Registration Dossier

Diss Factsheets

Toxicological information

Developmental toxicity / teratogenicity

Currently viewing:

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-guideline study.

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 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: M.A.F.F. in Japan notification 12 Nousan No 8147, (November, 2000)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): N-cyclopropyl-4-[(2-methoxybenzoyl)sulfamoyl]benzamide
- Analytical purity: 97.4%
- Lot/batch No.: 08466/0013

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River laboratories, St Germain-sur-l'Arbresle, France
- Age at study initiation: adult virgin, not further specified
- Weight at study initiation: 243-318 g (females selected for study)
- Fasting period before study: no data
- Housing: Pregnant females were housed individually in suspended, stainless steel, wire mesh cages.
- Diet (ad libitum): Certified rodent pelleted and irradiated diet A04C-10 from S.A.F.E. (Scientific Animal Food and Engineering, Augy, France). Feed was stored in an identified room, controlled for temperature and humidity. Diet was used only until the date of expiry.
- Water (ad libitum): Water from the municipal supply was provided with an automatic watering system.
- Acclimation period: at least 12 days prior to mating

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 40-70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: aqueous solution of methylcellulose 400 (Fluka, Mulhouse, France) at 0.5%
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The appropriate amount of test substance was suspended (w/v) in an aqueous solution of methylcellulose 400 (Fluka, Mulhouse, France) at 0.5% and stored at approximately 5 °C (± 3 °C). Formulations were prepared twice (F1 and F2) during the study. The suspensions were mixed continuously before and during treatment with an electromagnetic stirrer.

VEHICLE
- Justification for use and choice of vehicle (if other than water): 0.5% aqueous methylcellulose 400; methylcellulose is commonly used as thickener or emulsifier in various food and cosmetic applications and toxicological studies to generate appropriate consistency.
- Concentration in vehicle: 6, 25 and 100 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Homogeneity of the suspensions was checked on the first formulation (F1) for the lowest and the highest concentrations (6 and 100 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 study (SA03017), and was found to be stable for 28 days under similar conditions to those of the current study.

Homogeneity and concentration checks of dosing suspensions of the test substance were between 94 and 99% of nominal values, which was within the in-house target range of 90 to 110% of the nominal concentration.
Details on mating procedure:
- Impregnation procedure: [cohoused]
- If cohoused:
- M/F ratio per cage: 1/1
- Length of cohabitation: overnight
- Further matings after two unsuccessful attempts: not referred to
- Verification of same strain and source of both sexes: yes, stock males of same strain from same supplier
- 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:
until GD 21
Doses / concentrations
Remarks:
Doses / Concentrations:
60, 250, 1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
25
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The range of doses was selected in agreement based on results obtained in a range-finding study (SA 03017), where pregnant rats received up to 1000 mg/kg bw/day of the test substance. The high dose of 1000 mg/kg bw/day was a limit dose which was expected to cause possible slight maternal toxicity. The mid dose of 250 mg/kg bw/day provided an approximate four fold factor between the dose levels. The low dose of 60 mg/kg bw/day was expected to be a clear No Observed Effect Level (NOEL) in both dams and fetuses.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Clinical signs daily from GD 0 through GD 21; mortality twice daily (in the morning and in the afternoon, except at weekends and public holidays when check was carried out once daily).
- Cage side observations included: all clinical signs, mortality (dead or moribund animals)

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

FOOD CONSUMPTION: Yes
Full feeder weights were measured on GD: 1, 6, 8, 10, 12, 14, 16 and 18; Empty feeder weights were measured on GD: 6, 8, 10, 12, 14, 16, 18 and 21.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: Yes

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21, by inhalation of carbon dioxide
- Organs examined:
Examination of uterine content of all females was performed. Each female was first subjected to macroscopic examination of the visceral organs including examination of the kidney and urinary bladder for the presence of gritty material. Kidneys and urinary bladder were taken at necropsy from all dams, tissues and carcasses were then discarded. Histopathological examinations were performed on the kidney at all dose levels and on the urinary bladder at 1000 mg/kg/day and in the control group only. The reproductive tract was weighed (gravid uterine weight), dissected out and the caesarean parameters were recorded without knowledge of treatment group.
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: Sex of live fetuses, individual weight of live fetuses; if possible, sex and weight of dead fetuses were recorded and included in the study file, as well.
Uterine horn(s) without visible implantations were immersed in a 10% solution of ammonium sulfide according to the SALEWSKI method (1964) in order to visualize any sites which were not apparent.
Intra-uterine death was classified according to GLEICH and FROHBERG (1977) as:
- Early resorptions: macroscopic discrimination between fetal residues and placental material not possible.
- Late resorptions: distinct macroscopic discrimination between fetal and placental remains possible.

