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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
two-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
06 Oct 2003 to 07 Jul 2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to other study
Reason / purpose for cross-reference:
other: reference to review article

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report date:
2005

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Version / remarks:
adopted in 2001
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3800 (Reproduction and Fertility Effects)
Version / remarks:
adopted in 1998
Qualifier:
according to guideline
Guideline:
other: M.A.F.F. in Japan 12 Nousan N° 8147
Version / remarks:
adopted in 2000
GLP compliance:
yes
Limit test:
no
Justification for study design:
SPECIFICATION OF STUDY DESIGN
- Premating exposure duration for parental (P0) animals: approximately 12 weeks
- Basis for dose level selection: Dose selection was based on results of a one-generation pilot study, where rats received 0, 100, 600 and 3600 ppm in the diet. In this study the NOEL was 100 ppm. From 600 ppm onwards P0 males exhibited reduced body weights. P0 females showed reduced body weights at 3600 ppm during gestation and lactation. There was an increase in food intake at 3600 ppm in P0 rats. The pup weights were significantly reduced at 3600 ppm.
- Termination time for F2: after a 4-week lactation period
- Route of administration: dietary admix

Test material

Constituent 1
Chemical structure
Reference substance name:
(5-hydroxy-1,3-dimethyl-1H-pyrazol-4-yl)[2-(methylsulfonyl)-4-(trifluoromethyl)phenyl]methanone
EC Number:
609-256-3
Cas Number:
365400-11-9
Molecular formula:
C14H13F3N2O4S
IUPAC Name:
(5-hydroxy-1,3-dimethyl-1H-pyrazol-4-yl)[2-(methylsulfonyl)-4-(trifluoromethyl)phenyl]methanone
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Homogeneity and stability of the test substance in the diet: analytically confirmed

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Crl: (WI) WU BR
Details on species / strain selection:
no data provided
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River GmbH, Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: (P) 6 - 7 wks; (F1) 4 wks
- Weight at study initiation: (P) 109 - 149 g (males), 101 -137 g (females); (F1) 68 - 119 g (males), 65 - 106 g (females)
- Fasting period before study: no
- Housing: singly (except when co-housed for matings) in Makrolon® cages Type Illh on low-dust soft-wood shavings or nesting material (day 20 GD to 7 LD)
- Diet: Kliba 3883.9.25 supplied by Provimi Kliba SA, Kaiseraugst, Switzerland, ad libitum
- Water: tap water, ad libitum
- Acclimation period: approximately one week

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

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

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
PREPARATION OF DIETARY MIXTURES:
- Rate of preparation of diet (frequency): at least weekly
- Mixing appropriate amounts with: Kliba 3883.9.25
- Storage temperature of food: room temperature

Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: 12 nights during the three-week mating period
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged: individually
- Any other deviations from standard protocol: no second mating period with different males was initiated if first mating period was unsuccessful
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
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
Details on study schedule:
- P1 parental animals not mated until 10 weeks after selected from the F1 litters
- Selection of parents from F1 generation when pups were 28 days of age
- Age at mating of the mated animals in the study: P0: 18 weeks, P1: 13 weeks
Doses / concentrationsopen allclose all
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, where rats received 0, 100, 600 and 3600 ppm in the diet. In this study the NOEL was 100 ppm. From 600 ppm onwards F0 males exhibited reduced body weights. F0 females showed reduced body weights at 3600 ppm during gestation and lactation. There was an increase in food intake at 3600 ppm in F0 rats. Pup weights were significantly reduced at 3600 ppm. Therefore, this 2-generation reproduction study was dosed at 0, 30, 300 and 3000 ppm.
Positive control:
no

Examinations

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

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

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

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

Oestrous cyclicity (parental animals):
Estrus 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

