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

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: oral

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 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

GLP compliance:

yes

Specific details on test material used for the study:

Batch No.: M(S) 201170
Appearance/Color/State: Solid fine powder (whitish)
Content or purity: ≥99% (Nominal value)
99.50% (Measured result)
Stability: Stable at room temperature in closed containers under normal storage and handling conditions.

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

The number and sex of animals: Totally purchased animals were 124 rats (62 females, 62 males), and 104 rats (52 females, 52 males) were used. The females were nulliparous and non-pregnant.
Rats age: 4~5 weeks (28~35 days) on arrival; 5~6 weeks (38~45 days) on dosing.
Body weight range: 185.69~211.71 g for males and 162.90~188.15g for females at grouping. The weight variation of animals used was minimal and not exceed ±20% of the mean weight of each sex.

Husbandry : Animals were housed in Room D103 during acclimated period and test period in the barrier system of the facility. Animals were raised in suspended, stainless steel cages (L32.0cm×W28.0cm×H20.0cm) on cage racks (L167.0cm×W70.0cm× H171.0cm). There were 10 cages per layer and 4 layers per rack. Animals were housed two per cage for each sex for the convenient of food consumption amount calculation for each animal per day during the test. When study started, the cages were changed per 4 weeks that the lowest level animals' cages were changed to the top level and the top level animals' cages were changed to the next level.

Environmental Conditions: Temperature and humidity were controlled automatically and daily recorded. The actual values in the animal room were 22.0~25.0°C (target range: 20~25°C for temperature), and 44~66 % for humidity(target range: 40%~70% for humidity). Where the lighting sequence was 12 hours light, 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 oral exposure:

VEHICLE
- Justification for use and choice of vehicle (if other than water): The test item is not soluble in corn oil and poorly soluble in water, but can be prepared into uniform suspension in corn oil. At the same time, it is accredited vehicle in the Guideline, and so corn oil was selected as vehicle for making test suspension in this study.
- Lot/batch no. (if required): 20211019

Details on analytical verification of doses or concentrations:

The dose formulations (5, 15 and 50 mg/ml) were sampled accurately and diluted within linear range (5.000-100.0 mg/L), and determined by HPLC-PDA.

Duration of treatment / exposure:

Animals in the treatment groups (10/sex/group for low and mid dose and 16/sex/group for high dose) were administered the concentration of 5 mg/ml, 15 mg/ml, 50 mg/ml of test item suspension once daily for 91 days. The animals in the control group (16/sex/group) was administered the vehicle (corn oil) once daily for 91 days. Dosing volume was 4 ml/kg. The reversibility of effects was assessed after a recovery period of 28 days without exposure (6/sex/group for control and high dose).

Frequency of treatment:

daily

Dose / conc.:

20 mg/kg bw/day (nominal)

Dose / conc.:

60 mg/kg bw/day (nominal)

Dose / conc.:

200 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10/sex/group for low and mid dose and 16/sex/group for high dose and control group.

Control animals:

yes, concurrent no treatment

Details on study design:

Dose range finding studies: Prior to the start of this study, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test by Gavage with the test item in Rats was performed to provide a basis for the dose selection. There were three dose groups of 20, 80 and 200 mg/kg body weight/day and one vehicle control group with five female and five male rats in each group, respectively.
Based on above the results and OECD Guideline for the Testing of Chemicals “Repeated Dose 90-Day Oral Toxicity Study in Rodents” (No.408, adopted: 25 June 2018), three dose levels will be used in this study including 20, 60 and 200 mg/kg body weight/day, and a vehicle control group received corn oil was involved.

Observations and examinations performed and frequency:

Clinical obervations, measurement of body weight and food consumption, nerve functional observations and ophthalmological examination were performed during the test. Hematological examinations, coagulation, clinical chemistry, thyroid hormones analysis and urinalysis were carried out at the end of the dosing period and recovery period.

Sacrifice and pathology:

At necropsy, the oestrus cycle of all females were determined by taking vaginal smears and histopathological examinations were carried out on the preserved organs and tissues of all animals in the control and high dose groups at the end of treatment period and gross lesions found in all of the other animals. The concentrations of the formulations were determined by chemical analysis on the first preparation, one preparation in the seventh and thirteenth week of the exposure period, respectively.

