N,N''-(methylenedi-4,1-phenylene)bis[N'-(4-methylphenyl)urea]

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May 2017 - November 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May 2017 - November 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test

Version / remarks:

2000

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Crl: WI(Han)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: (P) 10 wks (at initiation of dosing); (F1) 13 wks (at initiation of dosing)
- Weight at study initiation: (P) Males: 259-296 g; Females: 189-236 g
- Fasting period before study: no
- Housing: On arrival and following the pre-test (females only) and pre-mating period, animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages (Macrolon, MIV type); During the mating phase, males and females were cohabitated on a 1:1 basis in Macrolon plastic cages (MIII type); During the post-mating phase, males were housed in their home cage (Macrolon plastic cages, MIV type) with a maximum of 5 males/cage. Females were individually housed in Macrolon plastic cages (MIII type); During the lactation phase, females were housed in Macrolon plastic cages (MIII type). Pups were housed with the dam.
- Diet: Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was provided ad libitum throughout the study, except during designated procedures.
- Water (e.g. ad libitum): Municipal tap water was freely available to each animal via water bottles.
- Acclimation period: 7 days

The feed was analyzed by the supplier for nutritional components and environmental contaminants. Results of the analysis were provided by the supplier and are on file at the Test Facility. It is considered that there were no known contaminants in the feed that would interfere with the objectives of the study.
Periodic analysis of the water is performed, and results of these analyses are on file at the Test Facility. It is considered that there were no known contaminants in the water that would interfere with the objectives of the study.
For psychological/environmental enrichment and nesting material, animals were provided with paper (Enviro-dri, Wm. Lilico & Son (Wonham Mill Ltd), Surrey, United Kingdom).

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22
- Humidity (%): 41-59
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 14 Jun 2017 To: 08 Aug 2017

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Remarks:

specific gravity 1.036

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared daily as a suspension and dosed within 6 hours after adding the vehicle to the test item.
Test item dosing formulations were kept at room temperature until dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing. Adjustment was made for specific gravity of the vehicle. No correction was made for the purity/composition of the test item. Any residual volumes were discarded.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The choice of the vehicle for preparation of the test item formulations in the current study was based on the vehicle used in a previous 28-day toxicity study.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose formulation samples were collected in week 2 for concentration measurements in all groups and for determination of homogeneity and stability in low and high dose groups.
Analyses were performed by HPLC using a validated analytical procedure.
Concentration analysis: Duplicate sets of samples (approximately 500 mg accurately weighed) for each sampling time point were sent to the analytical laboratory. Concentration results were considered acceptable if mean sample concentration results were within or equal to ±15% for suspensions of target concentration.
Homogeneity analysis: Duplicate sets of samples (approximately 500 mg accurately weighed) for each sampling time point were sent to the analytical laboratory. Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was below or equal to 10%.
Stability analysis: Stability of formulations was determined upon storage for 18 hours at room temperature under normal laboratory light conditions.
Duplicate sets of each sample (approximately 500 mg accurately weighed) were sent to the analytical laboratory. Stability results were considered acceptable if the sample analysis results were within or equal to ±10% of the concentration determined by the initial analysis of each formulation.

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/1
- Length of cohabitation: A maximum of 14 days was allowed for mating, after which females who had not shown evidence of mating were separated from their males
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 post-coitum

Duration of treatment / exposure:

The test item and vehicle were administered to the appropriate animals by once daily oral gavage 7 days a week for a minimum of 28 days. Males were treated for 29 days, up to and including the day before scheduled necropsy, i.e. 14 days prior to mating, during the mating period and up to the day before scheduled necropsy. Females were treated for 14 days prior to start of mating (with the objective to cover at least two complete estrous cycles), the variable time to conception, the duration of pregnancy and at least 13 days after delivery, up to the day before scheduled necropsy (50-55 days for the females with offspring and 41 or 54 days for the females without offspring).
Pups were not treated directly but were potentially exposed to the test item in utero, via maternal milk, or from exposure to maternal urine/feces.

Frequency of treatment:

daily

Duration of test:

total of 55 days

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose levels were selected based on a previously performed 28-day repeated dose toxicity study with KY-AF by oral gavage in rats (reference: Test Facility Study no. 276312), and in an attempt to produce graded responses to the test item. Increased levels of met-haemoglobin were observed in both males and females at 1000 mg/kg bw/day in this latter study. No mortality, severe toxicity or suffering was to be expected at the selected dose levels in the current study.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed individually on the first day of treatment (prior to dosing), and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13. A fasted weight was recorded on the day of necropsy.

FOOD CONSUMPTION: Yes
Food consumption was quantitatively measured weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.

WATER CONSUMPTION ): Yes
Water consumption was monitored on regular basis throughout the study by visual inspection of the water bottles. No quantitative investigation was introduced as no effect was suspected or noted at visual inspection of the water bottles.

POST-MORTEM EXAMINATIONS: Yes
SACRIFICE
- Male animals: All surviving animals following completion of the mating period (a minimum of 28 days of administration).
- Females which delivered: PND 14-16
- Females which failed to deliver : With evidence of mating: Post-coitum Day 25 or 26. Without evidence of mating: 26 days after the last day of the mating period.