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 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 were prepared for microscopic examination and weighed, respectively.
Statistics:
Statistical evaluation was performed on an Alpha 800 5/500 computer (TASC-system) using the following methods:
a) Analysis of Variance (ANOVA) and in case of significant results Dunnett's test as post hoc
test for: body weights and body weight gains, food consumption, number of implantation sites per female, number of viable pups per female, organ weights at necropsy, number of estruses, time to insemination, life birth, viability and lactation rate
b) 2 by N CHI2 test; in case of significant differences Fisher's exact test with Bonferroni correction for: number of viable pups per group based on the number of implantations, insemination, fertility, gestation and rearing rate
c) Kruskall-Wallis test and in case of significant differences Dunnett's test for: number of prenatal loss per litter
The sperm and spermatid count data were not evaluated statistically, because there were no meaningful differences occurred between the high dose and control groups.
Generally, differences between the control group and groups treated with the test substance groups were considered as statistically significant when p < 0.05. Significant differences from the control are indicated with * for p < 0.05 and **for p < 0.01.
Follicle counts were evaluated statistically using the Wilcoxon Mann-Whitney test.
Reproductive indices:
Insemination index (%) = (number of sperm positive females / number of females co-housed with a male) x 100
Fertility index (%) = (number of pregnant females / number ofsperm positive females) x 100
Gestation index (%) = (number of females completing delivery / number of pregnancies) x 100
Live birth index (%) = (number of live pups at birth / total number of pups born) x 100
Offspring viability indices:
Rearing index = (number of females rearing a litter to lactation day 21 / number of females delivering a litter) x 100
Viability index = (number of pups alive on lactation day 4 pre-culling / number of live pups born) x 100
Lactation index = (number of pups alive after 3 weeks / number of pups after four days (after culling)) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Eye opacities were noted in 15 males and 4 females at 3000 ppm (for details see table 5).
Dermal irritation (if dermal study):
not examined
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.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
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 table 5). These effects correlated with histopathological findings.
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
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.
Histopathological findings: neoplastic:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

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

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

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
general toxicity
Effect level:
30 ppm
Based on:
test mat.
Remarks:
equivalent to 26 and 33 mg/kg bw/day in P0 males and females, respectively
Sex:
male/female
Basis for effect level:
other: No adverse effects noted at 30 ppm
Key result
Dose descriptor:
LOAEL
Remarks:
general toxicity
Effect level:
300 ppm
Based on:
test mat.
Remarks:
equivalent to 26 and 33 mg/kg bw/day in P0 males and females, respectively
Sex:
male/female
Basis for effect level:
other: At 300 ppm increased liver / kidney weights were noted. Adverse effects in the following organs were noted: Liver, pituitary gland and kidney (males only).
Key result
Dose descriptor:
NOAEL
Remarks:
for reproductive function
Effect level:
3 000 ppm
Based on:
test mat.
Remarks:
equivalent to 272 and 346 mg/kg bw/day in P0 males and females, respectively
Sex:
male/female
Basis for effect level:
other: No adverse effects on reproductive parameters noted up to 3000 ppm.

Target system / organ toxicity (P0)

open allclose all
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
300 ppm
System:
endocrine system
Organ:
pituitary gland
Treatment related:
yes
Dose response relationship:
yes
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
300 ppm
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
300 ppm
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
yes
Key result
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
Key result
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

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Eye opacities were noted in 15 males and 7 females at 3000 ppm (for details see table 5).
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Two females at 3000 ppm were euthanized in moribund condition (for details see table 5). 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.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
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 table 5). These effects correlated with histopathological findings.
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
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.
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
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.
Histopathological findings: neoplastic:
no effects observed

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related effects on reproduction performance were noted at any dose.
Insemination and fertility indices as well as gestation length and number of litters born were not changed by the treatment up to 3000 ppm.

