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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20 Jun 2017 to 09 Nov 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:
20 Jun 2017 to 09 Nov 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 nulliparous and non-pregnant: yes
- Age at study initiation: males 10 weeks old, females 13 weeks old
- Weight at study initiation: (P) Males: between 264 and 300 g; Females: 204 and 248 g.
- Fasting period before study: no
- Housing: On arrival and following the pretest (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: Municipal tap water was freely available to each animal via water bottles.
- Acclimation period: 7 days prior to start of the pretest period (females) or 8 days before the commencement of dosing (males).

The feed was analyzed by the supplier for nutritional components and environmental contaminants. Results of the analysis were provided by the supplier. 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. 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): 21 to 22
- Humidity (%): 50 to 59
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 06 Jul 2017 To: 31 Aug 2017
Route of administration:
oral: gavage
Vehicle:
other: 0.5% Methylcellulose
Details on exposure:
Test item dosing formulations (w/w) were homogenized by grinding test item using a mortar and pestle and mixing formulations with an ultra-turrax to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared daily as a suspension and dosed within 5 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. No adjustment was made for specific gravity of the vehicle and test item. No correction was made for the purity/composition of the test item.

VEHICLE
Vehicle testing was performed at the Test Facility to select the suitable vehicle and to establish a suitable formulation procedure.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dose formulation samples were collected for analysis one day prior to start of the treatment: concentration (all dose groups), homogeneity and stability (low and high dose groups).
Stability samples were kept at room temperature under normal laboratory light conditions for 5 hours, and then placed on dry ice. All other samples were stored on dry ice immediately after sampling.
Analyses were performed by using a validated analytical procedure.
Concentration analysis: Duplicate sets of samples 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 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 = 10%.
Stability analysis: During the course of this study at one occasion during the treatment phase, stability of the prepared formulation was determined at 5 hours at room temperature.
Duplicate sets of each sample 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:
- M/F ratio per cage: 1:1
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as Day 0 post-coitum
Duration of treatment / exposure:
Males were treated for 29 days, up to and including the day before scheduled necropsy. This included a minimum of 14 days prior to mating and during the mating period. Females that delivered were treated for 50-56 days, i.e. 14 days prior to mating (with the objective to cover at least two complete estrous cycles), the variable time to conception, the duration of pregnancy and at least 13-15 days after delivery, up to and including the day before scheduled necropsy. Females which failed to deliver were treated for 42-53 days.
The first day of dosing was designated as Day 1.
Frequency of treatment:
daily
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 information provided by the Sponsor (28-day repeated dose toxicity study with oral exposure of KY-UN in rats, reference 626492 TSR), and in an attempt to produce graded responses to the test item. During the 28-day study dose levels of 150, 450 and 1000 mg /kg bw/day were tested. At 150 mg/kg bw/day, no overt signs of toxicity were noted.
At 450 mg/kg bw/day and 1000 mg/kg bw/day the females showed lower body weight gain during the first week, whereas higher body weight gain was recorded during the third week. Concurrent effects were noted on the food consumption of females at 450 mg/kg bw/day and 1000 mg/kg bw/day. Slightly higher cholesterol levels were noted in females at 450 mg/kg bw/day and 1000 mg/kg bw/day. No further treatment-related effects were seen in any of the other examinations and/or assessments.
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.

FOOD CONSUMPTION:
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
Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no effect was suspected.

Other: Haematology including Met-Hb measurement; Thyroid hormone measurement

Estrous cycles were evaluated by examining the vaginal cytology of samples obtained by vaginal lavage. Daily vaginal lavage was performed for all females beginning 15 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.

in parental males:Testis weight, epididymis weight, prostate gland weight, seminal vesicles weight, spermatogenic profiling, microscopic examination reproductive organs. For the testes of all control and high dose males, and all males that failed to sire, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle.

Reproductive parameters as included in section 7.8.1.
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
- Number of late resorptions: No
Fetal examinations:
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, plasma thyroid hormone measurement.