GROSS NECROPSY
- All animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs. The numbers of former implantation sites were recorded for all paired females.
In case no macroscopically visible implantation sites were present, nongravid uteri were stained using the Salewski technique in order to detect any former implantation sites and the number of corpora lutea was recorded in addition.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues according to Guidelines were prepared and weighed. Histological examinaniation on epididymis, thyroid gland, gross lesions/masses, ovaries and testes was performed for all control and high dose animals; from all low and mid dose females thyroid gland was examinated.
Males that failed to sire and females that failed to deliver pups: Cervix, epididymis, coagulation gland, prostate gland, ovaries, seminal vesicles, testes, uterus and vagina.

OTHER: CLINICAL PATHOLOGY
Blood of F0-animals (except for animals which were sacrificed in extremis) was collected on the day of scheduled necropsy (under anesthesia using isoflurane). F0-animals were fasted overnight with a maximum of 24 hours before blood sampling, but water was available. 5 animals/group were selected for haematology, including met-Hb assessment.
Haematology parameters included: White blood cells, Neutrophil, Lymphocyte, Monocyte, Eosinophil , Basophil, Red blood cells, Reticulocyte, Red Blood Cell Distribution Width, Haemoglobin, Met-haemoglobin, Haematocrit, Mean corpuscular volume, Mean corpuscular haemoglobin, Mean corpuscular haemoglobin concentration, Platelets.
Blood samples were processed for serum for possible analysis for the thyroid hormoneparameters total thyroxine (T4) and/or thyroid-stimulating hormone (TSH). These serum samples were stored until (possible) analysis in a freezer (≤-75°C). Measurement of serum for total thyroxine (T4) was conducted for F0-males and PND 13-15 pups.
For the F0-generation, assessment of T4 in females and Thyroid Stimulating Hormone (TSH) in males and females was considered not relevant because there were no treatment-related changes in T4 in males or in thyroid weights of males and females. Moreover, no adverse morphological changes were noted in the thyroid.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes / No / No data
Examinations included:
- Gravid uterus weight:No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: No data
- Number of late resorptions: No data

Fetal examinations:

sex ratio and early postnatal pup development (mortality, clinical signs, body weights, sex, anogenital distance, areola/nipple retention, serum level of thyroid hormone T4 in PND 13-15 pups, and macroscopy)

Statistics:

All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% and 5% levels. Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation (or %CV or SE when deemed appropriate) were reported whenever possible.
Inferential statistics were performed according to the matrix below when possible, but excluded semi-quantitative data, and any group with less than 2 observations.
The following pairwise comparisons were made:
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1

Parametric: Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).
Non-parametric: Datasets with at least 3 groups was compared using a Steel-test (many-to-one rank test).
Incidence: An overall Fisher’s exact test was used to compare all groups at the 5% significance level. The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant.

Indices:

Live birth index (%): Number of live offspring on Day 1 after littering / Total number of offspring born) x 100
Percentage live males at First Litter Check (%): (Number of live male pups at First Litter Check / Number of live pups at First Litter Check) x 100
Percentage live females at First Litter Check (%): (Number of live female pups at First Litter Check / Number of live pups at First Litter Check) x 100
Viability index (%): (Number of live offspring on Day 4 before culling / Number live offspring on Day 1 after littering)x 100
Lactation index (%): (Number of live offspring on Day 13 after littering / Number live offspring on Day 4 (after culling)) x 100

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related clinical signs were noted during the daily clinical observations.
Salivation noted in a few animals of all groups, the control group included, was considered to be a physiological response rather than a sign of systemic toxicity considering its slight severity and the time of occurrence (i.e. after dosing). Any other clinical signs noted incidentally occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and showed no dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

No mortality occurred during the study period that was considered to be related to treatment with the test item.
One male treated at 100 mg/kg bw/day was sacrificed for humane reasons on Day 20 of treatment. This animal showed laboured respiration, rales, a hunched posture, piloerection, a lean or emaciated appearance and markedly reduced body weight gain. There were no macroscopic or microscopic findings that could explain the moribund state of the animal. The moribundity of this single male of the lowest dose group was regarded as unrelated to treatment due to its incidental occurrence.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Body weight and body weight gain were considered not to be affected by treatment. In the absence of a dose-related response, the statistically significantly higher mean body weights noted at 100 mg/kg bw/day in females (Days 0-11 of the post-coitum period and Days 1-4 of the lactation period) were considered to be unrelated to treatment (mean body weight of 100 mg/kg bw/day females was already higher than that of controls at initiation of treatment).
Reduced weight gain and/or weight loss was noted in a few individual animals that survived until scheduled necropsy (one high dose male in the third week of treatment, and one mid dose females and one high dose female during lactation). These findings were not attributed to treatment as they were transient or occurred in only a single animal of a group.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Food consumption (before and after correction for body weight) was considered not to be affected by treatment. The statistically significantly higher mean food consumption noted in 1000 mg/kg bw/day females b
etween Days 0-4 post-coitum and the markedly reduced food consumption of a single 1000 mg/kg bw/day female between Days 1-4 of lactation were considered to be unrelated to treatment because food consumption of these females was normal at the other time points.

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Haematological parameters in F0-females were considered not to be affected by treatment. Any statistically significant variations noted in haematology parameters were considered unrelated to treatment due to the absence of a clear dose-related trend and slight magnitude of the differences from controls. Moreover, the control values in this study were slightly higher than normal. However, values in control and treated rats remained within normal limits.