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

Details on results (P1)

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

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

Effect levels (P1)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
general toxicity
Effect level:
30 ppm
Based on:
test mat.
Remarks:
equivalent to 3.68 and 4.18 mg/kg bw/day in P1 males and females, respectively
Sex:
male/female
Basis for effect level:
other: No adverse effects were noted at 30 ppm.
Key result
Dose descriptor:
LOAEL
Remarks:
general toxicity
Effect level:
300 ppm
Based on:
test mat.
Remarks:
equivalent to 34 and 39 mg/kg bw/day in P1 males and females, respectively
Sex:
male/female
Basis for effect level:
other: At 300 ppm reduced body weights and food consumption, increased liver / kidney weights were noted. Adverse effects in the following organs were noted: liver, pituitary gland and kidney (males only).
Key result
Dose descriptor:
NOAEL
Remarks:
for reproduction
Effect level:
3 000 ppm
Based on:
test mat.
Remarks:
equivalent to 272 and 346 mg/kg bw/day in males and females, respectively
Sex:
male/female
Basis for effect level:
other: No adverse effcts on reproductive parameters noted up to 3000 ppm.

Target system / organ toxicity (P1)

open allclose all
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
300 ppm
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
yes
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
300 ppm
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
300 ppm
System:
endocrine system
Organ:
pituitary gland
Treatment related:
yes
Dose response relationship:
yes
Key result
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
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
300 ppm
System:
endocrine system
Organ:
thyroid gland
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
At 3000 ppm more F1 pups (n = 12, in two litters) were found to be cold to touch than in the other groups (n = 5, 5 and 0 at 0, 30 and 300 ppm, respectively)
Dermal irritation (if dermal study):
not examined
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.
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.
Food efficiency:
not examined
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.
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
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.
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.
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.
Histopathological findings:
effects observed, treatment-related
Other effects:
not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)

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

Effect levels (F1)

open allclose all
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
300 ppm
Based on:
test mat.
Remarks:
equivalent to 22.25 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: No adverse effects were noted at 300 ppm.
Key result
Dose descriptor:
LOAEL
Generation:
F1
Effect level:
3 000 ppm
Based on:
test mat.
Remarks:
equivalent to 228.59 mg/kg bw/day
Sex:
male/female
Basis for effect level:
body weight and weight gain
gross pathology

Target system / organ toxicity (F1)

open allclose all
Key result
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
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
3 000 ppm
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
yes

Results: F2 generation

General toxicity (F2)

Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
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.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
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.
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
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.
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).
Histopathological findings:
not examined
Other effects:
not examined

Developmental neurotoxicity (F2)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:
not examined

Details on results (F2)

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

Effect levels (F2)

Key result
Dose descriptor:
NOAEL
Generation:
F2
Effect level:
300 ppm
Based on:
test mat.
Remarks:
equivalent to 23.24 mg/kg bw/day
Sex:
male/female
Basis for effect level:
viability
body weight and weight gain
gross pathology

Target system / organ toxicity (F2)

open allclose all
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
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
3 000 ppm
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
yes

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Any other information on results incl. tables

Table 3: Mean (±SD) test substance intake during premating (mg/kg body weight/day)

 

 

 

 

Observation in study weeks

 

Male

 

Female

 Dietary concentration (ppm)

 

30

 

300

 

3000

 

30

 

300

 

3000

 

P0    

(study week 1-12)

 

2.50

± 0.900

 

26.28

± 9.433

 

272.42

± 105.45

 

3.09

± 0.853

 

32.64

± 7.863

 

345.67

± 93.286

P1  

(week of age 5-14)

 

3.68

± 1.862

 

34.13

± 17.048

 

353.62

± 173.37

 

4.18

± 1.472

 

38.88

± 11.342

 

393.38

± 145.13

 

.