Statistics:

The data of Hematology, Coagulation, Clinical Chemistry, Thyroid Hormones Analysis, and Urinalysis were collected using verified softwares attached to the instruments verified and qualified.
Death and Moribund Observations, and nerve function examination were collected using paper records.
The data of body weight, food consumption, general clinical observations, detailed clinical observations, gross necropsy, organ weights and histopathology were collected by "Provantis System 10.2.3.1" (before the day of February 18, 2022) and "Provantis System 10.5.0.3" (after the day of February 18, 2022). software.
Urinalysis data was analyzed by SPSS17.0.
The nerve function examination data were analyzed by verified SPSS 17.0 software.
The data of clinical observation was analyzed by verified SPSS 17.0 software.
The chi-square test(χ2) was adopted. The Tables and statistics module of "Provantis System 10.5.0.3" software was used for the incidence of gross pathological findings and non-graded histopathological findings. Fisher's exact probability test (one-tailed probability level) was adopted.
All the tests were two-tailed probability level except the Fisher’s exact test.

Clinical signs:

no effects observed

Description (incidence and severity):

No toxicologically treatment-related clinical signs were observed throughout the study. No abnormality was observed in all animals.

Mortality:

no mortality observed

Description (incidence):

All animals survived during the test.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There was no treatment-related effect on body weights in both sexes in all treated groups during the test.
During the treatment period:
Male animals
Mean body weights of male in the high dose group were significantly (p<0.05, p<0.01, p<0.001) lower (4.4~7.9% less than control) than those of rats in the control group at weeks 1-9 and 11-13 .Total body-weight gains (0-91 day) were significantly (p<0.05) lower (11.3% less than control) than those of the control. Final body weights of males in the high dose group were reduced by 7.7% at the end of the treatment period. The differences from controls in final body weights were small (less than 10%), there was therefore no treatment-related effects on body weight in males at the high dose level.
There was no treatment-related effects on body weights in males at the low and middle dose level. No significant differences in mean body weights each week and total body-weight gains (0-91 day) were observed for males in the low and middle dose group compared with those in rats in the control group.
Female animals
Mean body weights of female in the high dose group were significantly (p<0.05) lower (6.5% less than control) than those of rats in the control group at week 5 . Total body-weight gains (0-91 day) were significantly (p<0.05) lower (12.8% less than control) than those of the control. Final body weights of females in the high dose group were reduced by 5.9% at the end of the treatment period. The differences from controls in final body weights were small (less than 10%), there was therefore no treatment-related effects on body weight in females at the high dose level.
There was no treatment-related effects on body weights in females at the low and middle dose level. No significant differences in mean body weights and total body-weight gains (0-91 day) were observed for females in the low and middle dose group compared with those in rats in the control group.

During the recovery period:
Male animals
Mean body weights in additional males in the high dose group were significantly (p<0.05, p<0.01) lower (9.0~10.2% less than control) than those of rats in the control group at weeks 13-17. Due to the change of animal number after necropsy, the decreases in the mean body weight and body weight gain during the recovery period was more than during the treatment period, meanwhile, there were no significant differences in total body-weight gains (91-119 day) in additional males compared with that of the control group during the recovery period, there was therefore no treatment-related effects on body weight in males at the high dose level during the recovery period.
Female animals
There were no significant differences in mean body weights at each week and total body-weight gains (91-119 day) in additional females at the high dose compared with that of the controls during the recovery period.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no treatment-related effects on food consumption in all treated groups during the test.
During the treatment period
Male animals
Mean food consumptions of males in the high dose group were significantly (p<0.05, p<0.01, p<0.001) lower at weeks 3-5 and 12-13 than those of rats in the control group. The differences were considered not to be toxicologically relevant since they were inconsistent, and lacked a clear dose–response relationship.
There were no significant differences in mean food consumptions at each week for males in the middle dose group compared with that of the controls during the treatment period.
Mean food consumptions of males in the low dose group were significantly (p<0.05) lower at week 4 and significantly (p<0.05, p<0.01) higher at week 7-10, 12 than those of rats in the control group. The above differences had no dose-response and were considered not to be toxicologically relevant since they were sporadic and inconsistent.
Female animals
Mean food consumptions of females in the high dose group were significantly (p<0.01, p<0.001) lower than those of rats in the control group at weeks 1, 4 and 5. The differences were considered not to be toxicologically relevant since they were inconsistent, and lacked a clear dose–response relationship.
Mean food consumptions of females in the middle dose group were significantly (p<0.01, p<0.001) lower than those of rats in the control group at weeks 1, 4 and 5. The differences were considered not to be toxicologically relevant since they were inconsistent, and lacked a clear dose–response relationship.
Mean food consumption of females in the low dose group were significantly (p<0.05) higher at week 11 and significantly (p<0.05, p<0.001) lower at week 2, 4 and 5 than those of rats in the control group. The differences were considered not to be toxicologically relevant since they were inconsistent, and lacked a clear dose–response relationship.
During the recovery period
Male animals
There were no significant differences in the mean food consumption in additional males between the high dose group and the control group during the recovery period.
Female animals
There were no significant differences in the mean food consumption in additional females betweenthe high dose group and the control group during the recovery period.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