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There were no test item-realted alterations in organ weights.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no test item-related gross observations. All of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain. These findings were therefore considered to be unrelated to treatment.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related microscopic observations in male rats. In female rats, a slightly increased incidence of follicular cell hypertrophy in the thyroid gland (minimal degree) was noted at 300 and 1000 mg/kg bw/day, with incodences of 1, 2, 5 and 4 in control, low, mid and high dose females, respectively.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

Serum levels of T4 in F0-males were considered not to be affected by treatment.
In the absence of a dose related trend, the statistically significantly higher mean T4 value noted at 100 mg/kg bw/day was considered unrelated to treatment and of no toxicological significance.

Number of abortions:

no effects observed

Description (incidence and severity):

Examination of cage debris of pregnant females revealed no signs of abortion or premature birth.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

Post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites) was not affected by treatment. The survival indices
were 94, 93, 94 and 93% at 0 (control), 100, 300 and 1000 mg/kg bw/day, respectively.
For one female dosed 100 mg/kg bw/day and one female dosed(300 mg/kg bw/day the number of pups born was slightly higher than the number of implantation sites. This phenomenon is observed from time to time and is caused by normal resorption of these areas during lactation.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

The mean number of living pups at first litter check (live litter size) was considered to be unaffected by treatment.
A relatively large litter size was observed in control females in comparison with that in the females treated groups. Since the litter size in treated females was comparable with the historical control values and in the absence of a dose response no toxicological significance was attached to this finding.

Early or late resorptions:

not specified

Dead fetuses:

no effects observed

Description (incidence and severity):

Live birth index (number of live offspring on PND 1 as percentage of total number of offspring born) was not affected by treatment. The live birth indices were 100% for the control and 100 mg/kg bw/day groups and 99% for the 300 and 1000 mg/kg bw/day groups.
At first litter check, one pup at 300 mg/kg bw/day and one pup at 1000 mg/kg bw/day were found dead. This incidental pup mortality was unrelated to treatment.

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

No signs of difficult or prolonged parturition were noted among the pregnant females.

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

Gestation index and duration of gestation were not affected by treatment. All pregnant females had live offspring (gestation index 100% for all groups).

Other effects:

no effects observed

Details on maternal toxic effects:

No deficiencies in maternal care were observed.
lactation index: No pups died or went missing after PND 4. The lactation index (number of live offspring on PND 13 as percentage of number of live offspring after culling on PND 4) was 100% for all groups.
Precoital time was not affected by treatment. All mated females showed evidence of mating within four days.
Serum levels of T4 in F0-males were considered not to be affected by treatment. In the absence of a dose related trend, the statistically significantly higher mean T4 value noted at 100 mg/kg bw/day was considered unrelated to treatment and of no toxicological significance.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Body weights of pups were considered not to be affected by treatment.
Slightly lower mean body weight of male and female pups of control female were observed in comparison with that of pups of treated females. This finding was considered to be related to the slightly higher litter size observed in control females, resulting in slightly smaller pups at birth. In the absence of a dose related affect and as normal growth was observed of pups of control and treated females during lactation, no toxicological significance was attached to this finding. As a consequence, the statistically significant differences apparent in some cases were not indicative of an effect by treatment.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

Live birth index (number of live offspring on PND 1 as percentage of total number of offspring born) was not affected by treatment. The live birth indices were 100% for the control and 100 mg/kg bw/day groups and 99% for the 300 and 1000 mg/kg bw/day groups.
At first litter check, one pup at 300 mg/kg bw/day and one pup at 1000 mg/kg bw/day were found dead. This incidental pup mortality was unrelated to treatment.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

Sex ratio was considered not to be affected by treatment.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

The mean number of living pups at first litter check (live litter size) was considered to be unaffected by treatment.
A relatively large litter size was observed in control females in comparison with that in the females treated groups. Since the litter size in treated females was comparable with the historical control values and in the absence of a dose response no toxicological significance was attached to this finding.

Changes in postnatal survival:

no effects observed

Description (incidence and severity):

Viability index (number of live offspring on PND 4 before culling as percentage of number of live offspring on PND 1) was not affected by treatment. The viability indices were 100% for the 1000 mg/kg bw/day group and 99% for the other groups.
One pup of the control group and one pup at 100 mg/kg bw/day were found dead on PND 2 or 3, and one pup at 300 mg/kg bw/day was sacrificed for humane reasons on PND 1. This post-natal loss was considered to be unrelated to treatment as it was incidental and showed no dose-related trend.

External malformations:

no effects observed

Description (incidence and severity):

No macroscopic findings were noted among pups that were considered to be related to treatment.
The nature and incidence of the macroscopic findings noted incidentally remained within the range considered normal for pups of this age, and were therefore considered to be unrelated to treatment.

