Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-2017
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 guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
Batch (Lot) No.: 160410-1
Receipt Date: 26 Aug 2016
Expiration Date: 03 Apr 2018
Physical Description: White crystalline powder
Purity: 96.0%
Correction Factor: 1.04. dose calculations were corrected for purity
Storage Conditions: Ambient in the dark

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
The Sprague Dawley rat was chosen as the animal model for this study as it is a rodent species accepted by regulatory agencies for toxicity testing.
The total number of animals used in this study was considered to be the minimum required to properly characterise the effects of the test item. This study was designed such that it did not require an unnecessary number of animals to accomplish its objectives.

At study assignment, each animal was identified using a subcutaneously implanted electronic cylindrical, ‘glass-sealed’ TROVAN microchip. Animals were allowed to acclimate to the Test Facility rodent toxicology accommodation from arrival until Day 6 of gestation (3-5 days).

Husbandry practices and environmental enrichment were carried out as per Test Facility SOPs and protocol. Each batch of diet, bedding and all environmental enrichment items were analysed by the suppliers. Water from the public supply is analysed at regular intervals for chemical and microbiological burden. Copies of certificates provided by the suppliers for all materials are retained at the Test Facility. It is considered that there were no contaminants in any of these materials that influenced the outcome of this study.

Animals were housed 2 per cage for psychological/environmental enrichment in appropriately sized suspended polycarbonate cages with stainless steel grid tops and solid bottoms.
Bedding material was sterilised white wood shavings.

Temperatures of approximately 21°C with a relative humidity of 46–63% were maintained. A 12 hour light/12 hour dark cycle was maintained, except when interrupted for designated procedures. Ten or greater air changes per hour were maintained in the animal rooms.

SDS VRF-1 breeder diet was provided ad libitum throughout the study, except during designated procedures.
The animals had access to water ad libitum from the public supply from water bottles which were changed as necessary throughout the course of the study. Veterinary care was available throughout the course of the study; however, no examinations or treatments were required.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 0.5% (w/w) Carboxymethylcellulose (400-800 cps) and 0.1% (w/w) TWEEN® 80 in deionised water
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Validation of Methodologies for the Formulation and Analysis of FR-720 in Oral (Gavage) Dosing Formulation was peormed at the testing facility prior to initiation of the study (Report No. 37705- attached).



Duration of treatment / exposure:
The test and control items were administered to the appropriate rats from Days 6-19 of gestation.
Frequency of treatment:
Once daily oral gavage
Duration of test:
20 days
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Control
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
24
Control animals:
yes, concurrent vehicle
Details on study design:
The test and control items were administered to the appropriate rats by once daily oral gavage from Days 6-19 of gestation. The prepared control item and dosing formulations were removed from the refrigerator and stirred for at least 2 hours before dosing and continuously during dosing. The dose volume for each animal was based on the most recent body weight measurement. The doses were given using a plastic syringe with attached gavage cannula. The dosing formulations were stirred continuously during dose administration.

Dose levels were chosen after review of data from a preliminary prenatal development study (Test Facility Study No. 4981742). On that study, target dose levels of up to 1000 mg/kg/day (actual values in the range 78% to 101% of nominal) were well tolerated and were not associated with any adverse findings. A minimal reduction in weight gain at the high dose level was considered not adverse and therefore did not preclude use of 1000 mg/kg/day as the high dose level for this study. The in-life procedures, observations, and measurements listed below were performed for all animals.

Animals were observed for general health/mortality and morbidity twice daily, once at the start and once towards the end of the working day, throughout the study. Animals were not be removed from the cage during observation, unless necessary for identification or confirmation of possible findings.
Animals were removed from the cage for daily examinations, beginning on Day 6 of gestation. Animals were examined for reaction to treatment regularly throughout dosing days. Animals were examined for reaction to treatment. The onset, intensity and duration of any signs was recorded (if appropriate), with particular attention being paid to the animals during and for the first hour after dosing.

