 and 8, as well as clinical signs observed at 100 and 300 mg/kg bw/day, the maternal NOAEL in this study was considered to be 10 mg/kg bw/day. The fetal effects observed included decreased fetal body weight and delays in ossification, and therefore the NOAEL was 10 mg/kg bw/day.

Developmental toxicity study in the rabbit

In this study - that was performed under GLP and according to OECD 414 (M-266702-01-2) - the test item was administered by oral gavage to groups of 25 pregnant female New Zealand white rabbits at doses of 0, 10, 75, and 250 mg/kg bw/day from gestations Days 6 through 28, inclusive. On Day 29 of gestation, the rabbits were sacrificed and subjected to macroscopic examination. Liver weight and the number of ribs were noted for each animal. The gravid uterine weight, number of corpora lutea, number of implantations, resorptions, and live and dead fetuses, and the individual weight of live fetuses, were recorded for each animal. Live fetuses were sacrificed and examined externally. The heads of approximately half the fetuses were fixed for later examination. All fetuses were dissected for visceral findings, fixed, and stained for skeletal examination. Fetal observations were classified as common variants (changes occurring in more than approximately 5% of the control population), minor anomalies (slight, relatively rare structural changes that are not obviously detrimental), and malformations (very rare or obviously lethal changes).

There were no mortalities during the study. Yellow or beige sediments in the urine of females at 75 and 250 mg/kg bw/day were noted on several occasions, with the incidence greater at 250 mg/kg bw/day. The test substance is excreted in the urine and forms a yellow sediment, thus although this sediment was clearly treatment-related it was seen as an indication of compound excretion rather than as an adverse effect. There was no effect on pregnancy rate, with rates ranging from 88% to 96%. Maternal body weight gain at 250 mg/kg bw/day was statistically significantly reduced between Days 8 and 10, with an overall reduction between Days 6 and 29 of 31% compared to controls. Corrected maternal body weight at 250 mg/kg bw/day was slightly but statistically non-significantly decreased compared to controls. At 250 mg/kg bw/day, food consumption was consistently reduced compared to controls, with the reduction achieving statistical significance on several occasions. Liver weight was statistically significantly increased at 250 mg/kg bw/day (125.3 g, p ≤ 0.01) compared to controls (102.6 g). Prominent lobulation of the liver was noted at 250 mg/kg bw/day in 2 out of 25 females. Counting ribs in adult female rabbits revealed that the most common number of ribs was 26 (58% to 78% of dams per treatment group, 70% of all animals). As rib count is established prior to birth, this cannot be due to administration of the test substance. Instead, this data shows that rib count in rabbits can vary widely among groups, but that the general tendency is toward 26 ribs. At 250 mg/kg bw/day, fetal body weight was statistically significantly decreased both for combined sexes and for the sexes taken separately. This decrease was considered to be treatment-related. Although combined-sexes body weight was reduced at both 10 and 75 mg/kg bw/day, these decreases were within historical control data and did not show a dose-response relationship, and were therefore considered not to be related to treatment (M-284605-01-1). There was no effect of treatment on the number of corpora lutea, implant sites, early or late resorptions, number of live or dead fetuses, or percent pre- or post-implantation losses per dam. There was also no effect at any group on the percentage of male fetuses.

There was no effect of treatment on the incidence of external malformations. Two fetuses were observed to have malformations, one at 75 mg/kg bw/day with umbilical hernia and one in the control group with gastroschisis median, spina bifida, hindlimb hyperflexion, and acaudate. As these findings showed no dose-relationship, they were considered not to be treatment-related. The incidence of runt fetuses was biologically significantly increased at 250 mg/kg bw/day when compared to control animals. There were no findings at visceral examination which indicated an effect of treatment in any group. There were no treatment-related skeletal malformations observed. Further, a number of treatment-related skeletal anomalies and variants were recorded, the relevance of which were discussed in regulatory position paper M-301557-01-1. The incidence of unilateral or bilateral 13th ribs was increased in all dose groups. However, the incidence of this same finding in the dams ranged from 77-96% in the four study groups with no effect on reproductive capability or normal life, thus observation of more than 12 ribs on each side of the rib cage was considered not to be an adverse finding. In addition, an assessment of historical control data demonstrated that the incidence of 13 thoracic ribs and of insertion of the pelvic girdle on the second sacral vertebra were well within historical control range. Presence of an extra ossification site between the atlas and axis was not noted in any of the studies making up the historical control database and thus a statement regarding its relevance couldn’t be made.

