Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
05 April 2013 - 27 May 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Test performed in accordance with official guidelines, and in accordance with GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
The weight range of the animals at initiation of dosing deviated from the target weight range of 200-240g in the protocol. As all animals were of the correct age at mating, this was considered not to have affected the outcome or integrity of the study.
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Test material form:
solid: crystalline
Details on test material:
- Name of test material (as cited in study report): BIS-MPA™
- Substance type:
- Physical state: White crystalline powder
- Expiration date of the lot/batch: 01 Dec 2013
- Storage condition of test material: Ambient, Dark

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River
- Age at study initiation: 9 weeks old at the time of mating
- Weight at study initiation: 218- 317g at the initiation of dosing
- Fasting period before study: No
- Housing: 1 per cage in suspended polycarbonate cages with stainless steel grid tops and solid bottoms, containing an integral food hopper and sterilised white wood shavings bedding material
- Diet (e.g. ad libitum): SDS Rat and Mouse (modified) No. 3 Diet SQC Expanded was provided ad libitum
- Water (e.g. ad libitum): The animals had access to water ad libitum from the public supply
- Acclimation period: from arrival until Day 6 of gestation (3-6 days)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-23°C
- Humidity (%): 53-61%
- Air changes (per hr): A minimum of 10 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark

IN-LIFE DATES: From: 08 Apr 2013 To: 24 Apr 2013

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 0.5% aqueous methyl cellulose
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
VEHICLE
- Concentration in vehicle: 0, 20, 50, 100 mg/ml
- Amount of vehicle (if gavage): 10 ml/kg
- Lot/batch no. (if required): 021M0067V
- Purity: 0.5% methyl cellulose
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dose formulation samples were collected for analysis on Week 1 and Week 2 for all groups. Concentration and homogeneity were analysed. Analyses were performed by reverse phase HPLC with ELSD Detection using a validated analytical procedure.
Details on mating procedure:
- Impregnation procedure: purchased timed pregnant
Duration of treatment / exposure:
From Day 6 to Day 19 of gestation
Frequency of treatment:
Once daily
Duration of test:
20 days
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
200 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
500 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
24 females per dose level
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were decided baed on a 28 day dose range finding study where dose levels of up to 1000 mg/kg/day were considered to be well tolerated. 1000 mg/kg/day was therefore chosen as the high dose level for this study.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were checked early morning and as late as possible each day for viability. All the animals were examined for reaction to treatment prior to dosing and regularly throughout the day.
- Cage side observations were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily, from the start of dosing

BODY WEIGHT: Yes
- Time schedule for examinations: pretrial once on Day 4 of gestation and then daily during the dosing period Day 6 – 20 of gestation.

FOOD CONSUMPTION: Yes
- Frequency: Daily from Day 4 of gestation (first measured quantity given on Day 3 of gestation).
- Procedure: Food consumption was quantitatively measured.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Frequency: Regular basis throughout the study.
- Procedure: Water consumption was monitored by visual inspection of the water bottles.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20
- Organs examined: evaluation of the carcass and musculoskeletal system; all external surfaces and orifices; cranial cavity and external surfaces of the brain; and thoracic, abdominal, and pelvic cavities with their associated organs and tissues.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: Placentae (size, color or shape) and live and dead foetuses
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter were examined by open dissection for abnormalities of the thoracic and abdominal viscera, and for sex. These viscera were then discarded. Half per litter were examined for soft tissue abnormalities and sex.
- Skeletal examinations: Yes: all per litter
- The body weight of each foetus was recorded
Statistics:
Means and standard deviations were calculated for body weight, food consumption and pregnancy data.
Where required to assist interpretations, tests were applied to determine the statistical significance of observed differences between Control and groups receiving test item. Unless otherwise stated, all statistical tests were two-sided and performed at the 5% significance level using in house software. Pairwise comparisons were only performed against the control group.
Body weight and food consumption, data were analysed for homogeneity of variance using the ‘F-Max' test. If the group variances appear homogeneous, a parametric ANOVA was used and pairwise comparisons were made using Fisher’s F protected LSD method via Student's t test ie pairwise comparisons were made only if the overall F-test is significant. If the variances were heterogeneous, log or square root transformations were used in an attempt to stabilise the variances. If the variances remain heterogeneous, then a Kruskal-Wallis nonparametric ANOVA was used and pairwise comparisons were made using chi squared protection (via z tests, the non-parametric equivalent of Student's t test).
Foetal weight data was subjected to the Kruskal-Wallis non-parametric analysis.
Indices:
Not relevant
Historical control data:
Historical control data were supplied by the laboratory to clarify the significance of the apparent increase in post-implantation loss

