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Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
08/2014 to 09/2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: microgranules
Details on test material:
- Name of test material (as cited in study report): BRUGGOLITE FF6 M
- Substance type: organic
- Physical state: solid
- Lot/batch No.: 11062802
- Expiration date of the lot/batch: 31.07.2021
- Storage condition of test material: room temperature
- Other: RTC numbers 12924, 14263

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Italy s.r.l., San Pietro al Natisone (UD), Italy
- Age at study initiation: 9 weeks (female rats) and 11 weeks (male rats)
- Weight at study initiation: 200-225 g (female rats) and >350 g (male rats)
- Fasting period before study: no
- Housing: limited access rodent facility
- Diet (e.g. ad libitum): laboratory rodent diet (4 RF 21, Mucedola S.r.l., Via G. Galilei, 4, 20019, Settimo Milanese (MI), Italy): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimatisation period: 18 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C +/- 2°C
- Humidity (%): 55% +/- 15%
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): preparation for 3 to 6 days
- Mixing appropriate amounts with (Type of food): water

VEHICLE
- Justification for use and choice of vehicle (if other than water): N/A
- Concentration in vehicle: 100 mg/ml
- Amount of vehicle (if gavage): 10 ml/kg body weight
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Validation of the method of analysis was carried out by the Test Site LAUS
GmbH, according to a iodometric titration method released by the Sponsor (Validation of an
Analytical Method for the determination of Bruggolite FF6 M in the matrix
demineralised water - Project No. 14041501G926). The stability was found
to be 48 hours at room temperature and 1 week at +4°C for the concentration
of 100 mg/mL.

Before treatment commenced, analysis was performed to confirm that the proposed
formulation procedure was acceptable. Samples of the formulations prepared
during Weeks 1 and 2 of treatment were also analysed to check the concentration.

At each analytical session, triplicate samples of approximately 10 mL
each (of vehicle and dose level) were taken, according to instructions from
the Test Site and sent to the attention of the Principal Investigator. The
actual date for each sample collection was documented and retained with the
study raw data.
Details on mating procedure:
The females were paired with male rats. Females were paired one to one in
the home cage of the male and left overnight. Vaginal smears were taken
daily in the morning from the day after pairing until a positive identification
of mating was made. The day of mating, as judged by the presence of sperm
in the vaginal smear or by the presence of a copulation plug, was considered
as Day 0 of gestation (or Day 0 post coitum). Full mating records were
maintained.
Duration of treatment / exposure:
All animals were dosed once a day, from Day 6 through Day 19 post coitum.
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
control group
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
24 mated female rats per dose
Control animals:
yes, concurrent vehicle
Details on study design:
The dose level of 1000 mg/kg/day was selected in agreement with the Sponsor, based on information from previous studies.

Examinations

Maternal examinations:
Mortality
Throughout the study, all animals were checked early in each working day and
again in the afternoon. At weekends and Public Holidays a similar procedure
was followed except that the final check was carried out at approximately
mid-day.

Clinical signs
All clinical signs were recorded for individual animals. Each animal was
observed at least once daily and any clinical signs recorded starting from
allocation until sacrifice.
Signs observed outside the scheduled time period and mass(es) are reported
only as individual data.

Body weight
All animals were weighed on Days 0, 3, 6, 9, 12, 15, 18 and 20 post coitum.

Food consumption
Food consumption was measured on Days 3, 6, 9, 12, 15, 18 and 20 post
coitum, starting from Day 0 post coitum.
The food consumption measured on Day 18 post coitum for cage no. 2
was considered unreliable, since it was more than the double of the food
consumption of the same cage and of the other cages in the same group,
during the study.

Euthanasia
The animals were killed on Day 20 post coitum and necropsied as detailed
below. All females which completed the scheduled test period were euthanised
with carbon dioxide. All foetuses were sacrificed by intraperitoneal injection
of Sodium Thiopental followed by hypothermia. All animals were subjected
to necropsy, supervised by a pathologist.

Necroscopy
The clinical history of the animals was studied and a detailed post mortem
examination was conducted (including examination of the external surface
and orifices). Changes were noted and the abnormalities preserved in 10%
neutral buffered formalin.
Ovaries and uterine content:
The ovaries and uteri were examined to determine:
– Gravid uterine weight;
– number of corpora lutea;
– number of implantation sites;
– number, sex and weight of all live foetuses;
– number and sex of dead foetuses (foetuses at term without spontaneous
movements and breathing);
– number of intra-uterine deaths;
– gross evaluation of placentae.

