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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:
2022
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2022
Report date:
2022

Materials and methods

Test guidelineopen allclose all
Qualifier:
equivalent or similar to guideline
Guideline:
other: Guidance Document on the Recognition, Assessment and Use of Clinical Signs as Humane Endpoints for Experimental Animals Used in Safety Evaluation. Environmental Health and Safety Publications Series on Testing and Assessment No 19, 2000
Deviations:
not specified
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
not specified
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Fluorobenzene
EC Number:
207-321-7
EC Name:
Fluorobenzene
Cas Number:
462-06-6
Molecular formula:
C6H5F
IUPAC Name:
fluorobenzene

Test animals

Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
Species: Wistar rats
Source :VELAZ, Czech Republic – Origin Crl:WI(Han)
Number and Sex of Animals :100 females and 30 males
Housing Condition : The animals were housed in plastic cages suspended on stainless steel
racks, up to 5 animals per cage, in a room equipped with central airconditioning.
The room temperature was within the range of 22±2°C. The
relative humidity will be 55±10%. The light regime was set to a 12-hour
light /12-hour dark cycle. The sanitation was performed according to
standard operation procedures.

Administration / exposure

Route of administration:
oral: gavage
Type of inhalation exposure (if applicable):
not specified
Vehicle:
olive oil
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
The Sponsor provided the test item as a colorless transparent liquid substance. Calculations for the doses preparation were based on the dose (100, 300 and 900 mg/kg bw), administered volume (2 ml/kg body weight),documented in spreadsheets and maintained in the study file as printouts.
The test item was dissolved in the required volume of vehicle (olive oil) in order to achieve the concentrations
of 50, 150 and 450 mg/mL and then homogenized using a magnetic stirrer.
Test item formulations were prepared every day and stored in tightly closed glass jars at room temperature in
the dark before administration. For the control group, the required volume per day of olive oil was placed in a
labeled jar. On the day of dosing each formulation was mixed thoroughly and transferred to the barrier zone
with a jar of control vehicle.
Details on mating procedure:
25 females/group was used for mating; 25 males of the same batch were used for mating (avoiding siblings mating).
The total number of pregnant females and actions with remainders were recorded in the study file and in the final report.
Females were cohabited with a male (avoiding siblings mating), 3:1 until mating. Positive evidence of mating
was confirmed by the presence of sperm following a vaginal lavage. Each mating female was examined daily
on the morning. The day when evidence of mating was identified it was termed as gestation day 0 (GD0).
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day
Remarks:
group number 1: Control-Vehicle
Dose / conc.:
100 mg/kg bw/day
Remarks:
group number 2 : Fluorobenzene
Dose / conc.:
300 mg/kg bw/day
Remarks:
group number 3 : Fluorobenzene
Dose / conc.:
900 mg/kg bw/day
Remarks:
group number 4 : Fluorobenzene
No. of animals per sex per dose:
The number of animals selected for this study is consistent with the OECD guideline for prenatal reproductive toxicity study (OECD TG 414). Approximately 25 females/group will be used for mating to obtain at least 16females per group with implantation sites at necropsy.
The route of administration was oral (gavage) since this is a potential route of exposure to humans and it is
acceptable under OECD guidelines for studies of this type.
Dosage levels 100, 300, and 900 mg/kg bw/day with 3-4-fold optimal interval were selected for the current
study based on dose range-finding study (DRF) (DRF 01-2020, UEFT CEM SAS). In the DRF study, repeated
administration of Fluorobenzene at the dose of 1000 mg/kg bw/day over the next 14 days caused slight signs of
toxicity in all three females. Excess vocalization was observed in all females after administration of substance
and we also observed decreased body weight gain.
Control animals:
yes
Details on study design:
The test item, Fluorobenzene, in the vehicle (olive oil), was administered by gavage once daily to Wistar female rats from day 5 to day 19 post-mating. Three groups received the test item at dose-levels of 100, 300 or 900 mg/kg bw/day. A concurrent vehicle control group received the vehicle (olive oil) on a comparable regiment and in the same volume of 2 mL/kg bw. Each group consists of at least 22 females with confirmed mating.
All females were observed twice daily for mortality and morbidity and for signs of toxicity following dose
administration. Body weights and food consumption were recorded at three-day intervals and on the day of
scheduled humane killing.
On day 20 post-mating, the dams were sacrificed and subjected to a macroscopic examination and enumeration of corpora lutea. The weight of the thyroid gland, histopathological assessment of the thyroid gland, and assay of serum concentration of thyroxin (T4), triiodothyronine (T3) and thyroid stimulating hormone (TSH) were done in dams to observe pathological changes in thyroid function.
Gravid uteri were weighed and uteri content examined to record implantation sites, early and late resorptions, dead and live foetuses. The foetuses were weighed, sexed with measurement of anogenital distance (AGD) and submitted to external examination. Half of the foetuses from each litter were subjected to a detailed examination of soft tissue by serial sectioning after fixation in Bouin’s solution while the other half underwent detailed skeletal examination following staining of bone with Alizarin red.