Results and discussion

Results: maternal animals

Maternal developmental toxicity

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

Details on maternal toxic effects:
There were no premature deaths. All clinical observations were considered to be incidental background findings commonly observed in this species. Group mean body weight and food consumption in treated groups was comparable with Controls. All gross necropsy findings noted in dams and foetuses on this study were of the type and distribution which did not indicate any association with treatment.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
LOAEL
Effect level:
500 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes. Remark: Increased numbers of early resorptions at 500 and 1000 mg/kg bw/d, but without any effect on litter size

Details on embryotoxic / teratogenic effects:
At 500 and 1000 mg/kg/day there was an increase in total fetal deaths, when compared with Controls, caused by and increase in early embryonic deaths. In both groups, the number of litters affected and number of early embryonic deaths per litter were also increased when compared with Controls. The number total number of early embryonic death and litters with early embryonic deaths at 200 mg/kg/day was comparable with Controls.
The group mean and percentage values of all other pregnancy performance parameters and fetal weights were similar between all groups. Slight intergroup differences were considered to be incidental and too small to be attributed to treatment with Bis-MPA™.
The type and distribution of major and minor fetal abnormalities and skeletal ossification parameters did not indicate any association with treatment. Slight intergroup differences were considered to be incidental and unrelated to treatment with Bis-MPA™.

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Summary of litter parameters

Parameter

Dose level (mg/kg bw/d)

0

200

500

1000

Corpora lutea (#)

13.6

14.1

15.6

14.8

Pre-implantation loss (%)

6

5

4

3

Implantations (#)

12.8

13.4

15.0

14.4

Post-implantation loss (%)

7

8

10

12

Early resorption (%)

7

8

10

12

Late resorption (%)

0.3

0.3

0

0

Litter size (#)

11.9

12.4

13.5

12.6

Foetal death (#)

0

0

0

0

Gravid uterus weight (g)

71

76

81

75

Foetal weight (g)

3.73

3.80

3.85

3.74

Applicant's summary and conclusion

Conclusions:
Treatment with Bis-MPA™ at dose levels up to 1000 mg/kg/day was not associated with any treatment effects or findings in dams at any dose level.
At 500 and 1000 mg/kg/day, there was an increase in early embryonic deaths when compared with Controls and 200 mg/kg/day. All other pregnancy performance parameters and fetal weights were comparable with Controls in all treated groups, and therefore showed no treatment-related effects of findings.
Based on the results of this study, the maternal No Observed Effect Level (NOEL) was considered to be 1000 mg/kg/day and the foetal NOEL was considered to be 200 mg/kg/day.
Executive summary:

A study was conducted to determine the effects of Bis-MPA™ when administered orally by gavage during the period of organogenesis to pregnant rats. Forty two time-mated female Sprague-Dawley rats per dose were administered Bis-MPA™ at the concentrations of 200, 500, or 1000 mg/kg bw/day. A concurrent vehicle control was run. All animals were dosed over Days 6-19, inclusive of gestation, where the day of detection of mating was designated Day 0. Animals were regularly monitored for clinical signs of toxicity, body weight and food consumption performance and were killed on Day 20 of gestation for examination of pregnancies and foetal development.

Treatment with Bis-MPA™ at dose levels up to 1000 mg/kg/day was not associated with any treatment related clinical signs, body weight or food consumption effects or gross necropsy findings in dams at any dose level. At 500 and 1000 mg/kg/day, there was a slight increase in early embryonic deaths when compared with Controls and 200 mg/kg/day. All other pregnancy performance parameters and foetal weights were comparable with Controls in all treated groups, and the type and distribution of foetal major abnormalities, minor visceral and skeletal abnormalities and skeletal ossification parameters did not indicate an association with treatment of Bis-MPA™.

Based on the results of this study, the maternal No Observed Effect Level (NOEL) was considered to be 1000 mg/kg/day and the foetal NOEL was considered to be 200 mg/kg/day.