Intra-uterine deaths were classified as:
– Early resorptions: only placental remnants visible.
– Late resorptions: placental and foetal remnants visible.

Uteri or individual uterine horns without visible implantations were immersed
in a 10-20% solution of ammonium sulphide to reveal evidence of embryonic
death at very early stages of implantation.
Fetal examinations:
All live foetuses were examined externally. Approximately one-half of the
foetuses (i.e., routinely, every second live foetus) in each litter was preserved
in Bouin’s solution for subsequent fixed-visceral examination. The remaining
foetuses were eviscerated after which the carcasses were fixed in 95% (v/v)
ethanol for subsequent skeletal examination after bone staining with Alizarin
Red S. Skeletal and fixed-visceral examinations were performed in both groups.
Structural deviations were classified as follows:

Malformations
Major abnormalities that are rare and/or affect the survival or health of the
species under investigation.

Anomalies
Minor abnormalities that are detected relatively frequently.

Variants
A change that occurs within the normal population under investigation and
is unlikely to adversely affect survival or health. This might include a delay
in growth or morphogenesis that would have otherwise followed a normal
pattern of development.
Statistics:
For continuous variables (body weight, body weight gain and food consumption),
the significance of the differences amongst group means was assessed
by Dunnett’s test or a modified t test, depending on the homogeneity of
data. Statistical analysis of litter data and sex ratio, terminal body weight,
gravid uterus weight and absolute weight gain was carried out by means of
the Kruskal-Wallis test and intergroup differences between the control and
treated groups assessed by a non-parametric version of the Williams test.
Indices:
Pre-implantation loss was calculated as a percentage from the formula:

Pre impl: Loss% = [(no. of corpora lutea - no. of implantations)/(no. of corpora lutea)] X 100


Post-implantation loss was calculated as a percentage from the formula:

Post impl: Loss% = [(no. of implantations - no. of live foetuses)/(no. of implantations)] X 100


Total implantation loss was calculated as a percentage from the formula:

Total impl: Loss% = [(no. of corpora lutea - no. of live foetuses)/(no. of corpora lutea)] X 100


Sex ratios of the foetuses were calculated as the percentage of males per litter.

All derived values (e.g., means, percentages, ratios) were first calculated
within the litter and the group values derived as a mean of individual litter
values. Foetal structural deviations were expressed as the percentage of
affected foetuses relative to all foetuses examined per group, as well as in
terms of the mean litter percentage of affected litters.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No signs of toxicological significance were noted during the study. Salivation
on single occasions, was seen in some treated females (1000 kg/kg/day).
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
No differences of toxicological relevance were noted in body weight of females
during the study, between control and 1000 mg/kg/day treated group.
Decreases in body weight gain were seen in 1000 mg/kg/day females on
Days 9, 15, 18 and 20 post coitum, statistically significant on Day 9.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Slight statistically significant reduction in food consumption was seen on Day
20 post coitum in treated females compared to controls.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
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

Maternal developmental toxicity

Number of abortions:
no effects observed
Description (incidence and severity):
Litter data, mean foetal weight and sex ratios were not affected by treatment.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
Litter data, mean foetal weight and sex ratios were not affected by treatment.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
Litter data, mean foetal weight and sex ratios were not affected by treatment.
Early or late resorptions:
not specified
Dead fetuses:
effects observed, non-treatment-related
Description (incidence and severity):
No abnormalities were seen in the control group; one dead foetus and a
total of 4 small foetuses (<2.7 g) were noted in the treated group. All these
findings were considered incidental.
Changes in pregnancy duration:
not specified
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 specified
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
Litter data, mean foetal weight and sex ratios were not affected by treatment.
Details on maternal toxic effects:
Maternal toxic effects:no effects

