Benzene-1,2,4,5-tetracarboxylic acid, compound with 4,5-dihydro-2-phenyl-1H-imidazole (1:1)

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

GLP compliance:

yes

Specific details on test material used for the study:

Active ingredient: Benzene-1, 2, 4, 5-tetracarboxylic acid, compound with 4,
5-dihydro -2-phenyl-1H-imidazole (1:1)
CAS No.: 54553-90-1
Batch No.: M(S) 201170
Physical Properties: Solid fine powder (whitish)
Purity: ≥99% (supplied by sponsor)

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

Animal age on arrival: 63 days old.
Animal age at the experimental starting: near 12 weeks old.
Body weight range on arrival: 219.53-278.59g for females and 293.60-348.38g for
males.
Animals were housed in plastic cages on cage racks with 5 cages per layer and 4 layers per rack.
Environmental conditions: Temperature and humidity in animal room were
controlled automatically and daily recorded. The temperature was controlled
within 22.1-25.2°C (Target temp: 19~25°C) and the relative humidity was
controlled within 47-71% (Target humidity: 30~70%) in the animal room.
Lighting was controlled with the sequence of 12 hours light and 12 hours dark.
Diet and water were available to the animals ad libitum during test.

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

VEHICLE - Justification for use and choice of vehicle (if other than water):
Reason for selection: The test item was not soluble in corn oil and poorly soluble
in water, but could be prepared into uniform suspension in corn oil. At the same
time, corn oil was accredited vehicle in the Guideline, so corn oil was selected as
vehicle for making test suspension in this study.

Details on mating procedure:

During the pre-mating period, animals were group housed with two rats in one
cage. For mating, one male and one female were cohabitated together in plastic
mating cage (size: L46.0cm×W35.0cm×H20.0cm). Mated females and females
without mated evidence at the end of mating period were housed individually in
plastic cages, and mated males were returned to plastic cages in groups of two rats
at most.

Analytical verification of doses or concentrations:

yes

Remarks:

The analytical check for the concentrations of prepared test item was analyzed at the first week and last week preparation. The results indicate that the concentrations of the prepared test item met the requirements in the study plan.

Details on analytical verification of doses or concentrations:

Based on the results of the preliminary test, where slightly slow growth of body
weights in males and females were observed at the dose level 200 mg/kg/d, but
low spontaneous motility was observed at the dose level 400 mg/kg/d, considering
in this study, longer exposure period were used including gestation and lactation
period for females, so three dose levels of 200, 80 and 20 mg/kg/d were selected
for this study. A solvent control group received corn oil was tested in parallel.

Duration of treatment / exposure:

All animals started to be administered from the next day of grouping. For males,
the administration was continued for 4 weeks during the pre-mating period of 2
weeks, mating period of 2 weeks, and up to and including the day before being
scheduled kill. For females, the administration was continued during the
pre-mating period of 2 weeks, mating period, gestation period, lactation period up
to and including the day before scheduled kill.

Frequency of treatment:

As the administration, the animal was dosed once daily using a gastric gavage
needle at the volume of 4 mL/kg.bw daily.

Dose / conc.:

200 mg/kg bw/day

Dose / conc.:

80 mg/kg bw/day

Dose / conc.:

20 mg/kg bw/day

No. of animals per sex per dose:

14 animals for the low and medium dose
20 animals for the high dose and the vehicle control.

Control animals:

yes, concurrent vehicle

Parental animals: Observations and examinations:

General clinical observations were made at least once a day (except for the
detailed clinical observation days), and the observation time was at about 1 ± 0.5
hour after each animals' dosing, and the health status and toxic signs of the
animals should be recorded.
Detailed clinical observations were made for all animals prior to the first exposure
(after grouping) and then once a week before dosing during the treatment periods,
and once a week during the recovery period. The animals were taken outside the
home cage for observation, and all the findings were detailed recorded.
Twice daily all animals were observed for mortality and moribundity (once on
non-working day).
Observation includes changes in skin, fur, eyes, mucous membranes, occurrence
of secretions and excretions and autonomic activity (e.g., lacrimation,
pilo-erection, pupil size, unusual respiratory pattern), changes in gait, posture and
response to handling as well as the presence of clonic or tonic movements,
stereotypes (e.g. excessive grooming, repetitive circling) or bizarre behaviors (e.g.
self-mutilation, walking backwards).

