Ethyl 4-[[(methylphenylamino)methylene]amino]benzoate

Repeated dose toxicity: oral

Administrative data

Endpoint:

repeated dose toxicity: oral, other

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From February 06, 2017 to February 27, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Ethyl 4-[[(methylphenylamino)methylene]amino]benzoate

EC Number:

260-976-0

EC Name:

Ethyl 4-[[(methylphenylamino)methylene]amino]benzoate

Cas Number:

57834-33-0

Molecular formula:

C17H18N2O2

IUPAC Name:

ethyl 4-[[(methylphenylamino)methylene]amino]benzoate

Test material form:

liquid

Details on test material:

- Name of test material (as cited in study report): Eversorb EP4
- Substance type: Liquid
- Composition of test material, percentage of components: 98.94%
- Lot/batch No.: 4023
- Storage condition of test material: Ambient

Test animals

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

2 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

10 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

50 mg/kg bw/day (nominal)

No. of animals per sex per dose:

Male: five
Female: five

Control animals:

yes, concurrent vehicle

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Instances of sustained hair loss on the forelimb(s) were observed in both control and dose groups. Two male animals also exhibited stained hair at the nose (ID No. 1020, Group 4) and the forelimbs (ID No. 1002, Group 1). These signs are probably attributable to animal stress response unrelated to the test article effect.
A subcutaneous mass was identified on the dorsal neck skin of animal ID No. 1004 (Group 1). The mass was first recorded on Day 24 and persistent to the end of the study period on Day 28. Since the animal was only dosed with vehicle control, the growth was unrelated to test article effect.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

All animals survived to scheduled termination on Day 29 except for animal ID No. 1016 (male; Group 4). During the Day 10 dosing of the Eversorb EP4, this animal bit off the plastic gavage tube5. About 5 cm of the tube was swallowed and located between stomach and esophagus, according to the veterinarian report. As the aftermath of the accident, decreased activity, hunched posture, and stained hair (red) at mouth was noted on the affected animal. Attempt to retrieve the broken gavage tube (with the animal under anesthesia) failed. In order to end its suffering, the animal was subject to euthanasia as described in Section 13 of the protocol.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

For females:
The statistically significant decrease in red blood cell count (RBC) and increase in mean corpuscular volume (MCV) were also seen in Group 4 animals versus the control animals. The corresponding values of these two parameters in male animals corroborated with the observed trends in females, although the differences did not reach statistical significance. These changes suggest altered hematopoiesis in dosed animals as a result of test article administration.
For males:
The statistically significant increases in average white blood cell count (WBC) were seen in both Group 2 (2 mg/kg/day) and Group 4 (50 mg/kg/day). Concurrent increases in percentage of lymphocytes (%LYMPH) were also observed in dosed animals, with the difference reaching statistical significance in Group 4 versus Group 1. These changes were not observed in female animals. Given the absence of dose correlation, the link of these changes to the test article effect is unclear. Both activated partial thromboplastin time (APTT) and prothrombin time (PT) decreased for male animals in a dose-proportionated manner with increasing dose levels, with the difference versus Group 1 reaching statistical significance for Group 4 animals.

All changes discussed above remained within their respective reference ranges established at the Testing Facility except for the reticulocyte count and percentage of male Group 4 animals, which went higher than the reference range.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

For Female:
Statistically significant decreases in average lactate dehydrogenase (LDH) and creatine phosphokinase (CPKL) levels were observed in female Group 4 (50 mg/kg/day) animals. However, the decreases were not observed in male animals. The changes seen in female animals could be attributed to individual variances unrelated to the test article.

Urinalysis findings:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

The only treatment-related organ weight change was increased splenic weight in Group 4 males administered Eversorb EP4 at 50 mg/kg/day which correlated with increased splenic extramedullary hematopoiesis microscopically; in the Group 4 females, the splenic weight increase was minimal. Increased splenic weight was also noted in the animal humane sacrificed on Day 10 but the weight increase was less than that seen in scheduled sacrificed animals.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Abnormal content (oral tube) was present in the esophagus and stomach of the humanely sacrificed animal, as well as red discoloration of the mandibular lymph node which correlated microscopically with hemorrhage and sinus erythrocytosis / erythrophagocytosis. These findings were all consequential to the gavage incident. Decreased prostate/seminal vesicle size was noted in a Group 2 male (ID No. 1010); it correlated with seminal vesicle atrophy. A subcutaneous mass was noted in a control male (ID No. 1004) which correlated with a lipomyxoma microscopically.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related histopathology findings was limited to the spleen, including increased splenic extramedullary hematopoiesis in the red pulp of both sexes administered Eversorb EP4 at 50 mg/kg/day, and increased brown (hemosiderin) pigment deposition in the red pulp of females administered Eversorb EP4 at 50 mg/kg/day.
Other microscopic findings were considered spontaneous background changes common in this species (McInnes, 2012), incidental, toxicologically irrelevant or biologically insignificant due to low incidence/severity grade or a lack of dose response.

Effect levels

Dose descriptor:

LOAEL

Effect level:

50 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

haematology

Target system / organ toxicity

Critical effects observed:

yes

Lowest effective dose / conc.:

50 mg/kg bw/day (nominal)

System:

haematopoietic

Organ:

spleen

Treatment related:

not specified

Dose response relationship:

yes

Relevant for humans:

not specified

Applicant's summary and conclusion

Conclusions:

According to OECD 407 test method, the NOAEL of Eversorb EP4 was 10 mg/kg/day.

Executive summary:

This test using the procedures outlined in the QPS Taiwan Study Plan for T65316001-GN and OECD 407 (OECD, 2008).　The test article was administered to the three treatment groups by oral gavage in a dose of 2, 10 and 50 mg/kg/day for 28 consecutive days.　There were five male and five female Sprague-Dawley rat in each group.　Excluding one male animal from Group 4 that was humanely sacrificed due to a dosing incident on Day 10, there were no mortalities. Decreased hemoglobin and mean corpuscular hemoglobin concentration concurrent with marked increases in reticulocyte count and percentage were noted in both male and female animals at 50 mg/kg/day. Decreased red blood cell count and increased mean corpuscular volume were also note for female animals at 50 mg/kg/day; similar changes in male animals did not reach statistical significance. Both activated partial thromboplastin time and prothrombin time decreased with increasing dose for males to reach statistical significance at 50 mg/kg/day. Eversorb EP4-related treatment effects were limited to the spleen, including increased splenic extramedullary hematopoiesis in the red pulp of both sexes at 50 mg/kg/day, increased brown (hemosiderin) pigment deposition in the red pulp of females at 50 mg/kg/day, and correlating splenic weight increase in the males at 50 mg/kg/day. There were no treatment-related gross changes in the study animals examined. No test article-related changes were identified in clinical observations; assessment of sensory reactivity to stimuli, grip strength, and motor activity; body weight measurements; food consumption measurements; serum chemistry parameters; and urinalysis parameters. The findings of changes in hematology and coagulation parameters, coupled with the altered histological features of the splenic tissues, suggest past or ongoing impacts of the test article administration on hematopoiesis that was most conspicuous at the highest dose level (50 mg/kg/day) observed in this study. The no-observed-adverse effect level (NOAEL) of the study is therefore set at 10 mg/kg/day, at which dose level the above-mentioned findings were not yet apparent.