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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
26 May to 19 August 2014
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study was carried out in accordance with an appropriate OECD test guideline and in compliance with GLP. Since this is a read-across from the structural analogue substance CAS 5089-70-3, the reliability was set from RL1 to RL2.

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
Test material name (as stated in study report): Y-15428

Test animals

Species:
rat
Strain:
other: Crl:WI(Han)
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Germany
- Age at study initiation: 11 to 12 weeks
- Weight at study initiation: 316 - 375 g males; 197 - 250 g females
- Fasting period before study: no
- Housing: individually in IVC cages, except during pre-mating and periods when 2 females or 2 females and 1 male in type III H, polysulphone cages
- Diet (ad libitum): Altromin 1324 maintenance diet
- Water (ad libitum): facility tap water
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 25
- Humidity (%): 45 - 65
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES:
From: 26 May 2014
To: not stated

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was weighed into a tared plastic vial on a precision balance.

The test item was dissolved in corn oil.

The vehicle was selected based on the test item’s characteristics and the testing guideline. The test item formulations were prepared once every ten days. Prepared test item formulations were stored in plastic falcon vials at room temperature until the day of administration procedure. The water content (if any) in the test item container was purged with liquid nitrogen after every use.

Homogeneity of the test item in the vehicle was maintained by vortexing the prepared solution thoroughly before every dose administration.

VEHICLE
- Concentration in vehicle: 25, 75 or 250 mg/mL
- Amount of vehicle: 4 mL/kg
- Lot/batch no.: MKBP7039V
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The assessment of homogeneity as well as a determination of the measured nominal concentration of the test item in the vehicle were was performed at various intervals.

Samples for analysis of the measured dose formulations of the test item in the vehicle (nominal concentration) were taken in the first and last week of the study for all doses (8 samples in total). Samples for homogeneity were taken from the top, middle and bottom of the high dose, medium dose and low dose preparation. Samples were taken in the first and last week of the study (18 samples in total). Samples for stability analysis were taken in the first week of the study, 0 hours after the preparation and another sample 10 days after the preparation (at room temperature), from high, medium, and low dose formulations (6 samples in total). Each sample was retained twice (sample A, sample B, each of at least 5 mL).

All formulation samples were stored at -15 to -35°C and the A samples were analysed after completion of the in-life phase of the toxicity study at BSL BIOSERVICE Scientific Laboratories GmbH. Samples were analysed by GC-FID.
Details on mating procedure:
- Impregnation procedure: cohoused
- M/F ratio per cage: 1/2
- Length of cohabitation: until positive evidence of mating
- Proof of pregnancy: sperm in vaginal smear (designated Gestation Day 0)
Duration of treatment / exposure:
Gestation Day 5 to Gestation Day 19
Frequency of treatment:
Once daily
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0, 100, 300, 1000 mg/kg bw/day
Basis:
other: nominal in vehicle
Remarks:
Doses / Concentrations:
0, 25, 75, 250 mg/mL
Basis:
nominal conc.
No. of animals per sex per dose:
24 females per group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: little or no toxicity was expected with the test susbtance so the high dose was selected as 1000 mg/kg bw/day, correpsonding to the limit dose. Doses of 100 and 300 mg/kg bw/day were selected to define a dose response.
- Rationale for animal assignment: random

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily (except weekends and public holidays when once daily)

BODY WEIGHT: Yes
- Time schedule for examinations: Gestation Days 0, 5, 8, 11, 14, 17 and 20

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/period: Yes
- Time schedule for examinations: Gestation Days 0, 5, 8, 11, 14, 17 and 20

WATER CONSUMPTION: No
- Time schedule for examinations:

