N,N'-(2-(4-(2-aminobenzamido)butyl)pentane-1,5-diyl)bis(2-aminobenzamide)

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:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 September 2021 (Study Initiation) to 30 November 2022 (Study Completion)

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)

Deviations:

yes

Remarks:

Study deviations were considered to neither affected the overall interpretation of study findings nor compromised the integrity of the study.

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Crl:WI (Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

Animal Information

Eighty-six male Crl:WI(Han) rats were received on 18 October or 20 December 2021 (Subgroup 2) from Charles River Laboratories, Raleigh, North Carolina. Males were allowed to acclimate for 7 or 8 days prior to dosing. At initiation of dosing, males were 11 to 12 weeks old, and body weights ranged from 251 to 360 g. Males were 14 weeks old at pairing, as applicable.

Forty-eight female Crl:WI(Han) rats were received on 04 October 2021 from Charles River Laboratories, Raleigh, North Carolina. Females were allowed to acclimate for 7 days prior to predose estrous evaluations. Females were 11 weeks old at the start of predose estrous evaluations, 13 weeks old at initiation of dosing, and 15 weeks old at pairing. Body weights ranged from 198 to 232 g for females at initiation of dosing.

Animal Care and Husbandry

Males and females were group housed by sex (two animals/cage) in polycarbonate cages with Diamond Soft® bedding, except during pairing, when one female was housed with one male. After mating occurred (for females) or at the end of the pairing phase (for males and non-confirmed females), animals were individually housed in polycarbonate cages with Diamond Soft® bedding for the remainder of the study. Animals were given various cage enrichment devices and dietary enrichment (that did not require analyses) as environmental enrichment.

Animals were fed Certified Rodent Diet #2016C (Envigo RMS, Inc.) ad libitum. A 50 g sample of each lot of diet utilized on study was collected and retained until report finalization. Water was provided ad libitum.

Environmental controls for the animal room were set to maintain a temperature range of 20 to 26°C, a relative humidity range of 30 to 70%, 10 or greater air changes/hour, and a 12 hour light/12-hour dark cycle. The light/dark cycle was interrupted for study-related activities. Any variations to these conditions are maintained in the raw data and had no effect on the outcome of the study.

Animal Identification and Assignment

Predose estrous cycles were evaluated by the Study Director prior to animals being assigned to study. After group assignment, the mean body weight for each group/sex was not statistically different at the 0.05 probability level, as indicated by analysis of variance F probability (P > 0.05). Additionally, Levene’s analysis returned that the data were homogeneous at the 0.05 level.

F0 animals were identified via implantable microchip identification devices, individual cage cards, and/or a tail mark, as applicable. F1 animals were identified via indelible ink on PND 1.

Route of administration:

oral: gavage

Vehicle:

other: The vehicle control item was 1% w/v methyl cellulose (400 cps) in reverse osmosis water.

Details on oral exposure:

Animals were dosed by oral gavage at a volume of 10 mL/kg. Doses were based on the most recently recorded scheduled body weight.

F0 males were dosed once daily for at least 14 days prior to pairing, throughout the 2-week pairing phase, and through the day prior to termination, for a minimum total of 28 days. F0 males (Subgroup 2) were dosed once daily for 35 days.

F0 females were dosed once daily for at least 14 days prior to pairing, throughout the pairing phase (as applicable), and through gestation and lactation, until Lactation Day (LD) 12. For females without confirmed mating, dosing ceased at the completion of the 2-week pairing phase, for a minimum total of 28 days (total female exposure up to 62 days).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Concentration Verification

Samples (1.00 mL each) were taken from the middle of the solutions of vehicle control item and each test item formulation prepared for administration on the first and last days of dosing. Two samples were taken for the control and six for the dose solutions.

Samples were stored protected from light in a refrigerator, set to maintain 2 to 8°C, until shipped.

Sample Analysis and Disposition

After each collection interval, samples were shipped on cold packs by overnight carrier to the test site for analysis.

For homogeneity, one set of duplicate samples (from each stratum) was analyzed for test item content. The second set was retained as backup samples.

