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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:
September 2019 - April 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report date:
2020

Materials and methods

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

Test material

Constituent 1
Reference substance name:
1,2,3 Benzotriazole
IUPAC Name:
1,2,3 Benzotriazole
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: Connect Chemicals GmbH, 19303
- Expiration date of the lot/batch: 02.2021
- Production date: 03.2019



STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
-Stability of the test item in the vehicle prepared at concentrations of 12 and 110 mg/mL was
confirmed following storage for 7 days at room temperature (the temperature range, 20 – 25
°C) during method validation study. In addition, homogeneity analysis was performed for
formulations of 12 and 110 mg/mL after 4 days of storage after re-mixing

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: The Lab Animals Breeding Center “Pushchino”, Branch of
Institute of Bioorganic Chemistry RAS:
Nauki 6, Puschino, Moscow region, Russia 142290
- Age at study initiation: Approximately 11-12 weeks old at the initiation of dose
administration on G5 (day 1)
- Weight at study initiation: 233 ± 15 g, N = 93
- Fasting period before study: no
- Housing: After identification and until mating, all females were housed by groups. Males were housed
alone, and females cohabited with a male in the home cage of the male (2:1). After confirmed
mating, dams were housed alone until euthanasia on gestation day 20
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: ca 7-8 weeks, The animals were received at the age of 4 weeks
by separate litters to avoid of sibling mating

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24 °C
- Humidity (%): 30-70%
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12/ 12

IN-LIFE DATES: From: To:

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The Sponsor provided the test item as a neat solid substance. Calculations for the preparation of formulations were based on the dose (36, 120 and 330 mg/kg bw) and administered volume (3 mL/kg body weight), were documented in Excel spreadsheets, and maintained in the study file as printouts.
The test item was dissolved in the required volume of the vehicle (Kollisolv® PEG E 400) in
order to achieve the concentrations of 12, 40, and 110 mg/mL and then homogenized using a magnetic stirrer.
Test item formulations were prepared every four days, aliquoted to the required volumes of days of the administration, and stored in tightly closed glass jars at room temperature in the
dark. For the control group, the required volume per day of Kollisolv® PEG E 400 was placed in a labeled jar.

VEHICLE
- Justification for use and choice of vehicle (if other than water): based on preliminary studies, the vehicle was chosen
- Concentration in vehicle: 12, 40, and 110 mg/mL
- Amount of vehicle (if gavage): 3 mL/kg body weight
- Lot/batch no. (if required): BCCB1456
- Purity: not stated
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability of the test item in the vehicle prepared at concentrations of 12 and 110 mg/mL was
confirmed following storage for 7 days at room temperature (the temperature range, 20 – 25
°C) during method validation study. In addition, homogeneity analysis was performed for
formulations of 12 and 110 mg/mL after 4 days of storage after re-mixing.
Analysis of formulations for homogeneity and concentration during dosing period was
conducted in the test facility at the beginning, in the middle and at the end of
in-life phase using a validated method.
For homogeneity analysis, quadruplicate samples (approximately 0.1 mL of each) were
collected from the top, middle and bottom strata of each dosing formulation prepared during
the study.
For concentration analysis, quadruplicate samples were collected from the middle stratum of
each dosing formulation (including the vehicle control group) prepared during the study.
Samples collected from the mean stratum for homogeneity analysis used for this purpose.
A pair of quadruplicate samples from each strata was used for analysis, the other pair was
stored as back-up samples at room temperature in tightly closed flasks, analyzed if necessary
based on primary assays to verify concentration and were discarded after the study director's
approval of the analytical results.
Acceptance criteria for the formulations analysis are based on the test item in vehicle
composition as a suspension. For stability, the mean concentration of formulation samples of
12 and 110 mg/mL after 4 days of storage and resuspension should be within the acceptable
range of the target concentration (85-115% with RSD<10%), and within the range 85-115% of
the time zero point. The actual concentration of analyzed samples collected from the mean
stratum of formulations should be within the range of 85% - 115% of the target concentration.
The acceptance criteria for homogeneity are RSD<10% with the mean concentration within
85% to 115% of the target concentration.
Details on mating procedure:
After identification, females were monitored to an estrous cyclicity daily during 3-5 days. Females with
clear stages of estrous cycle in vaginal smear were cohabited with a male (avoiding siblings mating),
2:1 until mating. Positive evidence of mating was confirmed by the presence of a vaginal copulatory
plug or 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 is termed as gestation day 0 (G0).
Duration of treatment / exposure:
15 days, from day 5 to day 19 (including) of post mating (G5-G19)
Frequency of treatment:
daily
Duration of test:
Until Gestation day 20
Doses / concentrationsopen allclose all
Dose / conc.:
36 mg/kg bw/day (nominal)
Dose / conc.:
120 mg/kg bw/day (nominal)
Dose / conc.:
330 mg/kg bw/day (nominal)
No. of animals per sex per dose:
ca 25
Control animals:
yes, concurrent vehicle
Details on study design:
Before the beginning of the treatment period, the females with confirmed mating were
allocated to the groups, according to a stratification procedure, so that the average body
weight of each group did not statistically differ. Females with the same day of gestation were
allocated to a different group.