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day Gestation Day 20
- Organs examined: general macroscopic examination for structural abnormalities or pathological changes which may have influenced pregnancy.
- Organs weighed: liver
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
- Number of viable foetuses: Yes
- Position and number of foetuses in each uterine horn: Yes
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter
Statistics:
A statistical assessment of the results of the body weight, food consumption, prenatal parameters and litter weight data was performed by comparing values of dosed with control animals using a one-way ANOVA and a post-hoc Dunnett Test. Foetal evaluation parameters like external, visceral, craniofacial and skeletal parameters were analysed using a Fisher’s exact test. Litter incidence was the primary unit for the statistical analysis and interpretation. The statistics were performed with GraphPad Prism V.6.01 software (p<0.05 is considered as statistically significant).
Historical control data:
Historical control data from the Test Faciltity from 2010 to 2012 were provided.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes. Remark: 1000 mg/kg bw/day: reduced body weight gain and reduced food consumption

Details on maternal toxic effects:
Group mean body weight at 1000 mg/kg bw/day was slightly reduced compared to the control group with a maximal, statistically significant difference of 6% on gestation day 20. Body weight gain was also slightly lower in this group compared to the control group on various intervals within the study period and achieved statistical significance on gestation days 14-17.

In correlation to the body weight and body weight change, food consumption in the 1000 mg/kg bw/day group was noted to be slightly lower compared to the control group after the beginning of the treatment. Statistical significance was noted over the study period from gestation day 0 to day 20 as well as on several treatment intervals.

A slightly, statistically significantly lower terminal body weight was observed in the 1000 mg/kg bw/day group when compared to the control group. The uterus weight in this group was also slightly but not statistically significantly lower than in the control group. This also resulted in a slightly lower adjusted maternal weight compared to the control females.

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes. Remark: 1000 mg/kg bw/day: delayed ossification and reduced litter weight

Details on embryotoxic / teratogenic effects:
A slightly but statistically significantly lower mean litter weight was observed in the 1000 mg/kg bw/day group, mean litter weight was marginally lower in the 300 mg/kg bw/day group but not statistically significant compared to the control group. Taking into account the marginally lower number of foetuses in the 1000 mg/kg bw/day group, a statistically significantly lower total litter weight of this group compared to control was noted. Consequently statistically significantly lower male litter weight and a slightly lower female litter weight were also noted in the 1000 mg/kg bw/day group.

No external, visceral or craniofacial abnormalities attributable to treatment were noted.

A statistically significant increase in litter incidences of incomplete ossification of interparietal bone, frontal bone, parietal bone, supraoccipital bone and zygomatic arch was observed in the 1000 mg/kg bw/day group when compared to the concurrent control group. For interparietal and frontal bones, incidences in this group were also higher compared to historical control data.

Furthermore, a statistically significant increase in unossified 4th sternebra was observed in the 1000 mg/kg bw/day group compared to the concurrent control group. Values were within the range of historical control data, but due to the clear dose-dependency this finding was considered related to the treatment with the test item. In each litter of the 1000 mg/kg bw/day group several foetuses were observed with unossified cervical vertebra centra. The incidence of this finding was statistically significant compared to the control group and was considered to be test item-related due to the dose-dependency.

There was a higher, but not statistically significant, incidence of unossified xiphoid at 1000 mg/kg bw/day compared to the concurrent controls and historical control, which was considered to be test item-related. The observed reduced ossification of those bones that normally exhibit rapid ossification in the last days of gestation indicates a generalized skeletal delay in this group. This delayed ossification was considered to be associated with the observed maternal toxicity (lower body weight and food consumption) and reduced foetal body weight of the 1000 mg/kg bw/day group. Generally delayed ossification is not regarded to persist postnatally and not associated with long term consequences on survival, general growth and development and therefore not considered to be adverse.

There were a few findings of bent scapula body and of incompletely ossified 4th sacral arch which were only observed at 1000 mg/kg bw/day without statistical significance. Wavy ribs were seen in most litters of the 1000 mg/kg bw/day group showing a higher incidence than in the concurrent control group. Though this finding was not statistically significant, it can be considered treatment-related, as the incidence of this group was higher compared to historical control data.

