Registration Dossier

Administrative data

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Effects on fertility

Description of key information

No information on fertility available for the substance.


As the former read-across to the proposed analogueous substance Benzotriazole (CAS 95-14-7) proofed to be scientifcly not adequate, further testing is proposed.

Effect on fertility: via oral route
Endpoint conclusion:
no study available (further information necessary)
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available

Effects on developmental toxicity

Description of key information

A prenatal developmental toxicity test was performed in 2019.

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26. September 2019 - 10. April 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: 190105
- Expiration date of the lot/batch: 12.2020
- Purity test date: Production date 01.2019

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature (20 ± 5 °C)
- Stability under storage conditions: assumed stable
- Stability under test conditions: assumed stable
- Solubility and stability of the test substance in the solvent/dispersant/vehicle/test medium:
Stability of the test item in the vehicle Kollisolv® PEG E 400 prepared at concentrations of 10 and 100 mg/mL was confirmed following storage for 7 days at room temperature (the
temperature range, 20 – 25 °C) during method validation study. Besides, homogeneity
analysis was performed for formulations of 10 and 100 mg/mL after 4 days of storage after re-mixing
- Reactivity of the test substance with the solvent/vehicle /test medium (if applicable):

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
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
(www.spf-animals.ru)
- Age at study initiation: Approximately 12 weeks old at the initiation of dose
administration on G5 (day 1)
- Weight at study initiation: 240 ± 19 g, N = 97
- 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): The animals were fed Laboratory Rodent Diet (SSNIFF V1534-300 autoclavable, Spezialdiaten GmbH, Ferdinand-Gabriel-Weg 16, DE-59494 Soest, Germany) ad libitum.
- Water (e.g. ad libitum): Filtered tap water was provided ad libitum in standard water bottles.
- Acclimation period: 8 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24 °C
- Humidity (%): 30 - 60 %
- Air changes (per hr): 12 times per hour
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: To:
Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Details on exposure:
Dose Preparation
The Sponsor provided the test item as a neat solid substance. Calculations for the preparation
of formulations were based on the dose (30, 90 and 300 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 10, 30, and 100 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.
On the day of dosing, the aliquot of each formulation was mixed thoroughly and transferred to
the barrier zone

Each female was dosed once a day, at approximately the same time each day at the first half of the
day (09:00 – 12:00), from day 5 to day 19 (including) of post mating (G5-G19).
The vehicle and the test item will be administered orally by gavage, via an appropriately sized
stainless steel ball-tipped dosing cannula connected with syringe once daily. A separate cannula for
each group will be used. The dosage volume for all groups will be 3 mL/kg body weight. Individual
dosages will be based on the last value body weights to provide the correct mg/kg bw/day dose.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The validated High-performance liquid chromatography (HPLC) method was used for the
detection of the test item concentration in-vehicle formulations ranging from 10 to 100 mg/mL.
The validation of the analytical method was performed before the first analysis of doses
formulations, where linearity, accuracy, precision/repeatability, specificity/selectivity, sensitivity
(LLOQ) were established, and stability of formulation samples was assessed for 7 days of
storage.
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:
Each female was dosed once a day, at approximately the same time each day at the first half of the
day (09:00 – 12:00), from day 5 to day 19 (including) of post mating (G5-G19).
Frequency of treatment:
daily
Duration of test:
On gestation day 20, all females were euthanized by anesthesia (Zoletil® / Xyla®, i.m.)
Dose / conc.:
30 mg/kg bw/day (nominal)
Dose / conc.:
90 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
No. of animals per sex per dose:
25
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dosage levels were based on a dose-range-finding study (BTL BIBC Study No. 685/19) with the treatment of four female Sprague-Dawley rats at a single dose 450 mg/kg bw with a followed 13-days repeated administration of 330 mg/kg bw.
- Rationale for animal assignment (if not random): 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.
- Other:
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
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, and longer until recovery, if necessary. In addition, the presence of findings at the time of dose administration was recorded for individual animals.

DETAILED CLINICAL OBSERVATIONS: Yes / No / No data
- Time schedule:

BODY WEIGHT: Yes
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 AND COMPOUND INTAKE (if feeding study): Yes
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).

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
- Time schedule for examinations:

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: 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 and weighed after fixation.

