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

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Eversorb EP4 (2, 10 or 50 mg/kg/day) was given orally by gavage once daily to male rats for 28 days and female rats for 40 to 61 days, depending on the time of copulation and gestation. Eversorb EP4 did not affect clinical observations, body weight, food consumption, male and female rats’ reproductive performance such as gonadal function, mating behavior, conception, development of the conceptus, and parturition and offspring parameters including litter weight, AGD and nipple retention. The no observed adverse effect level of Eversorb EP4 in rat’s reproductive performance was 50 mg/kg/day.

Link to relevant study records

Reference

Endpoint:

one-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 July 2017 - 23 March 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

GLP compliance:

yes (incl. QA statement)

Species:

rat

Strain:

other: Crl:CD(SD)

Details on species / strain selection:

Crl:CD (SD) rats (47 males and 50 females) used in this study were purchased from
BioLASCO Taiwan Co., Ltd., Taipei, Taiwan. A total of 46 males and 46 females were
selected for the study. Animals were approximately 12 weeks of age and weighed 406 to
505 g (males) and 243 to 295 g (females) at dosing and mated at an age of 14 weeks.
Animals were identified by an ear-notch and a cage tag. The rat was chosen since it is
an appropriate rodent experimental species for reproduction/developmental toxicity
studies with documented susceptibility to a wide range of toxic substances.

Sex:

male/female

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

The oral route was selected since it is the proposed exposure route to human. Dose formulations were stirred during the dosing session for drawing into dosing syringe
. All animals received vehicle or test article formulation via oral gavage once daily from pre-mating, mating, gestation (female only, Deviation 4) until 12 days after delivery (female only). The dosing period for males was 28 days and for females was 40-61 days, depending on the time of copulation and gestation.

Details on mating procedure:

There was a 12-day pre-exposure period from A12. A 2-week per-mating period followed a 14-day mating period. In mating period, all animals were copulated at
a ratio of 1:1. The female was placed with the same male until pregnancy occurred. When pairing was unsuccessful in a continued 6 days, re-mating of females with proven males of the same group was conducted.

The gestation period was 21 to 23 days. Males were sacrificed after a dosing period of four weeks (D29). Maternal females with pups were sacrificed on P13. Presumed pregnant but non-delivering females were sacrificed on G25, and one non-copulated female was sacrificed on D54.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The samples were analyzed using the method entitled “Validation of a Method for the Determination of Eversorb EP4 in Dose Formulation by HPLC-UV”, which
was validated at QPS Taiwan. The details of the assay procedure and validation data are listed and summarized in the assay validation report (QPS Taiwan T653-1601).

Eversorb EP4 was obtained from Everlight Chemical Industrial Corporation and was subsequently used to prepare standard solutions and check standard (CS) solutions for this study.

All sample results were reported from analysis runs that met the acceptability criteria of QPS’ standard operating procedures.

Duration of treatment / exposure:

The dosing period for males was 28 days and for females was 40-61 days, depending on the time of copulation and gestation.

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

group 1

Dose / conc.:

2 mg/kg bw/day (nominal)

Remarks:

group 2

Dose / conc.:

10 mg/kg bw/day (nominal)

Remarks:

group 3

Dose / conc.:

50 mg/kg bw/day (nominal)

Remarks:

group 4

No. of animals per sex per dose:

12 males/ 12 females

Control animals:

yes

Details on study design:

The high dose (50 mg/kg/day) was determined based on the results of previous studies (QPS Taiwan Study No.: T65316003-RP and T65316001-GN).

The mid (10 mg/kg/day) and low (2 mg/kg/day) doses represent a fifth decrements from the high dose and are included to better define the dose-response of test article effects and assure that a nonseverely toxic dose is identified with the extension to repeated dosing.

T65316003-RP is the reproduction screen study. Doese are 1000, 300, 100 and 0 mg/kg/day. The impact at 100 mg/kg/day was significant. Therefore, the highest dose was determined at 50 mg/kg/day.