GROSS EXAMINATION OF DEAD PUPS:
yes, for external abnormalities 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 poss
ible, defects or cause of death were evaluated.
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 or 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 3 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:
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

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:
no effects observed
Description (incidence and severity):
No treatment-related clinical signs were noted during daily detailed clinical observations. Incidental findings that were noted 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. At the incidence observed, these were not considered to be signs of toxicological relevance.
Mortality:
no mortality observed
Description (incidence):
No mortality occurred during the study period.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weights and body weight gain of treated animals remained in the same range as controls over the treatment period.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption before or after correction for body weight was similar to the control level over the treatment period.
The slightly lower mean food consumption and relative food consumption (not statistically significant) that was noted for females at 1000 mg/kg bw/day during the lactation phase was caused by one female, for which lower food intake was noted during this phase.This female had only one pup, which could have contributed to her lowered food consumption. As this was noted in a single female only and mean values remained within the normal range, this was considered not to represent a change of toxicological significance.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
Hematological parameters, including met-hemoglobin concentrations of treated rats were considered not to have been affected by treatment. The statistically significant lower white blood cell count in males at 100 mg/kg bw/day was not considered to be of toxicological relevance in the absence of a treatment-related distribution.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
At the end of the treatment period, serum levels of T4 in F0 males were considered not to have been affected by treatment. The slightly lower mean plasma T4 concentration in males at 1000 mg/kg bw/day was considered not toxicologically relevant as the effect was only minor (7% decrease compared to concurrent controls) and not statistically significant.
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-related alterations in organ weights.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no test item-related gross observations.
All recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.
Neuropathological findings:
not examined
Histopathological findings: neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related microscopic observations.
There were 3/10 couples of the control group and 2/10 couples of the group treated at 1000 mg/kg bw/day without offspring. Histopathology did not reveal any changes in the reproductive organs that could explain this reproductive failure. There were no morphological findings in the reproductive organs 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.
Other effects:
no effects observed
Description (incidence and severity):
No adverse effects have been observed on reproductive parameter.
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):
Number of implantation sites was not considered to be affected by treatment up to and including 1000 mg/kg bw/day.
The total number of offspring born compared to the total number of uterine implantations was not considered to be affected by treatment. For one control female, one female at 300 mg/kg bw/day and one female at 1000 mg/kg bw/day, the number of pups were slightly higher than the number of implantations. This phenomenon is observed from time to time and is caused by normal resorption of these areas during the 14-16 days of lactation. No toxicological relevance was attached to this finding in the current study.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
Mean litter size was slightly lower for females treated at 300 and 1000 mg/kg bw/day when compared to controls, but without reaching statistical significance. This was mainly caused by one female in each of these dose groups with a low number of pups (4 and 1, respectively; related to a lower number of implantation sites). As all other litters in these dose groups were of normal size (i.e. comparable to the historical control range), this finding was not considered to represent a sign of toxicity.
Early or late resorptions:
no effects observed
Dead fetuses:
effects observed, non-treatment-related
Description (incidence and severity):
The number of live offspring on Day 1 after littering compared to the total number of offspring born was not considered to be affected by treatment. One pup at 100 mg/kg bw/day and two pups at 300 mg/kg bw/day were found dead at first litter check. No toxicological relevance was attributed to these dead pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.