All animals were weighed at grouping, and once per week during the pre-mating
and mating period. Mated females were weighed on gestation day (GD) 0, 7, 14
and 20, and on day 0, 4 and 13 post-partum. The animals were weighed on their
scheduled necropsy date.

During the pre-mating period, the ration food of 200±20g was added once weekly
and remaining food were weighed one day (24h±1.5h) later in order to obtain the
food consumption of the animals. During the gestation and lactation period, the food consumption of females was
obtained in GD6-7, 13-14 and 19-20, PND3-4 and PND12-13 by the same way.
The results were expressed in gram per animal per day.

Oestrous cyclicity (parental animals):

For the parous females, the number of implantation sites in uterus was counted.
For the females without delivery, the implantation sites in uterus were observed and the uteruses without implantation sites by naked eyes were further examined by ammonium sulphide staining.

Litter observations:

During lactation period, all litters were observed once per day, which could be
done during the dosing of dams or on the cage side. Clinical signs and any
abnormalities were recorded.
On PND 0, the litters were observed for the number and sex of live pups,
stillbirths and the presence of grossly malformed pups, and then were weighed.
On PND 4, the litters were observed for the number and sex of live pups. Each
pup was weighed individually at the time of anogenital distance (AGD)
measurement.
On PND 13, the litters were observed for the number and sex of live pups. Each
pup was weighed individually.

On PND 4 prior to culling, the AGD of each pup was measured. The AGD was
normalized relative to the cube root of body weight of the pup on PND 4 for the
statistical analysis.

Postmortem examinations (parental animals):

Parental males were sacrificed after the mating period, and dams were sacrificed
during 14~16 days after delivery (LD 14-16, and the day after the pup has been
dissected). Parental females without successful copulation and females with
successful copulation but without delivery were sacrificed during 24~26 days
after the last day of the mating period.
Animals in the satellite group were sacrificed after a further 14 days after the first
scheduled kill of males and dams.
Satellite males of the control and high-dose groups were sacrificed after a further
14 days after the scheduled kill of parental males; Satellite females were
sacrificed after a further 14 days after the first scheduled kill of parental dams.
Animals were fasted overnight prior to necropsy, but water was available.
On the day of necropsy, all surviving parental and satellite rats were anesthetized
by CO2 inhalation and blood samples were taken by exsanguinations from
abdominal aorta for measurement of related indicators, therefore they were
subjected to a full necropsy and macroscopically examinations.

ORGAN WEIGHTS
At the time of termination, the weight of the following organs was determined:
For the parental males that include testes, epididymides, prostate including
seminal vesicles with coagulating glands, levator ani plus bulbocavernosus muscle
complex, Cowper's glands, glans penis, and thyroid.
For the parental females that include paired ovaries, uterus with cervix, and
thyroid.
In addition, five adult parental males and females with good vital signs were
selected from each group to determine the weight of the liver, kidneys, adrenals,
thymus, spleen, brain and heart.
The organs should be weighed as soon as possible after dissection to avoid drying.
Care was exercised when trimming the prostate complex to avoid puncture of the
fluid filled seminal vesicle. The thyroid was weighted after the fixation in 10%
neutral-buffered formalin.

Histopathology was examined on the following organs:
The ovaries, testes, epididymides and other preserved organs and tissues of the
selected five males and five females of the parental animals in the control and
high-dose groups, all gross lesions in the sacrificed animals.
Additional histopathological examination was performed according to the results
of thyroid hormone (T4) determination for male satellite animals.