OTHER:
Ovaries and uterine content:
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 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) 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.

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.

Approximately half of the live fetuses in each litter were 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 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.

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. Number of male and female fetuses per litter, 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.
Additionally, Yates corrected Chi-square test was applied to the % male fetuses data.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
The test item related clinical findings were observed in the 300 mg/kg bw/day dose group as
follows: hypokinesia (25 of 25 females), staggering gait (13 of 25 females) or creeping gate (2
of 25 females), flattened posture (9 of 25 females) or full immobility as a more severe behavior
disorder (3 females). Behavioral findings were temporary after dosing with a recovery period
of 1-4 hours; however, they were considered as adverse. The total number of females with
clinical observations in the 300 mg/kg bw/day dose group was significantly higher (p < 0.001 –
0.05, Fisher Exact test) compared to the control vehicle group for all days with a slight
increase of incidence to the end of the dosing period. Over the entire period of the test item
administration, the total number of females with test item related clinical findings in the 300
mg/kg bw/day dose group was 25 out of 25 examined (p < 0.0001, Fisher Exact test).
Non-behavioral clinical findings were observed in single females and considered to be not test
item-related or with unclear relation to the test item and were non-adverse. Slight
chromodacryorrhea was noted in one female from the 300 mg/kg bw/day dose group and one
female from the control vehicle group and can be due to the distress, as well as palpebral
closure in a single female in the 300 mg/kg bw/day dose group. Slight episodic diarrhea was
observed in single females in the high dose group and control vehicle dose group, which can
be caused by the vehicle (PEG 400) used. One female from the 300 mg/kg bw/day dose
group had episodic ptyalism can be caused by an adverse test item taste or a locally irritating
effect.
In one female (No. 59) from the 90 mg/kg bw/day dose group, starting from the study day 11
(gestation day 15), a rapidly growing solid, not painful mass was observed in the inguinal
region. The round mass was isolated during necropsy, was of subcutaneous localization and
had reached approximately 35 mm in diameter. Histopathologically, it was diagnosed as a
typical mammary adenocarcinoma of rats.This finding was considered to be not related with
the test item administration.
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:
no effects observed
Description (incidence and severity):
The body weight of pregnant females administered with the 30, 90, and 300 mg/kg bw/day
doses of the test item was not changed compared to the control vehicle group at all days of
registration (Table S5, Figure 1, A). The mean final body weight gain of pregnant females for
the period of treatment (gestation days 5-20) was slightly reduced in the 300 mg/kg bw/day
dose group; however, this change was not significant.
In the 300 mg/kg bw/day dose group, the mean value of gravid uterine weight was 9.2 % less
compared to the control vehicle group; however, this decrease in uterine weight was not
statistically significant. 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 30, 90, and 300 mg/kg bw/day dose treated groups 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:
not examined
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. In one
non-gravid female (No. 43) from the 30 mg/kg bw/day dose group the absence of one uterine
horn was observed, which was not test item related finding. One female (No. 59) from the 90
mg/kg bw/day dose group had the solid subcutaneous mass in the inguinal area. The round
mass was isolated during necropsy, was of subcutaneous localization and had reached
approximately 35 mm in diameter. Histopathologically, it was diagnosed as a typical mammary
adenocarcinoma of rats. This finding was considered to be not related with the test item
administration.
Females No.20 (group 1), No.42, No.43, No. 45, and No.81 (group 2), No.38, No.86, and
No.92 (group 3), No,37 and No. 51 (group 4) were without fetuses in the uterus. 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:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Weight of Thyroid Glands

The absolute and relative thyroid weight of pregnant females was not significantly changed in
all dose test item treated group (ANOVA Dunnett test). The mean values of absolute and
relative thyroid weight were increased in the 30 mg/kg bw/day dose group; however, this
change was slight, not significant, not dose-dependent, and so, considered as not test item
related.

Microscopic Observations in Thyroid Glands

There were no histological alterations in the thyroid gland, which considered to be clearly
associated with the test item treatment. C-cell hyperplasia of the minimal and slight grade was
observed in all groups, including control; the slight increase in the frequency of findings and its
grade can be noted for the 300 mg/kg bw/day dose group, but this change was not significant.
The statistically significant increase in the incidence of the ultimobranchial cyst was recorded
in the 300 mg/kg bw/day dose group. However, this finding is considered as congenital, not
treatment-related, as well as single findings of ectopic lymphoid tissue.