Examinations

Maternal examinations:
Mortality/Moribundity Checks- Animals were observed for general health/mortality and morbidity- twice daily, once at the start and once towards the end of the working day, throughout the study.

Clinical Observations- daily examinations, beginning on Day 6 of gestation.

Postdose Observations- Animals were examined for reaction to treatment regularly throughout dosing days, with particular attention being paid to the animals during and for the first hour after dosing.

Body weights- were recorded once pretreatment (Day 4 of gestation) and recorded daily during in the dosing period (Days 6 – 20 of gestation). Animals were individually weighed.

Food consumption per cage was quantitatively measured daily from Day 4 of gestation (first measured quantity given on Day 3 of gestation).

Necropsy- All adult animals were subjected to a complete necropsy examination.
Ovaries and uterine content:
Ovarian/ Uterine examinations were performed on all animals (both control and test).
Fetal examinations:
Examinations of Pregnancies
The reproductive tract was dissected from the abdominal cavity. The gravid uterus was weighed. The uterus was opened and the contents examined. The fetuses were removed from the uterus.
The ovaries and uterus were examined for number and distribution of:
• Corpora Lutea
• Implantation Sites
• Placenta – Size, shape and colour observed (only abnormalities were recorded)
• Live and Dead Fetuses
• Early and Late Embryonic Deaths

External Abnormalities
Fetuses were examined for external abnormalities. Late resorptions and dead fetuses were examined for external abnormalities to the extent possible.
Each implant was classified as being live, or a dead fetus (dead full term fetus that showed no sign of maceration), or a late embryonic death (macerated tissue identifiable as an embryo fetus, with recognizable external features such as tail, limbs, mouth and nares present; attached to distinct identifiable placenta), or an early embryonic death (discrete, formless, discoloured tissue mass attached to the internal uterine wall; may be of varying size).

Body Weights and Identification
The body weight of each fetus was recorded. Fetuses were individually identified within litters.

Visceral Examination and Sex
Half of the viable fetuses from each uterus were fixed in methylated ethyl alcohol, the remaining half in Bouin's fluid. The fetuses fixed in Bouin's fluid were examined for soft tissue abnormalities and sex using a freehand sectioning technique derived from that of Wilson3.

Skeletal Examination
Following initial fixation, the fetuses fixed in alcohol were sexed and then eviscerated, and those viscera were then discarded without examination.
The eviscerated carcasses were then macerated in potassium hydroxide, the skeletons stained with Alizarin Red S, then the fetuses cleared with aqueous glycerol solutions. These preparations were then examined for the presence of skeletal abnormalities and for the extent of ossification.
Statistics:
Levene’s test was used to assess the homogeneity of group variances.
Datasets with at least 3 groups were compared using an overall one-way ANOVA F test if Levene’s test was not significant or the Kruskal-Wallis test if it was. If the overall F test or Kruskal-Wallis test was found to be significant, then the above pairwise comparisons were conducted using Dunnett’s or Dunn’s test, respectively.
Datasets with 2 groups (the designated control group and 1 other group) were compared using a t-test if Levene’s test was not significant or Wilcoxon Rank-Sum test if it was.
Indices:
Reproductive Indices:
For all fetal and litter data, the litter was used as the unit of assessment. Group mean values for each parameter were calculated as a mean of litter percentages.