There were no treatment-related effects on ophthalmological examination in both sexes during the test.
All the animals in all groups before dosing, the animals in the control and high dose group before scheduled necropsy at the end of the treatment and recovery periods didn't show abnormal symptom of periocular area of eyes conjunctiva, iris, cornea, optic disk and blood vessel of the eye fundus.

Haematological findings:

no effects observed

Description (incidence and severity):

There were no treatment-related effects on haematology and coagulation in both sexes in all treated groups during the test.
At the end of treatment period
Male animals
There were no treatment-related, statistically significant changes in all parameters on Hematology at any dose level compared with those of the controls.
Female animals
There were no treatment-related, statistically significant changes in all parameters on Hematology at any dose level compared with those of the controls.
At the end of recovery period
Male animals
There were no treatment-related, statistically significant changes in all parameters on Hematology in additional males in high dose group compared with those of the controls.
Female animals
RET and RET% levels in additional females in the high dose group were significantly (p<0.05) increased compared with those of the controls. MONO% levels in additional females at the high dose were significantly (p<0.05) decreased compared with those of the controls. The above statistic differences occurred only at the end of the recovery period, which was therefore not considered to be toxicologically relevant.

COAGULATION
At the end of treatment period
Male animals
PT level in males in the high dose group was significantly (p<0.01) extended compared with those of the controls, the change was small (+5.4%) which was therefore not considered to be toxicologically significant.
Female animals
There were no treatment-related and statistically significant changes in all the parameters on Coagulation in females at any dose level.
At the end of recovery period
Male animals
There were no treatment-related, statistically significant changes in all the parameters on Coagulation in additional rats at the high dose level.
Female animals
There were no treatment-related, statistically significant changes in all the parameters on Coagulation in additional rats at the high dose level.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

There were no treatment-related effects on clinical chemistry in both sexes at all treated groups at the end of the treatment period, there were no treatment-related durable or tardive toxic effects on clinical chemistry in both sexes after the recovery.

At the end of treatment period
Male animals
ALP level in males in the high dose group was significantly (p<0.05) increased compared with those of the controls. The difference was considered not to be toxicologically relevant since it was within the normal range of reference values provided by the laboratory.
Na＋ levels in males in the middle and high dose group were significantly (p<0.01, p<0.001) decreased (0.8~1.1% less than control) compared with those of the controls. The change was small, which was therefore not considered to be toxicologically significant.
T-BIL level in males in low dose group was significantly (p<0.001) increased compared with those of the controls. The change was considered not to be toxicologically relevant since it lacked a clear dose–response relationship.
Female animals
T-BIL level in females in the low dose group was significantly (p<0.05) increased compared with those of the controls. The change was considered not to be toxicologically relevant since it lacked a clear dose–response relationship.
At the end of recovery period
Male animals
There were no treatment-related and statistically significant changes in all parameters on Clinical Chemistry in additional males at the high dose.
Female animals
ALP level in additional females in the high dose group was significantly (p<0.05) increased compared with those of the controls. ALB level in additional females in the high dose group was significantly (p<0.05) decreased compared with those of the controls. The above statistic differences occurred only at the end of the recovery period, which was therefore not considered to be toxicologically relevant.

Endocrine findings:

no effects observed

Description (incidence and severity):

There were no treatment-related effects on thyroid hormones in both sexes in all treated groups during the test.

At the end of treatment period
Male animals
T3 level in males in the low dose group was significantly (p<0.05) decreased compared with those of the controls. The above statistic differences was considered not to be toxicologically relevant since it lacked a clear dose–response relationship.
Female animals
There were no treatment-related and statistically significant changes in all parameters in females at any dose level.
At the end of recovery period
Male animals
There were no treatment-related, statistically significant changes in all parameters in additional males at the high dose level.
Female animals
There were no treatment-related, statistically significant changes in all parameters in additional females at the high dose level.