Postmortem examinations (offspring):

All selected pups were subjected to a careful general examination for gross abnormalities, with particular attention on the external reproductive genitals.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

For satellite animals, one female in the high-dose group (animal code: 2315) was
found with hair loss partly, emaciation and soiled perineal region at the end of
administration, and found smudge of perinasal area and dead on D55 (recovery
period). At necropsy, no apparent cause of death was found, so it was considered
to be spontaneous and not to be treatment-related. One female in the high-dose
group (animal code: 2316) was observed with hair loss partly at the end of
administration and during recovery.
The incidence of those sign above was low and without dose-relation, the result
was not considered to be treatment-related.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

For parental animals, one female (animal code: 2009) was found soiled perineal
region and dead on GD23, which was considered to be pregnant as with fetuses in
its uterus. At necropsy, no apparent cause of death was found, so its death was
considered to be spontaneous and not to be treatment-related.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

For male parental and satellite animals during dosing period, no significantly
difference was observed on the mean body weights, the mean body weight gain
and the total body weight gains in all dose groups compared with the vehicle
control group (p≥0.05).
For male satellite animals during recovery, there was a statistically significant
decrease on the mean body weight gain on D35-42 and the total body weight
gains in the high-dose group compared with the vehicle control group (p＜0.01),
but there were no adverse effects on other indexes at this dose group, so it was
considered that the abnormal weight gain was due to the adaptive or
compensatory responses when stopping administration, rather than the adverse
reaction caused by the treatment of the test item.
For female parental and satellite animals during pre-mating, no significantly
difference was observed on the mean body weights in all dose groups, but the
mean body weight gains and the total body weight gains in the high-dose group
had a statistically significant decrease compared with the vehicle control group (p
＜0.01 or p＜0.001); No significantly difference was observed on the mean body
weight gains and the total body weight gains in the low- and mid-dose groups
(p≥0.05) in the pre-mating period excepted for the mean body weight gain on
D7-14 had a statistically significant decrease compared with the vehicle control
group (p＜0.05) in the low-dose group.
For female satellite animals, no significantly difference was observed on the mean
body weights, the mean body weight gains and the total body weight gains in the
high-dose group compared with the vehicle control group (p≥0.05) during the
dosing period; but there was a statistically significant increase compared with the
vehicle control group (p＜0.01) on the mean body weight gain on D49-56 during
recovery, no significantly difference was observed on the mean body weights, the
mean body weight gain of other week and the total body weight gains in the
high-dose group compared with the vehicle control group (p≥0.05) during
recovery.
No significantly difference was observed on the mean body weights, the mean
body weight gains and the total body weight gains in all dose groups compared
with the vehicle control group (p≥0.05) in the gestation period and lactation
period.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

For male parental animals, the mean corpuscular volume (MCV) had a
statistically significant increase compared with the vehicle control group (p＜0.05)
in the high-dose group; the mean corpuscular hemoglobin (MCH) had a
statistically significant decrease compared with the vehicle control group (p＜0.05)
in the mid-dose group; However, there were no obvious abnormalities in other
related indicators, and the abnormal data were all within the background value
range of this facility, which was considered to have no toxicological significance.
No statistically significant was observed on all the detective haematology indexes
in the low-dose group and other detective haematology indexes in the mid- and
high-dose groups (p≥0.05).
For female parental animals, no statistically significant was observed on all the
detective haematology indexes in all dose groups (p≥0.05).
For male satellite animals, the immature reticulocyte fraction (IRF) and the high
fluorescence intensity reticulocyte fraction (HFR) had a statistically significant
decrease compared with the vehicle control group (p＜0.05 or p＜0.01); the low
fluorescence intensity reticulocyte fraction (LFR) had a statistically significant
increase compared with the vehicle control group (p＜0.01); However, there were
no obvious abnormalities in other related indicators, and the abnormal data were
all within the background value range of this facility, which was considered to
have no toxicological significance. No statistically significant was observed on
other detective haematology indexes (p≥0.05).
For female satellite animals, the RDW-SD had a statistically significant increase
compared with the vehicle control group (p＜0.05); However, there were no
obvious abnormalities in other related indicators, and the abnormal data were all
within the background value range of this facility, which was considered to have
no toxicological significance. No statistically significant was observed on other
detective haematology indexes.
No statistically significant was observed on
all the detective coagulation indexes in all dose groups.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

For female satellite animals, the albumin (ALB) had a statistically significant
increase compared with the vehicle control group (p＜0.05) in the high-dose
group; However, there were no obvious abnormalities in other related indicators,
and the abnormal data were all within the background value range of this facility,
which was considered to have no toxicological significance.