Skeletal malformations:

not examined

Visceral malformations:

not examined

Other effects:

no effects observed

Description (incidence and severity):

No clinical signs occurred among pups that were considered to be related to treatment. The clinical signs observed incidentally remained within the range considered normal for pups of this age, and were therefore regarded to be unrelated to treatment.
Anogenital distance (absolute and normalized for body weight) in male and female pups was considered not to be affected by treatment.
Treatment up to and including 1000 mg/kg bw/day had no effect on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13.
Serum T4 levels in male and female PND 13-15 pups were considered not to be affected by treatment.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Conclusions:

In conclusion, based on the results of this reproduction/developmental toxicity screening test, the following No-Observed-Adverse-Effect Levels (NOAELs) of KY-AF were established:
Parental NOAEL: at least 1000 mg/kg bw/day
Reproduction NOAEL: at least 1000 mg/kg bw/day
Developmental NOAEL: at least 1000 mg/kg bw/day

Executive summary:

KY-AF was tested for its reproduction/developmental toxicity potential in a reproduction/developmental toxicity screening study (OECD 421) Wistar Han rats were treated with KY-AF by daily oral gavage at

dose levels of 100, 300 and 1000 mg/kg bw/day (10 rats/sex/dose level). Concurrent controls (10 rats/sex) received the vehicle, propylene glycol, alone. Males were treated for two weeks prior to mating, during mating, and up to termination (for 29 days). Females that delivered offspring were treated for two weeks prior to mating, during mating, during post-coitum, and 13-15 days of lactation (for 50-55 days).

Females without offspring were treated for 41 or 54 days.

Formulation analysis showed that the dosing formulations were prepared accurately and homogeneously, and that the formulations were stable for at least 18 hours at room temperature under normal laboratory light conditions.

Parental results

No parental toxicity was noted up to and including the highest dose level tested (1000 mg/kg bw/day). There were no treatment-related changes in the in-life parameters examined in this study (i.e. mortality, clinical appearance, body weight and food consumption), serum level of thyroid hormone T4 (males), haematology parameters (females), organ weights (thyroid in both sexes and male reproductive organs) or findings at macroscopic examination. Microscopic examination revealed a minor increase in the incidence of follicular cell hypertrophy in the thyroid of females treated at 300 or 1000 mg/kg bw/day compared to concurrent controls. It cannot be excluded that this finding was related to treatment with the test item. The severity of follicular cell hypertrophy in these dose groups (minimal) did not exceed that recorded in control females. Follicular cell hypertrophy at low degrees can be seen as a background finding in rats of this age and strain and was also noted in one female of the concurrent control group. Therefore this finding was considered to be non-adverse. No treatment-related microscopic changes were observed in the other organs examined (thyroid of male rats, testes, epididymides, ovaries).

Reproductive results:

No reproduction toxicity was observed up to and including the highest dose level tested (1000 mg/kg bw/day). No treatment-related changes were noted in any of reproductive parameters investigated in this study (i.e. mating and fertility indices, precoital time, number of implantation sites, estrous cycle, spermatogenic profiling, and histopathological examination of reproductive organs).

Developmental results:

No developmental toxicity was observed up to and including the highest dose level tested (1000 mg/kg bw/day). No treatment-related changes were noted in any of the developmental parameters investigated in this study (i.e. gestation, viability and lactation indices, duration of gestation, parturition, sex ratio, maternal care and early postnatal pup development consisting of mortality, clinical signs, body weight, anogenital distance, areola/nipple retention, serum level of thyroid hormone T4 (PND 13-15) and macroscopic examination).

In conclusion, based on the results of this reproduction/developmental toxicity screening test, the following No-Observed-Adverse-Effect Levels (NOAELs) of KY-AF were established:

Parental NOAEL: at least 1000 mg/kg bw/day

Reproduction NOAEL: at least 1000 mg/kg bw/day

Developmental NOAEL: at least 1000 mg/kg bw/day

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

2016

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test

Version / remarks:

2000

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

429-380-1

EC Name:

-

Cas Number:

133336-92-2

Molecular formula:

C29H28N4O2

IUPAC Name:

1-(4-methylphenyl)-3-{4-[(4-{[(4-methylphenyl)carbamoyl]amino}phenyl)methyl]phenyl}urea

Test material form:

solid: particulate/powder

Details on test material:

- Appearance: white powder
- Storage conditions: at room temperature

Test animals

Species:

rat

Strain:

other: Crl: WI(Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: (P) 10 wks (at initiation of dosing); (F1) 13 wks (at initiation of dosing)
- Weight at study initiation: (P) Males: 259-296 g; Females: 189-236 g
- Fasting period before study: no
- Housing: On arrival and following the pre-test (females only) and pre-mating period, animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages (Macrolon, MIV type); During the mating phase, males and females were cohabitated on a 1:1 basis in Macrolon plastic cages (MIII type); During the post-mating phase, males were housed in their home cage (Macrolon plastic cages, MIV type) with a maximum of 5 males/cage. Females were individually housed in Macrolon plastic cages (MIII type); During the lactation phase, females were housed in Macrolon plastic cages (MIII type). Pups were housed with the dam.
- Diet: Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was provided ad libitum throughout the study, except during designated procedures.
- Water (e.g. ad libitum): Municipal tap water was freely available to each animal via water bottles.
- Acclimation period: 7 days

The feed was analyzed by the supplier for nutritional components and environmental contaminants. Results of the analysis were provided by the supplier and are on file at the Test Facility. It is considered that there were no known contaminants in the feed that would interfere with the objectives of the study.
Periodic analysis of the water is performed, and results of these analyses are on file at the Test Facility. It is considered that there were no known contaminants in the water that would interfere with the objectives of the study.
For psychological/environmental enrichment and nesting material, animals were provided with paper (Enviro-dri, Wm. Lilico & Son (Wonham Mill Ltd), Surrey, United Kingdom).

