Registration Dossier

Toxicological information

Developmental toxicity / teratogenicity

Currently viewing:

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
23 Nov 2016 -
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
adopted Jan 2001
Deviations:
yes
Remarks:
(purity of the test material not given)
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
not specified
Qualifier:
according to
Guideline:
other: Japanese Ministry of Agriculture, Forestry and Fisheries, Test Data for Registration of Agricultural Chemicals, 12 Nohsan No. 8147
Version / remarks:
24 Nov 2000
Deviations:
not specified
GLP compliance:
yes (incl. certificate)
Remarks:
The Department of Health of the Government of the United Kingdom
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

Test animals

Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS
- Age at study initiation: 18 - 22 weeks
- Weight at study initiation: 2.31 - 4.36 kg
- Housing: The animals were individually housed during acclimatization and gestation period, and co-housed (M/F ration: 1/1) during mating period in suspended cages fitted with perforated floor panels. An aspen chew block was provided to each cage as environmental enrichment.
- Diet: Teklad 2930 pelleted diet, initially 150 g/animal/day during acclimatization up to one week prior to the onset of mating and 200 g/animal/day thereafter. In addition, a small supplement of autoclaved hay was given on a daily basis and a small amount of chopped fresh vegetables were given twice weekly.
Water: Potable water, ad libitum
- Acclimation period: 19 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 15 - 21
- Humidity (%): 45 - 70
- Photoperiod (hrs dark / hrs light): 10/14

IN-LIFE DATES: From: 23 Nov 2016 To:

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
purified
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Dosing solutions were prepared weekly by dissolving appropriate amounts of the test material in purified water yielding a final concentration of 100 mg/mL. The remaining concentrations were prepared by serial dilution with further quantities of vehicle.

VEHICLE
- Amount of vehicle (if gavage): 5 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability of the test substance in water formulations at nominal concentrations of 2 mg/mL and 200 mg/mL was confirmed following refrigeration for 17 days at the same testing facility (Study No. KK40KJ)
Details on mating procedure:
- Impregnation procedure: cohoused
- M/F ratio per cage: 1/1
- Verification of same strain and source of both sexes: yes
- Other: The day of mating is considered to be Day 0 of Gestation. Where possible only females mating at least twice were allocated.
Duration of treatment / exposure:
Day 6 - 28 of gestation
Frequency of treatment:
daily, 7 days/week
Duration of test:
Day 29 post mating
Doses / concentrationsopen allclose all
Dose / conc.:
125 mg/kg bw/day (nominal)
Dose / conc.:
250 mg/kg bw/day (nominal)
Dose / conc.:
500 mg/kg bw/day (nominal)
No. of animals per sex per dose:
22
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Doses were chosen based on the results of a preliminary study for effects on embryo-fetal development in the New Zealand White rabbit at the same testing facility (Study No. SF87BJ). In that study, treatment with the test substance at 125 or 250 mg/kg/day was well-tolerated, with no effects apparent that precluded the use of these doses on the subsequent main study for effects on embryo-fetal development. Treatment at 500 mg/kg/day elicited effects of reduced body weight gain and food consumption on the does, and reduced litter and fetal weights amongst the litters of females which received the test substance at 500 mg/kg/day, the magnitude of which did not preclude the use of this dose on the subsequent main study for effects on embryo-fetal development where the highest dose was chosen with the aim to induce some developmental and/or maternal toxicity. Doses of 125, 250 and 500 mg/kg/day were considered appropriate for the subsequent main study for effects on embryo-fetal development.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes, all animals were inspected visually for evidence of clinical signs and mortality.
- Time schedule: At least twice daily

DETAILED CLINICAL OBSERVATIONS: Yes, a detailed phyisical examination was performed on all animals to monitor general health.
- Time schedule: On Days 0, 6, 12, 18, 24 and 29 after mating

BODY WEIGHT: Yes, body weights of all animals were recorded.
- Time schedule for examinations: Weekly during acclimatizatuin, on the day of mating and on Days 0, 3 and 6 - 29 post mating.

FOOD CONSUMPTION: Yes
- The weight of food supplied to each animal, that remaining and an estimate of any spilled was recorded daily from Day 1 after mating.

POST-MORTEM EXAMINATIONS: Yes, all adult animals were subject to a detailed necropsy. Animals surviving until the end of the scheduled study period were killed on Day 29 post mating. Females that exhibited pregnancy loss were killed on the day of detection.
- Organs examined: All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes, for females surviving to term.
- Number of corpora lutea: Yes, for all animals.
- Number of implantations: Yes, for all animals.
- Number of early resorptions: Yes, for all animals.
- Number of late resorptions: Yes, for all animals.
Fetal examinations:
- External examinations: Yes, all fetuses were weighed, and were subject to an external examination and a gross internal examination of the viscera of the neck, thorax and abdominal cavities and the sex of each fetus was recorded.
- Soft tissue examinations: Yes, serial sections were examined for soft tissue abnormalities.
- Skeletal examinations: Yes, the torsos of the decapitated fetuses and the remaining fetuses were eviscerated and fixed in Industrial Methylated Spirit.
- Head examinations: Yes, half per litter were decapitated and the heads were fixed in Bouin`s fluid.
Statistics:
The following data types were analysed at each timepoint separately:
- Body weight, using absolute values and gains over appropriate study periods
- Gravid uterine weight and adjusted body weight
- Food consumption, over appropriate study periods
- Litter size and survival indices
- Fetal, placental and litter weight

Detailed information on statistical information is provided under "any other information on materials and methods incl. tables".

