Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
05 August 2014 to 29 September 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, performed in accordance with valid guidelines and the study was conducted under GLP conditions.
Cross-reference
Reason / purpose for cross-reference:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report date:
2014

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Tetrasodium 7-[[4-[[4,6-bis[(3-sulfonatopropyl)thio]-1,3,5-triazin-2-yl]amino]-3-methoxyphenyl]azo]naphthalene-1,3-disulfonate
EC Number:
428-480-0
EC Name:
Tetrasodium 7-[[4-[[4,6-bis[(3-sulfonatopropyl)thio]-1,3,5-triazin-2-yl]amino]-3-methoxyphenyl]azo]naphthalene-1,3-disulfonate
Cas Number:
214559-61-2
Molecular formula:
C26H24N6O13S6Na4
IUPAC Name:
tetrasodium 7-{2-[4-({4,6-bis[(3-sulfopropyl)sulfanyl]-1,3,5-triazin-2-yl}amino)-3-methoxyphenyl]diazen-1-yl}naphthalene-1,3-disulfonate
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Appearance: Orange powder
- Storage condition of test material: Controlled room temperature (15 - 25 °C, <70 RH %) protected from light and humidity

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Strain: Crl:WI
- Age at study initiation: 10 - 11 weeks
- Weight at study initiation: 360 - 404 g (males); 212 - 256 g (females)
- Housing: Rodents were group-housed as practical, up to 3 animals of the same group per cage (during the mating and gestation/delivery period, they were paired or individually housed, respectively). Animals were housed in Type II and III polypropylene/polycarbonate cages with Lignocel® Hygienic Animal Bedding
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20.6 - 25.0 °C
- Humidity: 33 - 66 % (relative)
- Air changes: 15 - 20 air exchanges per hour
- Photoperiod: 12 hours of darkness / 12 hours of light (06:00 to 18:00)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
(distilled)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test material was formulated in the vehicle as a visibly stable homogenous formulation at the appropriate concentrations according to the dose level and volume selected. Formulations were prepared fresh prior to administration to animals or at the appropriate frequency to allow their use according to stability assessment results.

ADMINISTRATION OF DOSING SOLUTIONS:
The dosing solutions were administered using a bulb-tipped gavage needle attached to a syringe. A volume of 5 mL/kg bw was administered to all animals. The actual volume administered was calculated and adjusted based on each animal’s most recent body weight. Control animals were treated concurrently with the vehicle only.
Details on mating procedure:
- M/F ratio per cage: 1 male / 1 female
- Length of cohabitation: Females remained with the same male until the copulation occurred
- Proof of pregnancy: vaginal plug or sperm in vaginal smear was referred to as day 0 of pregnancy
- After 14 days of unsuccessful pairing, replacement of first male by another male
- After successful mating each pregnant female was caged individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Triplicate samples were taken from the top, middle and bottom of the test material formulations on 2 occasions, during the first and the last weeks of treatment. Two sets were taken for analysis (collected in replicates, as practical) and one set was taken as a back-up for any confirmatory analyses, as necessary. Similarly, one sample was taken in duplicate from the vehicle (control / Group 1) solution for concentration measurements.

- HPLC conditions for analysis of test material concentration:
Column: ACE 5 AQ 125 × 4.6 mm, 5 μm
Mobil Phase: Methanol/Buffer pH = 6 (5 mM TEA/5 mM NaH₂PO₄) = 4/6
Flow: 1.0 mL/min.
Injection volume: 10 μL
Temperature: 25 °C
Detector: UV at 255 nm
Retention time: 4.5 min ± 10 %
Duration of treatment / exposure:
Males were dosed for up to 34 days (14 days pre-mating and up to 20 days mating/post-mating), then they were euthanised and subjected to necropsy examination.
Females were dosed for 14 days pre-mating, for up to 16 days mating period, through gestation and up to and including the day before necropsy, 4 days post-partum dosing. The day of birth (viz. when parturition was complete) was defined as Day 0 post-partum.
Frequency of treatment:
Daily during the treatment period (on a 7 days/week basis)
Doses / concentrations
Remarks:
Doses / Concentrations:
0 (vehicle control), 100, 300, 1000 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
12 males and 12 females per group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected based on available data, including the results of a dose range finding study.
- Rationale for animal assignment (if not random): All parental (P) animals were sorted according to body weight by computer and divided into weight ranges. There were an equal number of animals from each weight group in each of the experimental groups assigned by randomisation to ensure that animals of all test groups were as close as practicable to a uniform weight. The grouping was controlled by SPSS/PC+4.0 software according to the actual body weight, thereby verifying the homogeneity/variability between/within the groups and cages. Males and females were randomised separately.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Parental animals were inspected for signs of morbidity and mortality twice daily (at the beginning and end of each day). General clinical observations were made once a day, after treatment, at approximately the same time as practical. Pertinent behavioural changes, signs of difficult or prolonged parturition and all signs of toxicity, including mortality, were monitored.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: More detailed examinations were made once before the first exposure (to allow for within-subject comparisons), and at least once a week thereafter. These observations were made outside the home cage in a standard arena. Signs evaluated included monitoring for any changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern), changes in gait, posture, and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling), difficult or prolonged parturition, or bizarre behaviour (e.g. self-mutilation, walking backwards). Special attention was directed towards the observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.