Examinations

Maternal examinations:
Cage Side and Clinical Observations
All rats were observed twice daily, once in the morning and once in the afternoon at the same time,
for morbidity and mortality. Each female was also observed for signs of toxicity approximately 15-45
minutes following dose administration. In addition, the presence of findings at the time of dose
administration was recorded for individual animals.

Body Weights (F0)
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 (gestation day 5), and
at three-day intervals thereafter (gestation days 8, 11, 14, 17, and 20 as the day of euthanasia). Body
weight value on gestation day 20 was corrected for gravid uterine weight to calculate maternal body
weight change.

Food Consumption
Food consumption was assessed for each female quantitatively as g/kg of body weight/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).

Scheduled Euthanasia
On gestation day 20, all females were euthanized by anesthesia (Zoletil® / Xyla®, i.m.)
followed by terminal blood sampling for hormones assay and subjected
to hysterectomy.
The weight of the gravid uterus was recorded for each pregnant female (with at least one live
fetus). Uterus were examined. Ovaries were examined to determined number of corpora lutea.
Each dam was examined macroscopically, thyroid gland was collected in 10% neutral formalin
(in complex with trachea and esophagus) and weighed after fixation.

Necropsy of Females and Examination of Uterine Content
A complete necropsy was conducted on all females at scheduled termination. Necropsy
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.
Hysterectomy and examination of uterine content was done for all females. Ovaries were
examined to determined number of corpora lutea. Gravid uterine weight was collected at
scheduled necropsy. Uteri, which appear non-gravid by macroscopic examination, were
opened and placed in 10 % ammonium sulfide solution for detection of early implantation loss.
Thyroid glands were preserved (in scheduled or euthanized in extremis females) and weighed
after fixation (scheduled euthanized).

Blood Sample Collection for Hormones Assay
Blood samples were collected from all surviving females at the scheduled necropsies
(as part of euthanasia on day 20 of post-mating).
Animals were not fasted prior to blood collection. The blood was collected terminally
following anesthesia (Zoletil® / XylaVET®) from the caudal vena cava after
laparotomy using a syringe with 23G needle. Blood collection was done on the first
part of the day (within 10:00 – 13:00 hours) in randomized order to avoid bias.
The blood sample 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 divided into 6 aliquots (for two aliquots for each of T4,
T3 and TSH analysis) and immediately frozen at –70 °C until assayed.

T4, T3 and TSH Assay
Thyroid hormones (thyroxin (T4), triiodothyronine (T3), and thyroid stimulating
hormone (TSH)) were assayed in serum from all pregnant females (with at least one
fetus) by competitive inhibition enzyme immunoassay technique using relevant ELISA
kits (see below) and Multiskan™GO Microplate Spectrophotometer (Termo Scientific)
and according to standard procedure of manufacturer and SOP of BTL BIBC RAS.

Microscopic Examination of Thyroid Gland
Thyroid gland of all females euthanized at the scheduled necropsy was trimmed,
embedded in paraffin, sectioned, stained with hematoxylin and eosin, and examined
microscopically.


Ovaries and uterine content:
The weight of the gravid uterus was recorded for each pregnant female (with at least one live
fetus). Uterus were examined. Ovaries were examined to determined number of corpora lutea.
Each dam was examined macroscopically, thyroid gland was collected in 10% neutral formalin
(in complex with trachea and esophagus) and weighed after fixation.
Fetal examinations:
After cesarean section, all fetuses were subjected to external examination. Half of the fetuses
from each litter was examined for skeletal abnormalities and the remaining for soft tissue
alterations.
The fetal 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.
The reproductive tract was examined with particular attention, and external sex was compared
with internal (gonadal) sex in all fetuses (examined for both skeletal and soft tissue
malformations). In addition, male fetuses were examined for undescended testes.