The number of live offspring on Day 4 before culling compared to the number of offspring on Day 1 was not considered affected by treatment. One pup of the control group and one pup at 1000 mg/kg bw/day were found dead on PND 3. No toxicological relevance was attributed to these dead pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.
The number of live offspring on Day 13 after littering compared to the number of live offspring on Day 4 (after culling) was not considered to be affected by treatment. One pup at 1000 mg/kg bw/day was sacrificed early on PND 8, based on its poor physical condition (i.e. lean appearance, no milk in the stomach). No toxicological relevance was attributed to this single early sacrificed pup since the mortality incidence remained within the range considered normal for pups of this age.
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
Gestation index and duration of gestation were not considered to be affected by treatment.
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
Fertility index was not considered to be affected by treatment. One control female and one female at 1000 mg/kg bw/day were not pregnant. Since these cases of non-pregnancy showed no dose-related incidence across the dose groups, and given the absence of any reproductive/developmental toxicity, this was not considered to be related to treatment.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Length and regularity of the estrous cycle were not considered to have been affected by treatment. All females had regular cycles of 4 days. Extended di-estrus occurred in one control female, which was not mated. Extended di-estrus also occurred in one female, which was scored sperm positive on Day 1 of mating. However less than 10 sperm cells were observed, therefore this female was considered not mated. Both females had a regular cycle during the pre-test and premating phase. For one female at 100 mg/kg bw/day, an extended di-estrus was scored as well. However, this female delivered, indicating that mating was overlooked. Given their incidental nature and absence of a dose-related incidence, these findings did not indicate a relation with treatment.
Mating index was not considered to be affected by treatment. One control female and one female at 1000 mg/kg bw/day did not show evidence of mating. At the low incidence observed and absence of dose response, this was considered to be unrelated to treatment.
Precoital time was not considered to be affected by treatment. All females showed evidence of mating within 5 days.
No signs of difficult or prolonged parturition were noted among the pregnant females. No deficiencies in maternal care were observed.
Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Description (incidence and severity):
Body weights of pups were not considered to be affected by treatment.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
The number of live offspring on Day 1 after littering compared to the total number of offspring born was not considered to be affected by treatment. One pup at 100 mg/kg bw/day and two pups at 300 mg/kg bw/day (in one litter) were found dead at first litter check. No toxicological relevance was attributed to these dead pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
Sex ratio was not considered to be affected by treatment.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
Mean litter size was slightly lower for females treated at 300 and 1000 mg/kg bw/day when compared to controls, but without reaching statistical significance. This was mainly caused by one female in each of these dose groups with a low number of pups (4 and 1, respectively; related to a lower number of implantation sites). As all other litters in these dose groups were of normal size (i.e. comparable to the historical control range), this finding was not considered to represent a sign of toxicity.
Changes in postnatal survival:
no effects observed
Description (incidence and severity):
The number of live offspring on Day 1 after littering compared to the total number of offspring born was not considered to be affected by treatment.
The number of live offspring on Day 4 before culling compared to the number of offspring on Day 1 was not considered affected by treatment.
The number of live offspring on Day 13 after littering compared to the number of live offspring on Day 4 (after culling) was not considered to be affected by treatment.
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 macroscopic findings remained within the range considered normal for pups of this age, and were therefore not considered to be related to treatment.
Skeletal malformations:
not examined
Visceral malformations:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Anogenital distance (absolute and normalized for body weight) in male and female pups was not considered 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 not considered to be affected by treatment. Serum T4 levels were slightly lower for male and female pups at 1000 mg/kg bw/day, when compared to controls, but as the mean values remained well within the range considered normal for rats of this age and strain3 and the changes were not statistically significant, these slight differences were considered not toxicologically relevant.
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