BODY WEIGHT: Yes
- Time schedule for examinations: All adult animals were weighed on Day -1, on Day 0 and at least weekly thereafter until termination. Parent females were weighed on gestation Days (GD) 0, 7, 14 and 20 and on post-partal Days (PPD) 0 (within 24 hours after parturition) and on PPD3. The females were additionally weighed on GD 4, 10 and 17 in order to give accurate treatment volumes.

FOOD CONSUMPTION: Yes
- Time schedule: Animal food consumption was determined by re-weighing the non-consumed diet with a precision of 1 g on Day 7 then at least weekly thereafter.

WATER CONSUMPTION: No

OTHER:
On gestation Day (GD) 13 or 14 the sperm positive females were examined for the presence of vaginal bleeding or “placental sign” (intrauterine extravasation of blood as an early sign of pregnancy in rat).
In addition, the duration of gestation was recorded and was calculated from Day 0 of pregnancy. Dams were observed for nesting behaviour (whether they made a nest from the bedding material, and covered their new-borns, or not). The efficiency of the suckling was observed by the presence of milk in the stomach. All observations were recorded.
Sperm parameters (parental animals):
Parameters examined in P male parental generation: testis and epididymis weight. Special attention was paid to evaluation of the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure after necropsy.
Litter observations:
PARAMETERS EXAMINED
Each litter was examined as soon as possible after delivery to establish the number and sex of pups, stillbirths, live births, runts (pups that are significantly smaller than normal pups) and the presence of gross abnormalities. Live pups were counted, sexed, weighed individually within 24 hours of parturition (on the first day after parturition was complete, i.e. Day 0 or 1 post-partum) and on Day 4 post-partum with an accuracy of 0.01 g.
All the litters were checked and recorded daily for the number of viable and dead pups. The dead pups found were subjected to necropsy with macroscopic examination. All observed abnormalities were recorded and reported. Some of the pups that were found dead were cannibalised, thus, they were counted and sex determined (when it was possible), but were not further examined macroscopically.
Postmortem examinations (parental animals):
SACRIFICE
Terminally, after completion of treatment, animals were euthanised under pentobarbital anaesthesia, followed by exsanguination.

GROSS NECROPSY
Gross necropsy was performed on all animals. After exsanguination the external appearance was examined, cranium, thoracic and abdominal cavities were opened and the appearance of the tissues and organs were observed macroscopically. Any abnormality was recorded with details of the location, colour, shape and size, as appropriate.
Special attention was paid to the organs of the reproductive system. The number of implantation sites and of corpora lutea was recorded.

ORGAN WEIGHTS
At the time of termination, body weight and weight of the following organs of all parental animals were determined:
- With a precision of at least 0.01 g: uterus (with cervix), testes, epididymides, prostate, seminal vesicles with coagulating glands, brain
- With a precision of at least 0.001 g: ovaries
Testes and epididymides were weighed individually; absolute organ weights were measured and reported. Total weight of paired organs was calculated and reported. Relative organ weights (to body and brain weight) were calculated and reported.
The weighed organs and all organs showing macroscopic lesions of all adult animals were preserved. Testes and epididymides were preserved in Bouin’s solution, all other organs in 10 % buffered formalin solution.

