Registration Dossier

Toxicological information

Toxicity to reproduction

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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:
2012-03-21 to 2012-10-10
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted by a GLP-compliant laboratory according to an OECD guideline.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): Y-4036
- Physical state: clear colourless liquid
- Analytical purity: 99.5%
- Purity test date: 2012-10-15
- Lot/batch No.: 11GSVE305
- Expiration date of the lot/batch: 2015-07-29
- Label: Y-4036 Lot 11GSVE305 0.907KG
- Date received: 2012-02-02

- Stability under test conditions:

- Storage condition of test material: room temperature in the dark
- Other:

Test animals

Species:
rat
Strain:
other: Wistar Han^TM:RccHan^TM:WIST
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories U.K. Ltd., Blackthron, Bicester, Oxon, UK.
- Age at study initiation: approximately (P) 12 wks
- Weight at study initiation: (P) Males: 334-402 g; Females: 197-237 g
- Fasting period before study:
- Housing: Initially, all animals were housed in groups of five in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, U.K). During the pairing phase, the animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one female basis. Following evidence of succesful mating, the males were returned to their original cages. Mated females were housed individually during gestation and lactation, in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes.

- Diet and Water: The animals were allowed free access to food and water. A pelleted diet (Rodent 2018C Teklad Global Certified Diet, Harlan Laboratories U.K. Ltd., Oson, UK) was used. Mains drinking water was supplied from polycarbonate bottles attached to the cage.
- Environmental enrichment: Was provided in the form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd., Cheshire, UK) expect for mated females during gestation and lactation. Mated females were also given softwood flakes, as bedding, throughout gestation and lactation.
- Acclimation period: Thirteen days during which time their health status was assessed.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): The rate of air exchange was at least fifteen air changes per hour
- Photoperiod (hrs dark / hrs light): The low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness

IN-LIFE DATES: From: To:

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: Polyethylene glycol 400
Details on exposure:
The test item was administered daily by gavage using a stainless steel cannula attached to a disposable plastic syringe. Control animals were treated in an identical manner with 4 ml/kg of Polyethylene glycol 400.
The volume of test and control item administered to each animal was based on the most recent scheduled body weight and was adjusted at regular intervals.
Details on mating procedure:
- M/F ratio per cage: Animals were paired on a 1 male: 1 female basis within each dose group
- Length of cohabitation: For a period of up to fourteen days
- Proof of pregnancy and After successful mating each pregnant female was caged (how): Cage tray-liners were checked each morning for the presence of ejected copulation plugs and each female was examined for the presence of a copulation plug in the vagina. A vaginal smear was prepared for each female and the stage of oestrus or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating (Day 0 of gestation) and the males were subsequently returned to their original holding cages (unless required for additional pairing). Mated females were housed individually during the period of gestation and lactation. Pregnant females were allowed to give birth and maintain their offspring until Day 5 post partum. Litter size, offspring weight and sex, surface righting and clinical signs were also recorded during this period.


Duration of treatment / exposure:
The first day of dosing was designated as Day 1 of the study.
The male dose groups were killed and examined macroscopically on Day 43.
At Day 5 postpartum, all surviving females and surviving offspring were killed and examined macroscopically.
Frequency of treatment:
Groups of ten male and ten female animals were treated daily at the appropriate dose level throughout the study (except for females during parturition where applicable).
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
100 mg/kg bw/day
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
300 mg/kg bw/day
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
1000 mg/kg bw/day
Basis:
nominal conc.
No. of animals per sex per dose:
10  per sex per dose group
Control animals:
yes
Details on study design:
The animals were allocated to dose groups using a randomisation procedure based on stratified body weights and the group mean body weights were then determined to ensure similarity between the dose groups.

Examinations

Parental animals: Observations and examinations:
Clinical Observations:
All animals were examined for visual signs of toxicity, ill-health and behavioural change immediately before dosing, soon after dosing, and one and five hours after dosing, during the working week. Animals were observed immediately before dosing, soon after dosing, and one hour after dosing at weekends and public holidays (except for females during parturition where applicable).