Accuracy and homogeneity test.

No test item was detected in the control group formulation.

Mean accuracy was 94.2%, 94.7% and 96.6% in the low, mid and high dose groups, respectively. The concentrations analyzed in the formulations of the low, mid and high dose groups were in agreement with the target concentrations (i.e. mean accuracies between 85% and 115%).

Homogeneity (coefficient of variation) was 2.6% and 2.1% in the low and high dose groups, respectively. The formulations of the low and high dose groups prepared were homogeneous (i.e. coefficient of variation = 10%).

Stability test.

The relative difference was 3.5% and -1.8% in the low and high dose group, respectively. Formulations at the entire range were stable when stored at room temperature under normal laboratory light conditions for at least 5 hours (i.e. relative difference = 10%).

Stability at storage conditions (23 days at =-70°C): mean recovery of 95.3% and 93.4% at high and low dose, respectively. The test item was stable in 0.5% methylcellulose over a storage period of at least 23 days at a temperature = -70°C.

Conclusions:
Based on the absence of any substance related adverse effect in the reproduction/developmental toxicity screening test, the NOAEL for parental, reproduction toxicity and developmental toxicity are at least 1000 mg/kg bw/day.
Executive summary:

KY-UN was studied in a reproduction/developmental toxicity screening test according to OECD guidelines and in accordance with GLP principles. Wistar Han rats were treated with KY-UN by daily oral gavage at dose levels of 100, 300 and 1000 mg/kg bw/day. The rats of the control group received the vehicle, 0.5% Methylcellulose, alone. Males were treated for 2 weeks prior to mating, during mating, and up to termination (for 29 days). Females that delivered offspring were treated for 2 weeks prior to mating, during mating, during post-coitum, and at least 13-15 days of lactation (for 50-56 days). Females that failed to deliver pups were treated for 42-53 days.

Test formulations prepared were considered homogeneous at the concentrations tested and analysis of the accuracy revealed acceptable levels. Test formulations prepared were considered stable, for at least 5 hours at room temperature.

Parental results

No parental toxicity was observed up to and including the highest dose level tested (1000 mg/kg bw/ day). No toxicologically significant changes were noted in any of the parameters investigated in this study (i.e. clinical appearance, body weight, food consumption, hematology investigations (including met-hemoglobin), male T4 thyroid hormone levels, macroscopic examination, organ weights, and microscopic examination).

Reproductive results

No reproduction toxicity was observed up to and including the highest dose level tested (1000 mg/ kg bw/day). No toxicologically significant changes were noted in any of the reproductive parameters investigated in this study (i.e. mating and fertility indices, precoital time, number of implantations, 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). In the control group, seven instead of the intended eight litters were available for evaluation. Sufficient information was available from control litters for a thorough toxicological evaluation. No toxicologically significant 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, T4 thyroid hormone levels and macroscopic examination).

In conclusion, based on the results of this reproduction/developmental toxicity screening test, a parental, reproduction and developmental No Observed Adverse Effect Level (NOAEL) of at least 1000 mg/kg bw/day for KY-UN was established.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
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

Test material

Constituent 1
Test material form:
solid: particulate/powder
Details on test material:
- Physical appearance: white powder
- Test item storage: at room temperature

Test animals

Species:
rat
Strain:
other: Crl: WI(Han)
Details on species / strain selection:
The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing by regulatory agencies. Charles River Den Bosch has general and reproduction/developmental historical data in this species from the same strain and source.
This animal model has been proven to be susceptible to the effects of reproductive toxicants. The total number of animals used in this study was considered to be the minimum required to properly characterize the effects of the test item. This study has been designed such that it does not require an unnecessary number of animals to accomplish its objectives.
At this time, studies in laboratory animals provide the best available basis for extrapolation to humans and are required to support regulatory submissions. Acceptable models which do not use live animals currently do not exist.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: males 10 weeks old, females 13 weeks old
- Weight at study initiation: (P) Males: between 264 and 300 g; Females: 204 and 248 g.
- Fasting period before study: no
- Housing: On arrival and following the pretest (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: Municipal tap water was freely available to each animal via water bottles.
- Acclimation period: 7 days prior to start of the pretest period (females) or 8 days before the commencement of dosing (males)

The feed was analyzed by the supplier for nutritional components and environmental contaminants. Results of the analysis were provided by the supplier. 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. 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): 21 to 22
- Humidity (%): 50 to 59
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 06 Jul 2017 To: 31 Aug 2017

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 0.5% Methylcellulose
Details on exposure:
Test item dosing formulations (w/w) were homogenized by grinding test item using a mortar and pestle and mixing formulations with an ultra-turrax to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared daily as a suspension and dosed within 5 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. No adjustment was made for specific gravity of the vehicle and test item. No correction was made for the purity/composition of the test item.