For concentration verification, one control group sample was analyzed to confirm the absence of test item. The second sample was retained as a backup sample. One set of triplicate samples was analyzed for test item content. The second set was retained as backup samples.

Results

Dose formulations met acceptance criteria for homogeneity (relative standard deviation for the mean concentration was ≤10% at concentrations within 100 ± 15% of the theoretical concentration) and concentration (mean concentration within 100 ± 15% of the theoretical concentration, with each individual sample concentration within ± 20%) for suspensions.

Duration of treatment / exposure:

For 35 consecutive days for male rats and for female rats up to 62 days (pre-mating, throughout gestation, and during the first 2 weeks of lactation)

Frequency of treatment:

Once a day 7 days per week.

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

Control

Dose / conc.:

500 mg/kg bw/day (actual dose received)

Remarks:

Low Dose

Dose / conc.:

750 mg/kg bw/day (actual dose received)

Remarks:

Mid Dose

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

High Dose

No. of animals per sex per dose:

20 males and 10 females per dose

Control animals:

yes, concurrent vehicle

Positive control:

None

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes

Males were weighed once prior to dose initiation at randomization, predose on the first day of dosing, and once weekly thereafter, including the day of termination.

Females were weighed three times during the predose phase (including once prior to dose initiation at randomization), predose on the first day of dosing, and once weekly until confirmation of mating, as applicable. Body weights were also recorded on GD 0, 7, 14, and 20 and on LD 1, 4, 7, 11, and 13 (day of termination).

For females without confirmation of mating, body weight measurements continued weekly until termination (these data are maintained in the raw data but not reported).



FOOD CONSUMPTION: YES
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes / No / No data


For males, food consumption was measured weekly during the pre-mating, post-pairing, and dosing phases, as applicable. Food consumption was not measured during the pairing phase.

For females, food consumption was measured weekly during the pre-mating phase, and at body weight collection intervals during gestation and lactation. Food consumption was not measured during the pairing phase. For females without confirmation of mating, food consumption measurements continued weekly until termination (these data are maintained in the raw data but not reported).

Wet and spilled feeders were recorded in the data and reported as such.


WATER CONSUMPTION AND COMPOUND INTAKE : Not Conducted

Sacrifice and pathology:

GROSS PATHOLOGY

MALES

One week following completion of the pairing phase, males were weighed prior to sacrifice and examined macroscopically for abnormalities of the external features of the carcass; external body orifices; cervical, cranial, abdominal, and thoracic viscera; organs; and tissues. Macroscopic abnormalities were noted. Abnormal tissues/viscera were retained in appropriate fixative. On Day 36 of the dosing phase, Subgroup 2 males were weighed, sacrificed, and discarded without necropsy following blood collection.

FEMALES

On GD 24 (females that failed to produce a viable litter), LD 13, or Post-Pairing Day 7 (non confirmed females, without positive signs of mating), surviving females were weighed prior to sacrifice and examined macroscopically for abnormalities of the external features of the carcass; external body orifices; cervical, cranial, abdominal, and thoracic viscera; organs; and tissues. Macroscopic abnormalities were noted. Abnormal tissues/viscera were retained in appropriate fixative.

Pregnancy status was determined. When no fetuses were in utero and implantation sites were not apparent when pressing the uteri between glass plates, the uterus was placed in ammonium sulfide solution. The number of implantation sites was recorded, as applicable.


HISTOPATHOLOGY

The following organs and tissues were collected, weighed, and/or preserved from five animals/sex/group (with exception to males in Subgroup 2) and all animals sacrificed at unscheduled intervals (number in brackets is number of organs taken per animal):

Adrenals (2)
Brain (including cerebrum, cerebellum, and pons)
Cecum
Cervix
Coagulating gland
Colon
Duodenum
Epididymides (2)
Eye (2)
Heart (including auricular and ventricular regions)
Gross lesions
Gut-associated lymphoid tissue (GALT/Peyer’s Patch)
Ileum
Jejunum
Kidney (2)
Liver
Lungs (with large bronchi)
Lymph node - mandibular
Lymph node - mesenteric
Skin with mammary glands/region (inguinal area)
Muscle, skeletal
Nerve, sciatic
Ovary (2)
Oviduct (2)
Pituitary
Prostate
Rectum
Seminal vesicles with coagulating glands (2)
Spinal cord, cervical
Spleen
Sternum (with marrow)
Stomach
Testes (2)
Thymus
Thyroid (2 lobes) with parathyroid
Trachea
Urinary bladder
Uterus
Vagina