Examinations

Maternal examinations:
Each female was dosed once a day, at approximately the same time each day at the first half of the day, from
day 5 to day 19 (including) of post mating (GD5-GD19).
The vehicle and the test item was administered orally by gavage, via an appropriately sized stainless steel balltipped
dosing cannula connected with syringe once daily. A separate cannula for each group was used. The
dosage volume for all groups was 2 mL/kg body weight. Individual dosages were based on the last value body
weights to provide the correct mg/kg bw/day dose.
Ovaries and uterine content:
Hysterectomy and examination of uterine content underwent all females. Ovaries were examined to determined number of corpora lutea. Gravid uterine weight was collected at scheduled necropsy. Uteri, which appeared non-gravid by macroscopic examination, were opened for detection of early implantation loss. Thyroid gland was preserved (in scheduled or euthanized in extremis females) and weighed after fixation (scheduled euthanized).
Blood sampling:
Blood samples were collected from all surviving females at the scheduled necropsies (as part of euthanasia on day 20 of post-mating) and from euthanized in extremis females (as possible).
Animals were not fasted prior to blood collection. The blood was collected terminally following anesthesia (Zoletil® / Xylariem) from the caudal vena cava after laparotomy using a syringe with 23G needle. The blood sample (at least 3.0 mL) was placed in a tube without anticoagulant. The blood was allowed to clot for 50 min and centrifuged (1600 x g, 4 °C, 15 min) for serum separation. Serum from each animal was immediately frozen at –70 °C until assayed.
Fetal examinations:
Examination of Foetuses
After cesarean section, all foetuses were subjected to external examination. Half of the foetuses from each litter
were examined for skeletal abnormalities and the remaining for soft tissue alterations.
The foetal findings were described according to the harmonized terminology of the International Federation of
Teratology Societies (IFTS) [1, 2] without categorization and classified as malformations or variations:
 malformation (major abnormality) refers to structural change considered detrimental to the animal (may
also be lethal) and is usually rare;
 variation (minor abnormality) refers to structural change considered to have little or no detrimental effect
on the animal; may be transient and may occur relatively frequently in the control population.
Statistics:
All statistical tests will be performed using Microsoft Excel (descriptive statistics) and statistical software Statistica for Windows v.7.1 to compare the treated groups to the control group. Descriptive statistics (mean, standard deviation (Std.Dev.), standard error of mean (Std.Err), and N are presented for all measurement data and shown in the summary tables. The litter was the experimental unit for statistical analysis.
Data variables were analysed by parametric one-way analysis of variance (ANOVA). If the results of the ANOVA were significant (p<0.05), Dunnett's test was applied to the data to compare the treated groups to the control group. Offspring sex ratio data and AGD value were subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup difference. If the results of the ANOVA were significant (<0.05), Dunn's test was applied to the data to compare the treated groups to the control group.
The foetal body weight was analysed by sex as well as for both sex combined using one-way analysis of variance (ANOVA) as described above. Additionally, statistical analysis for foetal body weight will be done using analysis of covariant with litter size as a covariant.
Descriptive data, percentage values, and pathomorphological data were analysed by Fisher's Exact Test.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Description (incidence and severity):
Each female was dosed once a day, at approximately the same time each day at the first half of the day, from
day 5 to day 19 (including) of post mating (GD5-GD19). All rats were observed daily for morbidity and mortality. We did not observe signs of toxicity during dosing period except slight diarrhea in high dose group (900 mg/kg) appearing approximately from 16th day of gestation. No animals died during study.
Dermal irritation (if dermal study):
not specified
Mortality:
no mortality observed
Description (incidence):
Each female was dosed once a day, at approximately the same time each day at the first half of the day, from
day 5 to day 19 (including) of post mating (GD5-GD19). All rats were observed daily for morbidity and
mortality. We did not observe signs of toxicity during dosing period except slight diarrhea in high dose group
(900 mg/kg) appearing approximately from 16th day of gestation. No animals died during study.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Individual female body weights were recorded during animal identification, at day of confirmed mating and
group assignment (gestation day 0), on the first day of dose administration (GD5), and at three-day intervals
thereafter (gestation days 8, 11, 14, 17, and 20 as the day of euthanasia).
the foetal and placental weight was significantly
decreased in all dose levels.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Food consumption was assessed for each female quantitatively as g/animal/day by weighing of feeder (cage lid)
at the beginning of the day and 24 hours after. Food consumption was recorded prior to the initiation of dose
administration (gestation days 0-1), and at three-day intervals thereafter (gestation days 4-5, 7-8, 10-11, 13-14,
16-17 and 19-20). Decreased food consumption was observed only in the Mid Dose group prior to the initiation
of dose administration [F(3,19)=3.386; p=0.039] (Table 3). The decrease in food consumption was not treatment
related and might have been caused by new environment (new cage) after mating.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Endocrine findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Gross pathological findings:
no effects observed
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Thyroid tissue with preserved histological structure, with smaller, focal follicles filled with colloid present, without more noticeable histomorphological changes.
Histopathological findings: neoplastic:
not specified
Other effects:
no effects observed
Description (incidence and severity):
Necropsy of Females and Examination of Uterine Content
No females died spontaneously or were euthanized in extremis. Necropsy at scheduled termination include examination of the external surface of the body, all orifices, the cranial cavity, the external surface of the brain, and the thoracic, abdominal and pelvic cavities including viscera. No treatment related pathological changes were observed in all pregnant females