 Table 4: Mean (±SD) test substance consumption - gestation and lactation

 

 

Observation in study days

 

Dietary concentration (ppm)

30

300

3000

P0 Females

 

Mean test substance consumption day 14 to 20 p.c. (mg/kg body weight/day)

 

2.04

±0.201

 

22.25

+3.080

 

228.59

±28.324

 

Mean test substance consumption day 0 to 4 p.p. (mg/kg body weight/day)

 

3.10

±0.779

 

30.91

+8.101

 

294.23

+51.915

P1 Females

 

Mean test substance consumption day 14 to 20 p.c. (mg/kg body weight/day)

 

2.20

±0.405

 

23.24

±4.054

 

250.39

+24.864

 

Mean test substance consumption day 0 to 4 p.p. (mg/kg bodv weight/dav)

 

3.15

±0.791

 

29.86

+11.380

 

263.15

+114.47

Table 5: Mortality, clinical signs and ophthalmology

 

Observations #

 

Dietary concentration (ppm)

 

0

 

30

 

300

 

3000

 

0

 

30

 

300

 

3000

 

 

F0 Males

 

F1 Males

 

Mortality

No. of unscheduled deaths

 

 

0

 

 

0

 

 

0

 

 

0

 

 

0

 

 

0

 

 

0

 

 

0

 

Clinical findings (premating + mating)

eye opacity

 

 

0

 

 

0

 

 

0

 

 

15

 

 

0

 

 

0

 

 

0

 

 

15

 

Ophthalmology (near or at nnecropsy)

 

 

 

 

 

 

 

 

diffuse cornea opacity (%)

0

0

6.0

48.0

0

0

10.0

60.0

reticulate cornea opacity (%)

0

0

4.0

34.0

0

0

12.0

60.0

cornea neo-vascularisation (%)

0

0

4.0

48.0

0

0

10.0

60.0

 

 

F0 Females

 

F1 Females

 

Mortality

No. of unscheduled deaths

 

 

0

 

 

0

 

 

0

 

 

0

 

 

0

 

 

0

 

 

0

 

 

2

 

Clinical findings (premating)

 

 

 

 

 

 

 

 

eye opacity

0

0

0

0

0

0

0

2

Clinical findings (gestation/lactation)

 

 

 

 

 

 

 

 

eye opacity

poor general condition a)

0

0

0

0

0

0

4

0

0

0

0

0

0

0

7

2

palpable mass on neck a)

0

0

0

0

0

0

0

1

 

Ophthalmology (near or at necropsy)

 

 

 

 

 

 

 

 

diffuse cornea opacity (%)

4.3

0

4.8

68.2

0.2

0

10.0

69.6

reticulate cornea opacity (%)

0

0

42.9

61.4

0

0

22.0

69.6

cornea neovascularisation (%)

0

0

4.8

59.1

0

0

0.6

65.0

#)  Numbers of animals affected is given, if not mentioned otherwise.

a) Pregnant females sacrificed unscheduled.

 

Table y: Mean absolute organ weights of parental F0 animals (± SD)

 

 

 

 

 

Observation

Absolute Weights (mg)

Dietary concentration (ppm)

0

30

300

3000

 

F0 Males

Body weight (g)

478.7

±29.05

473.0

±34.89

457.1*

 

±26.10

461.6

±29.86

Kidneys

3063

±237.2

3032

±277.6

3107

 

±225.4

3436**

±268.9

Liver

17010

±1658.9

17345

±1929.3

17487

 

±1775.1

19199**

±1722.2

Spleen

754

±62.5

802

±164.7

750

 

±76.0

784

±72.3

Epididymis (left)

805

±80.7

747

±120.8

702**

 

·±74.4

771

±77.0

Prostate

1270

±145.8

1197

±189.3

1145*

 

±191.2

1139*

±196,1

Seminal vesicles

1559

±287.2

1720

±294.3

1546

 

±267.1

1514

±350.3

Testes

3625

±221.2

3603

±580.3

3498

 

±468.4

3531

±258.8

Brain

1929

±56.7

1969

±69.1

1908

 

±67.5

1871**

±71.8

Adrenals

55

±8.0

47**

±8.4

45**

 

±7.1

51

±8.5

Pituitary a)

14

±2.5

16

±3.6

14

 

±2.4

15

±4.2

Thyroid b)

14

±3.1

16

±4.3

18**

 

±4.9

19**

±4.0

 

F0 Females

Body weight (g)

266.1

±17.21

256.8

±19.11

265.4

 

