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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:
24 August 2016 - 01 December 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
adopted 22 January 2001
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
August 1998
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Version / remarks:
30 May 2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Test material form:
liquid: viscous

Test animals

Species:
rat
Strain:
Sprague-Dawley
Remarks:
Sprague-Dawley Crl:CD (SD) IGS BR
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, Kent
- Weight at study initiation: 189 to 285 g
- Fasting period before study: no
- Housing: individually in solid-floor polypropylene cages with stainless steel mesh lids furnished with softwood flakes
- Diet (e.g. ad libitum): pelleted diet (Rodent 2018C Teklad Global Certified Diet, Envigo RMS (UK) Limited, Oxon, UK), ad libitum
- Water (e.g. ad libitum): mains drinking water, ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 ºC
- Humidity (%): 50 ± 20%
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: 26 August 2016 (first day of treatment) to 15 September 2016 (final day of necropsy)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
propylene glycol
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The stability and homogeneity of the test item formulations were previously determined by Envigo Research Limited, Shardlow, UK Analytical Services (Envigo Study Number YY43XT). Results showed the formulations to be stable for at least twenty-two days. Formulations were therefore prepared once and stored at approximately +4 °C in the dark and under nitrogen.

VEHICLE
- Concentration in vehicle: 4.17, 16.7, 41.7 mg/mL
- Amount of vehicle (if gavage): 6 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples were taken of the test item formulations and were analyzed for concentration of BADGE-IPD at Envigo Analytical Laboratory, Shardlow. The results indicate that the prepared formulations were within ± 3% of the nominal concentration.
Details on mating procedure:
- Impregnation procedure: purchased timed pregnant (Animals were delivered in two batches containing females prior to Day 3 of gestation.)
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
Duration of treatment / exposure:
Day 3 to Day 19 of gestation
Frequency of treatment:
daily
Duration of test:
18 d (aminals were sacrificed on day 20 of gestation)
Doses / concentrationsopen allclose all
Dose / conc.:
25 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
24 (control, low and mid dose) / 26 (high dose)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were chosen based on previous toxicity data (Envigo Research Limited, Study Number PM78LM).
- Rationale for animal assignment (if not random): randomly allocated to treatment groups using a randomization procedure based on stratified body weight to ensure similarity between the treatment groups

Examinations

Maternal examinations:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: all animals were examined for overt signs of toxicity, ill-health or behavioral changes once daily during the gestation period; during the dosing period, observations were recorded immediately before dosing, up to thirty minutes after dosing and one hour after dosing

BODY WEIGHT: Yes
- Time schedule for examinations: Day 3 (before the start of treatment) and on Days 4, 5, 8, 11, 14 and 17 of gestation. Body weights were also recorded for surviving animals at terminal kill (Day 20).

FOOD CONSUMPTION: Yes
Food consumption was recorded for each surviving individual animal at Day 3, 5, 8, 11, 14, 17 and 20 of gestation

WATER CONSUMPTION: Yes
- Time schedule for examinations: daily

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day #20
- Organs examined: ovaries and uteri

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: placental weight


Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: all per litter
Statistics:
The following parameters were analyzed statistically, where appropriate, using the test methods outlined below:
Female body weight change, food consumption and gravid uterus weight: Shapiro Wilk normality test and Bartlett’s test for homogeneity of variance and one way analysis of variance, followed by Dunnett’s multiple comparison test or, if unequal variances were observed, on alternative multiple comparison test.
All caesarean necropsy parameters and fetal parameters: Kruskal-Wallis non-parametric analysis of variance; and a subsequent pairwise analysis of control values against treated values using the Mann-Whitney ‘U’ test, where significance was seen.
Fetal evaluation parameters, including skeletal or visceral findings: Kruskal-Wallis non-parametric analysis of variance and Mann-Whitney ‘U’ test.
Historical control data:
attached as .pdf file