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
In maternal organisms no abortion was seen (females without foetuses but with implantations).
Total litter losses by resorption:
not specified
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Description (incidence and severity):
Each live foetus was weighed and sexed with measurement of anogenital distance (AGD). Results of findings
and descriptive statistics are depicted in Table 6. Administration of tested substance caused significant decrease
in body weight of male and female foetuses [Fmale(3,69)=4.723; p=0.0047]; [Ffemale(3,69)=4.220; p=0.0084]
foetuses overall [F(3,73)=5.338; p=0.0022] and placenta [F(3,73)=5.179; p=0.0027]. Dunnett’s post hoc test
further revealed that decrease appeared in all Fluorobenzene treated groups. Detailed external examination for
gross anomalies did not show any apparent malformations and variations or non-classified findings other than
reduction of size. No changes in anogenital distance (AGD) were recorded Compared to control group (Table 6).
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
no effects observed

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
900 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
clinical signs
food consumption and compound intake
mortality
necropsy findings
other:
Remarks on result:
not determinable due to absence of adverse toxic effects

Maternal abnormalities

Key result
Abnormalities:
no effects observed
Localisation:
not specified
Description (incidence and severity):
No females died spontaneously or were euthanized in extremis. Necropsy at scheduled termination included examination of the external surface of the body, all orifices, the cranial cavity, the external surface of the brain, and the thoracic, abdominal and pelvic cavities including viscera. No treatment related pathological changes were observed in all pregnant females.