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22
- Humidity (%): 41-59
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 14 Jun 2017 To: 08 Aug 2017

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Remarks:

specific gravity 1.036

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared daily as a suspension and dosed within 6 hours after adding the vehicle to the test item.
Test item dosing formulations were kept at room temperature until dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing. Adjustment was made for specific gravity of the vehicle. No correction was made for the purity/composition of the test item. Any residual volumes were discarded.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The choice of the vehicle for preparation of the test item formulations in the current study was based on the vehicle used in a previous 28-day toxicity study.

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: A maximum of 14 days was allowed for mating, after which females who had not shown evidence of mating were separated from their males
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 post-coitum.
A maximum of 14 days was allowed for mating, after which females who had not shown evidence of mating were separated from their males.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose formulation samples were collected in week 2 for concentration measurements in all groups and for determination of homogeneity and stability in low and high dose groups.
Analyses were performed by HPLC using a validated analytical procedure.
Concentration analysis: Duplicate sets of samples (approximately 500 mg accurately weighed) for each sampling time point were sent to the analytical laboratory. Concentration results were considered acceptable if mean sample concentration results were within or equal to ±15% for suspensions of target concentration.
Homogeneity analysis: Duplicate sets of samples (approximately 500 mg accurately weighed) for each sampling time point were sent to the analytical laboratory. Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was below or equal to 10%.
Stability analysis: Stability of formulations was determined upon storage for 18 hours at room temperature
under normal laboratory light conditions.
Duplicate sets of each sample (approximately 500 mg accurately weighed) were sent to the analytical laboratory. Stability results were considered acceptable if the sample analysis results were within or equal to ±10% of the concentration determined by the initial analysis of each formulation.

Duration of treatment / exposure:

The test item and vehicle were administered to the appropriate animals by once daily oral gavage 7 days a week for a minimum of 28 days. Males were treated for 29 days, up to and including the day before scheduled necropsy, i.e. 14 days prior to mating, during the mating period and up to the day before scheduled necropsy. Females were treated for 14 days prior to start of mating (with the objective to cover at least two complete estrous cycles), the variable time to conception, the duration of pregnancy and at least 13 days after delivery, up to the day before scheduled necropsy (50-55 days for the females with offspring and 41 or 54 days for the females without offspring).
Pups were not treated directly but were potentially exposed to the test item in utero, via maternal milk, or from exposure to maternal urine/feces.

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose levels were selected based on a previously performed 28-day repeated dose toxicity study with KY-AF by oral gavage in rats (reference: Test Facility Study no. 276312), and in an attempt to produce graded responses to the test item. Increased levels of met-haemoglobin were observed in both males and females at 1000 mg/kg bw/day in this latter study. No mortality, severe toxicity or suffering was to be expected at the selected dose levels in the current study.

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed individually on the first day of treatment (prior to dosing), and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13. A fasted weight was recorded on the day of necropsy.

FOOD CONSUMPTION: Yes
Food consumption was quantitatively measured weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.

WATER CONSUMPTION : Yes
Water consumption was monitored on regular basis throughout the study by visual inspection of the water bottles. No quantitative investigation was introduced as no effect was suspected or noted at visual inspection of the water bottles.

OTHER: CLINICAL PATHOLOGY
Blood of F0-animals (except for animals which were sacrificed in extremis) was collected on the day of scheduled necropsy (under anesthesia using isoflurane). F0-animals were fasted overnight with a maximum of 24 hours before blood sampling, but water was available. 5 animals/group were selected for haematology, including met-Hb assessment.
Haematology parameters included: White blood cells, Neutrophil, Lymphocyte, Monocyte, Eosinophil, Basophil, Red blood cells, Reticulocyte, Red Blood Cell Distribution Width, Haemoglobin, Met-haemoglobin, Haematocrit, Mean corpuscular volume, Mean corpuscular haemoglobin, Mean corpuscular haemoglobin concentration, Platelets.

Blood samples were processed for serum for possible analysis for the
thyroid hormoneparameters total thyroxine (T4) and/or thyroid-stimulating hormone (TSH). These serum samples were stored until (possible) analysis in a freezer (≤-75°C).
Measurement of serum for total thyroxine (T4) was conducted for F0-males and PND 13-15 pups.
For the F0-generation, assessment of T4 in females and Thyroid Stimulating Hormone (TSH) in males and females was considered not relevant because there were no treatment-related changes in T4 in males or in thyroid weights of males and females. Moreover, no adverse morphological changes were noted in the thyroid.

Oestrous cyclicity (parental animals):

Estrous cycles were evaluated by examining the vaginal cytology of samples obtained by vaginal lavage.
Daily vaginal lavage was performed for all females beginning 14 days prior to treatment (pretest period), the first 14 days of treatment and during mating until evidence of copulation was observed. Vaginal lavage was continued for those females with no evidence of copulation until termination of the mating period. On the day of necropsy, a vaginal lavage was also taken to determine the stage of estrus. This was done for all females.