±16.69

267.7

±14.15

Kidneys

2170

±170.6

2120

±182.3

2245

 

±169.4

2240

±143.1

Liver

13669

±1601.1

12676

±1835.4

13836

 

±1586.5

14290

±1423.7

Spleen

590

±78.5

600

±100.1

645

 

±176.4

694

±194.7

Ovaries

140

±17.6

131

±17.8

128

 

±16.7

135

±19.8

Uterus

480

±81.4

487

±88.2

459

 

±66.9

475

±123.8

Brain

1793

±64.2

1783

±62.3

1741*

 

±42.8

1756

±63.5

Adrenals

66

±9.4

64

±8.8

62

 

±7.7

66

±9.0

Pituitary a)

17

±4.5

17

±6.0

17

 

±2.8

16

±4.4

Thyroid b)

11

±1.8

12

±3.7

12

 

±2.8

12

2.6

a)       Weighed in a fixed stage.

b)      One thyroid with parathyroid weighed after fixation.

*   Statistically different from control, p ≤ 0.05

** Statistically different from control, p ≤ 0.01

 

TABLE : Mean relative organ weights of parental F0 animals (±SD)

 

 

 

 

 

 

 

Observation

 

Relative organ weights in mg/100 g body weight

Dietary concentration (ppm)

 

0

 

30

 

300

 

3000

 

 

 

 

F0 Males

 

 

 

Body weight (g)

478.7

±29.05

473.0

±34.89

457.1*

±26.10

461.6

±29.86

Kidneys

640.1

±36.05

641.6

±45.36

680.1**

±35.24

745.1**

±49.82

Liver

3551.8

±244.52

3664.1

±270.41

3820.9**

±254.76

4160.0**

±266.47

Spleen

157.8

±12.10

169.2

±29.93

164.2

±14.64

170.0

±14.62

Epididymis (left)

168.4

±16.61

157.7

±23.26

153.7*

±16.13

167.4

±17.37

Prostate

265.8

±30.07

253.2

±35.69

250.0

±35.82

247.3

±42.21

Seminal vesicles

325.9

±57.47

363.9

±60.34

340.0

±65.02

329.1

±77.52

Testes

759.7

±60.71

760.9

±122.61

766.1

±104.42

767.7

±71.16

Brain

404.5

±28.36

417.9

±27.45

418.6

±25.53

406.7

±25.41

Adrenals

11.5

±1.51

9.9**

±1.46

9.9**

±1.53

11.1

±1.59

Pituitary a)

2.8

±0.41

3.3

±0.84

3.1

±0.57

3.2

±0.86

Thvroid b)

2.9

±0.63

3.4

±0.83

4.0**

±1.09

4.0**

±0.86

 

F0 Females

Body weight (g)

266.1

±17.21

256.8

±19.11

265.4

±16.69

267.7

±14.15

Kidneys

816.1

±50.33

827.1

±57.32

848.1

±70.22

838.1

±57.07

Liver

5139.8

±561.78

4919.4

±503.04

5213.4

±497.40

5347.9

±567.01

Spleen

222.3

±30.88

233.8

±37.35

242.0

±59.50

260.6

±78.19

Ovaries

52.8

±7.73

51.3

±7.97

48.4

±6.32

50.6

±7.28

Uterus

181.3

±33.89

191.6

±43.21

173.3

±25.69

178.2

±49.96

Brain

676.0

±45.82

697.2

±46.29

658.1

±37.39

657.4

±37.42

Adrenals

25.0

±4.04

25.2

±3.72

23.3

±2.79

24.8

±3.40

Pituitary a)

6.3

±1.85

6.5

±2.42

6.4

±1.11

5.9

±1.66

Thvroid b)

4.1

±0.65

4.7

±1.46

4.6

±1.09

4.5

±0.90

a)       Weighed in a fixed stage.

b)      One thyroid with parathyroid weighed after fixation.

