Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
07 November 2017 - 15 March 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
Adopted 22 January 2001
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
August 1998
Deviations:
no
Qualifier:
according to
Guideline:
other: Japanese Ministry of Agriculture, Forestry and Fisheries Testing guideline for Toxicology studies, 12 NouSan No 8147
Version / remarks:
24 November 2000
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: PFW160721
- Expiration date of the lot/batch: 30 December 2018
- Purity : ca. 98.32%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Stored at ambient temperature/humidity in darkness; may be used/formulated in light
- Solubility and stability of the test substance in the solvent/vehicle: For the purpose of the study the test item was prepared at the appropriate concentrations as a solution in Distilled Water. The stability and homogeneity of the test item formulations were determined by Envigo Research Limited, Shardlow, UK Analytical Services. The formulations were stable for at least 21 days.

OTHER SPECIFICS: No correction for purity was made.

Test animals

Species:
rat
Strain:
Sprague-Dawley
Remarks:
Crl:CD (SD) IGS BR
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, Kent
- Age at study initiation: no data
- Weight at study initiation: At the start of treatment the females weighed 204 to 299g.
- Fasting period before study: no data
- Housing: The animals were housed individually in solid-floor polypropylene cages with stainless steel mesh lids furnished with softwood flakes (Datesand Ltd., Cheshire, UK).
- Diet (e.g. ad libitum): ad libitum; A pelleted diet (Rodent 2018C Teklad Global Certified Diet, Envigo RMS (UK) Limited, Oxon, UK) was used.
- Water (e.g. ad libitum): ad libitum, Mains drinking water was supplied from polycarbonate bottles attached to the cage.
- Acclimation period: no data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): at least 15 air changes per hour
- Photoperiod (hrs dark / hrs light): twelve hours continuous light and twelve hours darkness

IN-LIFE DATES: From: 01 December 2017 (first day of treatment) To: 20 December 2017 (final day of necropsy)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
distilled water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations are stable for at least 21 days and were therefore prepared twice and stored at approximately 4 °C in the dark.

VEHICLE
- Concentration in vehicle: 0, 2.5, 10 and 25 mg/mL
- Treatment volume 10 mL/kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The test item concentration in the test samples was determined by gas chromatography (GC) using an external standard technique. The test item gave a chromatographic profile consisting of a single peak.
The analytical procedure was successfully validated with respect to specificity of chromatographic analysis, linearity of detector response, method accuracy and precision.
The homogeneity and stability was confirmed for test item in distilled water formulations at nominal concentrations of 2.5 mg/mL and 25 mg/mL when stored refrigerated for 21 days.
The mean concentrations of test item in test formulations analyzed for the study were within ± 10% of nominal concentrations, confirming accurate formulation.
The results indicate that the prepared formulations were within 3% of the nominal concentration.
Details on mating procedure:
Animals were delivered in two batches containing females prior to Day 3 of gestation. The day that positive evidence of mating was observed was designated Day 0 of gestation.
Duration of treatment / exposure:
between Days 3 and 19 of gestation
Frequency of treatment:
daily
Duration of test:
From Day 3 to Day 20 of gestation.
Doses / concentrationsopen allclose all
Dose / conc.:
25 mg/kg bw/day (nominal)
Remarks:
low treatment group
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Intermediate treatment group
Dose / conc.:
250 mg/kg bw/day (nominal)
Remarks:
high treatment group
No. of animals per sex per dose:
24
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were chosen in collaboration with the Sponsor and were based on previous toxicity data (Envigo Research Limited, Study Number XX96MF). The preliminary oral (gavage) pre-natal development toxicity study in the rat was performed as dose range finder for the OECD 414 study. Following dose levels were recommended for use in the main prenatal developmental toxicity study: 0 (Control), 25, 100 and 250 mg/kg bw/day. Based on the results of the study, dose levels of 0 (Control), 25, 100 and 250 mg/kg bw/day were recommended for use in the planned prenatal developmental toxicity study.
The highest dose level should produce an effect on body weight gain and the low and intermediate dose levels follow the recommendations of the OECD 414 guideline for a 2 to 4-fold interval between dose levels.