External Examination, Body Weight and AGD
Each live fetus was weighed and sexed with measurement of anogenital distance
(AGD). All fetuses were subjected to a detailed external examination for gross
anomalies with a recording of malformations and variations or non-classified findings.
Fetuses were then anaesthetized by subcutaneous injection of Zoletil® + XylaVET®
mixture and fixed in 96 % ethanol (approximately one-half of litter) for skeleton
examination or Bouin’s fixative (remainder of litter) for soft tissues examination.

Skeletal Examination
Approximately half of the live fetuses in each litter was fixed in ethyl alcohol,
eviscerated and skinned for subsequent double staining of cartilage and bone with
alcian blue and alizarin red.
A detailed examination of the skeleton was done and included the observation of all
the bone and cartilage structure of the head, spine, rib cage, pelvis, and limbs.
During evisceration, the reproductive tract was examined and external sex was
compared with internal (gonadal) sex; an indication of incomplete testicular descent
was noted in male fetuses.

Soft Tissues Examination
The remaining live fetuses in each litter were fixed with Bouin’s fluid. A detailed soft
tissue examination was performed according to a free-hand serial sectioning
technique, which will include the observation of all the organs and structures of the
head, neck, thorax and abdomen.
Organs within the abdomen were examined in unsectioned abdominal region with
particular attention on reproductive tract; an indication of incomplete testicular
descent was noted in male fetuses.
Statistics:
All statistical tests were performed using Microsoft Excel (descriptive statistics) and statistical software
Statistica for Window v.7.1 to compare the treated groups to the control group. Descriptive statistics (mean,
standard deviation (S.D.), and N) are presented for all measurement data and shown in the summary
tables. The litter is accepted as an experimental unit for statistical analysis.
Continuous data variables (mean body weights and food consumption data) were analyzed by multi-factor
analysis of variance ANOVA-2, followed by the Duncan test, to determine inter-group differences. Former
implantation sites, number of corpora lutea, implantation loss indices, hormones concentration value,
uterine, and thyroid weights were analyzed by parametric one-way analysis of variance (ANOVA). If the
results of the ANOVA were significant (p<0.05), Dunnett's test is applied to the data to compare the treated
groups to the control group. The t-test was used additionally to compare each dose group with the control
value. Offspring sex ratio data, AGD value, and mean value of affected fetuses per litter 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 is applied to the data to compare the treated groups to the
control group.
The fetal body weight was analyzed by sex as well as for both sexes combined using a one-way analysis of
variance (ANOVA) as described above. Additionally, statistical analysis for fetal body weight was done
using analysis of covariant with litter size as a covariant.
Descriptive data, percentage values, and pathomorphological data were analyzed by Fisher's Exact Test
and additionally by Chi-square test (when a tendency to a difference was observed).
Indices:
Parameters Calculation / Note
Pregnant females alive at termination (N)
Body weight at necropsy (g)
Gravid uterine weight (abs, % of pregnant
female body weight)
Thyroid weight (abs, % of body weight without gravid uterine weight)
Females with total resorptions (N)
Females with all dead fetuses (N)
Females with live fetuses (N)
Corpora lutea (N per animal)
Implantation sites (N per animal)
Pre-implantation loss (N per animal, % of implantation sites) (Number of corpora lutea – Number of implantation sites) / Number of corpora lutea
Fetuses (N per animal)
Live fetuses (N per animal, % of implantation sites)
Dead fetuses (N per animal, % of implantation sites)
Resorptions + Scars (N per animal, % of implantation sites)
Implantation scars (N per animal, % of implantation sites)
Resorptions – early (N per animal, % of implantation sites)
Resorptions – late (N per animal, % of implantation sites)
Post-implantation loss (N per animal, % of implantation sites) (Number of implantation sites – Number of live fetuses) / Number of implantation sites
Historical control data:
none reported

