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Toxicological information

Developmental toxicity / teratogenicity

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

Data source

Reference Type:
study report
Report Date:

Materials and methods

Test guidelineopen allclose all
according to
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
according to
EU Method B.31 (Prenatal Developmental Toxicity Study)
according to
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes (incl. certificate)
Limit test:

Test material


Test animals

Details on test animals and environmental conditions:
- Strain: Crl:WI(Han) (outbred, SPF-Quality)
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at delivery: 10-14 weeks
- Housing: individually in Makrolon cages (MIII type) on sterilized sawdust as bedding material.
- Diet and water: ad libitum
- Acclimation period: at least 5 days prior to treatment

- Temperature (°C): 18 - 24°C
- Humidity (%): approximately 40-70 %
- Air changes (per hr): > 10 per hour
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
polyethylene glycol
specific gravity 1.125
Details on exposure:
Administration volume: 5 mL/kg bw; actual dose volumes were calculated according to the latest body weight;
Appearance of formulations: clear solutions
PREPARATION OF DOSING SOLUTIONS: Formulations (w/w) were prepared daily within 5 hours prior to dosing and were homogenized to a visually acceptable level. Adjustment was made for specific gravity of the vehicle and test substance. No correction was made for the purity/composition of the test substance.

Analytical verification of doses or concentrations:
Details on analytical verification of doses or concentrations:
Analytical investigations were performed in the course of the pilot and of the main study. Accuracy of dose formulations, homogeneity and stability of the test substance in formulations were confirmed.
No test substance was detected in the Group 1 formulations (control group). The concentrations analysed in the formulations of Groups 2, 3, and 4 were in agreement with the target concentrations (i.e. mean accuracies between 90% and 110%).
The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10%).
Formulations at the entire range were stable when stored at room temperature under normal laboratory light conditions for at least 5 hours (i.e. relative difference ≤ 10%). The long term storage samples were stable at ≤-70°C for 9 days.
Details on mating procedure:
Untreated females were mated at the Supplier, and were at Day 0 or 1 post-coitum on arrival at the Test Facility (Day 0 post-coitum is the day of
successful mating; confirmed by vaginal plug).
Duration of treatment / exposure:
Days 6 - 20 p. c., inclusive
Frequency of treatment:
Once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest
Duration of test:
from day 0 to necropsy at day 21 p.c.
Doses / concentrations
Doses / Concentrations:
100, 300, 1000 mg/kg bw
actual ingested
No. of animals per sex per dose:
22 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: In order to set the dose levels for the main teratology study, a dose range finding study was performed. Four groups of 6 females were exposed to 100, 300, or 1000 mg/kg bw/day for Days 5 to 19 post-coitum inclusive by oral gavage. These dose levels were based on a repeated-dose oral toxicity study (OECD 407) with Desmorapid VP.PU 59IF05 in which male and female rats received the test substance by oral gavage, as a solution in polyethylene glycol 400, at dose levels of 0, 100, 300 or 1000 mg/kg bw/day.
No toxicologically significant clinical signs were noted up to 100 mg/kg bw/day. Hunched posture, rales and piloerection were observed in one animal at 300 mg/kg bw/day. In one additional animal at 300 mg/kg bw/day piloerection was observed. At 1000 mg/kg bw/day rales were
noted in 5/6 females and piloerection in 4/6 animals. Salivation was observed in all animals of Group 3 and Group 4. No further effects were seen.
Based on the results of the dose range finding study, the dose levels for the main study were selected to be 100, 300 and 1000 mg/kg bw/day.


Maternal examinations:
- Time schedule: At least once daily from Day 2 post-coitum onwards up to the day prior to necropsy.

- Time schedule for examinations: Days 2, 6, 9, 12, 15, 18 and 21 post-coitum.

- Days 2-6, 6-9, 9-12, 12-15, 15-18 and 18-21 post-coitum.

- Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected.