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
125 mg/kg bw/d: 1/22 females had shown swaying and gasping respiration prior to death, and was found dead on Day 15 of gestation.
500 mg/kg bw/d: 1/22 females showed signs of respiratory distress and was getting very stressed when the technicians tried to touch her. The animal was despatched to necropsy on Day 9 of gestation (plase refer to "Mortality").
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Control: 1/22 females was found dead on Day 21 of gestation. The animal did not show any clinical signs prior to death, and there were no abnormalities detected at macroscopic examination.

125 mg/kg bw/d: 1/22 females was found dead on Day 15 of gestation. Clinical signs and a loss of body weight were observed prior to death. Macroscopic examination revealed abnormalities (please refer to "Gross pathological findings"). However, according to the study director the death was unclear and was not considered to be treatment-related. 1/22 females was despatched to necropsy on Day 24 of gestation following evidence of abortion. No clinical signs were observed prior to death and no abnormalities were detected at macroscopic examination.

500 mg/kg bw/d: 1/22 females was despatched to necropsy on Day 9 of gestation having shown signs of respiratory distress and a lost 410 g of body weight since the start of dosing. Macroscopic examination revealed abnormalitites (please refer to "Gross pathological findings"). According to the study director, the death was considered to be non-treatment related.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
There was no clear effect of treatment on the body weight performance of females receiving the test substance at 125 or 250 mg/kg bw/d when compared with that of the control group. The mean body weight loss of females receiving dosed at 500 mg/kg bw/d was slightly greater than that of the control group after the start of treatment (between Days 6 and 8 after mating), and body weight gain was slightly less than that of the control animals from Days 8 - 29 after mating.
When mean values of body weight and body weight gain were adjusted for the contributions of the gravid uterus, overall maternal mean body weight loss during Days 6 - 29 of gestation was greater at 500 mg/kg bw/d than in the control group.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
There was no clear effect of treatment on the food consumption of females receiving the test substance at 125 or 250 mg/kg bw/d when compared with that of the control group. The food consumption of females dosed at 500 mg/kg bw/d was slightly lower than that of the control group from the start of treatment (Day 6 after mating), resulting in a lower overall (Day 6 - 28 after mating) food consumption.
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:
not examined
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
125 mg/kg bw/d: The macroscopic examination of the animal found dead on Day 15 of gestation, revealed discoloured (dark) adipose tissue in the thoracic region, dark staining to the muzzle, dark fluid in the nasal turbinates, and dark right lungs.
500 mg/kg bw/d: The animal which was despatched to necropsy on Day 9 of gestation had poorly defined, dark areas on the lungs, gas in the jejunum and duodenum, and findings in the stomach consisting of a raised and firm antrum with a depressed area on the glandular mucosa at necropsy.

These findings were not considered to be treatment related or toxicologically relevant.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined

Maternal developmental toxicity

Number of abortions:
effects observed, non-treatment-related
Description (incidence and severity):
125 mg/kg bw/d: The animal which was despatched to necropsy on Day 24 of gestation following evidence of abortion, was confirmed as pregnant with 7 implantations, 5 of which were early resorptions, 1 late resorption and 1 aborted implantation. However, according to the study director, this single abortion is considered not to be related to treatment.
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:
effects observed, non-treatment-related
Description (incidence and severity):
2/22 females of the control group and of the highest dose group, respectively, 1/22 females of the low-dose group and 3/22 females of the mid-dose group were not pregnant.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: No adverse maternal toxicity was observed at the highest tested dose level.

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
500 mg/kg bw/d: Fetal weights were marginally lower than the fetal weight of the control group.
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:
not examined
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
effects observed, non-treatment-related
Description (incidence and severity):
500 mg/kg bw/d: Litter weights were marginally lower than the fetal weight of the control group.
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
500 mg/kg bw/d: There were two minor fetal pathology findings including a slightly high incidence of additional cranial sutures, and a slightly high incidence of delayed ossification of the cervical vertebrae. According to the study director, the slightly high incidence of additional cranial sutures is in isolation considered not to represent an adverse effect of treatment as cranial bone development was normal. The delayed ossification of the cervical vertebrae represents the results of an evaluation at a snapshot in time (ossification would be expected to continue as the animals matured) and may be linked to the marginally lower mean fetal weights. The incidence of 7th costal cartilage not connected to sternum was lower compared to concurrent controls. According to the study director, the values were slightly high in the control and low-dose group, respectively, and slightly low in the high-dose group when compared with historical control data, thus the finding was considered to be not toxicologically significant.
Visceral malformations:
no effects observed
Other effects:
no effects observed

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse embryo-fetal toxicity was observed at the highest tested dose level.

Fetal abnormalities

Key result
Abnormalities:
effects observed, non-treatment-related
Localisation:
skeletal: skull sutures
skeletal: vertebra
Description (incidence and severity):
Two minor fetal pathology findings including a slightly high incidence of additional cranial sutures, and a slightly high incidence of delayed ossification of the cervical vertebrae were observed at 500 mg/kg bw/d. According to the study director, the slightly high incidence of additional cranial sutures is considered not to represent an adverse effect of treatment as cranial bone development was normal. The delayed ossification of the cervical vertebrae represents the results of an evaluation at a snapshot in time (ossification would be expected to continue as the animals matured) and may be linked to the marginally lower mean fetal weights.

Overall developmental toxicity

Key result
Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
Under the conditions of the study the test substance did not induce any treatment-related biologically relevant malformations in the developing unborn organism in the presence of maternal toxicity. Therefore, the test substance does not meet the criteria for classification for prenatal developmental toxicity according to Regulation (EC) No 1272/2008. The available data is thus conclusive but not sufficient for classification.