Clinical signs:

no effects observed

Description (incidence and severity):

No test article-related death or clinical signs was observed in males and females during the study period.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The test article did not have effect on body weight and body weight gain.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

no effects observed

Description (incidence and severity):

The test article did not have effect in food consumption.

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

Immunological findings:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

There were no statistically significantly differences in T4 concentration determinate on D29 (male adult), P4 (pup), P13 (pup and maternal rat) between control and treated
groups.

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

The estrus cycles of all animals during 15 consecutive days of premating period. The sequence of the stages was: proestrus, estrus, metestrus and diestrus.
It was normal to observe extra diestrus and estrus stages. The estrus was cycling when the proestrus was followed by the estrus. A 4–5 day cycle was observed in almost all animals. Some animals showed 1 or 2 cycles during pre-mating, which included one control animal (ID# 0007), two animals of Group 2 (IDs# 0021 and 0023), three animals of Group 3 (IDs# 0024, 0029 and 0033) and three animals of Group 4 (ID# 0040, 0041, and 0045). Except for ID# 0007, other animals mentioned above were mated in 2–5 days (see section 5.6). There were no statistically significant differences in the number of estrus cycles between control and treated groups. The intergroup difference may be due to discrepancies caused by different examiners.

Overall, the test article did not have an effect of the estrus cycle.

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

In males, a positive indication of mating was obtained for 11 of 12, 10 of 11, 10 of 11 and 12 of 12 animals in Groups 1 to 4, resulting in a mating index of 92%, 91%, 91% and 100%, respectively, indicating that the test article did not have an effect in male mating behavior.

In females, 4-5 day cycles were observed in almost animals during the pre-mating period. Some animals showed a 3-day cycle, which included one in the control (ID#
0008), two in Group 2 (IDs# 0017 and 0019), two in Group 3 (IDs# 0027 and 0030) and one in Group 4 (ID# 0039). One Group 2 animal (ID# 0022) showed an 8-day cycle. One animal in Groups 2 and 4 (IDs# 0023 and 0045) showed no cycling.

A positive indication of mating was obtained for 11 of 12, 11 of 11, 10 of 10, 12 of 12 animals in Groups 1 to 4. The mating index was 92%, 100%, 100% and 100% for
Group 1 to 4, respectively. There was one animal (ID# 0017) in Group 2 that showed a pre-coital interval of longer than 5 days. In this female, the mating with the first male failed but the copulation with another male was successful. In Groups 1, 2 and 4, one animal with positive indications of mating was later found not to be pregnant (IDs# 0002, 0023 and 0041). All Group 3 animals were found to be pregnant. Thus, the fertility index was 91%, 91%, 100% and 92% in the control, low, mid and high dose group, respectively, indicating that the test article did not have an effect in female fertility. The average length of gestation was 22 days without dose-dependency.

Dose descriptor:

NOAEL

Effect level:

50 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effects seen at 50 mg/kg bw/day

Critical effects observed:

no

Clinical signs:

no effects observed

Description (incidence and severity):

No externally abnormal pups were observed at birth in the control and treated groups. At birth, 49.3% to 57.0% of pups were male and on P4, 50.3% to 56.9% pups were
male. The highest male pup rate at birth and on P4 was 57.0% and 56.9%, respectively and observed in Group 3. There was no dose-dependency in gender distribution.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

There were 1, 5 and 5 dead pups observed at birth in Groups 1, 3 and 4, respectively. The incidence of dams with dead pups at birth was 10%, 0%, 40% and 27% in Group 1, 2, 3 and 4, respectively. The dead pup incidence in Group 4 was slightly higher than in the control group. However, the number of stillborns per litter was 0.45 (5 stillborns divided by 11 litters), which was still within the historical data range. In this case, the slightly higher stillborn rate was not considered to be test article-related in absence of a dose-dependency. On P4, a decreased number of live pups was observed in all groups. The 4-day survival index was 99.3%, 98.7%, 96.6% and 99.4% in Groups 1 to 4, respectively, showing no dose-dependency.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no statistically significant differences in litter weight gain on P4 and P13 between control and treated groups. The weight gain between P0 and P4 relative to
weight on P0 was 61%, 56%, 49% and 59% in control and Groups 2–4, respectively, as well as between P4 and P13 relative to weight on P13 was 214%, 221%, 221% and 223%.