VEHICLE
Vehicle testing was performed at the Test Facility to select the suitable vehicle and to establish a suitable formulation procedure.
Details on mating procedure:
- M/F ratio per cage: 1:1
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as Day 0 post-coitum
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dose formulation samples were collected for analysis one day prior to start of the treatment: concentration (all dose groups), homogeneity and stability (low and high dose groups).
Stability samples were kept at room temperature under normal laboratory light conditions for 5 hours, and then placed on dry ice. All other samples were stored on dry ice immediately after sampling.
Analyses were performed by using a validated analytical procedure.
Concentration analysis: Duplicate sets of samples 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 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 = 10%.
Stability analysis: During the course of this study at one occasion during the treatment phase, stability of the prepared formulation was determined at 5 hours at room temperature.
Duplicate sets of each sample 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:
Males were treated for 29 days, up to and including the day before scheduled necropsy. This included a minimum of 14 days prior to mating and during the mating period. Females that delivered were treated for 50-56 days, i.e. 14 days prior to mating (with the objective to cover at least two complete estrous cycles), the variable time to conception, the duration of pregnancy and at least 13-15 days after delivery, up to and including the day before scheduled necropsy. Females which failed to deliver were treated for 42-53 days.
The first day of dosing was designated as Day 1.
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
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 information provided by the Sponsor (28-day repeated dose toxicity study with oral exposure of KY-UN in rats, reference 626492 TSR), and in an attempt to produce graded responses to the test item. During the 28-day study dose levels of 150, 450 and 1000 mg/kg bw/day were tested. At 150 mg/kg bw/day, no overt signs of toxicity were noted.
At 450 mg/kg bw/day and 1000 mg/kg bw/day the females showed lower body weight gain during the first week, whereas higher body weight gain was recorded during the third week. Concurrent effects were noted on the food consumption of females at 450 mg/kg bw/day and 1000 mg/kg bw/day. Slightly higher cholesterol levels were noted in females at 450 mg/kg bw/day and 1000 mg/kg bw/day. No further treatment-related effects were seen in any of the other examinations and/or assessments.

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.

FOOD CONSUMPTION:
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
Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no effect was suspected.

Other: Haematology including Met-Hb measurement; Thyroid hormone measurement
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 15 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.
Sperm parameters (parental animals):
Parameters examined in male parental generation:
testis weight, epididymis weight, prostate gland weight, seminal vesicles weight, spermatogenic profiling, microscopic examination reproductive organs

For the testes of all control and high dose males, and all males that failed to sire, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
To reduce variability among the litters, on PND 4 eight pups from each litter of equal sex distribution (if possible) were selected. Blood samples were collected from two of the surplus pups (if possible from one male and one female pup). Selective elimination of pups, e.g. based upon body weight or AGD, was not done. Whenever the number of male or female pups prevents having four of each sex per litter, partial adjustment (for example, five males and three females) was acceptable.

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, plasma thyroid hormone measurement.

GROSS EXAMINATION OF DEAD PUPS:
yes, for external abnormalities 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.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: no

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: no
Postmortem examinations (parental animals):
SACRIFICE
Animals surviving until scheduled euthanasia were weighed, and deeply anaesthetized using isoflurane and subsequently exsanguinated and subjected to a full post mortem examination.
Scheduled necropsies were conducted on the following days:
Males (which sired and failed to sire): 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); Without evidence of mating: 25 days after the last day of the mating period.

All animals surviving to scheduled necropsy were fasted (overnight with a maximum of approximately 24 hours) before their scheduled necropsy. Water was available.
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.

GROSS NECROPSY
All animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs.

HISTOPATHOLOGY / ORGAN WEIGHTS
Tissues were prepared for microscopic examination and weighed according to Guidelines.
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.