Urinalysis findings:

no effects observed

Description (incidence and severity):

There were no treatment-related effects on urinalysis in both sexes at all treated groups during the test.
Male animals
There were no treatment-related and statistically significant changes in all parameters in males at any dose.
Female animals
KET level was significantly (p<0.01) increased in females in the middle dose group compared with that of the controls. The change had no dose-resoponse and wasn't considered to be toxicologically relevant.
At the end of recovery period
Male animals:
URO level was significantly (p<0.01) reduced in additional males in the high dose group compared with that of the controls. The change was considered not to be toxicologically relevant since it was not adverse effects.
Female animals:
There were no treatment-related and statistically significant changes in all parameters in females at the high dose.

Behaviour (functional findings):

no effects observed

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There were no treatment-related effects on organ weights in both sexes at all treated groups during the test.
At the end of treatment period
Male animals
There were no significant differences in the absolute and relative organ weights in males between the control group and any treated group.
Female animals
There were no significant differences in the absolute and relative organ weights in females between the control group and any treated group.
At the end of recovery period
Male animals
The absolute organ weights of brain and kidneys were significantly (p<0.05, p<0.01) decreased compared with those of the controls. The absolute and relative organ weights of spleen were significantly (p<0.05, p<0.01) decreased compared with those of the controls. The absolute and relative organ weight of lung were significantly (p<0.05,p<0.01) decreased compared with those of the controls. The above statistic differences occurred only at the end of the recovery period, which was therefore not considered to be toxicologically relevant.
Female animal
There were no significant differences in the absolute and relative organ weights in additional females between the control group and the high dose group

Gross pathological findings:

no effects observed

Description (incidence and severity):

No treatment-related gross necropsy findings were observed during the test.

Neuropathological findings:

no effects observed

Description (incidence and severity):

There were no treatment-related effects on nerve function observation in both sexes in all treated groups during the test.
NERVE FUNCTION OBSERVATION
Male animals
Reactivity score (low in 7/10, moderately low in 3/10) in males in the low dose group was significantly (p<0.05) increased compared with that of the controls in the eleventh week of the exposure period. The above change had no dose-response and weren't considered to be toxicologically significant.
Female animals
Click response score (obvious reaction in 4/10 and slight or sluggish reaction 6/10) in females at the middle dose was significantly (p<0.01) increased compared with that of the controls in the eleventh week of the exposure period. The above change had no dose-response and weren't considered to be toxicologically significant.

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No treatment-related histological findings were observed during the test. Most histopathological findings were considered to be spontaneous lesions.

Terminal vaginal cytology
The number of females in proestrus for the control group, the low, middle and high groups were 1, 0, 1, 0, respectively.
The number of females in estrus for the control group, the low, middle and high groups were 1, 1, 2, 1, respectively.
The number of females in metestrus or diestrus for the control group, the low, middle and high groups were 8, 9, 7, 9, respectively.
Gross Autopsy
There were no treatment-related gross necropsy findings.
Histopathological Examination
The histopathological findings for male rat at the high dose included kidney tubular basophilia, liver focal necrosis, liver focal abscess, liver vacuolation, liver cystic degeneration, lung suppurative inflammation, lung alveolar macrophage aggregation, unilateral testis seminiferous tubule atrophy.
The histopathological findings for female rat at the high dose included kidney mineralization and ovaries corpus luteum degeneration, which severity degrees were from minimal to mild.
These findings were considered not to be toxicologically relevant, since there was no statistically significant differencein incidence and severity for histopathological findings above between the control group and the high dose
group.

Histopathological findings: neoplastic:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

200 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

gross pathology

haematology

histopathology: non-neoplastic

mortality

neuropathology

ophthalmological examination

organ weights and organ / body weight ratios

urinalysis

Critical effects observed:

no

Conclusions:

According to the results, no treatment-related terminal necropsy and histopathological findings were observed during the test. The no-adverse-observed-effect-level (NOAEL) for Benzene-1,2,4,5-tetracarboxylic acid, compound with 4, 5-dihydro-2-phenyl -1H-imidazole (1:1) in the repeated dose 90-day oral toxicity study in SD rats under the condition of the study was considered to be as following:
Male rats: 200 mg/kg body weight/day
Female rats: 200 mg/kg body weight/day

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

200 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

With a NOEL (90-day study, rat, oral gavage, OECD 408) of 200 mg/kg bw/day there is no requirement for classification in a STOT RE category