Food consumption and compound intake (if feeding study):

not examined

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

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

Other effects:

not examined

Dose descriptor:

NOAEC

Remarks:

teratogenicity

Generation:

F1

Effect level:

50 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Not teratogenic effects at highest dose tested 50 mg/kg bw/day

Critical effects observed:

no

Reproductive effects observed:

no

Eversorb EP4 (2, 10 or 50 mg/kg/day) was given orally by gavage once daily to malerats for 28 days and female rats for 40 to 61 days, depending on the time of copulationand gestation. Eversorb EP4 did not affect clinical observations, body weight, foodconsumption, male and female rats’ reproductive performance such as gonadal function,mating behavior, conception, development of the conceptus, and parturition andoffspring parameters including litter weight, AGD and nipple retention. The noobserved adverse effect level of Eversorb EP4 in rat’s reproductive performance was50 mg/kg/day.

Conclusions:

Eversorb EP4 (2, 10 or 50 mg/kg/day) was given orally by gavage once daily to male rats for 28 days and female rats for 40 to 61 days, depending on the time of copulation
and gestation. Eversorb EP4 did not affect clinical observations, body weight, food consumption, male and female rats’ reproductive performance such as gonadal function,
mating behavior, conception, development of the conceptus, and parturition and offspring parameters including litter weight, AGD and nipple retention. The no observed adverse effect level of Eversorb EP4 in rat’s reproductive performance was 50 mg/kg/day.

Executive summary:

The purpose of this study was to generate limited information on the effects of“Eversorb EP4” on male and female rats’ reproductive performance such as gonadalfunction, mating behavior, conception, development of the conceptus and parturition.

Methods

Animals were assigned to four groups and received vehicle (corn oil) or Eversorb EP4(Lot 4023) at dose levels of 2, 10 and 50 mg/kg/day by oral gavage at 5 mL/kg for 28consecutive days in males and 40–61 days in females. The parent males were sacrificedafter the mating period (Day 29). Females were sacrificed on Day 54 if un-copulated, onGestation Day 25 if copulated without delivery, or reared their offspring (F1), and thensacrificed on Postnatal Day 13. The pups were sacrificed on Postnatal Day 4 or 13.

Measurements

The following parameters were monitored: adult mortality and clinical observations,adult estrus cycle, adult and litter body weights, adult food consumptions, offspringparameters including number, sex, gross abnormalities, androgen-dependentdevelopmental markers (e.g., anogenital distance [AGD] and nipple retention), adultsand pups thyroid hormones (T4) determination, adults gross necropsy (includingimplantation site examination), organ weight and microscopic examinations of collectedtissues.

Results

The dose formulations were homogeneous and target concentrations were confirmed.Test article (0.7 mg/mL) was detected in the control group on the last preparation andwas subjected to be contaminant. The dose level could be adjusted to 0–3.6 mg/kg incontrol, 2–5.8 mg/kg in Group 2 (except ID# 0017) and 10–13.7 mg/kg in Group 3.Treatment of animals with Eversorb EP4 did not induce death, abortion or prematuredelivery. One female of Group 3 delivered as a result of incorrect mating determinationon Day 40 and found dead in parturition. The animal was clinically normal during thestudy period. Pleural effusion was observed at necropsy.Clinical signs during the pre-mating, gestation and lactation period, body weights andfeed consumptions were comparable across all groups. Mating and fertility indices andestrus cycle were un-effected by treatment with Eversorb EP4. F1 pup viability, litterbody weight, sex, AGD and nipple retention were not affected by treatment withEversorb EP4. T4 concentrations in adults and pups as well as reproductive evaluationin gross or histopathology in adult male and female and external gross examination inpups were unaffected by test article treatment.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

50 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

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no 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