Endocrine findings:

no effects observed

Description (incidence and severity):

For parental animals, the results of hormone determination showed that no
statistically significant effects on the thyroid hormone (T4) level was observed in
all dose groups (p≥0.05); for the thyroid hormone of pups, no statistically
significant effects was observed in all dose groups (p≥0.05) at either PND4 (P4T4)
or the termination (PTT4).
For satellite animals, the results of the thyroid hormone (T4) level showed that
there was a statistically significant decrease compared with the vehicle control
group (p＜0.05) in high-dose group males serum, but there were no adverse
effects on histopathological examination for satellite males’ thyroid at this dose
group, and no statistically significant effects on the thyroid hormone (T4) level
was observed for parental males, so it was considered that the abnormal thyroid
hormone (T4) level was due to the adaptive or compensatory responses when
stopping administration, rather than the adverse reaction caused by the treatment
of the test item; No statistically significant effect was observed in high-dose group
females’ serum (p≥0.05).

Urinalysis findings:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

In the whole pre-mating period, the mean cycle length and number of estrus
cycles of females in all dose groups had no significantly difference compared with
the vehicle control group (p≥0.05). 2 acyclic females was observed in the vehicle control group and mid-dose group, respectively; 3, 3, 5 and 5 females in the
vehicle control group, low-, mid-, high- dose groups respectively were observed
with prolonged estrus period, but the incidence had no statistically significant
difference in each dose group compared with the vehicle control group (p≥0.05).

Reproductive performance:

no effects observed

Description (incidence and severity):

The mating index had no significant difference in all dose groups compared with
the vehicle control group (p≥0.05).
The fertility index of females had no
significant difference in all dose groups compared with the vehicle control group
(p≥0.05).
The pregnancy index and gestation index of females had no difference in all dose
groups compared with the vehicle control group (p≥0.05).
No statistically significant difference was observed on mean precoital interval and
gestation length in all dose groups compared with the vehicle control group
(p≥0.05).

Dose descriptor:

NOAEL

Effect level:

200 mg/kg bw/day

Based on:

test mat.

Sex:

male

Basis for effect level:

mortality

Dose descriptor:

NOAEL

Effect level:

80 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

Dose descriptor:

LOAEL

Effect level:

200 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

Key result

Dose descriptor:

NOAEL

Effect level:

200 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive function (oestrous cycle)

reproductive performance

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Description (incidence and severity):

There were 2, 3 and 3 pups stillborn in the vehicle control group, low- and high-
dose groups, respectively. No statistically significant difference on the live birth
index was observed in all dose groups compared with the control group (p≥0.05).
There were 5, 2, 2 and 9 pups dead before culling in the vehicle control group and
low-, mid-, high- dose groups, respectively, among which, 3, 2, 1 and 4 pups of
them were found missing and presumed dead. No statistically significant
difference on the survival index at PND4 was observed in the all dose groups
(p≥0.05).
No pup died after culling to PND13 in each dose group. The survival index at
PND13 had no statistically significant effects in all dose groups (p≥0.05).

Body weight and weight changes:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

For male and female parental animals, the absolute and relative organ weights of
all weighed organs in each dose group were comparable with the vehicle control group (p≥0.05)..
For male satellite animals, the absolute brain weight in the high-dose group had a
statistically significant decrease compared with the vehicle control group (p＜
0.05), but the relative brain weight in the high-dose group had no statistically
different comparable with the vehicle control group (p≥0.05); The absolute and
relative organ weights of other weighed organs in the high-dose group were
comparable with the vehicle control group (p≥0.05).
For female satellite animals, the absolute and relative organ weights of all
weighed organs in the high-dose group were comparable with the vehicle control
group (p≥0.05).