Dead fetuses: defined as dead conceptuses showing distinct digits on fore- and hind-paws.
Runt fetuses: defined as live fetuses weighing less than 4 g at cesarean section of the dam.
Fetal examinations:
- External examinations: Yes: All the live fetuses were killed by subcutaneous injection (0.02 mL/fetus) of Dolethal (18.22 g/100 mL, sodium pentobarbital) and subjected to an external examination.
- Soft tissue examinations: Yes: Approximately half of the live fetuses from each litter were stained in Bouin's fluid for subsequent internal examination following free-hand sectioning.
- Skeletal examinations: Yes: The remaining half of the live fetuses from each litter were skinned, eviscerated and stained with alizarin red S and alcian blue for skeletal examination of bone and cartilage.
Structural deviations were classified as follows:
Malformations: Very rare or obviously lethal changes.
Minor anomalies: Slight, relatively rare structural changes not obviously detrimental.
Common variants: Structural changes occurring in more than approximately 5% of the control population.
- Head examinations: No
Statistics:
Mean and standard deviation for all maternal, litter and fetal parameters were calculated for each group. Statistical analyses were performed separately for all pregnant females and for all pregnant females with live fetuses, for body weight change and food consumption parameters. As the data were identical, only tables and appendices for all pregnant females are presented.

For details on statistics performed please refer to "Any other information on materials and methods incl. tables".
Indices:
Maternal body weight (BW) changes for interval periods; Corrected body weight change (CBWC); Average food consumption (FC) during intervals in g/day; Pre-implantation loss per litter; Post-implantation loss per litter; Number of live fetuses per litter; Number of dead fetuses per litter; Percentage of dead fetuses per litter; Percentage of male fetuses per litter; Mean fetal body weight per litter; Mean fetal body weight per litter and per sex; Mean fetal body weight per group; Mean fetal body weight per sex per group; Percentage of fetuses affected per litter; Mean percentage of fetuses affected per group; Percentage of litters affected per group
Historical control data:
Historical control data from studies conducted in-house were provided and referred to in order to allow comparison with concurrent controls.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes. Remark: body weight change was reduced by 32% (corrected body weight change was reduced by 11%); macroscopical and microscopical kidney changes

Details on maternal toxic effects:
Mortality:
There were no mortalities during the course of the study.

Clinical signs:
Two females at 1000 mg/kg bw/day and one female at 250 mg/kg bw/day regurgitated the dose suspension on one occasion. As these incidences occurred in isolation and in so few animals, this finding was considered to be incidental. One female at 1000 mg/kg bw/day had reduced motor activity, vaginal discharge and red traces on the cage tray on GD 21. These clinical signs were considered to be due to the commencement of parturition and not related to treatment. Another female at 1000 mg/kg bw/day had vaginal discharge on GD 14, however, this finding is common in this strain of rat at this stage of gestation and was therefore considered to be a normal occurrence.
No treatment-related signs were observed at 60 mg/kg bw/day.

Pregnancy rate:
There was no effect on pregnancy rate. The pregnancy rate was 100% at 1000 and 250 mg/kg bw/day and in the control group, and 96% at 60 mg/kg bw/day.

Body weights:
Body weight change was reduced by 32% between GD 6 to 8 at 1000 mg/kg bw/day, compared to the controls, though the effect was not statistically significant. Thereafter, body weight change was comparable to the controls at this dosage.
There was no effect on body weight change at 250 or 60 mg/kg bw/day.
Maternal corrected body weight change was reduced by 11% at 1000 mg/kg bw/day, compared to the controls.
There was no effect on maternal corrected body weight change at 250 or 60 mg/kg bw/day.