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Noisy respiration and increased salivation were evident in the majority of females treated with 250 mg/kg bw/day throughout the treatment period. One of these females also showed decreased respiratory rate whilst another two females were observed with pilo erection on Day 17 of gestation. Of the females that were sacrificed in extremis at this level, three showed increased salivation and all of them showed respiratory pattern changes (noisy respiration, gasping/labored respiration and/or decreased respiratory rate). In addition, the female that was sacrificed in extremis on Day 10 had hunched posture and lethargy and the female that was sacrificed in extremis on Day 19 had hunched posture, pilo-erection, dehydration, red/brown stained snout and tiptoe gait.
Instances of noisy respiration were evident in eight females treated with 100 mg/kg bw/day on Days 5-10, 12, 13 and/or 16-20 and two females treated with 100 mg/kg bw/day had increased salivation on Days 4, 6 and/or 10. The female found dead on Day 16 of gestation showed no clinical signs before death.
At 25 mg/kg bw/day, three females had noisy respiration on Days 15 or 17 and one female had increased salivation on Day 15 only.
Mortality:
mortality observed, treatment-related
Description (incidence):
Four females treated with 250 mg/kg bw/day were sacrificed in extremis on Days 10, 15 and 19 of gestation due to adverse clinical signs and in three of the females, excessive body weight losses were also noted.
One female treated with 100 mg/kg bw/day was found dead on Day 16 of gestation. Macroscopic examinations of this female revealed a hole in the esophagus, therefore this death was considered to be the result of a dosing trauma rather than a direct effect of the test item.
There were no further unscheduled deaths.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weight gain for females treated with 250 mg/kg bw/day between Days 3 and 8 of gestation was generally comparable to controls. Cumulative body weight gain from Day 11 onwards was lower and statistical significance (p<0.05) was achieved between Days 3 and 11, 3 and 14 and 3 and 17. Incidences of actual body weight losses were also evident in surviving females, sporadically throughout the treatment period. Three out of the four females that were sacrificed in extremis showed significant body weight loss prior to termination. Body weight gain for surviving females when adjusted for gravid uterus weight was also statistically significantly (p<0.01) reduced when compared to controls.
Body weight gain during gestation, including after adjustment for the contribution of the gravid uterus, was considered to be unaffected by treatment at 100 or 25 mg/kg bw/day.
Females treated with 25 mg/kg bw/day showed a statistically significant (p<0.05) increase in body weight gain between Days 5 and 8 of gestation. An increase in body weight gain is considered not to represent an adverse effect of treatment.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Females treated with 250 mg/kg bw/day showed a statistically significant reduction (p<0.05-0.001) in food consumption between Days 3 and 17 of gestation. Food consumption between Days 17 and 20 was also lower, although statistical significance was not achieved.
No differences as compared to the control group were detected on food consumption in females treated with 100 or 25 mg/kg bw/day.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Daily visual inspection of water bottles did not reveal any overt intergroup differences.
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:
not examined
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Three of the females treated with 250 mg/kg bw/day that were sacrificed in extremis had gaseous distension in the stomach and/or gastro-intestinal tract. One of the females treated with 250 mg/kg bw/day that was sacrificed in extremis on Day 15 also had a dark liver and dark patches on all lobes of the lungs.
The female treated with 100 mg/kg bw/day that was found dead on Day 16 of gestation had gaseous distension in the stomach and gastro-intestinal tract, a dark liver, dark red patches on the lungs and a hole in the esophagus (approximately 3mm x 3mm). Due to the observations evident in the esophagus, this death was most likely to be the result of a dosing trauma rather than a direct effect of the test item.
No toxicologically significant macroscopic abnormalities were detected in the surviving females treated with 250 mg/kg bw/day or in females treated with 100 or 25 mg/kg bw/day.
One female treated with 100 mg/kg bw/day had a small left adrenal and an enlarged right adrenal. A further female from this treatment group had patchy fur loss on both hindlimbs. One female treated with 25 mg/kg bw/day had gaseous distension in the duodenum and a control female had pale adrenals. These observations were considered to be low incidental findings and of no toxicological significance.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
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): no effects observed
Changes in number of pregnant:
no effects observed
Details on maternal toxic effects:
There was no obvious effect of maternal treatment on litter data as assessed by numbers of implantations, in-utero offspring survival (as assessed by the mean numbers of early or late resorptions), live litter size, sex ratio or pre- or post-implantation losses at 25, 100 or 250 mg/kg bw/day.
Intergroup differences for mean fetal, litter or placental weights did not indicate any obvious effects of maternal treatment at 25, 100 or 250 mg/kg bw/day.
Statistical analysis of the data did not reveal any significant intergroup differences.