Body Weight:
Individual body weights were recorded on Day 1 (prior to dosing) and then weekly for males until termination and weekly for females until mating was evident. Body weights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1 and 4 post partum.

Food Consumption:
During the pre-pairing period, weekly food consumption was recorded for each cage of adults until pairing. This was continued for males after the mating phase. For females showing evidence of mating, food consumption was recorded for the periods covering post coitum Days 0-7, 7-14 and 14-20. For females with live litters, food consumption was recorded during the lactation period (Days 1-4).

Weekly food efficiency (body weight gain/food intake) was calculated retrospectively for males and for females during the pre-pairing phase. Due to offspring growth and milk production for lactation, food efficiency for females could not be accurately calculated for females during gestation and lactation.

Water Consumption:
Water intake was observed daily by visual inspection of water bottles for any overt changes.

Pregnancy and Parturition:
Each pregnant female was observed at approximately 0830, 1230 and 1630 hours and around the period of expected parturition. Observations were carried out at approximately 0830 and 1230 hours at weekends and public holidays. The following was recorded for each female:
- Date of pairing
- Date of mating
- Date and time of observed start of parturition
- Date and time of observed completion of parturition






Litter observations:
On completion of parturition (Day 0 post partum), the number of live and dead offspring was recorded. Offspring were individually identified within each litter by tattoo on Day 1 post partum.

For each litter the following was recorded:
- Number of offspring born
- Number of offspring alive recorded daily and reported on Days 1 and 4 post partum
- Sex of offspring on Days 1 and 4 post partum
- Clinical condition of offspring from birth to Day 5 post partum
- Individual offspring weights on Days 1 and 4 post partum (litter weights were calculated retrospectively from this data)

All live offspring were assessed for surface righting reflex on Day 1 postpartum.
Postmortem examinations (parental animals):
Pathology:
Adult males were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination on Day 43. Adult females were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination on Day 5 post partum. Any females which failed to achieve pregnancy or produce a litter were killed on or after Day 25 post coitum.
For all females, the uterus was examined for signs of implantation and the number of uterine implantations in each horn was recorded. This procedure was enhanced; as necessary, by staining the uteri with a 0.5% ammonium polysulphide solution (Salewski 1964).
All adult animals, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

Organ Weights:
The epididymides and testes were removed from terminal kill adult males dissected free from fat and weighed before fixation.

Histopathology:
Samples of the following tissues were preserved from all animals from each dose group, in buffered 10% formalin, except where stated:
Coagulating gland Prostate
Epididymides* Seminal vesicles
Ovaries Testes*
Mammary gland (females only) Uterus/Cervix
Pituitary Vagina
* = preserved in Bouin's fluid then transferred to 70% Industrial Methylated Spirits (IMS) approximately forty-eight hours later

The tissues from control and 1000 mg/kg bw/day dose group animals, any animals dying during the study, and any animals which failed to mate or did not achieve a pregnancy were prepared as paraffin blocks, sectioned at a nominal thickness of 5 pm and stained with Haematoxylin and Eosin for subsequent microscopic examination. In addition, sections of testes and epididymides from all control and 1000 mg/kg bw/day males were also stained with Periodic Acid-Schiff (PAS) stain and examined.




Postmortem examinations (offspring):
Surviving offspring were terminated via intracardiac overdose of sodium pentobarbitone.

All offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

Statistics:
Where considered appropriate, quantitative data was subjected to statistical analysis to detect the significance of intergroup differences from control; statistical significance was achieved at a level of p<0.05. Statistical analysis was performed on the following parameters:
Body Weight, Body Weight Change, Food Consumption during gestation and lactation, Gestation Length, Litter Size, Litter Weight, Sex Ratio, Corpora Lutea, Implantation Sites, Implantation Losses, Viability Indices, Offspring Body Weight, Offspring Body Weight Change, Offspring Surface Righting, Absolute Organ Weights, Body Weight-Relative Organ Weights.
Data were analysed using the decision tree from the ProvantisTM Tables and Statistics Module as detailed in section "Any other infromation on materials and methods incl. tables" under the headline "Statistical Analysis"
Reproductive indices:
Mating Performance and Fertility:
The following parameters were calculated from the individual data during the mating period of the parental generation:
- Pre-coital Interval: Calculated as the time elapsing between initial pairing and the observation of positive evidence of mating.
- Fertility Indices: For each group the following were calculated:
Mating Index (%) = (Number of animals mated/Number of animals paired) x 100
Pregnancy Index (%) = (Number of pregnant females/Number of animals mated) x 100