Food consumption:
Food consumption was comparable between the three treated groups and the controls throughout treatment.

Maternal necropsy and microscopic findings:
At autopsy, one female had yellow sediment in both kidneys and prominent lobulation of the liver, one female was found to have an enlarged liver with white foci on the left lobe and one female had a white area on the left lobe, at 1000 mg/kg bw/day. The few other macroscopic findings were considered to be incidental as they occurred in isolation or not in a dose-related manner.
At the histopathological examination of the kidney and urinary bladder, one female from the 1000 mg/kg bw/day treatment group was found to have a moderate bilateral papillary necrosis in the kidney, associated with mixed cell infiltrate. This change correlated with the macroscopic finding of yellow bilateral sediment in the kidney of this female at autopsy.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOEL
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
LOEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Litter data:
No litter parameters were affected by treatment including early and late resorptions, fetal death status, percentage of male fetuses and fetal body weights (by sex and combined sex).

Fetal evaluation:
At least 24 litters with live fetuses were present in each group and fetal evaluations were performed through external, visceral and skeletal examinations.
External observations:
There were three fetuses observed with malformations, two occurred at 250 mg/kg bw/day and one at 60 mg/kg bw/day. At 250 mg/kg bw/day, one fetus had an umbilical hernia and one fetus had anasarca, whilst at 60 mg/kg bw/day one fetus had a cleft palate. In isolation and in the absence of a dose response, these malformations were considered to be incidental.
There were no treatment-related anomalies or variant findings at the external fetal observation.
The number of runt fetuses observed was very low with no indication of a treatment-related effect.
Visceral observations:
At 1000 mg/kg bw/day, one fetus had the malformation retinal fold associated with a small lens, whilst a second fetus had the malformation right-sided aortic arch and pulmonary trunk.
As these two malformations occurred in isolation, they were considered to be incidental. In addition, the incidence of the malformation absent renal papilla (unilateral) was marginally higher in terms of both mean percentage of fetuses affected and percentage of litters affected, compared with the control group. However, as the incidence was not statistically significant for either parameter, when compared to the control group, and the incidence in terms of litters (the litter being regarded as the experimental unit), was within the in-house historical control range, this finding was considered to be fortuitous.
Skeletal observations:
Two fetuses with malformations were noted, one occurred at 1000 mg/kg bw/day and comprised of hemivertebra in the atlas vertebra and malpositioned axis vertebra. The second malformation occurred at 60 mg/kg bw/day, where one fetus had a small and split palatine (the same fetus which had a cleft palate at the external observation). In isolation, these malformations are considered to be incidental.
The following two variants were found at a higher incidence at 1000 mg/kg bw/day; incomplete parietal ossification and incomplete interparietal ossification. In addition, the incidence of 13th costal cartilage absent (unilateral/bilateral), classified as a variant, was marginally higher at 1000 mg/kg bw/day, compared with the controls. However, as in all three cases the incidence was found not to be statistically significantly different from the control group at both the fetal and litter level, these findings were considered to be fortuitous.

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
There were no mortalities or treatment-related signs in any dose group. At 1000 mg/kg bw/day, body weight change was reduced by 32% between GD 6 to 8 in dams, thereafter, body weight change was comparable to the controls. Maternal corrected body weight change was reduced by 11% compared to the controls. At autopsy, one female was noted with yellow sediment in both kidneys and prominent lobulation of the liver, one female had an enlarged liver with white foci on the left lobe and one female had a white area on the left lobe. At the microscopic examination, one female was found to have a moderate bilateral papillary necrosis in the kidney, associated with mixed cell infiltrate. The change correlated with the macroscopic finding of yellow bilateral sediment in the kidney of this female.
No litter parameters were affected; including the number of live fetuses, early and late resorptions, fetal death status, percentage of male fetuses and mean fetal body weights.
At the external, visceral and skeletal fetal examination, no treatment-related findings were observed.
At 250 and 60 mg/kg bw/day, there were no treatment-related maternal, litter based or fetal findings at either dosage.
In conclusion, a dose level of 1000 mg/kg bw/day test substance caused toxic effects to the dam and was a NOEL in terms of fetal development. A dose level of 250 mg/kg bw/day was a NOEL for maternal toxicity.