*   Statistically different from control, p ≤ 0.05

**Statistically different from control, p ≤ 0.01

Table xyz: Mean absolute organ weights of parental F1 animals (±SD)

 

Observation

 

Absolute Weights (mg)

 

 

Dietary concentration (ppm)

 

 

0

30

300

3000

 

F1 Males

Body weight (g)

457.7

 

±41.49

461.5

±34.29

436.2

±26.74

407.9**

±23.61

Kidneys

2883

 

±273.5

3071*

±324.1

3090*

±183.6

2957

±252.4

Liver

16878

 

±1916.0

17619

±1916.7

17048

±1757.8

16581

±1804.3

Spleen

771

 

±118.0

783

±86.1

700*

±69.7

662*

±76.2

Epididymis (left)

735

 

±73.9

762

±66.5

738

±135.0

653**

±103.0

Prostate

1203

 

±241.8

1217

±147.7

1080

±164.5

970**

±165.9

Seminal vesicles

1603

 

±246.0

1502

±290.5

1415

±305.9

1535

±378.5

Testes

3574

 

±317.6

3788*

±207.0

3568

±335.0

3289**

±308.9

Brain

1931

 

±80.7

1999**

±63.7

1904

±64.8

1796**

±78.5

Adrenals

49

 

±8.1

47

±10.0

48

±7.3

46

±5.4

Pituitary a)

11

 

±3.7

12

±2.2

12

±2.4

10

±2.4

Thyroid b)

12

 

±3.0

16**

±3.9

16**

±5.4

15

±3.8

 

F1 Females

Body weight (g)

257.7

 

±13.54

268.8

±19.74

261.2

±17.57

251.0

±14.09

Kidneys

1975

 

±167.4

2150

±181.0

2091

±198.0

2210

±478.2

Liver

13048

 

±1167.2

13519

±1664.5

13147

±1478.2

13503

±1688.4

Spleen

519

 

±45.8

587

±65.1

541

±51.2

543

±141.1

Ovaries

121

 

±12.8

135

±19.6

130

±17.2

128

±15.2

Uterus

455

 

±86.0

498

±95.5

504

±130.1

506

±162.6

Brain

1799

 

±56.2

1842

±76.4

1760

±67.4

1722*

±106.6

Adrenals

59

 

±7.7

64

±7.7

63

±6.0

68**

±9.8

Pituitary a)

14

 

±2.6

15

±2.5

15

±2.5

13

±3.0

Thyroid b)

12

 

±3.5

12

±3.5

11

±2.9

11

±2.9

a)       Weighed in a fixed stage.

b)      One thyroid with parathyroid weighed after fixation.

*   Statistically different from control, p ≤ 0.05

**Statistically different from control, p ≤ 0.01

Table aaa: Mean relative organ weights of parental F1 animals (±SD)

 

 

Relative organ weights in mg/100 g body weight

 

Dietary concentration (ppm)

 

Observation

 

0

 

30

 

300

 

3000

 

               

 

 

"\;

F1 Males

 

 

 

 

Body weight (g)

457.7

±41.49

461.5

±34.29

436.2

 

±26.74

407.9**

±23.61

Kidneys

630.7

±35.47

665.3*

±44.93

709.6**

 

±41.03

725.5**

±51.45

Liver

3688.3

±253.79

3813.4

±235.37

3908.9*

 

±332.50

4069.1**

±400.95

Spleen

168.3

±17.81

169.8

±15.47

160.5

 

±14.29

162.8

±19.76

Epididymis (left)

161.1

±13.73

165.3

±12.30

169.9

 

±35.19

160.3

±25.26

Prostate

263.6

±50.88

264.4

±31.00

247.8

 

±36.70

237.9

±37.83

Seminal vesicles

351.3

±50.20

327.7

±68.94

324.1

 

±66.41

377.1

±92.65

Testes

785.0

±80.28

824.9

±73.58

819.1

 

±74.01

807.7

±76.63

Brain

424.3

±32.19

434.9

±26.12

437.8

 

±28.69

441.2

±22.12

Adrenals

10.6

±1.51

10.2

±1.86

11.0

 