Statistics:

Various methods of statistical analysis (ANOVA, ANCOVA, Levene’s test, Dunnett’s test, Fisher’s exact test, Wilcoxon Rank Sum Test) and computer programs were used to analyze data in this study. Only data collected on or after the first day of dosing were analyzed statistically.


Mean number of days to confirmation for each group was calculated but was not statistically evaluated.


Data from non-pregnant animals were excluded from statistical analysis.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Clinical observations observed were noted in either singular occurrences or lacked dose response, and were, therefore, considered incidental.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

A number of deaths occurred but were considered to result from test item administration, or were singular occurrences.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No test item-related alterations in body weight or body weight gain were noted for F0 males or females administered up to 1000 mg/kg/day.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No test item-related alteration to food consumption was noted for F0 males or females administered up to 1000 mg/kg/day.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Minimally decreased total calcium was noted on Post-Pairing Day 7 in two males administered 1000 mg/kg/day. This was considered unlikely related to the test item because it was noted in only two males.

Endocrine findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

No statistically significant differences between groups were noted for quantitative assessment of grip strength, latency, rearing, or number of urine pools or fecal boli. In addition, neurobehavioral observations of auditory startle response, approach response, pain response, or pinna response were comparable with controls for F0 males and females.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Test item-related organ weight differences were noted for females administered ≥500 mg/kg/day and sacrificed on LD 13. Increased adrenal weights were noted in females administered ≥500 mg/kg/day and sacrificed on LD 13, which correlated with the minimal microscopic finding of adrenal cortical hypertrophy in females administered ≥500 mg/kg/day. Increased weights were also noted for the heart, liver, and spleen of females administered 1000 mg/kg/day and sacrificed on LD 13, without a microscopic or clinical pathology correlate. These organ weight effects were not noted in males sacrificed on Post-Pairing Day 7.

The absolute weight for testes and epididymides of one male administered 1000 mg/kg/day and sacrificed on Post-Pairing Day was of notably lesser weight, compared to the weights for other males administered 1000 mg/kg/day or other males sacrificed on Post Pairing Day 7; however, tissues of this male were not examined microscopically. Prostate and seminal vesicle/coagulating gland weights of this male were comparable to other males, including controls.

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

The test item-related microscopic finding of adrenal cortical hypertrophy was noted in F0 females sacrificed on Lactation Day (LD) 13 and administered ≥500 mg/kg/day, which correlated with increased adrenal weights.

Other test item related microscopic findings included minimal to slight pyogranulomatous inflammation associated with minimal fibrosis, which was noted in the thyroid of three F1 male pups sacrificed on Postnatal Day (PND) 13 from dams administered 750 or 1000 mg/kg/day. In addition, a non-dose responsive increase in thyroid weights (absolute and relative to body weight) was observed for F1 male pups sacrificed on PND 13 from the group with females administered ≥500 mg/kg/day. The thyroid weights for F1 female pups were comparable to control values.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other:

Remarks on result:

other: No effects related to test item administration which could be considered adverse were observed during the study.

Key result

Critical effects observed:

no

Conclusions:

The study no observed adverse effect level (NOAEL) determined for general toxicity and reporductive effects is 1000 mg/kg/day for males and females.

Executive summary:

Introduction 

 

The study was designed to investigate the systemic toxicity and potential adverse effects of N,N’-(2-(4-(2-aminobenzamido)butyl)pentane-1,5-diyl)bis(2-aminobenzamide) on reproduction (including offspring development), to evaluate some endocrine disruptor relevant endpoints and is designed to meet the requirements of the OECD Guidelines for Testing of Chemicals No. 422 “Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test” (adopted 29 July 2016). 