HISTOPATHOLOGY
Detailed histological examination was performed on the selected list of retained organs in the control and high dose groups and any macroscopic findings observed in all animals.
Special attention was paid to evaluation of the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure. Detailed histological examination of the ovaries covered the follicular, luteal, and interstitial compartments of the ovary, as well as the epithelial capsule and ovarian stroma.
The retained tissues and organs were embedded in paraffin wax, and sections were cut (4-6 µm) and transferred to slides. Tissue sections were stained with haematoxylin-eosin/phloxine and examined by light microscopy.
Postmortem examinations (offspring):
Pups euthanised at PND 4 were carefully examined at least externally for gross abnormalities.
Reproductive indices:
The following reproductive indices were investigated:

Male Mating Index = (Number of males with confirmed mating / Total number of males cohabited) x 100
Female Mating Index = (Number of sperm-positive females / Total number of females cohabited) x 100
Male Fertility Index = (Number of males impregnating a female / Total number of males cohabited) x 100
Female Fertility Index = (Number of pregnant females / Number of sperm-positive females) x 100
Gestation Index = (Number of females with live born pups / Number of pregnant females) x 100
Offspring viability indices:
The following offspring viability indices were investigated:

Survival Index = (Number of live pups (at designated time) / Number of pups born) x 100
Pre-implantation Mortality = [(Number of corpora lutea - Number of implantations) / Number of corpora lutea] x 100
Intrauterine Mortality = [(Number of implantations - Number of liveborns) / Number of implantations] x 100
Total Mortality = [(Number of implantations - Number of viable pups (D4)) / Number of implantations] x 100
Sex ratio = [(Number of pups examined - Number of males) / Number of pups examined] x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
There was no test material related mortality during the study. One female from the mid dose group (3509) was euthanised pre-terminally due to animal welfare reasons, as the animal was diagnosed with severe prolapse of uterus.
No test material-related adverse effects or systemic clinical signs were noted. Piloerection, abnormal skin colour (paleness of both pinnas, tail, both fore paws, both hind paws, both eyes) and red liquid around the urogenital area was noted in female 3502. Red liquid and vaginal bleeding was noted in animal 3509. All signs detailed above are not considered to be test material related, but ascribed to prolapsed uterus observed in both females. The prolapse was so severe in animal 3509, that euthanasia was performed; female 3502 remained on study until scheduled necropsy.
In male 3008, piloerection, abnormal skin colour (tail, both pinnas), laboured respiration (moderate) prostration and red discharge (both eyes) were observed, associated with perforated oesophagus found at necropsy, therefore the signs are considered to be the result of gavage error and were not considered to be test material-related.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
No test material-related effects were noted on group man body weights or body weight gains.
Sporadic statistical differences between groups were not considered to be treatment related. Compared to the control, statistically significantly higher body weight gains were noted in high dose group (1000 mg/kg bw/day) in males in the post-mating period (p<0.01 Day 14 to 21 and p<0.05 Day 0 to 27). The statistical significance is ascribed to a relatively lower control rather than a real test material related effect. In females, statistically significantly lower body weight group mean value was observed in the mid dose group (300 mg/kg bw/day) on PPD0 compared to the control. No dose response was noted however, and it was therefore considered incidental without toxicological significance. Statistically significantly lower mean body weight gains were noted sporadically in females in the low and mid dose groups (100 and 300 mg/kg bw/day, respectively) during the study, but no dose response was noted, and they were therefore considered incidental with no toxicological relevance.
There were no test material-related differences in the mean daily food consumption in any treated group compared to the control.
Statistically significantly higher than control group mean values were noted in males in the low and high dose groups (p<0.05 and p<0.01 respectively) at different measuring points during mating period, however the statistical significance is ascribed to a relatively low control value rather than a test material related effect. Statistically significantly lower than control group mean values were sporadically observed in females during the gestation period in the low and mid dose group; however with the lack of dose response it is considered incidental without toxicological relevance. Statistically significantly higher than control food consumption group mean values were noted in mid and high dose females from PPD0 to PPD3, but were considered to have no toxicological relevance.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
There were no significant differences between the control and test material-treated groups with regard to reproductive ability or in the mating or gestation indices.
Lower than control male fertility index was noted in low and mid dose groups, but not in high dose group, therefore in the lack of dose response it is considered incidental. A reduced fertility index was noted in low dose group females, compared to controls. However, in the absence of a dose response, it was also considered as incidental, and without toxicological relevance. A gestation index significantly lower than the control index was observed only in mid dose group females; however, in this dose group, two females had prolapse of uterus, from which one was euthanised pre-terminally (before delivery), and the second delivered but no live born pups were found. Prolapse of uterus and its effect on gestation index is therefore considered incidental, and not test material related.
Test material administration was considered to have no impact on the duration of the mating period. Successful coitus (sperm positive vaginal smears and/or vaginal plugs) generally occurred within one oestrus cycle (approximately 5 days) in all dose groups with a very limited number of exceptions (1-1 cases in the mid and high dose groups, where pseudo-pregnancy was observed in the females). In both cases, successful coitus was noted directly after the end of the pseudo-pregnancy.
There were no effects on the duration of gestation, or abnormalities in gestation outcome, that could be ascribed to treatment.
The mean duration of gestation (21 to 23 days) was similar between the control and treated groups. All parturitions were normal.
The number of corpora lutea and implantation sites in the treated groups was comparable with the values recorded for the control group. In comparison with the control, a statistically significant decrease was noted in mean pre-implantation loss in the low dose group, which was considered incidental without toxicological relevance.