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Basis for effect level:
clinical signs
mortality
gross pathology

Maternal abnormalities

Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
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:
no effects observed
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
A statistically significant reduction (p<0.01) in the number of fetuses/litters showing unossified areas of the occipital (supra-occipital) was evident at 100 mg/kg bw/day. The group mean value was within historical control range and the observation of one variant at a lower incidence compared with controls is not significant when evaluated in isolation. In the absence of a similar effect at the high dose group or any particular pattern of abnormal skeletal development of skeletal structures affecting treated fetuses, the observation of one affected skeletal structure can be considered unlikely to represent true developmental abnormality. This takes account of this finding being seen regularly on this study type amongst control group fetuses.
Visceral malformations:
no effects observed
Description (incidence and severity):
Neither the type, incidence nor the distribution of findings observed during external examination of the fetuses at necropsy on gestation Day 20 and subsequent detailed visceral and skeletal examination indicated any adverse effect of maternal exposure on fetal development.
Statistical analysis did not reveal any significant intergroup differences.

Effect levels (fetuses)

Dose descriptor:
NOEL
Effect level:
>= 250 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: no effects observed

Fetal abnormalities

Abnormalities:
no effects observed

Overall developmental toxicity

Developmental effects observed:
no

Any other information on results incl. tables

results tables are attached as .pdf file below

Applicant's summary and conclusion

Conclusions:
Treatment at 250 mg/kg bw/day was associated with increased salivation and respiratory pattern changes in the majority of females, the early termination of four females on Days 10, 15 and 19 and treatment-related effects on body weight performance and food consumption in the remaining females. The resulting lower overall body weight gain in surviving females remained lower than controls even when adjusted for the contribution of the gravid uterus.
No macroscopic abnormalities were detected in the surviving females of the high dose group however three of the females from the high dose group that were sacrificed in extremis had gaseous distension in the stomach and/or gastrointestinal tract. One of the females that was sacrificed in extremis on Day 15 also had a dark liver and dark patches on all lobes of the lungs. Despite the observed maternal effects, there were no obvious effects of maternal treatment on the survival, growth or morphological development of the offspring and therefore this dosage is considered to represent a No Observed Effect Level (NOEL) for the developing conceptus.
At 100 and 25 mg/kg bw/day, treatment was associated with instances of noisy respiration and increased salivation (at a lesser extent than at 250 mg/kg bw/day). There were no obvious effects on body weight or food intake during gestation and no treatment-related macroscopic necropsy findings were apparent for adult animals at these dosage. Given the transient nature of the clinical signs evident, 100 mg/kg bw/day is considered to represent a ‘No Observed Adverse Effect Level’ (NOAEL) for the pregnant female.
Executive summary:

The study was performed to investigate the effects of BADGE-IPD on embryonic and fetal development following repeated administration by gavage to the pregnant female during gestation including the period of organogenesis. The study was designed to comply with OECD Guideline 414 (adopted 22 January 2001).

The test item was administered by gavage, between Days 3 and 19 of gestation, to three groups at dose levels of 25, 100, and 250 mg/kg bw/day. The low and intermediate dose groups contained twenty-four time mated Sprague-Dawley Crl:CD (SD) IGS BR strain rats, and the high dose group contained twenty-six time mated Sprague-Dawley Crl:CD (SD) IGS BR strain rats. A further group of twenty-four time mated females was exposed to the vehicle only (Propylene Glycol) to serve as a control.

Clinical signs, body weight change, food and water consumptions were monitored during the study. 

All surviving females were terminated on Day 20 of gestation and subjected to gross necropsy including examination of the uterine contents. The number of corpora lutea, number, position and type of implantation, placental weights, fetal weights, sex and external and internal macroscopic appearance were recorded. Half of each litter were examined for detailed skeletal development and the remaining half were subjected to detailed visceral examination.

Mortality

Four females treated with 250 mg/kg bw/day were sacrificed in extremis on Days 10, 15 and 19 of gestation due to adverse clinical signs and in three of the females, excessive body weight losses were also noted. 