Results (fetuses)

Fetal body weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
The mean total body weight of foetuses was significantly decreased in all dose groups ranging from 11% to 13% reduction compared to the control group (p<0.01).
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The percentage of male foetuses was not changed compared to control group).
Changes in litter size and weights:
effects observed, non-treatment-related
Anogenital distance of all rodent fetuses:
no effects observed
Description (incidence and severity):
No changes in anogenital distance was noted in both sexes.
Changes in postnatal survival:
no effects observed
External malformations:
no effects observed
Description (incidence and severity):
There were no test item related external malformations in foetuses.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Analysis of skeletal malformations reveal only delayed ossification of the cervical vertebrae in MID dose group, which might be connected to lower body weight of foetuses.
Visceral malformations:
no effects observed
Description (incidence and severity):
Increased incidence of visceral anomalies was not observed in either treatment group.
Other effects:
no effects observed
Details on embryotoxic / teratogenic effects:
The mean total body weight of foetuses was significantly decreased in all dose groups ranging from 11% to 13% reduction compared to the control group (p<0.01). Similar findings were observed in the mean placental weight (ranging from 20% to 22% reduction; p<0.01) Placental weight in MID dose group was decreased by 14% which was on the edge of significance (p=0.059). No changes in anogenital distance was noted in both sexes. The percentage of male foetuses was not changed compared to control group). There were no test item
related external malformations in foetuses. Analysis of skeletal malformations reveal only delayed ossification of the cervical vertebrae in MID dose group, which might be connected to lower body weight of foetuses.Increased incidence of visceral anomalies was not observed in either treatment group.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reduction in number of live offspring
changes in sex ratio
fetal/pup body weight changes
changes in postnatal survival
skeletal malformations
visceral malformations
Remarks on result:
other: foetal and placental growth retardation

Fetal abnormalities

Key result
Abnormalities:
no effects observed
Localisation:
other:

Overall developmental toxicity

Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
100 mg/kg bw/day (actual dose received)
Treatment related:
no
Relation to maternal toxicity:
developmental effects in the absence of maternal toxicity effects
Dose response relationship:
no
Relevant for humans:
not specified

Any other information on results incl. tables

The test substance did not cause mortality or signs of toxicity in the dams. In maternal organisms no abortion
was seen (females without foetuses but with implantations). The health condition of females was good during
the whole study. No significant changes were recorded during examination of reproductive parameters. The
result of the administration of the test substance stated the following: No toxicity effect on maternal animals.
The developmental effect of the test substance on foetal organism was assessed according to the results of
weighing, careful necropsy and skeletal and visceral examination of foetuses. The number of foetuses at all dose
levels were in balance with the control group. However, the foetal and placental weight was significantly
decreased in all dose levels. The test substance application did not induce external and/or visceral malformations


(permanent structural change that may adversely affect survival). However, some variations in skeletal
ossification were detected.
According to the guidance for interpretation of skeletal variations published by Carney and Kimmel (2007)
these delayed changes represent rather insignificant findings – isolated statistical increases in a few variations,
but no consistent pattern indicative of delayed ossification. The key factors to consider in a weight of evidence
approach for interpreting delayed ossification is summarized in Table C-1.


Table C-1. Key factors to consider in a weight of evidence approach for interpreting delayed ossification (Carney and
Kimmel, 2007)























Examples of insignificant findings:
Isolated statistical increases in a few variations, but no consistent pattern indicative of delayed ossification
Incidences are within recent historical control ranges
Examples of low significance findings (may not be adverse):
Pattern of ossification is consistent with a slight generalized delay (e.g., limited to effects on bones such as phalanges,
sternebrae 5/6, cervical, thoracic sacral and caudal vertebral centra, and/or calvarium)
Normal cartilage is present
Delayed ossification associated with maternal toxicity
Findings that may warrant increased attention:
Unusual pattern of ossification that does not follow normal biologic sequence
Specific delays involving bones that normally are well ossified in the term foetus (e.g., ribs, clavicle, long bones of
the limbs, lumbar vertebral centra)
Abnormally-shaped cartilage template
Delayed ossification without decreases in foetal body weight
Delayed ossification in the absence of maternal toxicity
Delayed ossification associated with teratogenesis