Thyroid Hormons Assay Data

No significant changes were revealed in the mean values of triiodothyronine and thyroxine in
all dose test item treated groups. The mean level of thyroid stimulating hormone was slightly
decreased in the 300 mg/kg bw/day dose group (by 24.5% compared to the control group
using t-test, p < 0.05).
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 control vehicle group, two females (No. 5 and No. 41) were with the high level of post-
implantation loss. In female No. 41 was only one fetus found during necropsy and 83.3 % of
post-implantation loss due to the early resorptions. Data on the uterine content of female No.
41 notably differed from historical control data (% of post-implantation loss, Min-Max, 0.0 –
35.7 %, N = 101, Appendix 21) and were not included in the statistics. Female No.5 had 7
fetuses and 50 % of post-implantation loss due to early and late resorptions. The data from
this female increased the mean values of the early and late resorption and post-implantation
loss in the 0 mg/kg bw/day dose group.
In the 30 and 90 mg/kg bw/day dose groups, values of early and late resorptions, as well as
post-implantation loss and number of females with post-implantation loss, did not significantly
differ from the values in the control vehicle group. In the 300 mg/kg bw/day dose group, the
tendency to the increase in the relative value of early resorptions and post-implantation loss
was observed. The number of females with post-implantation loss was statistically increased
(17 of 23 compared to the 8 of 22 in the control group, p < 0.05, Fisher Exact test). The mean
number of fetuses per female was not significantly changed in all dose test item treated
groups.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
Females No.20 (group 1), No.42, No.43, No. 45, and No.81 (group 2), No.38, No.86, and
No.92 (group 3), No,37 and No. 51 (group 4) were without fetuses in the uterus. There were
no implantation sites and scars were observed in non-pregnant females after staining of their’s
uterus by ammonium sulfide.
Early or late resorptions:
effects observed, treatment-related
Description (incidence and severity):
In the control vehicle group, two females (No. 5 and No. 41) were with the high level of post-
implantation loss. In female No. 41 was only one fetus found during necropsy and 83.3 % of
post-implantation loss due to the early resorptions. Data on the uterine content of female No.
41 notably differed from historical control data (% of post-implantation loss, Min-Max, 0.0 –
35.7 %, N = 101, Appendix 21) and were not included in the statistics. Female No.5 had 7
fetuses and 50 % of post-implantation loss due to early and late resorptions. The data from
this female increased the mean values of the early and late resorption and post-implantation
loss in the 0 mg/kg bw/day dose group.
In the 30 and 90 mg/kg bw/day dose groups, values of early and late resorptions, as well as
post-implantation loss and number of females with post-implantation loss, did not significantly
differ from the values in the control vehicle group. In the 300 mg/kg bw/day dose group, the
tendency to the increase in the relative value of early resorptions and post-implantation loss
was observed. The number of females with post-implantation loss was statistically increased
(17 of 23 compared to the 8 of 22 in the control group, p < 0.05, Fisher Exact test). The mean
number of fetuses per female was not significantly changed in all dose test item treated
groups.
Dead fetuses:
no effects observed
Description (incidence and severity):
no dead fetuses observed
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
no effects reported
Changes in number of pregnant:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
90 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
clinical signs
early or late resorptions
pre and post implantation loss
other: effects on Thyroids
Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
In the 30 and 90 mg/kg bw/day dose groups, the body weight of male and female fetuses did not
significantly differ from the values in the control vehicle group. However, in the 90 mg/kg bw/day dose
group, a slight tendency to decrease in fetal body weight was already noticeable (by 4.9 %, males, and 4.1
%, females). In the 300 mg/kg bw/day dose group, this decrease was significant: 11.5 % for male, 12.7 %
for female fetuses (p < 0.0001). The mean fetal body weight in this group was decreased by 12.1 %
compared to the control vehicle group (p < 0.0001 using analysis of covariant with litter size as a covariant).
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
effects observed, treatment-related
Description (incidence and severity):
The percentage of male fetuses per litter was decreased in the 30 and 300 mg/kg bw/day dose group (39.0