On PND 4, the surplus pups (> 8 pups per litter) were euthanized by decapitation. From two surplus pups per litter, blood was collected, if possible. All remaining pups were euthanized on PND 13-15. Sex was determined both externally and internally. 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 (if possible 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 or 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 3 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:
no effects observed
Description (incidence and severity):
No treatment-related clinical signs were noted during daily detailed clinical observations. Incidental findings that were noted 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. At the incidence observed, these were not considered to be signs of toxicological relevance.
Mortality:
no mortality observed
Description (incidence):
No mortality occurred during the study period.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weights and body weight gain of treated animals remained in the same range as controls over the treatment period.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption before or after correction for body weight was similar to the control level over the treatment period.
The slightly lower mean food consumption and relative food consumption (not statistically significant) that was noted for females at 1000 mg/kg bw/day during the lactation phase was caused by one female, for which lower food intake was noted during this phase.This female had only one pup, which could have contributed to her lowered food consumption. As this was noted in a single female only and mean values remained within the normal range, this was considered not to represent a change of toxicological significance.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
Hematological parameters, including met-hemoglobin concentrations of treated rats were considered not to have been affected by treatment.
The statistically significant lower white blood cell count in males at 100 mg/kg bw/day was not considered to be of toxicological relevance in the absence of a treatment-related distribution.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
At the end of the treatment period, serum levels of T4 in F0 males were considered not to have been affected by treatment.
The slightly lower mean plasma T4 concentration in males at 1000 mg/kg bw/day was considered not toxicologically relevant as the effect was only minor (7% decrease compared to concurrent controls) and not statistically significant.
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.
There were 3/10 couples of the control group and 2/10 couples of the group treated at 1000 mg/kg bw/day without offspring. Histopathology did not reveal any changes in the reproductive organs that could explain this reproductive failure. There were no morphological findings in the reproductive organs 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.
Histopathological findings: neoplastic:
no effects observed

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 not considered to have been affected by treatment.
All females had regular cycles of 4 days. Extended di-estrus occurred in one control female, which was not mated. Extended di-estrus also occurred in one high dose female female, which was scored sperm positive on Day 1 of mating. However less than 10 sperm cells were observed, therefore this female was considered not mated. Both females had a regular cycle during the pre-test and premating phase. For one low dose female, an extended di-estrus was scored as well. However, this female delivered, indicating that mating was overlooked. Given their incidental nature and absence of a dose-related incidence, these findings did not indicate a relation with treatment.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
No toxicologically significant changes were noted in spermatogenic profiling.
Reproductive performance:
no effects observed
Description (incidence and severity):
No reproduction toxicity was observed up to and including the highest dose level tested (1000 mg/kg bw/day).
Mating index was not considered to be affected by treatment. One control female and one female at 1000 mg/kg bw/daydid not show evidence of mating. At the low incidence observed and absence of dose response, this was considered to be unrelated to treatment.
Precoital time was not considered to be affected by treatment. All females showed evidence of mating within 5 days.
Number of implantation sites was not considered to be affected by treatment up to and including 1000 mg/kg bw/day.
Fertility index was not considered to be affected by treatment.
One control female and one female at 1000 mg/kg bw/day were not pregnant. Since these cases of non-pregnancy showed no dose-related incidence across the dose groups, and given the absence of any reproductive/developmental toxicity, this was not considered to be related to treatment.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
general 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
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

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):
For the pup of one control female who was found dead on Day 3, absence of milk in the stomach was noted at last litter check. In addition, no milk in the stomach and a lean appearance was noted for one pup (surviving until scheduled necropsy) of one high dose female on Day 7 only. The nature and incidence of these and other clinical signs remained within the range considered normal for pups of this age, and were therefore not considered to be toxicologically relevant.
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
The total number of offspring born compared to the total number of uterine implantations was not considered to be affected by treatment. For one control female, one mid dose female no. 69 and one high dose female, the number of pups were slightly higher than the number of implantations.This phenomenon is observed from time to time and is caused by normal resorption of these areas during the 14-16 days of lactation. No toxicological relevance was attached to this finding in the current study.
Mean litter size was slightly lower for females treated at 300 and 1000 mg/kg bw/day when compared to controls, but without reaching statistical significance. This was mainly caused by one female in each of these dose groups with a low number of pups (4 and 1, respectively; related to a lower number of implantation sites). As all other litters in these dose groups were of normal size (i.e. comparable to the historical control range), this finding was not considered to represent a sign of toxicity.
The number of live offspring on Day 1 after littering compared to the total number of offspring born was not considered to be affected by treatment. One pup at 100 mg/kg bw/day and two pups at 300 mg/kg bw/day (both in one litter) were found dead at first litter check. No toxicological relevance was attributed to these dead pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.
The number of live offspring on Day 4 before culling compared to the number of offspring on Day 1 was not considered affected by treatment. One pup of the control group and one pup at 1000 mg/kg bw/day were found dead on PND 3. No toxicological relevance was attributed to these dead pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.
The number of live offspring on Day 13 after littering compared to the number of live offspring on Day 4 (after culling) was not considered to be affected by treatment. One pup at 1000 mg/kg bw/day was sacrificed early on PND 8, based on its poor physical condition (i.e. lean appearance, no milk in the stomach). No toxicological relevance was attributed to this single early sacrificed pup since the mortality incidence remained within the range considered normal for pups of this age.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weights of pups were not considered to be affected by treatment.
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 not considered to be affected by treatment. Serum T4 levels were slightly lower for male and female pups at 1000 mg/kg bw/day, when compared to controls, but as the mean values remained well within the range considered normal for rats of this age and strain and the changes were not statistically significant, these slight differences were considered not toxicologically relevant.
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 macroscopic findings remained within the range considered normal for pups of this age, and were therefore not considered to be related to treatment.
Histopathological findings:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Sex ratio was not considered to be affected by treatment.
Anogenital distance (absolute and normalized for body weight) in male and female pups was not considered 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.