- Rationale for animal assignment: The animals were randomly allocated to treatment groups using a randomization procedure based on stratified body weight to ensure similarity between the treatment groups.

Examinations

Maternal examinations:
DETAILED CLINICAL OBSERVATIONS: Yes
Following arrival, all animals were examined for overt signs of toxicity, ill-health or behavioral changes once daily during the gestation period. Additionally, during the dosing period, observations were recorded immediately before and soon after dosing and one hour post dosing. All observations were recorded.

BODY WEIGHT: Yes
Individual body weights were recorded on Day 3 (before the start of treatment) and on Days 5, 6, 7, 8, 11, 14 and 17 of gestation. Body weights were also recorded for animals at terminal kill (Day 20).

FOOD CONSUMPTION: Yes
Food consumption was recorded for each individual animal at Day 3, 5, 8, 11, 14, 17 and 20 of gestation.

WATER CONSUMPTION: Yes
Water intake was observed daily by visual inspection of the water bottles for any overt changes.

POST-MORTEM EXAMINATIONS: Yes
All animals were killed by carbon dioxide asphyxiation followed by cervical dislocation on Day 20 of gestation. All animals were subjected to a full external and internal examination and any macroscopic abnormalities were recorded.

Ovaries and uterine content:
The ovaries and uteri of pregnant females were removed, examined and the following data recorded:
i) Number of corpora lutea
ii) Number, position and type of intrauterine implantation
iii) Fetal sex
iv) External fetal appearance
v) Fetal weight
vi) Placental weight
vii) Gravid uterus weight
The uteri of any apparently non-pregnant females were immersed in 0.5% ammonium polysulphide solution to reveal evidence of implantation.

Implantation types were divided into:
Early Death: No visible distinction between placental/decidual tissue and embryonic tissue
Late Death: Separate embryonic/fetal and placental tissue visible
Dead Fetus: A fetus that had died shortly before necropsy. These were included as late deaths for reporting purposes

All implantations and viable fetuses were numbered according to their intrauterine position as follows (as an example):
Left Horn Cervix Right Horn

L1 L2 L3 L4 L5 L6 L7 L8 R1 R2 R3 R4 R5 R6 R7 R8
V1 V2 V3 V4 V5 V6 V7 V8 V9 V10 V11 V12 V13 V14 V15 V16
V = viable fetus
Fetal examinations:
The fetuses were killed by subcutaneous injection of a suitable barbiturate agent. Fetuses from each litter were divided into two groups and examined for skeletal alterations and soft tissue alterations. Alternate fetuses were identified using an indelible marker and placed in Bouin’s fixative. Fetuses were subsequently transferred to distilled water and examined for visceral anomalies under a low power binocular microscope and then stored in 10% Buffered Formalin. The remaining fetuses were identified using cardboard tags marked with chinagraph pencil and placed into 70% IMS in distilled water. The fetuses were subsequently eviscerated, processed and the skeletons stained with alizarin red S before being transferred to 50% glycerol for examination of skeletal development and anomalies and storage.
Statistics:
The following parameters were analyzed statistically, where appropriate, using the test methods outlined below:
Female body weight change, food consumption and gravid uterus weight: Shapiro Wilk normality test and Bartlett’s test for homogeneity of variance and one way analysis of variance, followed by Dunnett’s multiple comparison test or, if unequal variances were observed, on alternative multiple comparison test.
All caesarean necropsy parameters and fetal parameters: Kruskal-Wallis non-parametric analysis of variance; and a subsequent pairwise analysis of control values against treated values using the Mann-Whitney ‘U’ test, where significance was seen.
Fetal evaluation parameters, including skeletal or visceral findings: Kruskal-Wallis non-parametric analysis of variance and Mann-Whitney ‘U’ test.
Probability values (p) are presented as follows:
p<0.001 ***
p<0.01 **
p<0.05 *
p≥0.05 (not significant)
Indices:
Percentage pre-implantation loss was calculated as:
[(number of corpora lutea-number of implantations)/number of corpora lutea] x 100