% and 43.4 % compared to 52.4 % in the control group). The mean value of % males in the medium-dose
group was also slightly reduced (47.9 %). This decrease in the ratio of male and female fetuses was also
notable as compared to the historical control value (50.5 % males). The absolute and normalized anogenital
distance was statistically reduced in male fetuses from the 300 mg/kg bw/day dose group (by 2.5 % for
normalized value, p < 0.01). For female fetuses, the anogenital distance was not significantly changed.
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
No test item related external malformations were revealed. The external fetal findings recorded as
malformation according to IFTS terminology were observed in hindlimbs, and the face of some fetuses in
the test item treated groups. The disproportionately small hindlimbs of the minimal score did not correlate to
the skeletal and cartilage findings in a part of examined fetuses. However, in other fetuses, small hindlimbs
were associated with multiple incomplete ossifications and small body weight. The short snout in one fetus
from 300 mg/kg bw/day dose group and small mandible in the other one did not correlate to the findings in
the skull. So, the external malformations in hindlimbs and the face of some fetuses are considered of
unclear relationship to the test item administration. The fetal and litter incidence of small fetuses was 5.7 %
and 23.8 % in the 90 mg/kg bw/day dose group (p < 0.05 for litter incidence) and 1.7 % and 14.7 % in the
300 mg/kg bw/day dose group (non-significant, p = 0.054 for litter incidence) compared 0.0 % of incidence
in the control group. This external observation is considered test item-related.
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
he skeletal malformations were observed in the 300 mg/kg bw/day dose group. Absence of one 13th rib
was noted for two fetuses from two litters; there were no alterations in thoracic vertebrae in these fetuses.
Because of the low incidence and absence of dose-dependence, this finding is considered to be not test
item-related. Also, in the 300 mg/kg bw/day dose group only, the supernumerary thoracolumbar ribs as
skeletal variation were observed in 4 fetuses from two litter. Supernumerary ribs were noted as rudimentary
for three fetuses, and as extra ribs without cartilage for one fetus. Extra and rudimentary supernumerary ribs
may reflect separate developmental phenomena; however, due to the low incidence and absence of
significant difference from the control group, this finding is considered as of unclear relationship with the test
item.
Fetal skeletal variations which supposed to be related to the test item were revealed in the 30, 90, and 300
mg/kg bw/day dose groups and were as follows.
In the 30 mg/kg bw/day dose group, the incidence of incomplete ossification in 1st and/or 2nd thoracic
vertebra was increased (7.5 % and 30.0 % for fetal and litter incidence versus 1.5 % and 4.5 % in the
control group, p < 0.05). An unossified 6th sternebra was recorded with the increased fetal incidence (7.5 %
versus 1.5 % in the control group, p < 0.05), and with a higher, but non-significant litter incidence (25.0 %
versus 9.1 % in the control group). An unossified 5th metatarsal was recorded with an increased but not
significant frequency (2.5 % and 5.0 % for fetal and litter incidence versus 0.0 % in the control group). Also,
a single incident of proportionally small bones of hindlimb was noted, which correlated to the external
observation in hindlimbs (disproportionally small hindlimbs) and was associated with alterations of
ossification in multiple bones. The percentage of total affected fetuses in hindlimbs was increased in 30
mg/kg bw/day dose group (3.3% for fetal incidence, p < 0.05, and 10.0% for litter incidence, not significant).
In the 90 mg/kg bw/day dose group, the high incidence of following variations was revealed: unossified 5th
metacarpal (fetal incidence, 36.6% versus 19.9 % in the control group, p < 0.01), unossified 2nd to 5th
sternebrae (13.0 % versus 1.5 % for fetal incidence, p < 0.0001, and 42.9 % versus 9.1 % for litter
incidence, p < 0.05), unossified 6th sternebra (fetal incidence, 16.8 % versus 1.5 %, p < 0.0001, and 33.3 %
versus 9.1 % for litter incidence, non-significant), incomplete ossification in 2nd to 4th sternebra (fetal and
litter incidence, 7.6 % and 33.3 % compared to the control values of 2.2 %, p < 0.05, and 13.6 %, non-
significant).
In the 300 mg/kg bw/day dose group, the skeletal variations were observed as followed. In skull, the
incidence of incomplete ossification was increased for frontal bone (fetal and litter incidence, 29.4 %, p <
0.05, and 60.9 %, non-significant) compared to the control group (fetal and litter incidence, 14.0 % and 40.9