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
he test item related clinical findings were observed in the 330 mg/kg bw/day dose group as
follows: hypokinesia (12 females), staggering gait (11 females) or flatness as a more severe
behavior disorder (6 females), excessive vocalization (2 females), chromodacryorrhea (2
females), ptyalism (2 females), nasal discharge (2 females); eyelid closure, hyphosis, and
diarrhea was noted for single females.
These observations were episodic with the recovery of animals within approximately 1.5 – 3
hours after dosing. The higher frequency of clinical signs was noted after first dosing
(gestation day 5), as well as at the end of the administration period (gestation days 14, 15, 17,
and 19). Over the entire period of the test item administration, the total number of females with
clinical findings in the 330 mg/kg bw/day dose group was 14 out of 26 examined (p < 0.001,
Fisher Exact test).
In the 120 mg/kg bw/day dose group, one female (No. 18) had focal alopecia on the forelimbs
observed beginning from day 9 of dosing, which supposed to be not test item related.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
There was no morbidity and mortality of females caused by the test item administration.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The body weight of pregnant females administered with the dose of 330 mg/kg bw/day was
slightly decreased compared to the control vehicle group at the gestation day 20 (by 5.0%, p <
0.05). The body weight gain in 330 mg/kg bw/day dose group was statistically reduced (p <
0.01) the entire period of administration compared with the control group as well as lower
doses.
The reduced body weight gain of pregnant females in the 330 mg/kg bw/day dose group was
associated with a smaller weight of the pregnant uterus (by 16 % compared to the control
group, p < 0.05). The final maternal body weight without a gravid uterus did not statistically
differ from the body weight of control females
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Mean food consumption in the 36, 120 and 330 mg/kg bw/day group was similar to that in the
vehicle control group during all study days.

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
Description (incidence and severity):
The absolute and relative thyroid weight of pregnant females was not significantly changed in
all dose test item treated group.

Gross pathological findings:
no effects observed
Description (incidence and severity):
All treated females were sacrificed during a scheduled necropsy on post-mating day 20.
During necropsy, there were no gross findings related to the test item administration. One
female (No. 94) from the 330 mg/kg bw/day dose group had the uterus hydrometra, which is
supposed to be not test item related.
Females No.38 (group 1), No.16, No.42, and No.56 (group 2), No.8, No.33, and No.94 (group
4) were without fetuses in the uterus. In female No.16, two early resorptions were revealed;
there were no implantation sites, and scars were observed in non-pregnant females after
staining of their’s uterus by ammonium sulfide.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
C-cell hyperplasia considered as test item related finding was observed in 3 of 24 females from
120 mg/kg bw/day dose group, and in 4 of 23 females from 330 mg/kg bw/day groups. C-cell
hyperplasia was of minimum grade; however, it was not found in the control group, and the
increase in occurrence in the high dose group was significant (p < 0.05, Fisher Exact test)
compared with the control group. Although there is no data on the serum level of calcitonin and
calcium in pregnant females treated with benzotriazole, C-cell hyperplasia may be considered
potentially adverse.
Remaining histological findings (congenital cysts, ectopic lymphoid tissue, and one finding of
follicular cell vacuolization) were considered to be incidental not treatment-related findings.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
Thyroid Hormons Assay Data
Benzotriazole at the doses of 36, 120, and 330 mg/kg bw/day did not significantly affect the
level of thyroid hormones (Thyroxine, Triiodothyronine, and Thyroid Stimulating Hormone) in
the serum of pregnant females.

Maternal developmental toxicity

Number of abortions:
no effects observed
Description (incidence and severity):
none reported
Pre- and post-implantation loss:
effects observed, treatment-related
Description (incidence and severity):
The mean number of corpora lutea and pre-implantation loss did not statistically differ in the
test item treated groups compared to the control vehicle group. The mean number of
implantation sites was approximately the same among groups.