Pre-implantation loss (%) per litter was calculated as:
[(Number of corpora lutea – Number of implantations per female)/ Number of corpora lutea per female] x100

Post-implantation loss (%) per litter was calculated as:
[(Number of implantations – Number of live fetuses per female)/Number of implantations per female] x100

Group mean implantation losses were expressed on a per litter basis according to the following:
Total individual litter pre or post implantation loss (%)/ Number of litters per group

Fetal abnormalities were classified as follows:
Major abnormalities: Rare, probably lethal or detrimental to the fetus
Minor abnormalities: Minor differences from ‘normal’ that are not lethal
Variants: Alternative structures occurring regularly in the control population which may be permanent or transient observations

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
There were no unscheduled deaths during the study.
At 300 or 100 mg/kg/day, increased incidences of soft/pale faeces, erect and hunched posture were noted, when compared with controls, and there were also incidences of decreased activity at 1000 mg/kg/day.
At 300 or 1000 mg/kg/day, reduced mean body weight gain over Days 6-20 of gestation (-7% and -12%, respectively) was noted when compared with controls, with the reduced weight gain being most obvious over the first approximate week of treatment (Days 6-13 of gestation). There were no test item-related effects on body weight at 100 mg/kg/day.
At 1000 mg/kg/day, mean food consumption was approximately 20-40% lower than controls over Days 9 to 12 of gestation, with recovery noted from Day 13 of gestation. There were no test item-related effects on consumption at 100 or 300 mg/kg/day.
There were no test item-related gross necropsy findings.
Uterine findings and fetal weights were comparable between control and treated groups, and the type and distribution of major or minor fetal abnormalities, variants and skeletal ossification parameters did not indicate any association with treatment.
Maternal findings at 300 mg/kg/day were considered not to be adverse due to low magnitude and no overall effect on pregnancy or fetal development. At 1000 mg/kg/day, findings were considered to be adverse to dams due to increased incidence and severity.
Tables of the results are attached in the appropriate section.
Dermal irritation (if dermal study):
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 300 or 1000 mg/kg/day, reduced mean body weight gain over the study period (-7% or 12% respectively, over Days 6-20 of gestation) was noted when compared with controls, with the reduced gain being most obvious over the first approximate week of treatment (Days 6-13 of gestation).
At 100 mg/kg/day, mean body weights and weight gains were comparable to controls, throughout the study.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg/day, mean food consumption was approximately 20-40% lower than controls over Days 9 to 12 of gestation (p<0.01 or p<0.005 on Days 9-11 of gestation). From Day 13 of gestation, consumption was comparable with controls for the remainder of the study.
At 100 or 300 mg/kg/day, group mean food consumption was generally comparable with controls throughout the study. Occasional low values at those levels were considered not to be treatment related since they were sporadic and did not follow clear treatment related patterns.
Summary of data are attached.
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:
no effects observed
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed

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:
no effects observed
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
Other effects:
no effects observed
Details on maternal toxic effects:
There were no unscheduled deaths during the study.

Maternal clinical signs showed a dose related increased incidence of soft/pale faeces and erect fur in all treated groups, when compared with controls; and there were also isolated incidences of hunched posture at 300 or 1000 mg/kg/day, and decreased activity at 1000 mg/kg/day.

At 300 or 1000 mg/kg/day, reduced mean body weight gain over Days 6-20 of gestation (-7% and -12%, respectively) was noted when compared with controls, with the reduced weight gain being most obvious over the first approximate week of treatment (Days 6-13 of gestation). There were no test item-related effects on body weight at 100 mg/kg/day.

At 1000 mg/kg/day, mean food consumption was approximately 20-40% lower than controls over Days 9 to 12 of gestation, with recovery noted from Day 13 of gestation. There were no test item-related effects on consumption at 100 or 300 mg/kg/day.

There were no test item-related gross necropsy findings.
Uterine findings and fetal weights were comparable between control and treated groups, and the type and distribution of major or minor fetal abnormalities, variants and skeletal ossification parameters did not indicate any association with treatment.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
>= 300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
clinical signs
body weight and weight gain
food consumption and compound intake
Remarks on result:
other: clinical signs, body weight, food consumption

Results (fetuses)