%), and for nasal bone (fetal incidence, 7.0 % (p < 0.05), and litter incidence, 17.4 % (non-significant)
versus 0.7 % and 4.5 % in the control group). In forelimb, a significant increase in incidence of unossified
5th metacarpal was observed: 64.3 % versus 19.9 % for fetal incidence, p < 0.01, and 95.7 % versus 63.6
% for litter incidence, p < 0.05. In sternum, the significant increase in incidence of unossified 2nd to 5th
sternebra was revealed with the 17.5 % of fetal incidence (p < 0.0001), and 56.5 % of litter incidence (p <
0.01) compared to the control values of 1.5 % and 9.1 %, respectively. Also, the increased incidence was
observed for an unossifed 6th sternebra (19.6 %, p < 0.0001, and 52.2 %, p < 0.01, for fetal and litter
incidence, versus 1.5 % and 9.1 % control values), for incomplete ossification in 2nd to 4th sternebra (for
fetal and litter incidence, 10.5 % (p < 0.01) and 47.8 %, compared to the control values of 2.2 % and 13.6
%), and for incomplete ossification in 6th sternebra (p < 0.001 for fetal incidence). In vertebrae, an unossifed
1st and/or 2nd thoracic vertebra was observed with an increased fetal incidence of 6.3 % (p < 0.01); the
incidence of incomplete ossification in these vertebrae was also increased (11.2 % and 30.4 % for fetal and
litter incidence, p < 0.01 and p < 0.05 compared 1.5 % and 4.5 % in the control group). Besides, in the 300
mg/kg bw/day dose group, the significantly higher incidence of incomplete ossification was noted in the 10th
to 13th thoracic vertebrae (6.3 % versus 0.0 % for fetal incidence, p < 0.01). An unossified 5th metatarsal
was recorded with an increased frequency of 4.9 % for fetal incidence (p < 0.05), and 8.7 % for litter
incidence (not significant), versus 0.0 % of incidence in the control group. The incidence of incomplete
ossification in 5th metacarpal was also slightly increased in this group.
In general, the fetal incidence for total affected fetuses with skeletal and cartilage variation was significantly
increased in the 90 mg/kg bw/day dose group (71.8 %, p < 0.05) and 300 mg/kg bw/day dose group (86.7
%, p < 0.0001) compared to the control group with fetal incidence of 56.6 %.
Visceral malformations:
effects observed, treatment-related
Description (incidence and severity):
No malformations of soft tissues were found in any of the examined fetuses in all groups. The incidence of
total affected fetuses with variations and other alterations in soft tissues was increased in the 90 mg/kg
bw/day dose group (p < 0.05, fetal and litter incidence) and in the 300 mg/kg bw/day dose group (p < 0.05
for litter incidence). The incidence of the pale spotted liver was increased in the 90 and 300 mg/kg bw/day
dose groups for fetal value (p < 0.01) as well as for litter value (p < 0.05). In the 300 mg/kg bw/day dose
group, the percentage of intra-abdominal hemorrhage and enlarged blood-filled heart atrium was increased
for fetal incidence (p < 0.05 and p < 0.01) and litter incidence (p < 0.05). Alterations of unclear relation to the
test item were observed in the 90 mg/kg bw/day dose group in the brain and testis. The incidence of dilation
of lateral brain ventricle (4 fetuses in the three litters) was not significantly changed compared to the control
group. Additionally, in the 90 mg/kg bw/day dose group, the fetal and litter incidence of testes malposition
was statistically increased compared to the control group (p < 0.05). The malpositioned testis was found in
one fetus from the 30 mg/kg bw/day dose group and two fetuses from the 300 mg/kg bw/day dose group. In
most fetuses, this observation was expressed as a unilateral undescended testis at half the distance from
the kidney. Considering that undescended testes may occur with low frequency in control rat fetuses on
gestation day 20 and absence of dose-dependence, this finding in the 90 mg/kg bw/day dose group is seen
as with unclear relation to the test item.
Key result
Dose descriptor:
LOAEL
Effect level:
30 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
changes in sex ratio
fetal/pup body weight changes
skeletal malformations
visceral malformations
Abnormalities:
no effects observed
Description (incidence and severity):
no treatment related malformations obseved
Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
30 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects in the absence of maternal toxicity effects
Dose response relationship:
yes
Relevant for humans:
not specified
Conclusions:
Under the conditions of this study, the no-observed-adverse-effect-level (NOAEL) for maternal
toxicity of 4(or 5)-methyl-1H-benzotriazole considered to be 90 mg/kg bw/day. For embryo-fetal
development, the dose of 30 mg/kg bw/day is considered to be as lowest-observed-adverse-effect-level
(LOAEL).
Executive summary:

Objective

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

item, 4(or 5)-methyl-1H-benzotriazole (CAS 29385-43-1, Tolyltriazole), on pregnancy and on embryo-fetal

development in rats following daily oral (gavage) administration at doses 30, 90 and 300 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 Tolyltriazole and

data from a structurally closely related substance (Benzotriazole) were considered for dose selection.

The test item, Tolyltriazole, 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 30, 90 or 300 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 24-25 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 300 mg/kg bw/day dose group after dosing and were as

follows: hypokinesia, staggering or creeping gait, flattened posture, or full immobility as a more severe

behavior disorder. Clinical observations were episodic with the recovery of animals within approximately 1 –

4 hours after dosing; they were considered as adverse. Over the entire period of the test item

administration, the total number of females with test item related clinical findings in the 300 mg/kg bw/day

dose group was 25 out of 25 examined. One female from the 300 mg/kg bw/day dose group had episodic

ptyalism can be caused by an adverse test item taste or a locally irritating effect. In one female from the 90

mg/kg bw/day dose group, starting from the gestation day 15, a rapidly growing solid, not painful mass was

observed in the inguinal region diagnosed histopathologically as a typical mammary adenocarcinoma of

rats. This finding was considered to be not related to the test item administration.

The body weight of pregnant females administered with the 30, 90, and 300 mg/kg bw/day doses of the test

item was not changed compared to the control vehicle group at all days of registration. In the 300 mg/kg

bw/day dose group, the body weight gain was slightly decreased for the period of gestation day 5-8 (p <

0.05); however, the final body weight gain for overall treatment period (gestation days 5-20) was not

significantly changed. In the 300 mg/kg bw/day dose group, the mean value of gravid uterine weight was 9.2

% less compared to the control vehicle group; however, this decrease in uterine weight was not statistically

significant. 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 30, 90, and 300 mg/kg bw/day dose treated groups

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 absolute and relative thyroid weight of pregnant females was not significantly changed in all dose test

item treated group. There were no histological alterations in the thyroid gland, which considered being

clearly associated with the test item administration. The slight increase in the frequency and grade of C-cell

hyperplasia can be noted in the 300 mg/kg bw/day dose group, but this change was not significant. No

significant changes were revealed in the mean serum level of triiodothyronine and thyroxine in all dose test

item treated groups. The mean level of thyroid stimulating hormone was slightly decreased in the 300 mg/kg

bw/day dose group (by 24.5% compared to the control group using t-test, p < 0.05).

In the 30 and 90 mg/kg bw/day dose groups, values of early and late resorptions, as well as post-

implantation loss and number of females with post-implantation loss, did not significantly differ from the

values in the control vehicle group. In the 300 mg/kg bw/day dose group, the tendency to the increase in the

relative value of early resorptions and post-implantation loss was observed (respectively, 8.8 % and 10.3 %

versus 4.8 % and 6.9 % in the control group). The number of females with post-implantation loss was

statistically increased (17 of 23 versus 8 of 22 in the control group, p < 0.05, Fisher Exact test). The mean

number of fetuses per female was not significantly changed in all dose test item treated groups.

In the 30 and 90 mg/kg bw/day dose groups, the body weight of male and female fetuses did not

significantly differ from the values in the control vehicle group. However, in the 90 mg/kg bw/day dose

group, a slight tendency to decrease in fetal body weight was already noticeable (by 4.9 %, males, and 4.1

%, females). In the 300 mg/kg bw/day dose group, this decrease was significant: 11.5 % for male, 12.7 %

for female fetuses (p < 0.0001). The mean fetal body weight in this group was decreased by 12.1 %

compared to the control vehicle group (p < 0.0001 using analysis of covariant with litter size as a covariant).  