In the 120 and 330 mg/kg bw/day dose groups, the relative post-implantation loss was
increased compared to the control vehicle group (p < 0.01 and p < 0.05, respectively). The
increase of post-implantation loss in these groups was associated with the increase in the
absolute and percentage values of early resorptions. Also, the total number of females with
post-implantation loss was increased (non-significantly) in the 120 and 330 mg/kg bw/day
dose groups. The mean number of fetuses per female was slightly decreased in the 330
mg/kg bw/day dose group (by 6.2 % compared to the control group), but this change was not
significant.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
no total litter loss was reported .
Early or late resorptions:
effects observed, treatment-related
Description (incidence and severity):
a significant differnce in early resorptions is reported for the 120 and 330 mg/ kg bw/day dose group (p < 0.01 resp. p< 0.05).
Dead fetuses:
no effects observed
Description (incidence and severity):
none reported
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
none reported
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
There was no difference in number of mated to number of pregnant females when compared to control group
Other effects:
no effects observed
Description (incidence and severity):
no other effects repoted
Details on maternal toxic effects:
maternal toxicity is noted

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
36 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
clinical signs
histopathology: non-neoplastic
pre and post implantation loss

Maternal abnormalities

Abnormalities:
effects observed, treatment-related
Localisation:
uterus
Description (incidence and severity):
increase in Post-implantation Loss.

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
In the 330 mg/kg bw/day dose group, body weight of male and female fetuses as well as mean
fetuses weight of litter was significantly decreased compared to the control vehicle group
(respectively, by 11.2 %, 11.8 %, and 11.5 %).
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The fetuses sex ratio in all dose treated groups was not significantly changed
compared to the control vehicle group.
Changes in litter size and weights:
effects observed, treatment-related
Description (incidence and severity):
In the 330 mg/kg bw/day dose group, body weight of male and female fetuses as well as mean
fetuses weight of litter was significantly decreased compared to the control vehicle group
(respectively, by 11.2 %, 11.8 %, and 11.5 %).
Changes in postnatal survival:
not examined
External malformations:
effects observed, treatment-related
Description (incidence and severity):
There were no fetuses with external abnormalities recognized as malformations in the 36
mg/kg bw/day dose group.
In the 120 mg/kg bw/day dose group, three fetuses from one litter (No.90-2m, No. 90-3m and
No. 90-4m) were with inward rotated hindlimbs. No skeletal and visceral malformations were
observed for these fetuses; however, the altered ossifications and soft disintegrating
consistency of skeleton can be noted for No.90-2m and No. 90-3m fetuses. Besides, these
fetuses had a domed head as an external observation with unknown importance. In fetus
No.90-4m, dilated ventricles of the brain were noted, which can be an associated finding. In
the 120 mg/kg bw/day dose group, the increase in the incidence of brain ventricles dilation was
noted (Section 11.5.3) during a visceral examination. However, in the 330 mg/kg bw/day dose
group, this alteration with unknown importance was not observed. Also, in 120 mg/kg bw/day
dose group, there was no significant increase in fetal and litter incidence of skeletal
observations, so these findings in one litter can be considered as with unclear relationship with
the test item.
In the 330 mg/kg bw/day dose group, one fetus (52-7f) was with following malformations:
inward rotated hindlimbs, absent of some digits on the left hindlimb, thread-like tail, and absent
of anus (confirmed during evisceration). This fetus also had a large abdomen, probably as a
result of the absence of the anus. During evisceration, no other malformations of soft tissues
were found. The external malformations in the fetus No. 52-7f were associated with the
skeletal malformations (see Section 11.5.4), so the treatment relation of this finding cannot be
excluded despite its uniqueness.
For the fetuses in 330 mg/kg bw/day dose group, an increase of small fetus in fetal and litter
incidence was observed. This increase was not significant (p = 0.053, Fisher Exact test),
however, correlated to the decrease in fetal body weight in this group, and considered as test-
item related.
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
Skeletal malformations were found in one fetus (No.52-7f) from the 330 mg/kg bw/day group.
This fetus had no vertebrae after the thoracic T4, had only 7 ribs on the right and left, of which
the 5th, 6th, and 7th pairs were unossified. During the external observation after the cesarean
section, the few digits were recorded in the left hindpaw. Due to the low body weight of this
fetus and soft consistency of limbs, the skin was not completely removed from the phalanges
of hindlimbs to avoid damage and loss of structure during fetus processing. Phalanx cartilages
of hindpaws were not stained appropriately; however, for left hindpaw, the absence of distal
phalanges in 1st to 3d digits, absence of all phalanges in 4th digit, and absence of distal and
medial phalanges in 5th digit are supposed; four metatarsals (Mt2-Mt5) were ossified. These
malformations were associated with external observations (thread-like tail). Despite the
uniqueness of this case, it considered being test item-related.
Fetal skeletal variations which supposed to be related to the test item were observed in the
330 mg/kg bw/day dose group. The statistical significant increase in incidence compared to
the control vehicle group was noted for incomplete ossification of 2nd - 4th sternebra (fetal
incidence, p < 0.05), unossification of 6th sternebra (fetal and litter incidence, p < 0.05),
unossification in 5th metacarpal (fetal and litter incidence, p < 0.001 and p < 0.01) or its
incomplete ossification (fetal incidence, p < 0.05).
The total affected fetuses with alteration in ossification was statistically increased for sternebra
(fetal incidence, and mean of fetus per litter, p < 0.01), for metacarpal (fetal and litter
incidence, and mean of fetus per litter, p < 0.01), and for total variations (fetal incidence, p <
0.001, and mean of fetus per litter, p < 0.01).
Visceral malformations:
no effects observed
Description (incidence and severity):
No test item related malformations of fetal soft tissues were found. Findings of unilateral
absent of pair organs (eye, and testis with epididymis and vas deferens) were considered as
incidental, not test item related.
The statistical increase in the incidence of variations was observed in the 330 mg/kg bw/day
dose group for the liver (discoloration - pale, spotted), kidney (discolored due to the
hemorrhage), and uterus (thin uterine horns). The percentage increase was observed for the
fetal incidence (liver and kidney), as well as for the litter incidence (kidney, uterus, and liver
(non-significant)). Discoloration of renal pelvis content had a low incidence; however, it was
associated with the hemorrhage in the kidney. The slight non-statistical dose-dependence
was noted for these findings, so, they considered as test item related. It should be noted that
thin uterine horns are regarded as of unknown importance and may be due to the small
fetuses in the high dose group, recorded during an external examination and discussed
above.
Alterations of unknown importance were observed in the 120 mg/kg bw/day dose group in the
brain and kidney. The fetal and litter incidence of dilated brain ventricles was statistically
increased compared to the control group as well as the dilated renal pelvis. In the 330 mg/kg
bw/day dose group, the dilation of the fourth brain ventricle was found in 4 fetuses from 2
litters, and the dilation of the renal pelvis was found in 1 fetus, which did not significantly
higher compared to the control group. So, the dilated brain ventricles and dilated renal pelvis
in the 120 mg/kg bw/day dose group are considered to be with unclear relation to the test
item administrations.
Other findings in soft tissues presented in Table S16-4 were recorded with approximately
equal or/and low frequency in all groups and considered to be as not treatment-related. The
fetal and litter incidence of testis malposition was the same in the test item treated and control
groups. In most fetuses, this observation was expressed as a unilateral undescended testis at
half the distance from the kidney.
The total affected fetuses (incidence value and mean per litter value) were not significantly
changed in test item treated groups compared to the control vehicle group.
Other effects:
effects observed, treatment-related
Description (incidence and severity):
The absolute and normalized anogenital
distance was not changed in male fetuses of all dose test item groups. For female fetuses, the
normalized anogenital distance was statistically increased in the 330 mg/kg bw/day dose
group. The fetuses sex ratio in all dose treated groups was not significantly changed
compared to the control vehicle group.
Details on embryotoxic / teratogenic effects:
effects at 120 mg/kg bw/day are of unclear relation to the test item.
Therefore these effects are not considered for Effect level table below.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
120 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes
changes in litter size and weights
external malformations
skeletal malformations