Fetal body weight changes:
not examined
Description (incidence and severity):
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:
not specified
Changes in litter size and weights:
not examined
Changes in postnatal survival:
no effects observed
External malformations:
no effects observed
Skeletal malformations:
no effects observed
Visceral malformations:
no effects observed
Other effects:
no effects observed
Details on embryotoxic / teratogenic effects:
The type and distribution of major fetal abnormalities and minor skeletal or visceral abnormalities and variants did not indicate any association with treatment with FR-720.
There were also no changes in skeletal ossification which were considered to be related to treatment with FR-720. Intergroup differences in ossification parameters were considered to be incidental since there were no obvious treatment related patterns and/or overall trends to indicate an association with treatment.
Fetal abnormalities, variants and skeletal ossification were within Test Facility historical control ranges for the past approximate 5 years (historical ranges presented in the relevant tables), with the exception of the occasional finding. Where findings were not within the historical ranges, they were considered to be of the nature and incidence which did not indicate any association with treatment.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
ca. 1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other:
Remarks on result:
other: No effects

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no
Treatment related:
no
Dose response relationship:
no
Relevant for humans:
not specified

Applicant's summary and conclusion

Conclusions:
Based on the study results, it was concluded that the maternal No-Observed-Adverse-Effect-Level (NOAEL) was 300 mg/kg/day based on increased incidences of clinical observations, reduced weight gain and lower food consumption at 1000 mg/kg/day.
The fetal No-Observed-Adverse-Effect-Level (NOAEL) was considered to be 1000 mg/kg/day.
Executive summary:

SUMMARY

The objective of this study was todetermine the potential toxicity of the chemical FR-720when the material was administered to pregnant rats during the period of organogenesis.

Time-mated female Sprague Dawley rats were randomised into 3 treated groups and 1 control group. The study design was as follows:

Group No.

Test Item

Dose Level

(mg/kg/day)

Dose Volume

(mL/kg)

Dose Concentration

(mg/mL)

No. of Animals

Females

1

Control*

0

10

0

24

2

FR-720

100

10

10

24

3

FR-720

300

10

30

24

4

FR-720

1000

10

100

24

*The control item and vehicle for formulation was 0.5% (w/w) Carboxymethylcellulose (400-800 cps) and 0.1% (w/w) TWEEN® 80 in deionised water

Animals were dosed over Days 6-19, inclusive, of gestation (where Day 0 of gestation was the day of detection of mating). They were regularly monitored for clinical signs of toxicity, body weight and food consumption, and were killed on Day 20 of gestation for examination of pregnancies and embryo-fetal development.

There were no unscheduled deaths during the study.

At 300 or 100 mg/kg/day, increased incidences of soft/pale faeces, erect and hunched posture were noted, when compared with controls, and there were also incidences of decreased activity at 1000 mg/kg/day.

At 300 or 1000 mg/kg/day, reduced mean body weight gain over Days 6-20 of gestation (-7% and -12%, respectively) was noted when compared with controls, with the reduced weight gain being most obvious over the first approximate week of treatment (Days 6-13 of gestation). There were no test item-related effects on body weight at 100 mg/kg/day.

At 1000 mg/kg/day, mean food consumption was approximately 20-40% lower than controls over Days 9 to 12 of gestation, with recovery noted from Day 13 of gestation. There were no test item-related effects on consumption at 100 or 300 mg/kg/day.

There were no test item-related gross necropsy findings.

Uterine findings and fetal weights were comparable between control and treated groups, and the type and distribution of major or minor fetal abnormalities, variants and skeletal ossification parameters did not indicate any association with treatment.

Maternal findings at 300 mg/kg/day were considered not to be adverse due to low magnitude and no overall effect on pregnancy or fetal development. At 1000 mg/kg/day, findings were considered to be adverse to dams due to increased incidence and severity.

Based on the study results, it was, therefore, concluded that the maternal No-Observed-Adverse-Effect-Level (NOAEL) was 300 mg/kg/day based on increased incidences of clinical observations, reduced weight gain and lower food consumption at 1000 mg/kg/day. The fetal No-Observed-Adverse-Effect-Level (NOAEL) was considered to be 1000 mg/kg/day.