Sperm parameters (parental animals):

For the testes of all males of the controla and high dose groups, and all males that failed to sire or died before mating detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Any cell- or stage-specificity of testicular findings were noted.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter; From two surplus pups per litter, blood was collected, if possible.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), presence of nipples/areolae in male pups, other: blood was collected from two pups per litter and the thyroid from two pups per litter (one male and one female pup) was preserved in 10% buffered formalin. The pups selected for blood sampling were the same pups as selected for thyroid preservation.

GROSS EXAMINATION OF DEAD PUPS:
yes, for external abnormalities; possible cause of death was determined for pups born or found dead.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: no

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: no

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals following completion of the mating period (a minimum of 28 days of administration).
- Females which delivered: PND 14-16
- Females which failed to deliver : With evidence of mating: Post-coitum Day 25 or 26. Without evidence of mating: 26 days after the last day of the mating period.

GROSS NECROPSY
- All animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs. The numbers of former implantation sites were recorded for all paired females.
In case no macroscopically visible implantation sites were present, nongravid uteri were stained using the Salewski technique in order to detect any former implantation sites and the number of corpora lutea was recorded in addition.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues according to Guidelines were prepared and weighed. Histological examinaniation on epididymis, thyroid gland, gross lesions/masses, ovaries and testes was performed for all control and high dose animals; from all low and mid dose females thyroid gland was examinated.
Males that failed to sire and females that failed to deliver pups: Cervix, epididymis, coagulation gland, prostate gland, ovaries, seminal vesicles, testes, uterus and vagina.

Postmortem examinations (offspring):

SACRIFICE
Pups, younger than 7 days were euthanized by decapitation.
All remaining pups (PND 7-15), except for the two pups per litter selected for blood collection, were euthanized by an intraperitoneal injection of sodium pentobarbital (Euthasol® 20%).
The pups selected for blood collection on PND 13-15 were anesthetized using isoflurane followed by exsanguination.

Pups that died or were euthanized before scheduled termination were examined externally and sexed (both externally and internally).
The stomach of pups not surviving to the scheduled necropsy date was examined for the presence of milk, if possible. If possible, defects or cause of death were evaluated.

GROSS NECROPSY
All remaining pups were euthanized on PND 13-15. Sex was determined both externally and internally. Descriptions of all external abnormalities were recorded. Particular attention was paid to the external reproductive genitals to examine signs of altered development. In addition, blood was collected from two pups per litter, and the thyroid from two pups per litter (one male and one female pup) was preserved in 10% buffered formalin. The pups selected for blood sampling were the same pups as selected for thyroid preservation.

Statistics:

All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% and 5% levels. Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation (or %CV or SE when deemed appropriate) were reported whenever possible.
Inferential statistics were performed according to the matrix below when possible, but excluded semi-quantitative data, and any group with less than 2 observations.
The following pairwise comparisons were made:
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1

Parametric: Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).
Non-parametric: Datasets with at least 3 groups was compared using a Steel-test (many-to-one rank test).
Incidence: An overall Fisher’s exact test was used to compare all groups at the 5% significance level. The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant.

Reproductive indices:

Mating (%): (Number of females mated / Number of females paired) x 100
Precoital time: Number of days between initiation of cohabitation and confirmation of mating
Fertility index (%): (Number of pregnant females / Number of females mated) x 100
Gestation index (%): (Number of females with living pups on Day 1 / Number of pregnant females) x 100
Duration of gestation: Number of days between confirmation of mating and the beginning of parturition
Post-implantation survival index (%): (Total number of offspring born / Total number of uterine implantation sites) x 100

Offspring viability indices:

Live birth index (%): Number of live offspring on Day 1 after littering / Total number of offspring born) x 100
Percentage live males at First Litter Check (%): (Number of live male pups at First Litter Check / Number of live pups at First Litter Check) x 100
Percentage live females at First Litter Check (%): (Number of live female pups at First Litter Check / Number of live pups at First Litter Check) x 100
Viability index (%): (Number of live offspring on Day 4 before culling / Number live offspring on Day 1 after littering)x 100
Lactation index (%): (Number of live offspring on Day 13 after littering / Number live offspring on Day 4 (after culling)) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related clinical signs were noted during the daily clinical observations.
Salivation noted in a few animals of all groups, the control group included, was considered to be a physiological response rather than a sign of systemic toxicity considering its slight severity and the time of occurrence (i.e. after dosing). Any other clinical signs noted incidentally occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and showed no dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

No mortality occurred during the study period that was considered to be related to treatment with the test item.
One male treated at 100 mg/kg bw/day was sacrificed for humane reasons on Day 20 of treatment. This animal showed laboured respiration, rales, a hunched posture, piloerection, a lean or emaciated appearance and markedly reduced body weight gain. There were no macroscopic or microscopic findings that could explain the moribund state of the animal. The moribundity of this single male of the lowest dose group was regarded as unrelated to treatment due to its incidental occurrence.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Body weight and body weight gain were considered not to be affected by treatment. In the absence of a dose-related response, the statistically significantly higher mean body weights noted at 100 mg/kg bw/day in females (Days 0-11 of the post-coitum period and Days 1-4 of the lactation period) were considered to be unrelated to treatment (mean body weight of 100 mg/kg bw/day females was already higher than that of controls at initiation of treatment).
Reduced weight gain and/or weight loss was noted in a few individual animals that survived until scheduled necropsy (one high dose male in the third week of treatment, and one mid dose females and one high dose female during lactation). These findings were not attributed to treatment as they were transient or occurred in only a single animal of a group.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Food consumption (before and after correction for body weight) was considered not to be affected by treatment.
The statistically significantly higher mean food consumption noted in 1000 mg/kg bw/day females between Days 0-4 post-coitum and the markedly reduced food consumption of a single 1000 mg/kg bw/day female between Days 1-4 of lactation were considered to be unrelated to treatment because food consumption of these females was normal at the other time points.