±1.70

11.2

±1.38

Pituitary a)

2.5

±0.83

2.6

±0.47

2.7

 

±0.52

2.4

±0.61

Thyroid b)

2.7

±0.62

3.6**

±0.86

3.7**

 

±1.17

3.7**

±0.90

 

 

 

 

F1 Females

 

 

 

 

Body weight (g)

257.7

±13.54

268.8

±19.74

261.2

 

±17.57

251.0

±14.09

Kidneys

766.8

±55.07

800.7

±47.51

800.5

 

±57.88

880.6**

±185.92

Liver

5060.9

±306.82

5031.2

±507.80

5041.0

 

±539.58

5387.2

±646.22

Spleen

201.5

±14.14

218.9

±24.47

207.0

 

±14.77

216.4

±56.21

Ovaries

47.1

±5.42

50.3

±6.66

50.0

 

±7.82

51.3

±6.49

Uterus

176.5

±31.19

185.5

±35.89

193.8

 

±51.50

200.2

±56.85

Brain

699.3

±33.01

688.3

±50.21

676.0

 

±43.21

687.5

±47.68

Adrenals

22.7

±2.80

23.9

±2.40

24.1

 

±2.73

27.2**

±3.56

Pituitary a)

5.6

±0.89

5.7

±1.00

5.6

 

±0.88

5.2

±1.10

Thyroid b)

4.6

+1.45

4.5

+1.44

4.2

 

+1.14

4.5

±1.17

a)       Weighed in a fixed stage.

b)      One thyroid with parathyroid weighed after fixation.

*   Statistically different from control, p ≤ 0.05

**Statistically different from control, p ≤ 0.01

Table bbb: Ophthalmology in F1 and F2 weanlings (28 p.p.)

 

 

 

Ophthalmology observations

Males and females pooled

 

Dietary concentration (ppm)

 

Dietary concentration (ppm)

 

0

 

30

 

300

 

3000

 

0

 

30

 

300

 

3000

Fl Pups

F2 Pups

 

Diffuse cornea opacity (%)

 

0

 

0

 

0

 

10.5

 

0

 

0

 

2.6

 

12.5

Reticulate cornea opacity (%)

0

0

4.9

30.2

0

0

0

8.9

Cornea neovascularisation (%)

0

0

0

2.3

0

0

14.5

50.0

Table 1: Sexual maturation of F1 post weanlings (means and ±SD)

Observation

Dietary concentration (ppm)

 

0

30

300

3000

Balano-preputial separation

 

at mean age (days)

 

 

at mean body weight (g)

 

 

 

41.0

± 2.21

 

159.1

± 15.77

 

 

41.8

± 2.01

 

170.1*

± 14.17

 

 

44.2**

± 2.03

 

176.9**

± 10.01

 

 

46.3**

± 2.88

 

170.5**

± 12.25

Vaginal opening

 

at mean age (days)

 

 

at mean body weight (g)

 

 

32.6

± 1.58

 

90.9

± 6.98

 

 

32.6 

± 1.60

 

96.1

± 8.47

 

 

33.6 

± 2.24

 

96.2

± 10.74

 

 

34.2*

± 2.71

 

89.9

± 8.24

* p ≤ 0.05,  ** p ≤ 0.01

Table ccc: Mean absolute organ weights of F1 and F2 weanlings (±SD)

 

 

 

 

 

Observation

 

Absolute weights (mg)

Dietary concentration (ppm)

0

30

300

3000

 

F1 Weanlings - males

Body weight (g)

74.9

± 9 .32

78.8

± 8.03

77.3

± 8.22

71.0

± 12.17

Brain

1442

± 79.3

1440

± 62.1

1424

± 60.3

1345**

± 79.4

Spleen

291

± 34.0

286

± 41.3

276

± 29.7

247**

± 44.5

Tbymus

341

± 77.7

374

± 56.8

363

± 86.2

323

± 76.7

 