ORGAN WEIGHTS (PARENTAL ANIMALS)
There were no toxicologically significant effects on organ weights.

GROSS PATHOLOGY (PARENTAL ANIMALS)
No test material-related findings were observed at necropsy. Dilation of the uterine horns (found in one low, one mid and one high dose females); perforation of the oesophagus, yellow liquid material in the thoracic cavity and yellow discolouration of the gastric mucosa and testes, red material on the periorbital region and dark red discolouration of the lungs (in one mid dose male found dead) were regarded as incidental.

HISTOPATHOLOGY (PARENTAL ANIMALS)
No test material-related microscopic findings were observed in the parental animals. Luminal dilatation (observed in one low dose and one mid dose female) and intraluminal haemorrhages of the uterine horns (observed in one mid and one high dose female), focal haemorrhage in the wall of the oesophagus and congestion/haemorrhage in the lungs (3008 mid dose male) correlated with necropsy, and the spermatocele in one control male, were considered as incidental or background.
The follicular, luteal and interstitial compartments of the ovary as well as epithelial capsule and stroma were similar in histological structure in both control and high dose females. The primordial, secondary and tertiary follicles and corpora lutea were also bilaterally presented.
No test material related microscopic changes were noted in testes, epididymides, seminal vesicles and prostate. The spermatogenic cells representing different phases of the development and differentiation of the spermatozoons as well as interstitial cell structure were similar in control and high dose males.

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Details on results (F1)

VIABILITY (OFFSPRING)
The number of viable pups on PND4 as well as pups survival indices on PND0 and PND4 were comparable to control values at up to, and including, 1000 mg/kg bw/day. Overall there were no treatment-related effects on pup mortality, and there were no treatment related effect on the viability of pups on PND0 and PND 4.

CLINICAL SIGNS (OFFSPRING)
No adverse effects were observed in the F1 generation. The pups found cannibalised were counted and the sex determined where possible, but were not subjected to a necropsy. A few surviving pups were cold, not suckled, or haemorrhagic. No external abnormalities ascribable to treatment were detected at the clinical or external macroscopic examinations of the pups. The incidence of any findings was low, and within the physiological range expected in Wistar rats, hence they were considered incidental, and unrelated to treatment.

BODY WEIGHT (OFFSPRING)
There were no adverse effects on the weight or weight gain of offspring. When evaluated on a per-litter basis, the mean litter body weights (PND 0 and 4) and/or weight gains of pups (PND 4) showed no statistically or toxicologically significant differences compared to controls.
When evaluated on a whole group mean basis, statistically significant decrease (p≤0.05) in the mid dose group (300 mg/kg bw/day) on PND0, and a statistically significant increase (p≤0.01) in the high dose group on PND4 was noted in mean body weight group mean value in comparison with the control, which is considered incidental with no toxicological relevance. Also, a statistically significant elevation in comparison with the control was noted in body weight gain group mean value in the high dose group (1000 mg/kg bw/day) on PND4, considered incidental and without toxicological meaning.

SEXUAL RATIO (OFFSPRING)
A statistically significantly higher number of male pups was noted in all treated dose groups on PND0. A similar ratio was noted on PND4, no higher rate of mortality was noted in females. The numbers of female pups and male pups were in the normal historical range. No dose response was noted, therefore it is considered incidental with no toxicological relevance.

GROSS PATHOLOGY (OFFSPRING)
No test material related macroscopic changes were seen in the F1 offspring generation examined at scheduled termination. One pup from the control group was observed with generalised oedema, total situs inversus, discoloured liver, large pancreas, yellow-white intestinal content, dark red adrenal gland, and a defect was noted atrioventricular septum in the heart. It was regarded incidental with no toxicological relevance.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Dose Formulation Analysis

Test material content and homogeneity of the dosing formulations was determined two times during the treatment period (during the first and sixth weeks of the treatment). No test material was detected in the control solution samples. All formulations were shown to be homogeneous. All formulations were found to be in the range of 101 to 104 % of nominal concentrations. Based on these results, the formulations were considered acceptable.