Detected skeletal anomalies do not constitute anomalies dangerous for further development of individuals and
represents transitional findings. These may be upgraded to "malformation" or downgraded to "variation" status,
depending on severity and/or frequency of occurrence. In our opinion these findings represent variations from
development which are generally reversible or transitory, such as wavy ribs. Should temporary/transient effects
be viewed differently than permanent effects? The Segment II protocol currently does not include a postnatal
component. However, we know that in the postnatal period, wavy ribs resolve (Nishimura et al. 1982; Hayasaka
et al. 1985; Kast, 1994).
There is less variability in the development of the foetal skeleton the closer to term it is evaluated (i.e., if the
sacrifice is on GD 21 for rats, rather than on GD 20). But the risk of delivery before scheduled sacrifice is much
greater. In addition, the sacrifice order must be random or se1ection of one from each dose group in rotation. If
the dams and their foetuses are necropsied by group, there will be uncorrectable confounding. If done in the
order of high, mid, low, and control groups, then there will be a "dose"-related reduction in foetal body weight
and skeletal ossification due to the timing of sacrifice (independent of any treatment-related effects). Since late
gestation, even a few hours close to term, is the time of rapid growth and development, especially of the foetal
ossified skeleton (Rodweil, 2000).
Based on the results, the NOAEL for maternal toxicity was determined to be 900 mg/kg body weight/day (actual
dose received). Although we did not find any malformations and abnormalities of skeletal and visceral
development, but rather transient changes and anomalies in development, the NOAEL for developmental toxicity was determined to be ≤100 mg/kg body weight/day (actual dose received) due to foetal and placental
growth retardation observed in all treatment groups.


 

Applicant's summary and conclusion

Conclusions:
Based on the results, the NOAEL for maternal toxicity was determined to be 900 mg/kg body weight/day (actual dose received). Although we did not find any malformations and abnormalities of skeletal and visceral development, but rather transient changes and anomalies in development, the NOAEL for developmental toxicity was determined to be ≤100 mg/kg body weight/day (actual dose received) due to foetal and placental growth retardation observed in all treatment groups.
Executive summary:

The objective of this prenatal developmental toxicity study was to evaluate the potential effects of the test item,
Fluorobenzene, on pregnancy and on embryo-foetal development in rats following daily oral (gavage)
administration at doses 100, 300 and 900 mg/kg body weight/day from implantation to the day prior to scheduled
caesarean section (day 5 to day 19 post-mating inclusive).
No treatment-related clinical observations in any dose group were observed, except slight diarrhea in high dose
group (900 mg/kg). All females were schedule sacrificed on gestation day 20. The final body weight of pregnant
females was not influenced by administration of tested substance and similarly the weight gain did not differ
compared to control females. Neither the netto weight of females (without uterus) or uterine weight was changed
compared to control group. Post-implantation loss was similar in all groups ranging from 2.4% up to 6.8% with
no statistical significance among groups. The weight of thyroid gland was not influenced by the treatment of
tested substance and we found significant decrease (-14%) in T4 hormone in MID dose group (p<0.05). No
other changes in thyroidal hormones were observed. Histological examination of thyroid gland did not reveal
obvious histomorphological changes.
The mean total body weight of foetuses was significantly decreased in all dose groups ranging from 11% to
13% reduction compared to the control group (p<0.01). Similar findings were observed in the mean placental
weight (ranging from 20% to 22% reduction; p<0.01) Placental weight in MID dose group was decreased by
14% which was on the edge of significance (p=0.059). No changes in anogenital distance was noted in both
sexes. The percentage of male foetuses was not changed compared to control group). There were no test item
related external malformations in foetuses. Analysis of skeletal malformations reveal only delayed ossification
of the cervical vertebrae in MID dose group, which might be connected to lower body weight of foetuses.
Increased incidence of visceral anomalies was not observed in either treatment group.
Based on the results, the NOAEL for maternal toxicity was determined to be 900 mg/kg body weight/day (actual
dose received). Although we did not find any malformations and abnormalities of skeletal and visceral
development, but rather transient changes and anomalies in development, the NOAEL for developmental
toxicity was determined to be ≤100 mg/kg body weight/day (actual dose received) due to foetal and placental
growth retardation observed in all treatment groups.