The percentage of male fetuses per litter was decreased in the 30 and 300 mg/kg bw/day dose group (39.0

% and 43.4 % compared to 52.4 % in the control group). The mean value of % males in the medium-dose

group was also slightly reduced (47.9 %). This decrease in the ratio of male and female fetuses was also

notable as compared to the historical control value (50.5 % males). The absolute and normalized anogenital

distance was statistically reduced in male fetuses from the 300 mg/kg bw/day dose group (by 2.5 % for

normalized value, p < 0.01). For female fetuses, the anogenital distance was not significantly changed.

No test item related external malformations were revealed. The external fetal findings recorded as

malformation according to IFTS terminology were observed in hindlimbs, and the face of some fetuses in

the test item treated groups. The disproportionately small hindlimbs of the minimal score did not correlate to

the skeletal and cartilage findings in a part of examined fetuses. However, in other fetuses, small hindlimbs

were associated with multiple incomplete ossifications and small body weight. The short snout in one fetus

from 300 mg/kg bw/day dose group and small mandible in the other one did not correlate to the findings in

the skull. So, the external malformations in hindlimbs and the face of some fetuses are considered of

unclear relationship to the test item administration. The fetal and litter incidence of small fetuses was 5.7 %

and 23.8 % in the 90 mg/kg bw/day dose group (p < 0.05 for litter incidence) and 1.7 % and 14.7 % in the

300 mg/kg bw/day dose group (non-significant, p = 0.054 for litter incidence) compared 0.0 % of incidence

in the control group. This external observation is considered test item-related.

No malformations of soft tissues were found in any of the examined fetuses in all groups. The incidence of

total affected fetuses with variations and other alterations in soft tissues was increased in the 90 mg/kg

bw/day dose group (p < 0.05, fetal and litter incidence) and in the 300 mg/kg bw/day dose group (p < 0.05

for litter incidence). The incidence of the pale spotted liver was increased in the 90 and 300 mg/kg bw/day

dose groups for fetal value (p < 0.01) as well as for litter value (p < 0.05). In the 300 mg/kg bw/day dose

group, the percentage of intra-abdominal hemorrhage and enlarged blood-filled heart atrium was increased

for fetal incidence (p < 0.05 and p < 0.01) and litter incidence (p < 0.05). Alterations of unclear relation to the

test item were observed in the 90 mg/kg bw/day dose group in the brain and testis. The incidence of dilation

of lateral brain ventricle (4 fetuses in the three litters) was not significantly changed compared to the control

group. Additionally, in the 90 mg/kg bw/day dose group, the fetal and litter incidence of testes malposition

was statistically increased compared to the control group (p < 0.05). The malpositioned testis was found in

one fetus from the 30 mg/kg bw/day dose group and two fetuses from the 300 mg/kg bw/day dose group. In

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

the kidney. Considering that undescended testes may occur with low frequency in control rat fetuses on

gestation day 20 and absence of dose-dependence, this finding in the 90 mg/kg bw/day dose group is seen

as with unclear relation to the test item.  

The skeletal malformations were observed in the 300 mg/kg bw/day dose group. Absence of one 13th rib

was noted for two fetuses from two litters; there were no alterations in thoracic vertebrae in these fetuses.

Because of the low incidence and absence of dose-dependence, this finding is considered to be not test

item-related. Also, in the 300 mg/kg bw/day dose group only, the supernumerary thoracolumbar ribs as

skeletal variation were observed in 4 fetuses from two litter. Supernumerary ribs were noted as rudimentary

for three fetuses, and as extra ribs without cartilage for one fetus. Extra and rudimentary supernumerary ribs

may reflect separate developmental phenomena; however, due to the low incidence and absence of

significant difference from the control group, this finding is considered as of unclear relationship with the test

item.

Fetal skeletal variations which supposed to be related to the test item were revealed in the 30, 90, and 300

mg/kg bw/day dose groups and were as follows.  