Percentage post-implantation loss was calculated as:
[(number of implantations - number of live fetuses)/number of implantations] x 100

Sex ratio:
Sex ratio was calculated as:
% male fetuses (sex ratio) = (number of male fetuses/total number of fetuses) x 100

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
There were no significant adverse clinical signs evident in treated females.
One female treated with 250 mg/kg bw/day showed increased salivation post dosing on Day 5 only. A further female from this treatment group had pilo-erection on Day 8 only. In isolation, these findings were considered to be incidental and of no toxicological importance.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
There were no unscheduled deaths.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Females treated with 250 mg/kg bw/day showed a statistically significant reduction (p<0.01-0.001) in body weight gain between Days 4 and 11 of gestation. Body weight for these females was also statistically significantly reduced (p<0.05-0.01) from Day 8 onwards. Cumulative body weight gain was statistically significantly reduced (p<0.05-0.001) in these females throughout the treatment period and body weight and body weight gain when adjusted for gravid uterus weight was also statistically significantly reduced (p<0.05 and p<0.001 respectively) when compared to controls.
A statistically significant reduction (p<0.05) in body weight gain was evident in females treated with 100 mg/kg bw/day between Days 8 and 11 of gestation. A statistically significant reduction (p<0.05) in cumulative body weight gain was also evident in these females between Days 3 and 11 and Days 3 and 17. Body weight gain for these females during the remaining periods was comparable to controls.
Body weight gain during gestation, including after adjustment for the contribution of the gravid uterus, was considered to be unaffected by treatment at 25 mg/kg bw/day. See section 'Additional information on results' for more details. Historical control data for the parameters is described, when available.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Females treated with 250 mg/kg bw/day showed a statistically significant reduction (p<0.01-0.001) in food consumption throughout the treatment period.
No differences as compared to the control group were detected for food consumption in females treated with 100 or 25 mg/kg bw/day.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
No differences as compared to the control group were detected for water consumption.
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment related macroscopic abnormalities were detected in females treated with 250, 100 or 25 mg/kg bw/day.
One female treated with 100 mg/kg bw/day had a scab on the right shoulder and one control female had an enlarged right kidney. These findings were considered to be incidental findings.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not specified
Details on results:
At 100 mg/kg bw/day, treatment was associated with slightly lower body weight gains between Days 8 and 11 of gestation and lower cumulative body weight gain between Days 3 and 11 and Days 3 and 17 of gestation. Body weight gain for these females during the remaining periods was comparable to controls. No macroscopic necropsy findings were apparent for adult females at this dosage. Given the transient nature of the effects on body weight gain, this dosage is considered to represent a No Observed Adverse Effect Level (NOAEL) for the pregnant female.