Conclusions

Based on decreased maternal body weight and food consumption, the maternal LOAEL was 250 mg/kg bw/day. The maternal NOAEL in the rabbit was 75 mg/kg bw/day. Due to decreased ossification and other minor skeletal findings, the fetal LOAEL was 75 mg/kg bw/day, while the NOAEL was 10 mg/kg bw/day. The effects on fetal body weights and skeletal findings were considered to be related - as was demonstrated in the rat - to increased maternal plasma tyrosine concentration after administration of an HPPDase inhibitor.

Supplementary Study: Effects of tyrosinaemia on pregnancy and embryo-fetal development in the rat

The objectives of this study were to assess the relationship between increase in blood tyrosine level and potential effects on pregnancy and embryo-fetal development. The study was conducted under GLP and according to the OECD guideline 414 (M-263626-01-3), with the exception of visceral fetal examinations.

Tyrosinaemia was induced by a co-administration to sperm-positive females Sprague-Dawley rats of 2-(2-Nitro-4-trifluoromethylbenzoyl) cyclohexane-1,3-dione(NTBC), an inhibitor of 4-hydroxyphenylpyruvic acid dioxygenase, at 10 µg/kg bw/day by gavage from gestation day (GD) 6 to 20 and L-Tyrosine at 2% (w/w) in the diet from GD 6 to 21. Similar groups were co-administered vehicle alone (demineralized water) by gavage plus untreated diet (control group), or NTBC by gavage plus untreated diet, or vehicle alone by gavage plus diet supplemented with L-Tyrosine at the same dosage. The sperm-positive females were allocated to four groups (23 females per group); the sperm-positive day being GD 0. The volume of administration of NTBC preparation was 10 ml/kg bw based on the most recent body weight recorded.

Clinical observations were recorded daily. A detailed physical examination including careful examination of the eyes was conducted on GD 1, 8, 15 and 21. Maternal body weights were recorded for all females on GD 0, 6, 8, 10, 12, 14, 16, 18 and 21. Food consumption was also measured for all the females during the intervals GD 1-6, 6-8, 8-10, 10-12, 12-14, 14-16, 16-18 and 18-21. At scheduled sacrifice, on GD 21, blood samples were collected for determination of L-Tyrosine level. Liver was weighed fresh for pregnant females. Liver, thyroid gland and pancreas from all females were preserved in 10% neutral buffered formalin, whilst eyes were preserved in Davidson's fixative for possible histological examination. The gravid uterine weight was recorded and the dams were evaluated for number of corpora lutea, number and status of implantations (resorptions, dead and live fetuses). Live fetuses were removed from the uteri, counted, weighed, sexed and examined externally. All live fetuses were eviscerated, fixed in absolute ethanol and stained according to a modification of the Tyl and Marr technique, for skeletal examination of bone and cartilage on approximately half of the live fetuses from each litter.

There were no treatment-related mortalities or clinical signs during the course of the study. The pregnancy rate was unaffected by treatment with L-Tyrosine at 2% in the diet, or oral administration of NTBC at 10 µg/kg bw/day, or co-administration of L-Tyrosine at 2% in the diet plus NTBC at 10 µg/kg bw/day by gavage.

L-Tyrosine at 2% (group 2)

In dams, mean body weight gains, mean body weights, mean corrected body weight change and mean food consumption were unaffected by treatment. At the macroscopic examination, mottled kidney (bilateral) was noted in 1/23 females. Mean maternal liver weight was unaffected by treatment. Blood tyrosine level at autopsy was increased by a factor of 4.7, compared to the control group. Hence, administration of diet containing 2% w/w L-Tyrosine between GD 6 to 21 (equivalent to 1433 mg/kg bw/day) provoked a limited tyrosinaemia, in comparison to the control group.

At caesarean section, uterine content examination did not reveal any treatment-related effects on any litter parameter assessed, including fetal body weights, number of live fetuses, early and late resorptions, fetal death status and percentage of male fetuses.

No treatment-related findings were noted at the external fetal and fetal skeletal examinations.

NTBC at 10 µg/kg bw/day (group 3)

One female was sacrificed prematurely on GD 13 after reduced motor activity and tilting head were observed on GD 12 and 13. This female lost 21 g in body weight between GD 10 and 12, together with reduced food consumption, compared with the control group. At autopsy, no macroscopic changes were noted. No clear relationship to the treatment could be established.