 

 This study was also designed to be compatible with the following guidelines:

• United States Environmental Protection Agency - Office of Prevention, Pesticides & Toxic Substances (OPPTS) 870.3700
  
  


• United States Environmental Protection Agency 40 Code of Federal Regulations § 798.490 Developmental Toxicity
  
  


• Japanese Ministry of Agriculture, Forestry and Fisheries, Test Data for registration of Agricultural Chemicals, 12 Nohsan No. 8147, Agricultural Production Bureau, November 24, 2000 Animal Welfare and Veterinary Care

 

 Method 

 

The test item was administered by gavage to three dose groups, each of 20 male and 10 female Crl:WI(Han) strain rats, for 35 days for males and up to 62 days for females (pre-mating, throughout gestation, and during the first 2 weeks of lactation).  Control groups were administered the vehicle 1% w/v methyl cellulose.  The dosing regimen is outlined in the following table:

 

Group ^a	Dose Level ^b (mg/kg/day)	Dose Concentration ^b (mg/mL)	No. of Animals
Males ^c
1 (Control)	0	0	20
2 (Low)	500	50	20
3 (Mid)	750	75	20
4 (High)	1000	100	20
Females
1 (Control)	0	0	10
2 (Low)	500	50	10
3 (Mid)	750	75	10
4 (High)	1000	100	10
a      Group 1 was administered vehicle control item only. b      Animals were dosed at a volume of 10 mL/kg. c      Additional males (Subgroup 2; 10 animals/group) were added to the study.

Assessment of toxicity was based on mortality, clinical observations, neurobehavioral evaluation, body weights, food consumption, and necropsy findings. Blood samples were collected for clinical pathology evaluation.

Results 

 

 Mortality 

No test item-related deaths were observed during the study.

 

Clinical Observations 

No test item-related clinical observations were noted in F0 animals administered up to 1000 mg/kg/day.

 

Behavioural Assessment 

There were no treatment-related changes in the behavioural parameters observed during the study. 

 

Functional Performance Tests 

No treatment related changes in neurobehavior were noted in F0 animals administered up to 1000 mg/kg/day. 

 

Body Weight 

No test item-related alterations in body weight  were noted for F0 males or females administered up to 1000 mg/kg/day.

 

Food Consumption 

No test item-related alterations in food consumption were noted for F0 males or females administered up to 1000 mg/kg/day.

 

Haematology and Clinical Chemistry

No test item-related effects on haematology, coagulation, or clinical chemistry test results were identified in animals administered up to 1000 mg/kg/day.

 

Endocrine effects

No effects on endocrine function or changes to thyroid T4 levels were observed.

 

Necropsy 

No toxicologically significant effects were detected in treated adult animals of either sex. 

 

Organ Weights 

Increased organ weights were noted for the heart, liver, and spleen of females administered 1000 mg/kg/day, without a microscopic or clinical pathology correlate. No test item‑related organ weight differences or microscopic findings were noted in males sacrificed on Post‑Pairing Day 7.

 

Histopathology 

The following treatment-related microscopic abnormalities were detected: 

 

The test item-related microscopic finding of adrenal cortical hypertrophy was noted in F0 females sacrificed on Lactation Day (LD) 13 and administered ≥500 mg/kg/day, which correlated with increased adrenal weights.

 

Other test item‑related microscopic findings included minimal to slight pyogranulomatous inflammation associated with minimal fibrosis, which was noted in the thyroid of three F1 male pups sacrificed on Postnatal Day (PND) 13 from dams administered 750 or 1000 mg/kg/day. In addition, a non-dose responsive increase in thyroid weights (absolute and relative to body weight) was observed for F1 male pups sacrificed on PND 13 from the group with females administered ≥500 mg/kg/day. The thyroid weights for F1 female pups were comparable to control values.

 

Conclusion

The study no observed adverse effect level (NOAEL) determined for general toxicity and reporductive effects is 1000 mg/kg/day for males and females.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Justification for classification or non-classification

No treatment related effects that were considered adverse were observed during the study and hence the No Observed Adverse Effect Level (NOAEL) in both males and females was considered to be the study top dose of 1000 mg/kg bw/day for general toxicity and reproductive effects.