Maternal developmental toxicity

Number of abortions:
no effects observed
Description (incidence and severity):
There was no treatment-related effect observed in the pre- and post-implantation loss in all dose groups, when comparing to the control group:- preimplantation loss: 13.3%, 13.6%, 10.5%, 13.2% at 0, 25, 100 and 250 mg/kg bw/day-post-implantation loss: 0.5%, 0.9%, 1.4%, 1.2% at 0, 25, 100 and 250 mg/kg bw/day.All data was within the historical control range available for this species and strain. See section 'Additional information on results' for more details
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
There was no treatment-related effect observed in the pre- and post-implantation loss in all dose groups, when comparing to the control group:
- preimplantation loss: 13.3%, 13.6%, 10.5%, 13.2% at 0, 25, 100 and 250 mg/kg bw/day
- post-implantation loss: 0.5%, 0.9%, 1.4%, 1.2% at 0, 25, 100 and 250 mg/kg bw/day.
All data was within the historical control range available for this species and strain. See section 'Additional information on results' for more details.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
There were no total litter losses by resorption observed. See Section 'Additional information on results' for more details.
Early or late resorptions:
no effects observed
Description (incidence and severity):
There were no treatment-related effects on early or late resorptions observed. See Section 'Additional information on results' for more details.
Dead fetuses:
no effects observed
Description (incidence and severity):
There were no treatment-related effects on the number of dead fetuses observed. See Section 'Additional information on results' for more details.
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
There was no treatment-related, toxicologically relevant effect on the number of pregnant dams per dose group. The number of pregnant females per dose group were 24/24, 23/24, 23/24 and 22/24 at 0, 25, 100 and 250 mg/kg bw/day, resp. No historical control data is available for this parameter. See section 'Additional information on results' for more details
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
There was no treatment-related, toxicologically relevant effect on the number of pregnant dams per dose group. The number of pregnant females per dose group were 24/24, 23/24, 23/24 and 22/24 at 0, 25, 100 and 250 mg/kg bw/day, resp. No historical control data is available for this parameter. See section 'Additional information on results' for more details
Other effects:
not specified
Details on maternal toxic effects:
There was no obvious effect of maternal treatment on litter data as assessed by numbers of implantations, in-utero offspring survival (as assessed by the mean numbers of early or late resorptions), live litter size, sex ratio or pre- or post-implantation losses at 25, 100 or 250 mg/kg bw/day.
Intergroup differences for mean fetal, litter or placental weights did not indicate any obvious effects of maternal treatment at 25, 100 or 250 mg/kg bw/day.
Statistical analysis of the data did not reveal any significant intergroup differences.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
body weight and weight gain

Maternal abnormalities

Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
There is no treatment-related effect on the mean fetal body weight, mean male fetal body weight or mean female fetal body weight. See section 'Additional information on results' for further details. Available historical control data is described.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
There is no treatment-related effect on the number of live offspring. The percentage of live offspring was calculated as the number of live implants - the number of implants x 100. This means for the dose groups: 99.3%, 99.3%, 98.6% and 98.5% for the control, 25, 100 and 250 mg/kg bw/day. See section 'Additional information on results' for further details. The available historical control data is described.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
There was no treatment-related effect observed on sex ratio. See Section 'Additional information on results' for more details. Historical control data is described, when available.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
There was no treatment-related effects observed on litter weight or litter size. See Section 'Additional information on results' for more details. Historical control data is described, when available.
Changes in postnatal survival:
no effects observed
Description (incidence and severity):
There was no treatment-related effects observed on postnatal survival. See Section 'Additional information on results' for more details. Historical control data is described, when available.
External malformations:
no effects observed
Description (incidence and severity):
No treatment-related effect on external malformations was observed in all dose groups. See section 'additional information on results' for further details. Available historical control data is described.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
A statistically significant increase (p<0.05-0.01) in the number of fetuses/litters showing incomplete ossification of the parietal, interparietal, hyoid, sacral arch, sternebra and femur were evident at 250 mg/kg bw/day. The effect on the sternebra also extended to fetuses/litters at 100 and 25 mg/kg bw/day. A statistically significant increase (p<0.05) in the number of fetuses/litters showing incomplete ossification of the zygomatic process of squamosal were evident at 25 and 250 mg/kg bw/day. A statistically significant increase (p<0.05) in the number of fetuses/litters showing incomplete ossification of the hyoid was evident at 100 mg/kg bw/day. With the exception of the sacral arch and sternebra, true dose related responses were not evident in the remaining parameters and group mean values were generally within historical control ranges (excluding interparietal, sacral arch and femur). In the absence of any particular pattern of abnormal skeletal development of skeletal structures affecting treated fetuses and in the absence of an effect on mean fetal weight, the observation of isolated affected skeletal structures can be considered unlikely to represent true developmental abnormalities.
See Section 'Additional information on results' for further details. Available historical control data is described.
Visceral malformations:
no effects observed
Description (incidence and severity):
No treatment-related effect on visceral malformations was observed in all dose groups. See section 'additional information on results' for further details. Available historical control data is described.
Other effects:
not specified
Details on embryotoxic / teratogenic effects:
No treatment-related changes were detected in the offspring parameters measured or on embryofetal development. The ‘No Observed Adverse Effect Level’ (NOAEL) for developmental toxicity was therefore considered to be 250 mg/kg bw/day.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no treatment-related changes up to 250 mg/kg bw/day