Gestation and Parturition Data:
The following parameters were calculated from individual data during the gestation and parturition period of the parental generation:
- Gestation Length: Calculated as the number of days of gestation including the day for observation of mating and the start of parturition.
- Parturition Index: The following was calculated for each group:
Parturition Index (%) = (Number of females delivering live offspring/Number of pregnant females) x 100

Offspring viability indices:
The standard unit of assessment was considered to be the litter, therefore values were first calculated for each litter and the group mean was calculated using their individual litter values. Group mean values included all litters reared to termination (Day 5 of age).

- Implantation Losses (%): Group mean percentile pre-implantation and post-implantation loss were calculated for each female/litter as follows:
% pre-implantation loss = (Number of corpora lutea – Number of implantation sites/Number of corpora lutea) x 100
% post-implantation loss = (Number of implantation sites – Total number of offspring born/Number of implantation sites) x 100

- Live Birth and Viability Indices: The following indices were calculated for each litter as follows:
Live Birth Index (%) = (Number of offspring alive on Day 1/Number of offspring born) x 100
Viability Index (%) = (Number of offspring alive on Day 4/Number of offspring alive on Day 1) x 100

- Sex Ratio (% males): Sex ratio was calculated for each litter value on Days 1 and 4 post partum, using the following formula:
(Number of male offspring/Total number of offspring) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg bw/day, lower body weight gain of males
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg bw/day, lower body weight gain of males
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed

Reproductive function / performance (P0)

Reproductive performance:
no effects observed

Details on results (P0)