Gross pathological findings:

no effects observed

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

200 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

Reproductive effects observed:

no

Conclusions:

Under the conditions of combined repeated dose toxicity study with the reproduction/developmental toxicity screening test in rats for exposure to
Benzene-1,2,4,5-tetracarboxylic acid, compound with 4,
5-dihydro-2-phenyl-1H-imidazole (1:1) by gavage, the Lowest Observed Adverse
Effect Level (LOAEL) for toxicity to females, is considered to be 200 mg/kg/d
based on the decreased body weight gain during pre-mating; the No Observed
Adverse Effect Level (NOAEL) for toxicity to females is considered to be 80
mg/kg/d.
The NOAEL for toxicity to males is considered to be 200 mg/kg/d.
The NOAEL for reproductive toxicity effects to males and females and for
developmental toxicity effects to pups after parental exposure is considered to be
200 mg/kg/d.

Executive summary:

According to the method in OECD Guideline for the Testing of Chemicals, “Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test” (No.422, adopted on July 29, 2016), this study was conducted in SD rats. Fourteen parental rats/sex/group and six satellite rats/sex in
the vehicle control and high-dose groups, additional were exposed by oral administration via gavage to 0, 20, 80 and 200 mg/kg/d
Benzene-1,2,4,5-tetracarboxylic acid, compound with 4,5-dihydro-2-phenyl-1H-imidazole (1:1) respectively for two weeks prior to
mating, two weeks of the mating period, and the gestation period and part of lactation period for parental females. Satellite males were treated from the next day of grouping up to, and including the day before scheduled kill of the parental males, satellite females were treated from the next day of grouping up to, and including the day before the first scheduled kill of parental dams.
During the test, no adverse effect on the clinical observation and food consumption was observed in the different periods for the parental and satellite animals.
No adverse effect on the mean body weight or body weight gain was observed for the parental and satellite males at each dose group during dosing and recovery.
There was a statistically significant decrease on the mean body weight gain of parental and satellite females at the dose of 200mg/kg/d during pre-mating, but no adverse effect on the body weight or body weight gain was observed for the satellite females at the dose of 200mg/kg/d during recovery, so it is considered
that the test item had a certain adverse effect on the weight gain of female rats during dosing, but it could be recovered after drug withdrawal. No adverse effect on the body weight or body weight gain was observed for the male and female parental at the dose of 80 and 20mg/kg/d during dosing and recovery. 

No adverse effect on the reproductive ability of parental animals and the development of pups was observed at any dose level in this study. And no significant changes were observed about estrus cycles of female parental rats in any dose groups.
No significant changes in the absolute and relative organ weights were observed in the parental and satellite animals. And no treatment-related macroscopic findings and microscopic findings of organs were detected.
No significant changes were observed about the thyroid hormone in serum of both male and female parental rats and pups in any dose groups. No treatment-related adverse effect on the thyroid hormone in serum of both male and female satellite rats was observed at the dose of 200mg/kg/d in this study.
No adverse effects were observed about the blood haematology of both male and female parental rats in any dose groups and of both male and female satellite rats at the dose of 200mg/kg/d.
No adverse effects were observed about the coagulation of both male and female parental rats in any dose groups and of both male and female satellite rats at the dose of 200mg/kg/d.
No adverse effects were observed about the clinical chemistry of both male and female parental rats in any dose groups and of both male and female satellite rats at the dose of 200mg/kg/d.

 

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

200 mg/kg bw/day

Study duration:

subacute

Species:

rat

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

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

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

Specific details on test material used for the study:

Batch No.: M(S)201170
Physical Properties: Solid fine powder (whitish)
Purity: ≥99% (supplied by sponsor)
Norminal value: 99.50%

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

Animal age on arrival: 11-14 weeks old
The health status of all animals were checked for health within 24 hours after arrival at this lab. The animals meeting the quality requirements were acclimated to the laboratory conditions for five days prior to the experimental start date. All animals were weighed and identified with the special animal markers on fur, and were signed on the cage cards at the same time. The body weight range was 230.82~324.64g. Clinical observations were performed daily during acclimatization period, and were continued until the start of the test. All animals were weighed on the first day of paring; the body weight range was 240.69~329.85g.