In dams, mean body weight gain was reduced by 58% between GD 6 to 8 in comparison to the controls. Thereafter, mean body weight gains were comparable to the control values, resulting in an overall body weight gain between GD 6 to 21 similar to the control group. Mean corrected body weight change and mean food consumption were unaffected by treatment. At the macroscopic examination, mottled kidney (bilateral) was noted in 2/23 females. Mean maternal liver weight was unaffected by treatment. Blood tyrosine level at autopsy was increased by a factor of 8.4, compared to the control group. Hence, oral gavage administration of NTBC at 10 µg/kg bw/day between GD 6 to 20 provoked a slight tyrosinaemia, in comparison to the control group.

At caesarean section, the only litter parameter affected was mean fetal body weight, where there was a slight 2% reduction at both the combined and separate sex level.

No treatment-related findings were noted at the external fetal and fetal skeletal examinations.

L-Tyrosine at 2% + NTBC at 10 µg/kg bw/day (group 4)

In dams, mean body weight gain was reduced by 55% between GD 6 to 8 in comparison to the controls. Thereafter, mean body weight gains were similar to the control values, but the overall body weight gain between GD 6 to 21 was still slightly reduced by 6% in comparison to the control group. Mean corrected body weight change was unaffected by treatment, whilst mean food consumption was slightly decreased by 8% between GD 18 to 21, compared to the control group. At the macroscopic examination, mottled kidney (bilateral) was noted in 3/23 females and minimal corneal opacity (unilateral or bilateral) was noted in 4/23 animals. Mean maternal liver weight was unaffected by treatment. Blood tyrosine level at autopsy was increased by a factor of 63, compared to the control group. Hence, administration of diet containing 2% w/w L-Tyrosine between GD 6 to 21 (equivalent to 1 424 mg/kg bw/day) plus oral gavage administration of NTBC at 10 µg/kg bw/day between GD 6 to 20 provoked a marked tyrosinaemia, in comparison to the control group.

At caesarean section, the only litter parameter affected was mean fetal body weight, where there was a 7% reduction at both the combined and separate sex level, the effect being statistically significant in each case.

No treatment-related findings were noted at the external fetal examination. At the fetal skeletal examination, an increase in the incidence of no or incomplete ossification was noted for the 7th cervical centrum, 5th and 6th sternebrae, 3rd and/or 4th proximal phalanges of forepaws, 5th metacarpal, 1st metatarsal and sacrocaudal vertebrae, in comparison to the controls. In addition, higher incidences than in the control group were noted for short 14th rib and extra ossification points on the 14th thoracic vertebra. All these findings were classified as variant findings.

In conclusion, co-administration of L-Tyrosine at 2% w/w in the diet between GD 6 to 21 plus NTBC by oral gavage at 10 µg/kg bw/day between GD 6 to 20 provoked a marked maternal tyrosinaemia which caused a general delay of ossification in fetuses.

 

The findings from this supplementary study correlate with the findings seen in the developmental toxicity studies in rat and rabbit for (5-hydroxy-1,3-dimethylpyrazol-4-yl)(2-mesyl-4-trifluoromethylphenyl) methanone, where general delays in fetal maturation were observed, which were predominantly characterized by reduced body weights and skeletal findings such as delayed ossification. Thus, the findings of delayed fetal maturation seen in the developmental toxicity studies for (5-hydroxy-1,3-dimethylpyrazol-4-yl)(2-mesyl-4-trifluoromethylphenyl) methanone can be attributed to increases in maternal plasma tyrosine levels caused by the substances ability to inhibit of 4-hydroxyphenylpyruvic acid dioxygenase, an enzyme of the tyrosine catabolic pathway.

 

Mode of Action Analysis / Human Relevance Framework

Data on the mode of action and human relevance for HPPDase inhibitors, specifically in relation to effects seen in the eye and thyroid gland are presented in the section Additional Toxicological Information, as well as being discussed in detail within the endpoint summary for Repeated Dose Toxicity (Additional Information field). Furthermore, the relevance of the findings of delayed fetal development for HPPDase inhibitors is discussed within the Developmental Toxicity: Additional Information field.

Justification for classification or non-classification

The available data on toxicity to reproduction do not meet the criteria for classification according to Regulation (EC) 1272/2008, and are therefore conclusive but not sufficient for classification.