- Mortality
There were no unscheduled deaths considered to be related to the test item. One female (No.: 35) receiving 100 mg/kg bw/day was found dead and partially cannibalised on Day 7. No clinical signs had been apparent for this animal prior to this event. Necropsy examination revealed clear fluid in the chest cavity indicating an intubation error may have occurred during dosing on the previous day. In the absence of any similar deaths at higher dosages, there is no reason to suspect that this death was attributable to toxicity.
- Clinical Observations
No clinically observable signs of toxicity were detected. Clinical signs were confined to the presence of post-dose increased salivation for animals of either sex treated with 1000 and 300 mg/kg bw/day. This finding is commonly observed following the oral administration of a slightly unpalatable or slightly irritant test item formulation. In the absence of any supporting data to suggest irritancy, this isolated finding was considered not to represent an adverse health effect. No clinical observations were evident at 100 mg/kg bw/day.
- Body Weight
At 1000 mg/kg bw/day, body weight gain of males tended to be lower than control throughout the treatment period, attaining statistical significance during Weeks 4 and 6. Although differences from control at stages of the study were slight, overall gain at the end of the study was only 68% of the control and differences attained statistical significance. There was no obvious adverse effect on body weight gain for males at 100 and 300 mg/kg bw/day. For males at 300 mg/kg bw/day body weight gain during Week 2 to 4 was lower than control but differences did not attain statistical significance. In the absence of any effect on overall gain these differences were considered to be of no toxicological significance.
- Food Consumption and Food Efficiency
No adverse effects on dietary intake were detected for females during the pre-pairing, gestation or lactation phases of the study. At 1000 mg/kg bw/day, food consumption of males tended to be slightly lower than control throughout treatment. This finding was consistent with the lower body weight observed at this dosage and there was no adverse effect of treatment on food conversion efficiency. There was no consistent pattern of food intake or food utilisation for males at 100 and 300 mg/kg bw/day that indicated any adverse effect of treatment. There was no adverse effect of treatment on food consumption or food conversion efficiency for females during the pre-pairing treatment period at 100, 300 and 1000 mg/kg bw/day. Subsequent food intake for females during the gestation and lactation phases of the study was also unaffected by treatment at these dosages.
- Water Consumption
Daily visual inspection of water bottles did not reveal any overt intergroup differences in water intake.
- Mating
There was no adverse effect on mating performance at 100, 300 or 1000 mg/kg bw/day. The majority of animals on the study mated within the first four days of pairing (i.e. at the first oestrus opportunity).
- Fertility
There was no adverse effect on fertility at 100, 300 or 1000 mg/kg bw/day with the majority of matings within each dosage group leading to a successful pregnancy.
- Gestation Length
Intergroup differences in gestation lengths did not indicate any adverse effect of treatment at 100, 300 or 1000 mg/kg bw/day.
- Pathology
Necropsy examination of animals surviving to scheduled necropsy did not indicate any adverse effect of maternal treatment at 100, 300 or 1000 mg/kg bw/day. One male at 100 and one at 300 mg/kg bw/day showed small, flaccid testes and small epididymides and one control male showed small testes and epididymides. The control male failed to induce a pregnancy as did the male at 300 mg/kg bw/day. The remaining male at 100 mg/kg bw/day was scheduled to be paired with the female that died on the study and therefore its fertility was not assessed. These findings were considered to be incidental and unrelated to treatment.
- Organ weights
Assessment of testicular and epididymal absolute and body weight relative organ weights did not indicate any adverse effect of treatment at 100, 300 or 1000 mg/kg bw/day.
- Histopathology
Microscopic examination of tissues from control animals and animals receiving 1000 mg/kg bw/day did not indicate any adverse effect of treatment.
One control male, one male at 100 mg/kg bw/day and one male at 300 mg/kg bw/day showed testicular and epididymal findings at macroscopic necropsy and all these animals showed tubular degeneration in the testes and azoospermia in the epididymides. This apparent infertility for males was incidental and unrelated to treatment.





Effect levels (P0)

open allclose all
Dose descriptor:
NOEL
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Dose descriptor:
NOEL
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Body weight

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:
no effects observed
Gross pathological findings:
no effects observed

Details on results (F1)

In total, nine females from the control, 100 and 300 mg/kg bw/day dose groups and ten females treated with 1000 mg/kg bw/day gave birth to a live litter and successfully reared young to Day 5 of age. The following assessment of litter response is based on all litters reared to termination on Day 5 of lactation/age.
- Offspring Litter Size, Sex Ratio and Viability
There was no obvious adverse effect of treatment on corpora lutea and implantation counts, number of offspring born, litter size and sex ratio on Day 1 and subsequent offspring survival to Day 4 at 100, 300 or 1000 mg/kg bw/day.
- Offspring Growth and Development
There was no obvious adverse effect of treatment on offspring body weights and body weight change or litter weights at 100, 300 or 1000 mg/kg bw/day. Performance during surface righting assessments on Day 1 did not indicate any underlying effect of treatment on offspring development.
The low incidence of clinical signs observed for the offspring were typical of the age observed and did not indicate any obvious adverse effect of treatment.
- Pathology
Necropsy examination of offspring dying during the study and those surviving to terminations did not indicate any adverse effect of maternal treatment at 100, 300 or 1000 mg/kg bw/day.

Effect levels (F1)

Dose descriptor:
NOEL
Generation:
F1
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: clinical signs; mortality; body weight; gross pathology; implantation losses; live birth index; viability index; sex ratio (males); litter size; litter weights

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

TABULAR SUMMARY REPORT OF EFFECTS ON REPRODUCTION/DEVELOPMENT

Observations

Dose Level (mg/kg bw/day)

0 (Control)