Fetal abnormalities

Abnormalities:
effects observed, treatment-related
Localisation:
external: anogenital distance
skeletal: sternum
other: fetus size
Description (incidence and severity):
incomplete or absent ossification in highest dose group
small fetuses in highest dose group
increased anogenital distance in female fetus in highest dose group

Overall developmental toxicity

Developmental effects observed:
yes
Lowest effective dose / conc.:
330 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to maternal toxicity:
not specified
Dose response relationship:
not specified
Relevant for humans:
not specified

Applicant's summary and conclusion

Conclusions:
Under the conditions of this study, the no-observed-adverse-effect-level (NOAEL) for maternal
and developmental toxicity (post-implantation loss) of 1,2,3-Benzotriazole was considered to be 36 mg/kg bw/day.
The no-observed-adverse-effect-level (NOAEL) for embryo-fetal developmental toxicity of 1,2,3-Benzotriazole was considered to be 120 mg/kg bw/day
Executive summary:

Objective

The objective of this prenatal developmental toxicity study was to evaluate the potential effects of the test

item, 1,2,3-Benzotriazole (CAS No. 95-14-7), on pregnancy and on embryo-fetal development in rats

following daily oral (gavage) administration at doses 36, 120 and 330 mg/kg body weight/day from

implantation to the day prior to scheduled caesarean section (day 5 to day 19 post-mating inclusive).