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Haematological parameters in F0-females were considered not to be affected by treatment.
Any statistically significant variations noted in haematology parameters were considered unrelated to treatment due to the absence of a clear dose-related trend and slight magnitude of the differences from controls. Moreover, the control values in this study were slightly higher than normal. However, values in control and treated rats remained within normal limits.

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related microscopic observations in male rats.
In female rats, a slightly increased incidence of follicular cell hypertrophy in the thyroid gland (minimal degree) was noted at 300 and 1000 mg/kg bw/day, with incodences of 1, 2, 5 and 4 in control, low, mid and high dose females, respectively.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

Serum levels of T4 in F0-males were considered not to be affected by treatment.
In the absence of a dose related trend, the statistically significantly higher mean T4 value noted at 100 mg/kg bw/day was considered unrelated to treatment and of no toxicological significance.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

effects observed, non-treatment-related

Description (incidence and severity):

Length and regularity of the estrous cycle were considered not to be affected by treatment. Most females had regular cycles of four days. Extended di-estrus during pairing occurred in one female at 100 mg/kg bw/day (mated unsuccessfully) and one female at 300 mg/kg bw/day had an irregular cycle during the pre-mating period (it delivered a normal litter). These findings were considered to be unrelated to treatment due to their incidental occurrence and lack of a dose-related trend.

Description (incidence and severity):

One 100 mg/kg bw/day male was emaciated and showed a marked sperm granuloma in the left epididymidis, which probably attributed to the poor health condition and the fact the animal didn’t successfully mate. A unilateral sperm granuloma in itself, is no histopathological correlate to the lack of offspring for this couple, as normal sperm was present in both epididymides.
In addition, there were no morphological findings in the reproductive organs of the other microscopically examined animals of either sex which could be attributed to the test item and stage aware evaluation of the testes did not show any indication for abnormal spermatogenesis.

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

Two females were not pregnant, one of the control group and one of the 300 mg/kg bw/day group. Histopathology of these males and females did not reveal any changes in the reproductive organs that could explain the nonpregnancy in these females.
In another female of the 100 mg/kg bw/day group mating was not confirmed during the 14 days mating period. Histopathology did not reveal any changes in the reproductive organs in this female which could explain the non-mating and subsequent failure of delivering offspring. During the mating period, this female had been cohabitated with a male until day 6 of mating, followed by another male from Day 6 to Day 15 of mating.

mating index: Mating index was considered not to be affected by treatment. Except for one female at 100 mg/kg bw/day, all females showed evidence of mating. The mating indices were 90% for the 100 mg/kg bw/day group and 100% for the other groups.
On study Day 20, one male was sacrificed for humane reasons. This male had been cohabitated with one female for six days by then, but mating by the couple was not confirmed until the moment of removal of the male. Immediately after removal of male anothe male of the same dose group which previously mated successfully was cohabitated with the female for the remaining mating period. Also for this couple no successful mating was confirmed. In the absence of a dose relationship, the incidence of not mating was considered a fortuitous finding and not related to treatment.

Precoital time was not affected by treatment. All mated females showed evidence of mating within four days.

The number of implantation sites was considered not to be affected by treatment.
A relatively high mean number of implantation sites was observed in control females in comparison with that in the females treated groups. Since the mean number of implantation sites in treated females was comparable with the historical control values and in the absence of a dose response no toxicological significance was attached to this finding.

Except for one control female and one female at 300 mg/kg bw/day, all mated females were pregnant. These cases of non-pregnancy, which occurred in absence of related histopathology changes in reproductive organs, were considered to be unrelated to treatment due to their incidental occurrence and lack of a dose-related trend.

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Remarks:

reproduction toxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

2

Key result

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

no effects were observed on met-Hb

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs occurred among pups that were considered to be related to treatment.
The clinical signs observed incidentally remained within the range considered normal for pups of this age, and were therefore regarded to be unrelated to treatment.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

Viability index (number of live offspring on PND 4 before culling as percentage of number of live offspring on PND 1) was not affected by treatment. The viability indices were 100% for the 1000 mg/kg bw/day group and 99% for the other groups.
One pup of the control group and one pup at 100 mg/kg bw/day were found dead on PND 2 or 3, and one pup at 300 mg/kg (litter 64, with a large wound) was
sacrificed for humane reasons on PND 1. This post-natal loss was considered to be unrelated
to treatment as it was incidental and showed no dose-related trend.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Body weights of pups were considered not to be affected by treatment.
Slightly lower mean body weight of male and female pups of control female were observed in comparison with that of pups of treated females. This finding was considered to be related to the slightly higher litter size observed in control females, resulting in slightly smaller pups at birth. In the absence of a dose related affect and as normal growth was observed of pups of control and treated females during lactation, no toxicological significance was attached to this finding. As a consequence, the statistically significant differences apparent in some cases were not indicative of an effect by treatment.