F1 Weanlings - females

Body weight (g)

69.8

± 8.41

72.1

± 6.55

74.8

± 10.11

66.6

± 7.95

Brain

1393

± 63.2

1382

± 63.8

1408

± 58.7

1312**

± 58.9

Spleen

263

± 35.6

250

± 27.3

263

± 34.0

226**

± 32.9

Thymus

338

± 73.0

362

± 52.3

373

± 69.6

320

± 63.1

Uterus

73

± 22.3

63

± 19.0

72

± 21.1

62

± 17.0

 

F2 Weanlings - males

Body weight (g)

80.9

± 4.92

83.5

± 7.56

81.1

± 10.21

77.5

± 9.29

Brain

1474

± 40.6

1489

± 49.5

1437

± 76.0

1408**

± 41.6

Spleen

288

± 37.2

308

± 38.9

284

+ 41.4

263

± 42.0

Thymus

382

± 77.7

395

± 54.2

372

± 69.9

355

± 70.9

 

F2 Weanlings - females

Body weight (g)

73.1

± 7.76

78.5

± 6.33

74.3

± 7.92

66.6

± 9.31

Brain

1387

± 80.9

1441

± 61.5

1397

± 69.5

1315*

± 71.4

Spleen

243

± 34.0

272

± 40.5

248

± 34.5

221

± 40.4

Thymus

347

± 62.1

370

± 50.9

386

± 60.6

329

± 64.3

Uterus

81

+ 19.9

83

± 26.7

67

± 15.2

69

± 28.4

* p ≤ 0.05,  ** p ≤ 0.01

Table ccc: Mean relative organ weights of F1 and F2 weanlings (±SD)

 

 

 

 

 

Observation

 

Organ weights in mg per 100 g body weight

Dietary concentration (ppm)

0

30

300

3000

 

F1 Weanlings - males

Body weight (g)

74.9

± 9 .32

78.8

± 8.03

77.3

± 8.22

71.0

± 12.17

Brain

1944.4

±189.61

1844.4

±185.25

1856.3

±144.62

1948.5

±353.66

Spleen

390.0

±39.35

363.7

±39.13

360.7

±54.20

350.1**

±38.66

Tbymus

452.2

±77.82

473.5

±46.54

467.8

±89.97

452.4

±63.04

 

F1 Weanlings - females

Body weight (g)

69.8

± 8.41

72.1

± 6.55

74.8

± 10.11

66.6

± 7.95

Brain

2051.5

±182.66

1927.9

±136.63

1907.1

±196.73

1991.8

±206.73

Spleen

377.4

±34.20

348.3

±40.18

355.4

±53.17

342.4

±56.82

Thymus

483.9

±79.83

502.5

±58.74

498.9

±70.90

479.6

±78.67

Uterus

105.4

±32.04

87.8

±24.51

97.2

±28.83

93.8

±26.43

 

F2 Weanlings - males

Body weight (g)

80.9

± 4.92

83.5

± 7.56

81.1

± 10.21

77.5

± 9.29

Brain

1827.9

±102.01

1794.3

±149.74

1793.6

±200.08

1839.1

±212.29

Spleen

356.0

+44.89

370.0

+51.03

351.9

±47.07

339.6

+37.29

Thymus

469.3

±75.72

475.1

±72.25

460.1

±78.58

455.8

±54.38

 

F2 Weanlings - females

Body weight (g)

73.1

± 7.76

78.5

± 6.33

74.3

± 7.92

66.6

± 9.31

Brain

1911.0

+156.97

1844.1

±147.38

1892.5

±134.21

2010.8

+304.53

Spleen

331.5

±26.56

345.9

±43.27

334.4

±311.7

332.0

±39.58

Thymus

473.1

±54.42

471.5

±57.33

522.0

±83.18

493.7

±60.74

Uterus

112.1

±33.75

105.9

±34.64

91.8

±23.42

103.2

±38.32

* p ≤ 0.05,  ** p ≤ 0.01

Applicant's summary and conclusion