No historical control data is available for the incompletely ossified 4th sacral arch or bent scapula findings, however, incomplete ossification of sacral arch is considered as variation. Wavy ribs are common findings in rodent studies and together with bent scapula are part of chondrodystrophy syndrome in rats (characteristic multiple skeletal abnormalities, such as bent and/or short bones) which has been demonstrated to be post-natally reversible (Carney and Kimmel (2007), De Schaepdrijver et al. (2014), Mitchard and Stewart) and therefore considered as variations and not adverse.

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: teratogenicity

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Dose Formulation Analysis - tables to be added.

Table - Litter Data Summary

Group

 

Mean Litter Weight

Number of Males

Number of Females

Total No. of Foetuses

Total Litter Weight

Male Litter Weight

Female Litter Weight

Control

Mean

3.80

5.60

4.90

10.50

41.79

23.19

18.60

SD

0.49

2.60

2.20

3.35

10.82

10.85

6.84

N

19

20

20

20

19

19

19

100 mg/kg/day

Mean

3.81

4.20

5.50

9.70

38.50

16.84

21.66

SD

0.70

1.99

2.16

2.89

8.02

6.53

7.93

N

19

20

20

20

19

19

19

300 mg/kg/day

Mean

3.59

6.36

5.41

11.77

42.10

23.22

18.88

SD

0.46

1.94

2.06

2.02

9.09

8.09

7.13

N

22

22

22

22

22

22

22

1000 mg/kg/day

Mean

3.12***

4.76

4.71

9.48

29.62***

15.60*

14.02

SD

0.39

2.57

2.22

3.28

10.50

8.66

6.48

N

21

21

21

21

21

21

21

Asterisks indicate statistically significant differences to control group C, with* p<0.05, ** p<0.01 and *** p<0.001.

Table - Selected Foetal Skeletal Findings Summary

Skeletal Finding

Dose (mg/kg/day)

Control

100

300

1000

Scapula body (B) bent

No of Incidences

0

0

0

3

Total No of Observed Foetuses

109

101

135

103

% Fetal incidence of Abnormality

0.00

0.00

0.00

2.91

No of Litters with at least 1 incidence

0

0

0

2

Total No of Observed Litters

19

19

22

21

% Litter incidence of Abnormality

0.00

0.00

0.00

9.52

Scapula body (L) bent

No of Incidences

0

0

0

1

Total No of Observed Foetuses

109

101

135

103

% Fetal incidence of Abnormality

0.00

0.00

0.00

0.97

No of Litters with at least 1 incidence

0

0

0

1

Total No of Observed Litters

19

19

22

21

% Litter incidence of Abnormality

0.00

0.00

0.00

4.76

Scapula body (R) bent

No of Incidences

0

0

0

3

Total No of Observed Foetuses

109

101

135

103

% Fetal incidence of Abnormality

0.00

0.00

0.00

2.91

No of Litters with at least 1 incidence

0

0

0

3

Total No of Observed Litters

19

19

22

21

% Litter incidence of Abnormality

0.00

0.00

0.00

14.29

Asterisks indicate statistically significant differences to control group C, with* p<0.05, ** p<0.01 and *** p<0.001.

Applicant's summary and conclusion

Conclusions:
Administration of Y-15428 at doses of 0, 100, 300 or 1000 mg/kg bw/day by oral gavage to pregnant female Wistar rats from gestation days 5 to 19 resulted in lower body weight, body weight gain and food consumption at 1000 mg/kg bw/day and were considered to be adverse. Lower terminal body weight, uterus weight and mean total litter weight were also noted at this dose.

Increased incidences of skeletal findings such as delayed ossification and lower litter weight recorded at 1000 mg/kg bw/day were considered to be attributable to the maternal effects and not adverse. An increased incidence of wavy ribs and bent scapula was also noted at this dose and these findings are recognised as part of chondrodystrophy syndrome in rats and have been demonstrated to be post-natally reversible and therefore also not adverse. However, the increased incidence of skeletal variations noted together with the lower litter weight was considered to be adverse at 1000 mg/kg bw/day.

Therefore the maternal and foetal No-Observed-Adverse-Effect-Levels (NOAEL) were both considered to be 300 mg/kg bw/day.