Table 1: Summary of Fertility Data

Dose (mg/kg bw/day)

Control

100

300

1000

Paired males - females

12/12

12/12

12/12

12/12

Mated males - females

12/12

12/12

11/12

12/12

Male Fertility Index (%)

100

75

92

100

Female Fertility Index (%)

100

75

100

100

Non-pregnant females

0/12

3/12

0/12

0/12

Gestation Index (%)

100

100

67

92

Pregnant females, but not delivered

0/12

0/12

3*/12

1/12

*1 of 3 had prolapse of the uterus

Table 2: Summary of Gestation/ Delivery Data

Females

Dose (mg/kg bw/day)

Control

100

300

1000

Number of pregnant females

12/12

9/12

12/12

12/12

Number of females which delivered

12/12

9/12

9/12

11/12

Mean duration of gestation (days)

22.23

22.00

22.33

22.18

Mean number of corpora lutea

12.58

13.67

14.08

14.33

Mean number of implantations

12.17

10.83

13.08

13.67

Mean pre-implantation loss (%)

0.42

2.83*

1.00

0.67

Mean number of pups delivered

11.42

14.44

10.83

12.83

Intrauterine mortality (%)

12.80

2.21*

37.37

12.16

*p ≤ 0.05

Table 3: Body Weights (Offspring)

Dose (mg/kg bw/day)

Control

100

300

1000

Mean for all pups

Mean body weight (g), PND0

124 pups

127 pups

119 pups

154 pups

6.521

6.450

6.346*

6.610

Mean body weight (g), PND4

118 pups

127 pups

114 pups

149 pups

9.892

9.634

9.738

10.356**

Body weight gain (g), PND0-4

3.363

3.184

3.379

3.737**

*p ≤ 0.05

**p ≤ 0.01

Table 4: Sex Ratio (Offspring)

Dose (mg/kg bw/day)

Control

(12 dams)

100

(9 dams)

300

(8 dams)

1000

(11 dams)

Sex ratio PND0

56.28

40.35*

46.16

42.42*

Sex ratio PND4

57.04

40.35*

45.68

42.01*

*p ≤ 0.05

Applicant's summary and conclusion

Conclusions:
Under the conditions of the study the No Observed Adverse Effect level was determined to be 1000 mg/kg bw/day, the highest dose level tested.
Executive summary:

The reproductive toxicity of the test material was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guideline OECD 421.

During the study groups of 12 male and 12 female Wistar rats were administered with test material in distilled water at doses of 0 (vehicle control), 100, 300 and 1000 mg/kg bw/day. Males were dosed for up to 34 days (14 days pre-mating and up to 20 days mating/post-mating), then they were euthanised and subjected to necropsy examination. Females were dosed for 14 days pre-mating, for up to 16 days for the mating period, through gestation and up to and including the day before necropsy, 4 days post-partum dosing. The day of birth was defined as Day 0 post-partum.

Adult animals were inspected for signs of morbidity and mortality twice daily. Clinical observations were performed once daily, with detailed examination performed weekly. Special attention was paid to evaluation of the mating, pregnancy, parturition and postpartum

periods, and relevant parameters and/or indices were measured and/or calculated. Body weight and food consumption were measured at least weekly.

After delivery, all pups from each litter were counted, sex established, weighed on post-natal days (PND) 0 and 4, offspring evaluated for presence of stillbirths, live births, runts (pups that are significantly smaller than normal pups) or any gross

abnormalities, then examined clinically at least daily.

Gross necropsy of the adult parental animals was conducted at the end of the treatment period and the weights of a standard list of organs were measured. Pups euthanized at PND 4 were carefully examined at least externally for gross abnormalities. Detailed histological examination was performed on the selected list of retained organs (ovaries, testes, epididymides,) in the control and High dose and all macroscopic findings (abnormalities) from all animals. Special attention was paid to evaluation of the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure. Detailed histological examination of the ovaries covered the follicular, luteal, and interstitial compartments of the ovary, as well as the epithelial capsule and ovarian stroma.

Under the conditions of the study, daily administration of Y-1189 by oral gavage to Wistar rats at up to 1000 mg/kg bw/day did not result in test item related mortality, clinical adverse effects, or changes in body weight or food consumption.

No test item related, or adverse effects were noted at evaluation of the reproductive parameters, either during the mating, gestation, delivery or post-partum/lactation period up to post-partum Day 4.

There were no adverse effects ascribable to test item administration on the F1 offspring viability, clinical signs, development or at observation following euthanasia.

There were no test item-related changes in organ weights, gross findings or histopathology of the adult animals.

In conclusion, under the conditions of this study, the no observed adverse effect level (NOAEL) for Y-1189 for parental/adult and F1 effects is considered to be 1000 mg/kg bw/day.