Design

Results from the separate dose-range-finding study as well as published toxicity data of 1,2,3-Benzotriazole

and data from a structurally closely related substance (Methyl-1H-benzotriazole, CAS No. 29385-43-1) were

considered for dose selection.

The test item, 1,2,3-Benzotriazole, in the vehicle (Kollisolv® PEG E 400) was administered by gavage once

daily to Sprague-Dawley female rats from day 5 to day 19 post-mating (inclusive). Three groups received

the test item at dose-levels of 36, 120 or 330 mg/kg bw/day. A concurrent vehicle control group received the

vehicle (Kollisolv® PEG E 400) on a comparable regiment and in the same volume of 3 mL/kg bw. Each

group consists of 24-26 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 are recorded at three-day intervals. 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 are examined to record implantation sites, early and late resorptions, dead, and live fetuses. The

fetuses were weighed, sexed with measurement of anogenital distance (AGD), and submitted to external

examination. Approximately half of the fetuses 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 and cartilage with alcian blue.

The fetal findings were described according to the harmonized terminology of the International Federation of

Teratology Societies (IFTS) and classified as malformations or variations. Fetal incidence, litter incidence,

and affected fetuses per litter were calculated for external, visceral, and skeletal alterations. The

reproductive tract was examined with particular attention, and external sex was compared with internal

(gonadal) sex in all fetuses. In addition, male fetuses were examined for undescended testes.  

Results

There was no morbidity and mortality of females caused by the test item administration. The test item

related clinical findings were observed in the 330 mg/kg bw/day dose group after dosing and were as

follows: hypokinesia, staggering gait or flatness as a more severe behavior disorder, excessive vocalization,

chromodacryorrhea, ptyalism, nasal discharge. Clinical observations were episodic with the recovery of

animals within approximately 1.5 – 3 hours after dosing. The higher frequency of clinical signs was noted

after first dosing (gestation day 5), as well as at the end of the administration period (gestation days 14, 15,

17, and 19). Over the entire period of the test item administration, the total number of females with clinical

findings in the 330 mg/kg bw/day dose group was 14 out of 26 examined (p < 0.001, Fisher Exact test). The

body weight of pregnant females administered with the dose of 330 mg/kg bw/day was slightly decreased

compared to the control vehicle group at the gestation day 20 (by 5.0%, p < 0.05), and the body weight gain

was statistically reduced (p < 0.01) the entire period of administration compared with the control group as

well as lower doses. The reduced body weight gain of pregnant females in the 330 mg/kg bw/day dose

group was associated with a smaller weight of the pregnant uterus (by 16 % compared to the control group,

p < 0.05). The final maternal body weight without a gravid uterus did not statistically differ from the body

weight of control females. Mean food consumption in the 36, 120 and 330 mg/kg bw/day group was similar

to that in the vehicle control group during all study days.

During necropsy, there were no gross findings related to the test item administration. The mean number of

corpora lutea and pre-implantation loss did not statistically differ in the test item treated groups compared to

the control vehicle group. The mean number of implantation sites was approximately the same among

groups.  

The test item did not affect the observed thyroid function of pregnant females. The absolute and relative

thyroid weight was not significantly changed in all dose test item treated group. Test item related C-cell

hyperplasia was observed in 3 of 24 females from 120 mg/kg bw/day dose group, and in 4 of 23 females

from 330 mg/kg bw/day groups (p < 0.05, Fisher Exact test). This histological alteration was of minimum

grade and can be associated with the calcitonin exchange (as speculation). Although there is no data on the

serum level of calcitonin and calcium in pregnant females treated with benzotriazole, C-cell hyperplasia may

be considered potentially adverse. The level of thyroid hormones T3, T4, and TSH in the serum of pregnant

females was not significantly changed in the test item treated groups.  

The relative post-implantation loss was increased in the 120 and 330 mg/kg bw/day dose groups compared

to the control vehicle group (p < 0.01 and p < 0.05, respectively). The increase of post-implantation loss in

these groups was associated with the increase in the absolute and percentage values of early resorptions.