The temperature was controlled within 22.2-25.0°C and the relative humidity was controlled within 48-70% in the animal room. Lighting was controlled with the sequence of 12 hours light and 12 hours dark.
Diet and water were available to the animals ad libitum during test.

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on analytical verification of doses or concentrations:

The analytical check for the concentrations of prepared test item was analyzed at the first week and last week preparation.
As the analysis, the prepared test item of different concentrations and the vehicle control were sampled and analyzed using the validated method.

Details on mating procedure:

Female rats should be paired with adult male rats in this facility. The male rats should be 10-14 weeks old when purchased, and the study batch for the male rats was RM202102(Certificate number of animals: 110324210102929565). For each mating, two (or one) female rats and one male rat were paired at afternoon and separated in the next morning. Then the female rats were examined for presence of the sperm using vaginal smear method. The day of finding the sperms was defined as the day 0 of pregnancy (GD0). The female rats which were failed to mate were paired again until the number of mated females met the requirements of the test. In the test, there were twenty-eight mated females in each group

Duration of treatment / exposure:

All animals were dosed daily during the day 5~19 of pregnancy (GD5~19).

Frequency of treatment:

Daily.

Dose / conc.:

20 mg/kg bw/day (nominal)

Dose / conc.:

80 mg/kg bw/day (nominal)

Dose / conc.:

200 mg/kg bw/day (nominal)

No. of animals per sex per dose:

There were twenty-eight mated female rats in each group, which resulted in 25, 25, 26 and 27 pregnant rats in the vehicle control group and the low-, mid- and high- group, respectively

Maternal examinations:

Each animal was observed daily which could be done during dosing. The observation contained the appearance, fur, activity, breathing, posture, excrement and urine. All abnormalities, signs of ill health and reactions were recorded.
For animals of non-dosing, cage-side observation daily was done.

All pregnant rats were weighed on GD0, then were weighed once per 3 days during the dosing period (GD5~19), and on the day of scheduled kill (GD20).

Ovaries and uterine content:

After euthanasia at D20, removed the uterus of each animal immediately, and certained the pregnancy status, weighed gravid uterine and thyroid glands, counted the number of corpora lutea, absorptive fetuses, dead fetuses and viable fetuses for each pregnant animal.

Blood sampling:

Blood samples of pregnant females were collected and used for analysis of T3, T4 and TSH.

Fetal examinations:

Each viable fetus was examined for external alterations, and the sex, body weight and anogenital distance(AGD) were determined at the same time. The fetus was examined for viscera or skeleton after being prepared.

Statistics:

Calculated the average and standard deviation of the data for each group including body weight, food consumption, body weight change corrected for gravid uterine weight, number of corpora lutea and implantations, number of viable fetuses, thyroid glands weight, T3, T4, and body weight of fetuses, etc. AGD of fetuses was normalized relative to the cube root of body weight, and calculated the average and standard deviation. As the data statistics, variance homogeneity test was P≥0.05, Anova was used. and Dunnett's multiple comparison test was further used when significance was found. The data about abnormal clinical sign, ratio of absorptive fetus, dead fetus and viable fetus, and incidence of individual anomalies were evaluated by the chi-square test. In this study, Anova analysis was done with Provantis 10.2.3.1 software, the chi-square test was done with SPSS 17.0 software.

Clinical signs:

no effects observed

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One female of the control group was found dead on GD17, which was observed with soiled perineal region before their death, and at necropsy, no apparent cause of death was found. No accidental death and clinical signs was observed in the other groups.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

During the administration period, body weight gain in GD5-8 of the pregnant rats in the high-dose group had a significant decrease compared with the control group, and the food consumption of the pregnant rats in the mid- and high- dose groups had a significant decrease compared with the control group. No significant effect in the mean body weights and food consumption of the pregnant rat during the treated period was observed in the low-dose group.

Endocrine findings:

no effects observed

Description (incidence and severity):

No toxicologically relevant change was observed in macroscopic examination and histopathological examination of thyroid glands, and no statistically significant difference was observed in the mean weights of thyroid glands, thyroid hormones T4 and T3 compared in all dose groups with the control group.

