Registration Dossier

Administrative data

Endpoint:
developmental toxicity
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 well 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:
other: 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
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).
Duration of test:
43 days
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

Maternal examinations:
Clinical Observations:
All animals were examined for overt 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 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. 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 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






Ovaries and uterine content:
For all females, the uterus was examined for signs of implantation and the number of uterine implantations in each horn was 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"
Indices:
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:
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: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:no effects

Effect levels (maternal animals)

Dose descriptor:
NOEL
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity

Results (fetuses)

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental 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:
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 nomarked adverse effects of treatment being observed for either sex. For both sexes at300 and 1000 mg/kg bw/day an increased incidence of post-dosing salivation wasapparent but this was considered to reflect palatability of the dosing formulations ratherthan evidence of systemic toxicity.

For males at 1000 mg/kg bw/day, body weight gain and food consumption tended to beslightly lower than control throughout treatment, with overall gain at the end of the studybeing only 68% of the control. Animals of the age used on this study design are nolonger on a growth curve and the differences observed may, to a large extent, reflectnormal biological variation for animals of this age. Whether these differences fromcontrol 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 conclusionis 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 intakewere reported at 1000 mg/kg bw/day.

There were no effects of treatment observed on body weight gain or food consumption offemales 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 dosagessuccessfully mated and produced offspring. Litter data and assessment of offspringsurvival, growth and development did not indicate any treatment-related effects.