Also, the total number of females with post-implantation loss was increased (non-significantly) in the 120

and 330 mg/kg bw/day dose groups. The mean number of fetuses per female was slightly decreased in the

330 mg/kg bw/day dose group (by 6.2 % compared to the control group), but this change was not

significant.

The body weight of male and female fetuses, as well as mean fetuses weight of litter, was significantly

decreased in the 330 mg/kg bw/day dose group (respectively, by 11.2 %, 11.8 %, and 11.5 %). The

absolute and normalized anogenital distance was not changed in male fetuses; however, for female fetuses,

the normalized anogenital distance was statistically increased in the 330 mg/kg bw/day dose group. The

fetal sex ratio in all dose treated groups was not significantly changed compared to the control vehicle

group.

In the 36 mg/kg bw/day dose group, there were no fetuses with external observations, soft tissue, and

skeletal and cartilage alterations recognized as malformations; there were no variations or other alterations

which are considered as test item related.    

In the 120 mg/kg bw/day dose group, three fetuses from one litter were with inward rotated hindlimbs during

an external examination. No skeletal and visceral malformations were observed for these fetuses; however,

the altered ossifications were noted for two of them. Besides, these fetuses had a domed head as an

external observation with unknown importance, and the dilated ventricles of the brain were noted for one of

them. The fetal and litter incidence of dilated brain ventricles (fourth, third, lateral and ventricle) was

statistically increased in the 120 mg/kg bw/day dose group compared to the control group (p < 0.01 for

ventricle) as well as the incidence of the dilated renal pelvis (p < 0.01). These findings were not dose-

dependent. In the 330 mg/kg bw/day dose group, the dilation of the fourth brain ventricle was found in 4

fetuses from 2 litters, and the dilation of the renal pelvis was found in 1 fetus, which was not significantly

higher compared to the control group. So, in the 120 mg/kg bw/day dose group, the dilated brain ventricles

and dilated renal pelvis as soft tissue alterations of unknown importance are considered to be with unclear

relation to the test item administrations. In 120 mg/kg bw/day dose group, there was no significant increase

in the fetal and litter incidence of skeletal and cartilage observations.

In the 330 mg/kg bw/day dose group, one female fetus had following external malformations: inward rotated

hindlimbs, absent of some digits on the left hindlimb, thread-like tail, and absent of anus confirmed during

evisceration. This fetus also had a large abdomen, probably as a result of the absence of the anus; no other

malformations of soft tissues were found during evisceration. The external malformations in the fetus were

associated with the skeletal malformations: the absence of vertebrae after the thoracic T4, and the presence

of only seven ribs on the right and left, of which the 5th, 6th, and 7th pairs were unossified, and unilateral

absence of hindpaw phalanges. Despite the uniqueness of this finding, it considered being test item-related.

In this group, the increase in the incidence of altered fetal ossification was observed. The statistical

significant increase in incidence compared to the control vehicle group was noted for incomplete ossification

of 2nd - 4th sternebra (fetal incidence, p < 0.05), unossification of 6th sternebra (fetal and litter incidence, p

< 0.05), unossification in 5th metacarpal (fetal and litter incidence, p < 0.001 and p < 0.01) or its incomplete

ossification (fetal incidence, p < 0.05). The total affected fetuses with alteration in ossification was

statistically increased for sternebra (fetal incidence, and mean of fetus per litter, p < 0.01), for metacarpal

(fetal and litter incidence, and mean of fetus per litter, p < 0.01), and for total variations (fetal incidence, p <

0.001, and mean of fetus per litter, p < 0.01). Moreover, in the 330 mg/kg bw/day dose group, the statistical

increase in the incidence of soft tissue variations was observed for the liver (discoloration - pale, spotted),

kidney (discolored due to the hemorrhage), and uterus (thin uterine horns). The percentage increase was

observed for the fetal incidence (liver and kidney), as well as for the litter incidence (kidney, uterus, and liver

(non-significant)). The slight non-statistical dose-dependence was noted for these findings, so, they are

considered as test item related. It should be noted that thin uterine horns are regarded as of unknown

importance and may be due to the small fetuses in the high dose group, recorded during an external

examination.

The fetal and litter incidence of testis malposition was the same in the test item treated and control groups.

In most fetuses, this observation was expressed as a unilateral undescended testis at half the distance from

the kidney.