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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report date:
2006

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
adopted in 2001
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Version / remarks:
adopted in 1992
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
adopted in 1998
Qualifier:
according to guideline
Guideline:
other: M.A.F.F. in Japan, notification 12 Nousan N°8147 guideline
Version / remarks:
adopted in 2000
GLP compliance:
yes (incl. QA statement)
Remarks:
Groupe Interministeriel des Produits Chimiques, Paris, France
Limit test:
no

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
- Solubility and stability of the test substance in the solvent/vehicle: analytically demonstrated

Test animals

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

Administration / exposure

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
Duration of test:
15 days of treatment
Doses / concentrationsopen allclose all
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, 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.

Examinations

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 apparently 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.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

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.
Dermal irritation (if dermal study):
not examined
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.
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.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
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):
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
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Maternal developmental toxicity

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):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not examined
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.

Effect levels (maternal animals)

open allclose all
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

Maternal abnormalities

Abnormalities:
no effects observed
Description (incidence and severity):
No organ changes were noted.

Results (fetuses)

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.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): not examined
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
Changes in postnatal survival:
not examined
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.
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.
Visceral malformations:
no effects observed
Other effects:
no effects observed

Effect levels (fetuses)

open allclose all
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

Fetal abnormalities

Key result
Abnormalities:
no effects observed
Description (incidence and severity):
No treatment-related fetal malformations were noted.

Overall developmental toxicity

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

Applicant's summary and conclusion