- Sacrifice on gestation day 21 with subsequently exsanguinated and subjected to an external, thoracic and abdominal examination, with special attention being paid to the reproductive organs.
Ovaries and uterine content:
Each ovary and uterine horn of animals surviving to planned necropsy was dissected and examined as quickly as possible to determine:
- The number of corpora lutea.
- The weight of the (gravid) uterus.
- The number and distribution of live and dead fetuses.
- The number and distribution of embryo-fetal deaths (early and late resorptions).
- The weight of each fetus.
- The sex of each fetus from the ano-genital distance (during necropsy) and also from gonadal
inspections (during further fetal examination).
- Externally visible macroscopic fetal abnormalities.
Fetal examinations:
External, visceral, and skeletal findings were recorded as developmental variations (alterations in anatomic structure that are considered to have no significant biological effect on animal health or body conformity and/or represent slight deviations from normal) or malformations (those structural
anomalies that alter general body conformity, disrupt or interfere with normal body function, or may be incompatible with life).
Each viable fetus was examined in detail, weighed and sexed. All live fetuses were euthanized by administration of approximately 0.05 mL (=10mg) of sodium pentobarbital into the oral cavity using a small flexible plastic or metal feeding tube.
Nonviable fetuses (the degree of autolysis was minimal or absent) were examined and weighed. For late resorptions a gross external examination performed (if possible).
Visceral (Internal):
Approximately one-half of the fetuses (live and dead) in each litter (all groups)were examined for visceral anomalies by dissection in the fresh (non-fixed) state. The thoracic and abdominal cavities were opened and dissected using a technique described by Stuckhardt and Poppe (Ref. 2). This
examination included the heart and major vessels. Fetal kidneys were examined and graded for renal papillae development as described by Woo and Hoar (Ref. 3). The sex of all fetuses was confirmed by internal examination.
The heads were removed from these fetuses and placed in Bouin's solution. Tissues were then transferred to a 70% aqueous ethanol solution for subsequent processing and soft-tissue examination of all groups using the Wilson sectioning technique. After examination, the tissues were stored in 10% buffered formalin. Any remaining tissues (from the fetuses used for fresh visceral examination) was discarded. The carcasses were processed and stained with Alizarin Red S (as described below), but not examined in first instance.
From the other one-half of the fetuses (live and dead) in each litter (all groups), the sex was confirmed by internal examination. All fetuses were eviscerated, fixed in 96% aqueous ethanol, macerated in potassium hydroxide and stained with Alizarin Red S by a method similar to that described by Dawson (Ref. 5). Skeletal examination was done for one-half of the fetuses (i.e. the fetuses with heads). The specimens were archived in glycerin with bronopol as preservative. A few bones were not available for skeletal examination because they were accidentally damaged or lost during processing. The missing bones were listed in the raw data; evaluation by the fetal pathologist and study director determined there was no influence on the outcome of the individual or overall skeletal examinations, or on the integrity of the study as a whole.
The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control group.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
- The Mann Whitney test was used to compare mean litter proportions (percent of litter) of the number of viable and dead fetuses, early and late resorptions, total resorptions, pre- and postimplantation loss, and sex distribution.
- Mean litter proportions (percent per litter) of total fetal malformations and developmental variations (external, visceral and skeletal), and each particular external, visceral and skeletal malformation or variation were subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunn’s test was used to compare the compound-treated groups to the control group.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.
Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations might be rounded off before printing. Therefore, two groups might display the same printed means for a given parameter, yet display different test statistics values.
No statistics were applied for data on maternal survival, pregnancy status, group mean numbers of dead fetuses, early and late resorptions, and pre- and post-implantation loss.
For each litter the following calculations were performed:
Percent Pre-implantation loss, percent Post-implantation loss
The fetal developmental findings were summarized by: 1) presenting the incidence of a given finding both as the number of fetuses and the number of litters available for examination in the group; and 2) considering the litter as the basic unit for comparison, calculating the number of affected fetuses as a
mean litter proportion on a total group basis, i.e. percent Viable fetuses affected/litter
The following definitions were applicable for implantation data:
- Fetal (late) resorptions were defined as a dead fetus with external degenerative changes and presence of distinguishable features such as head or limbs.
- Embryonic (early) resorptions were defined as evidence of implantation without presence of distinguishable features such as head or limbs.
- Dead fetus was defined as a non-viable fetus without external degenerative changes and presence of distinguishable features such as head or limbs.
- Post-implantation loss included embryonic (early) resorptions, fetal (late) resorptions and dead fetuses.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
Although the incidence and persistence of salivation showed a dose related increase, no related findings were noted. Therefore, the salivation is likely attributed to the taste of the test item and of no toxicological relevance.
Laboured respiration and/or rales for one to several days were noted in a few females in Group 2 and 3 and several animals in Group 4. In Group 3 and 4, for one and three females, respectively, these findings were concurrent with other signs of ill health, including hunched posture and decreased body weight gain. Therefore, these findings were considered toxicologically relevant and adverse in Group 3 and 4. In Group 2 no other signs of toxicity were present in the females for which the rales were noted and were considered of no toxicological significance.
Three of the above mentioned animals with laboured respiration showed ill health towards the end of the study (one female at 300 mg/kg bw/day and two females at 1000 mg/kg bw/day). For two of these females (one in each dose group) macroscopic examination showed a distended GI tract filled with gas and a small spleen, which was confirmed by a low spleen weight in both animals. Although at low incidence, the distended GI tract filled with gas was considered to be of toxicological relevance due to similar findings in a repeated-dose oral toxicity study (OECD 407) with Desmorapid VP.PU 59IF05.
Liver and kidney weights were unaffected by treatment up to 1000 mg/kg bw/day.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
Effect level:
1 000 mg/kg bw/day
Basis for effect level:
other: developmental toxicity
Dose descriptor:
dose level: 300 and 1000 mg/kg bw
Basis for effect level:
other: maternal toxicity

Results (fetuses)

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

Details on embryotoxic / teratogenic effects:
There were no treatment related effects on litter size, sex ratio, fetal body weights, fetal mophological examination, external malformations and variations, visceral malformations and variations, sceletal malformations and variations

Fetal abnormalities

not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

Executive summary:

In a prenatal developmental toxicity study performed according to OECD TG 414 mated female Wistar rats were assigned to four dose groups, each containing twenty-two animals. The test item was administered once daily by gavage from Day 6 to 20 post-coitum at doses of 100, 300 and 1000 mg/kg bw/day. The rats of the control group received the vehicle, Polyethylene glycol 400, alone.

One female of the 300 mg/kg bw group and three females of the 1000 mg/kg bw group showed laboured respiration concurrent with other signs of ill health, including hunched posture and decreased body weight gain. For two of these females (one in each dose group) macroscopic examination showed a distended GI tract filled with gas and a small spleen, which was confirmed by a low spleen weight in both animals. Although at low incidence, the distended GI tract filled with gas was considered to be of toxicological relevance due to similar findings in a repeated-dose oral toxicity study (OECD 407) with the test item.

No treatment-related effects on the fetal development were observed in all Desmorapid 01 treatment groups. In detail, litter size, sex-ratio, fetal body weight, and fetal morphological examinations (external, visceral and skeletal) were not affected by the treatment. In conclusion, based on the results in this prenatal developmental toxicity study the maternal No Observed Adverse Effect Level (NOAEL) for the test item was established as being 100 mg/kg bw/day. The developmental NOAEL was established as being at least 1000 mg/kg bw/day.