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:

no effects observed

Description (incidence and severity):

Serum T4 levels in male and female PND 13-15 pups were considered not to be affected by treatment.

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

No macroscopic findings were noted among pups that were considered to be related to treatment.
The nature and incidence of the macroscopic findings noted incidentally remained within the range considered normal for pups of this age, and were therefore considered to be unrelated to treatment.

Histopathological findings:

not examined

Description (incidence and severity):

viability index: Viability index (number of live offspring on PND 4 before culling as percentage of number of live offspring on PND 1) was not affected by treatment. The viability indices were 100% for the 1000 mg/kg bw/day group and 99% for the other groups. One pup of the control group and one pup at 100 mg/kg bw/day were found dead on PND 2 or 3, and one pup at 300 mg/kg bw/day was sacrificed for humane reasons on PND 1. This post-natal loss was considered to be unrelated to treatment as it was incidental and showed no dose-related trend.

lactation index: No pups died or went missing after PND 4. The lactation index (number of live offspring on PND 13 as percentage of number of live offspring after culling on PND 4) was 100% for all groups.

sex ratio: Sex ratio was considered not to be affected by treatment.

anogenital distance: Anogenital distance (absolute and normalized for body weight) in male and female pups was considered not to be affected by treatment.

areola/nipple retention: Treatment up to and including 1000 mg/kg bw/day had no effect on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Any other information on results incl. tables

Dose Formulation Analyses

-accuracy: The mean accuracy of the analyzed concentrations in the formulations were 92, 90 and 90 % in the low, mid and high dose, respectively.

-homogeneity coefficient of variation was 0.42 and 5.6 in the low and high dose groups, respectively.

Formulations of the low and high dose groups were stable when stored at room temperature under normal laboratory light conditions for at least 18 hours.

Applicant's summary and conclusion

Conclusions:

In conclusion, based on the results of this reproduction/developmental toxicity screening test, the following No-Observed-Adverse-Effect Levels (NOAELs) of KY-AF were established:
Parental NOAEL: at least 1000 mg/kg bw/day
Reproduction NOAEL: at least 1000 mg/kg bw/day
Developmental NOAEL: at least 1000 mg/kg bw/day

Executive summary:

KY-AF was tested for its reproduction/developmental toxicity potential in a reproduction/developmental toxicity screening study (OECD 421) Wistar Han rats were treated with KY-AF by daily oral gavage at dose levels of 100, 300 and 1000 mg/kg bw/day (10 rats/sex/dose level). Concurrent controls (10 rats/sex) received the vehicle, propylene glycol, alone. Males were treated for two weeks prior to mating, during mating, and up to termination (for 29 days). Females that delivered offspring were treated for two weeks prior to mating, during mating, during post-coitum, and 13-15 days of lactation (for 50-55 days). Females without offspring were treated for 41 or 54 days.

Formulation analysis showed that the dosing formulations were prepared accurately and homogeneously, and that the formulations were stable for at least 18 hours at room temperature under normal laboratory light conditions.

Parental results

No parental toxicity was noted up to and including the highest dose level tested (1000 mg/kg bw/day).

There were no treatment-related changes in the in-life parameters examined in this study (i.e. mortality, clinical appearance, body weight and food consumption), serum level of thyroid hormone T4 (males), haematology parameters (females), organ weights (thyroid in both sexes and male reproductive organs) or findings at macroscopic examination.

Microscopic examination revealed a minor increase in the incidence of follicular cell hypertrophy in the thyroid of females treated at 300 or 1000 mg/kg bw/day compared to concurrent controls. It cannot be excluded that this finding was related to treatment with the test item. The severity of follicular cell hypertrophy in these dose groups (minimal) did not exceed that recorded in control females. Follicular cell hypertrophy at low degrees can be seen as a background finding in rats of this age and strain and was also noted in one female of the concurrent control group. Therefore this finding was considered to be non-adverse. No treatment-related microscopic changes were observed in the other organs examined (thyroid of male rats, testes, epididymides, ovaries).

Reproductive results:

No reproduction toxicity was observed up to and including the highest dose level tested (1000 mg/kg bw/day). No treatment-related changes were noted in any of reproductive parameters investigated in this study (i.e. mating and fertility indices, precoital time, number of implantation sites, estrous cycle, spermatogenic profiling, and histopathological examination of reproductive organs).

Developmental results:

No developmental toxicity was observed up to and including the highest dose level tested (1000 mg/kg bw/day). No treatment-related changes were noted in any of the developmental parameters investigated

in this study (i.e. gestation, viability and lactation indices, duration of gestation, parturition, sex ratio, maternal care and early postnatal pup development consisting of mortality, clinical signs, body weight, anogenital distance, areola/nipple retention, serum level of thyroid hormone T4 (PND 13-15) and macroscopic examination).

In conclusion, based on the results of this reproduction/developmental toxicity screening test, the following No-Observed-Adverse-Effect Levels (NOAELs) of KY-AF were established:

Parental NOAEL: at least 1000 mg/kg bw/day

Reproduction NOAEL: at least 1000 mg/kg bw/day

Developmental NOAEL: at least 1000 mg/kg bw/day