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Remarks:
developmental toxicity
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

Accuracy and homogeneity test.

No test item was detected in the control group formulation.

Mean accuracy was 94.2%, 94.7% and 96.6% in the low, mid and high dose groups, respectively. The concentrations analyzed in the formulations of the low, mid and high dose groups were in agreement with the target concentrations (i.e. mean accuracies between 85% and 115%).

Homogeneity (coefficient of variation) was 2.6% and 2.1% in the low and high dose groups, respectively. The formulations of the low and high dose groups prepared were homogeneous (i.e. coefficient of variation = 10%).

Stability test.

The relative difference was 3.5% and -1.8% in the low and high dose group, respectively. Formulations at the entire range were stable when stored at room temperature under normal laboratory light conditions for at least 5 hours (i.e. relative difference = 10%).

Stability at storage conditions (23 days at =-70°C): mean recovery of 95.3% and 93.4% at high and low dose, respectively. The test item was stable in 0.5% methylcellulose over a storage period of at least 23 days at a temperature = -70°C.

Applicant's summary and conclusion

Conclusions:
Based on the absence of any substance related adverse effect in the reproduction/developmental toxicity screening test, the NOAEL for parental, reproduction toxicity and developmental toxicity are at least 1000 mg/kg bw/day.
Executive summary:

KY-UN was studied in a reproduction/developmental toxicity screening test according to OECD guidelines and in accordance with GLP principles.

Wistar Han rats were treated with KY-UN by daily oral gavage at dose levels of 100, 300 and 1000 mg/kg bw/day. The rats of the control group received the vehicle, 0.5% Methylcellulose, alone. Males were treated for 2 weeks prior to mating, during mating, and up to termination (for 29 days). Females that delivered offspring were treated for 2 weeks prior to mating, during mating, during post-coitum, and at least 13-15 days of lactation (for 50-56 days). Females that failed to deliver pups were treated for 42-53 days.

Test formulations prepared were considered homogeneous at the concentrations tested and analysis of the accuracy revealed acceptable levels. Test formulations prepared were considered stable, for at least 5 hours at room temperature.

Parental results

No parental toxicity was observed up to and including the highest dose level tested (1000 mg/kg bw/day). No toxicologically significant changes were noted in any of the parameters investigated in this study (i.e. clinical appearance, body weight, food consumption, hematology investigations (including met-hemoglobin), male T4 thyroid hormone levels, macroscopic examination, organ weights, and microscopic examination).

Reproductive results

No reproduction toxicity was observed up to and including the highest dose level tested (1000 mg/kg bw/day). No toxicologically significant changes were noted in any of the reproductive parameters investigated in this study (i.e. mating and fertility indices, precoital time, number of implantations, 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). In the control group, seven instead of the intended eight litters were available for evaluation. Sufficient information was available from control litters for a thorough toxicological evaluation. No toxicologically significant 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, T4 thyroid hormone levels and macroscopic examination).

In conclusion, based on the results of this reproduction/developmental toxicity screening test, a parental, reproduction and developmental No Observed Adverse Effect Level (NOAEL) of at least 1000 mg/kg bw/day for KY-UN was established.

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