One female treated with 100 mg/kg bw/day was found dead on Day 16 of gestation. Macroscopic examinations of this female revealed a hole in the esophagus, therefore this death was considered to be the result of a dosing trauma rather than a direct effect of the test item.

There were no further unscheduled deaths.

Clinical Observations

Noisy respiration and increased salivation were evident in the majority of females treated with 250 mg/kg bw/day throughout the treatment period. In addition to increased salivation and respiratory pattern changes, one of the females that were sacrificedin extremisalso showed hunched posture and lethargy and another one of these females showed dehydration, hunched posture, pilo-erection, tiptoe gait and a red/brown stained snout.

Instances of noisy respiration and increased salivation were also evident in some females treated with 100 and 25 mg/kg bw/day albeit to a lesser extent.

Body Weight

Body weight gain for females treated with 250 mg/kg bw/day between Days 3 and 8 of gestation was generally comparable to controls. However cumulative body weight gain from Day 11 onwards was lower and incidences of actual body weight losses were evident in surviving females throughout the treatment period. Three out of the four females that were sacrificedin extremisshowed significant body weight loss prior to termination. Body weight gain for surviving females when adjusted for gravid uterus weight was also reduced when compared to controls.

No such effects were evident in females treated with 100 or 25 mg/kg bw/day.

Food Consumption

Females treated with 250 mg/kg bw/day showed a reduction in food consumption throughout the treatment period.

No differences as compared to the control group were detected on food consumption in females treated with 100 or 25 mg/kg bw/day.

Water Consumption

No differences as compared to the control group were detected on water consumption.

Post Mortem Studies

Three of the females treated with 250 mg/kg bw/day that were sacrificedin extremishad gaseous distension in the stomach and/or gastro-intestinal tract. One of the females treated with 250 mg/kg bw/day that was sacrificedin extremison Day 15 also had a dark liver and dark patches on all lobes of the lungs.

The female treated with 100 mg/kg bw/day that was found dead had gaseous distension in the stomach and gastro-intestinal tract, a dark liver, dark red patches on the lungs and a hole in the esophagus (approximately 3mm x 3mm). Due to the observations evident in the esophagus, this death was most likely to be the result of a dosing trauma rather than a direct effect of the test item.  

No toxicologically significant macroscopic abnormalities were detected in the surviving females treated with 250 mg/kg bw/day or in females treated with 100 or 25 mg/kg bw/day.

Litter Data and Litter Placental and Fetal Weights

The number of implantations, subsequent embryofetal survival, live litter size and sex ratio on Day 20 of gestation were considered to be unaffected by maternal treatment at 25, 100 or 250 mg/kg bw/day. Mean fetal, placental and litter weights were also considered to have been unaffected by maternal treatment at 25, 100 or 250 mg/kg bw/day.

Fetal Examination

External examination of fetuses on Day 20 of gestation did not indicate any obvious effect of maternal treatment on fetal development at 25, 100 or 250 mg/kg bw/day. Findings at detailed skeletal and visceral examinations of fetuses on Day 20 of gestation did not indicate any obvious effect of maternal treatment on fetal development at 25, 100 or 250 mg/kg bw/day.

Conclusion

The oral (gavage) administration of BADGE-IPD to pregnant rats during gestation at dose levels of 25, 100 and 250 mg/kg bw/day, resulted in treatment related effects at 250 mg/kg bw/day. A reduction in cumulative body weight gain and food consumption was evident at this level together with adverse clinical signs detected and the early sacrifice of four females (Days 10, 15 and 19 of gestation).

Although one female treated with 100 mg/kg bw/day was found dead on Day 16, macroscopic observations revealed a hole in the esophagus, therefore this death was most likely to be the result of a dosing trauma rather than a direct effect of the test item. No adverse effects were evident in body weight gain or food consumption at 100 mg/kg bw/day, therefore 100 mg/kg bw/day was considered to represent the ‘No Observed Adverse Effect Level’ (NOAEL) for the pregnant female.

No treatment-related changes were detected in the offspring parameters measured or on embryofetal development. The ‘No Observed Effect Level’ (NOEL) for developmental toxicity was therefore considered to be 250 mg/kg bw/day.