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Details on maternal toxic effects:

In all dosed groups, no statistically significant difference was observed in the mean numbers of corpora lutea and implantation sites compared with the control group. The gravid-uterine weights had no statistically significant difference in all dosed groups. In the high-dose group, the rate of embryo loss was significantly higher than the control group, and in the low- and mid- dose groups, no dose-related significant difference was observed in the rate of embryo loss compared with the control group.

Dose descriptor:

NOAEL

Effect level:

200 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

changes in number of pregnant

dead fetuses

number of abortions

Key result

Dose descriptor:

NOAEL

Effect level:

200 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

clinical signs

Abnormalities:

effects observed, non-treatment-related

Fetal body weight changes:

no effects observed

Changes in sex ratio:

no effects observed

External malformations:

no effects observed

Skeletal malformations:

effects observed, non-treatment-related

Details on embryotoxic / teratogenic effects:

The fetal examination showed that no adverse effect in body weight, sex distribution and AGD of viable fetuses was observed in all dosed groups. No adverse effect attributable to treatment was observed across all dose groups with respect to external, soft-tissue and Skeleton malformations or variations. Some examined fetuses had less than six sternal ossification points, but there was no adverse effect attributable to treatment.

Dose descriptor:

LOAEL

Effect level:

200 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: embryotoxicity

Key result

Dose descriptor:

NOAEL

Effect level:

80 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: embryotoxicity

Developmental effects observed:

no

Conclusions:

Under the conditions of this study, Benzene-1,2,4,5 -tetracarboxylic acid, compound with 4,5-dihydro-2-phenyl-1H- imidazole(1:1) administered to pregnant SD rats by gavage in corn oil, had produced embryotoxicity at the dose of 200 mg/kg/d, and had not produced any embryotoxicity at the dose of 80 mg/kg/d and below, had not produced any maternal toxicity and teratogenicity at the dose of 200 mg/kg/d and below.
Based on these findings, the following effect levels, including Lowest Observed Adverse Effect Level (LOAEL) and No Observed Adverse Effect Level (NOAEL) were derived:
NOAELmaternal toxicity: 200 mg/kg/d
LOAELembryotoxicity: 200 mg/kg/d
NOAELembryotoxicity: 80 mg/kg/d
NOAELteratogenicity: 200 mg/kg/d.

Executive summary:

The exposure of gestating rats to different doses of Benzene-1,2,4,5 -tetracarboxylic acid, compound with 4,5-dihydro-2-phenyl-1H-imidazole(1:1) by gavage during GD5~GD19 did not result in any death or significant specific toxicity at the highest dose of 200mg/kg/d and below. The mean body weight gain had a significant decrease in the high-dose group at the beginning of the administration period, but it returned to normal in a short time and no symptom was observed on the animals of the highest dose of 200mg/kg/d. So it's considered that no treatment related effect on the body weight gain of pregnant rats was observed. The food consumption in some test weeks of the pregnant rats in both mid- and high- dose groups had a significant decrease compared with the control group. The results showed that the test item may affect animal food consumption, but no obvious adverse effects. Based on these results, it is concluded that the test item had no maternal toxicity at the highest dose of 200mg/kg/d.

The prenatal reproductive parameters showed that at the dose of 200mg/kg/d group the percent of absorbed fetuses, dead fetuses and the post-implantation loss were statistically significantly increased, while the same time, the percent of live fetus was statistically significantly decreased. The fetal examination showed that no adverse effect in the body weight, sex distribution and AGD of viable fetuses was observed in all dosed group, additionally, no adverse effect attributable to treatment was observed across the dose groups with respect to external, soft-tissue and skeleton malformations or variations. Based on these results, it is concluded that the test item had embryotoxicity at 200mg/kg/d dose level, the main expressions were embryo survival rate decreased. But the above abnormalities of fetuses are considered to be possible to recover later, so it is concluded that the test item had no teratogenicity at this dose level.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

80 mg/kg bw/day

Study duration:

chronic

Species:

rat

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Justification for classification or non-classification

At the found effect level in an OECD TG 422 study and an OECD 414 there is no requirement for classification.