100

300

1000

Paired animals

n

10

9

10

10

Females showing evidence of copulation

n

10

9

10

10

Pregnant females

n

9

9

9

10

Conception Days 1-5

n

9

8

9

10

Conception Days 12-13

n

1

1

1

0

Gestation = 22 Days

n

1

3

3

2

Gestation = 22 1/2 Days

n

6

3

4

2

Gestation = 23 Days

n

1

2

0

3

Gestation = 23 1/2 Days

n

1

1

2

3

Dams with live young born

n

9

9

9

10

Dams with live young at Day 4post partum

n

9

9

9

10

Corpora lutea/dam

m

16.1

16.0

15.2

14.6

Implants/dam

m

14.4

14.9

14.0

12.6

Live offspring/dam at Day 1post partum

m

13.3

13.7

12.8

11.9

Live offspring/dam at Day 4post partum

m

12.8

13.0

12.4

11.0

Sex ratio: % males at Day 1post partum

m

50.1

58.4

47.7

55.3

Sex ratio:% males at Day 4post partum

m

50.0

58.7

47.7

54.5

Litter weight (g) at Day 1post partum

m

75.16

78.59

73.37

69.70

Litter weight (g) at Day 4post partum

m

101.81

107.11

104.89

95.11

Male offspring weight (g) at Day 1post partum

m

5.73

5.90

5.94

6.16

Male offspring weight (g) at Day 4post partum

m

8.04

8.44

8.71

9.12

Female offspring weight (g) at Day 1post partum

m

5.54

5.64

5.66

5.88

Female offspring weight (g) at Day 4post partum

m

7.89

8.09

8.34

8.79

LOSS OF OFFSPRING/DAM

 

 

Pre-implantation (corpora lutea minus implantations)

 

 

0

n

3

4

3

4

1

n

1

3

2

1

2

n

2

0

3

2

3

n

2

1

1

1

4

n

1

1

0

0

5

n

0

0

0

1

7

n

0

0

0

1

Pre-natal (implantations minus live births)

 

 

0

n

3

3

4

5

1

n

2

1

1

3

2

n

4

5

2

2

3

n

0

0

2

0

Post natal (live births minus offspring alive on Day 4postpartum)

 

 

0

n

5

7

8

6

1

n

3

1

0

3

2

n

1

0

0

0

3

n

0

0

1

0

5

n

0

1

0

0

n

0

0

0

1

n = Number; m = Mean

Applicant's summary and conclusion

Conclusions:
The study did not find any effect of treatment by Y-4036 on reproduction. At 1000 mg/kg bw/day, male body weight gain and food consumption was slightly lower than control but, at the level observed, was insufficient to preclude this dosage from representing a no observed adverse effect level (NOAEL) for adult toxicity. A dosage of 300 mg/kg bw/day represents a clear NOEL for adult toxicity.
A dosage of 1000 mg/kg bw/day was considered to be the no observed effect level (NOEL) for reproduction and offspring survival, growth and development.
Executive summary:

Treatment with Y-4036 at dosages up to 1000 mg/kg bw/day was well tolerated with no marked adverse effects of treatment being observed for either sex. For both sexes at 300 and 1000 mg/kg bw/day an increased incidence of post-dosing salivation was apparent but this was considered to reflect palatability of the dosing formulations rather than evidence of systemic toxicity.

For males at 1000 mg/kg bw/day, body weight gain and food consumption tended to be slightly lower than control throughout treatment, with overall gain at the end of the study being only 68% of the control. Animals of the age used on this study design are no longer on a growth curve and the differences observed may, to a large extent, reflect normal biological variation for animals of this age. Whether these differences from control are sufficient to prevent this dosage being a no observed adverse effect (NOAEL) level is equivocal and this dosage level probably represents a NOAEL. This conclusion is supported by the results from the ninety day oral toxicity study for the test item (supplied by the Sponsor), where no adverse effects on body weight and food intake were reported at 1000 mg/kg bw/day.

There were no effects of treatment observed on body weight gain or food consumption off males during the pre-pairing, gestation or lactation phases of the study.

There was no effect of treatment on reproduction. The majority of animals at all dosages successfully mated and produced offspring. Litter data and assessment of offspring survival, growth and development did not indicate any treatment-related effects.