In the 30 mg/kg bw/day dose group, the incidence of incomplete ossification in 1st and/or 2nd thoracic

vertebra was increased (7.5 % and 30.0 % for fetal and litter incidence versus 1.5 % and 4.5 % in the

control group, p < 0.05). An unossified 6th sternebra was recorded with the increased fetal incidence (7.5 %

versus 1.5 % in the control group, p < 0.05), and with a higher, but non-significant litter incidence (25.0 %

versus 9.1 % in the control group). An unossified 5th metatarsal was recorded with an increased but not

significant frequency (2.5 % and 5.0 % for fetal and litter incidence versus 0.0 % in the control group). Also,

a single incident of proportionally small bones of hindlimb was noted, which correlated to the external

observation in hindlimbs (disproportionally small hindlimbs) and was associated with alterations of

ossification in multiple bones. The percentage of total affected fetuses in hindlimbs was increased in 30

mg/kg bw/day dose group (3.3% for fetal incidence, p < 0.05, and 10.0% for litter incidence, not significant).

In the 90 mg/kg bw/day dose group, the high incidence of following variations was revealed: unossified 5th

metacarpal (fetal incidence, 36.6% versus 19.9 % in the control group, p < 0.01), unossified 2nd to 5th

sternebrae (13.0 % versus 1.5 % for fetal incidence, p < 0.0001, and 42.9 % versus 9.1 % for litter

incidence, p < 0.05), unossified 6th sternebra (fetal incidence, 16.8 % versus 1.5 %, p < 0.0001, and 33.3 %

versus 9.1 % for litter incidence, non-significant), incomplete ossification in 2nd to 4th sternebra (fetal and

litter incidence, 7.6 % and 33.3 % compared to the control values of 2.2 %, p < 0.05, and 13.6 %, non-

significant).

In the 300 mg/kg bw/day dose group, the skeletal variations were observed as followed. In skull, the

incidence of incomplete ossification was increased for frontal bone (fetal and litter incidence, 29.4 %, p <

0.05, and 60.9 %, non-significant) compared to the control group (fetal and litter incidence, 14.0 % and 40.9

%), and for nasal bone (fetal incidence, 7.0 % (p < 0.05), and litter incidence, 17.4 % (non-significant)

versus 0.7 % and 4.5 % in the control group). In forelimb, a significant increase in incidence of unossified

5th metacarpal was observed: 64.3 % versus 19.9 % for fetal incidence, p < 0.01, and 95.7 % versus 63.6

% for litter incidence, p < 0.05. In sternum, the significant increase in incidence of unossified 2nd to 5th

sternebra was revealed with the 17.5 % of fetal incidence (p < 0.0001), and 56.5 % of litter incidence (p <

0.01) compared to the control values of 1.5 % and 9.1 %, respectively. Also, the increased incidence was

observed for an unossifed 6th sternebra (19.6 %, p < 0.0001, and 52.2 %, p < 0.01, for fetal and litter

incidence, versus 1.5 % and 9.1 % control values), for incomplete ossification in 2nd to 4th sternebra (for

fetal and litter incidence, 10.5 % (p < 0.01) and 47.8 %, compared to the control values of 2.2 % and 13.6

%), and for incomplete ossification in 6th sternebra (p < 0.001 for fetal incidence). In vertebrae, an unossifed

1st and/or 2nd thoracic vertebra was observed with an increased fetal incidence of 6.3 % (p < 0.01); the

incidence of incomplete ossification in these vertebrae was also increased (11.2 % and 30.4 % for fetal and

litter incidence, p < 0.01 and p < 0.05 compared 1.5 % and 4.5 % in the control group). Besides, in the 300

mg/kg bw/day dose group, the significantly higher incidence of incomplete ossification was noted in the 10th

to 13th thoracic vertebrae (6.3 % versus 0.0 % for fetal incidence, p < 0.01). An unossified 5th metatarsal

was recorded with an increased frequency of 4.9 % for fetal incidence (p < 0.05), and 8.7 % for litter

incidence (not significant), versus 0.0 % of incidence in the control group. The incidence of incomplete

ossification in 5th metacarpal was also slightly increased in this group.  

In general, the fetal incidence for total affected fetuses with skeletal and cartilage variation was significantly

increased in the 90 mg/kg bw/day dose group (71.8 %, p < 0.05) and 300 mg/kg bw/day dose group (86.7

%, p < 0.0001) compared to the control group with fetal incidence of 56.6 %.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LOAEL
30 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
one study available
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available

Mode of Action Analysis / Human Relevance Framework

a mode of action cannot be identified based on the available information.

Justification for classification or non-classification

The available information is conclusive and sufficient for classification as reproductive toxic, cat. 2: suspected of damaging the unborn child.

Additional information