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: developmental toxicity

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
Litter data, mean foetal weight and sex ratios were not affected by treatment.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): Litter data, mean foetal weight and sex ratios were not affected by treatment.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
Litter data, mean foetal weight and sex ratios were not affected by treatment.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
Litter data, mean foetal weight and sex ratios were not affected by treatment.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
Litter data, mean foetal weight and sex ratios were not affected by treatment.
Changes in postnatal survival:
no effects observed
Description (incidence and severity):
Litter data, mean foetal weight and sex ratios were not affected by treatment.
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
No abnormalities were seen in the control group; one dead foetus and a
total of 4 small foetuses (<2.7 g) were noted in the treated group. All these
findings were considered incidental.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related changes were seen at skeletal examination of foetuses
performed in control and treated group, since the incidences of the findings
observed were comparable to the controls.
Metacarpal of the forepaw incompletely ossified (variant) or unossified (anomaly)
were seen in treated and control foetuses with different incidences. No
toxicological importance was attributed to the higher incidence of incompletely
ossified metacarpals seen in treated foetuses, since it should be considered in
association with the higher incidence of metacarpals unossified observed in
the control.
Other changes in foetuses of treated group were: pube incompletely ossified
(anomaly), seen in one small foetus; sternebrae unossified (anomaly) seen
in few foetuses, one of which was small; sternebrae incompletely ossified
(variant) seen in some foetuses comprising the small ones.
In addition, to the experience of the testing facility, these changes can be considered as a common
spontaneous alteration in animals of this strain.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related changes were seen at visceral examination of foetuses
performed in control and treated group, since the incidences of findings
observed were comparable to the controls. No toxicological significance was
attributed to the enlarged lateral ventricles of brain and the haemorrhagic
lungs, since they were seen only in one small foetus in the treated group.
In addition, to the experience of the testing facility, the changes observed can be considered as a
common spontaneous alteration in animals of this strain.
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
reduction in number of live offspring
changes in sex ratio
fetal/pup body weight changes
changes in litter size and weights
changes in postnatal survival
external malformations
skeletal malformations
visceral malformations

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The dose level of 1000 mg/kg bw/day was considered the NOAEL (No Observed Adverse Effect Level) for maternal and developmental toxicity.
Executive summary:

Study design

The effects of BRUGGOLITE FF6 M were investigated after oral administration

in Wistar Hannover female rats during pregnancy and on embryo-foetal

development. The study design was as follows:

Group 1: 0 mg/kg bw/day; 24 females

Group 2: 1000 mg/kg bw/day; 24 females

All animals were administered during the gestation period, starting from

Day 6 through Day 19 post coitum at the dose volume of 10 mL/kg. Body

weight, daily clinical signs and food consumption were recorded during the

in vivo phase. All females were caesarean-sectioned on Day 20 post coitum

and subjected to post mortem examination. The number of corpora lutea,

implantations, early and late intrauterine deaths, live and dead foetuses,

uterus weight, foetal weight and sex were recorded. All foetuses were examined

for external abnormalities. Approximately one half of the foetuses in each

litter was examined for fixed-visceral and skeletal abnormalities.

Mortality and fate of females

No animals died during the study. The number of females with live foetuses

on gestation Day 20 was 24 in the control and 23 in the 1000 mg/kg/day

dose group.

Clinical signs

No signs of toxicological significance were noted during the study. Salivation

on single occasions, was seen in some treated females (1000 kg/kg/day).

Body weight and body weight gain

No differences of toxicological relevance were noted in body weight of females

during the study, between control and 1000 mg/kg/day treated group.

Decreases in body weight gain were seen in 1000 mg/kg/day females on

Days 9, 15, 18 and 20 post coitum, statistically significant on Day 9.

Food consumption

Slight statistically significant reduction in food consumption was seen on Day

20 post coitum in treated females compared to controls.

Terminal body weight, uterus weight and absolute weight gain

No significant differences in terminal body weight, gravid uterus weight and

absolute weight gain were observed in the treated group, when compared to

the control group.

Litter data and sex ratios

Litter data, mean foetal weight and sex ratios were not affected by treatment.

Macroscopic examination

No treatment-related changes were observed at post mortem examination in

treated females, when compared to the controls.

External examination of foetuses

One dead foetus was detected in one treated female and 4 small foetuses were

noted in 4 treated females. All these findings were considered incidental.

Skeletal examination of foetuses

No relevant changes were recorded at the skeletal examination of foetuses in

the treated group compared to controls.

Visceral examination of foetuses

No treatment-related differences were seen at visceral exmination of foetuses

between the control and the treated group.