Fetal abnormalities

Abnormalities:
no effects observed

Overall developmental toxicity

Developmental effects observed:
no

Any other information on results incl. tables

Table 1: Summary of Female Performance: number of pregnant and non-pregnant dams per dose group

Category

Number of Females at Dose Level (mg/kg bw/day)

0 (Control)

25

100

250

Initial Group Size

24

24

24

24

Non-Pregnant

0

1

1

2

Pregnant

24

23

23

22

° No historical data available

Table 2: Group Mean Litter Data Values: pre- and postimplantation loss, number and percent

Dose Level (mg/kg bw/day)

 

Number of Corpora Lutea

Number of Implants

Number of Embryonic/Fetal Deaths

Implantation Loss
%

       Number of live implants

Early

Late

Total

Pre

Post

 Male  Female  Total

0 (Control)

mean

16.3

14.0

0.0

0.0

0.1

13.3

0.5

 6.0 7.9  13.9 

sd#

2.1

1.6

0.2

0.2

0.3

8.2

1.8

 1.8 2.0  1.5 

number of animals

24

24

24

24

24

24

24

 24 24  24 

25

mean

16.7

14.4

0.1

0.0

0.1

13.6

0.9

 6.7 7.6  14.3 

sd#

1.8

1.8

0.3

0.0

0.3

7.9

2.5

 1.7 1.9  1.9 

number of animals

23

23

23

23

23

23

23

 23 23  23

100

mean

15.9

14.2

0.2

0.0

0.2

10.5

1.4

 7.3 6.7  14.0 

sd#

1.5

1.5

0.5

0.2

0.6

7.6

3.9

 1.9 1.7 1.5

number of animals

23

23

23

23

23

23

23

 23 23  23 

250

mean

15.9

13.7

0.1

0.1

0.2

13.2

1.2

 6 7.5  13.5 

sd#

2.1

2.0

0.3

0.3

0.5

8.9

3.2

 2.6 2.2  1.9 

number of animals

22

22

22

22

22

22

22

 22 22  22 
Historical data  range$ 11 - 20  9 - 18 0 - 2  0 - 4 0 - 4  0 - 31.8  0 - 6.9   3 - 10 3 - 10  10 - 17 
 mean 15   14 11.4  1.3   6 13 
 sd# 2  0 10.2  2.8   2
 number of animals 194  200  210 210  210  203  198   201 196  203 

# sd: range = minimum to maximum

$ range = mean + / - 2 standard deviations

pre-implantation loss = number of corpora lutea - number of implants; pre-implantation loss % = pre-implantation loss / number of corpora lutea x 100; post-implantation loss = number of implants - number of live implants (total); postimplantation loss % = post implantation loss / number of implants x 100

Table 3: Group Mean Body Weight Values:

Dose Level (mg/kg bw/day)

 

Body Weight (g) on Day of Gestation

3

4

5

8

11

14

17

20

0 (Control)

mean

252.8

257.2

263.0

276.1

292.8

311.9

339.1

382.5

sd

23.1

24.4

25.8

26.7

28.8

29.2

31.8

33.6

number of animals

24

24

24

24

24

24

24

24

25

mean

251.8

258.4

263.7

276.7

293.3

312.8

338.0

383.3

sd

16.4

19.4

19.0

18.7

20.4

21.0

21.2

24.4

number of animals

23

23

23

23

23

23

23

23

100

mean

248.6

253.4

257.7

269.1

283.1

302.4

326.8

370.7

sd

13.2

15.3

15.3

15.5

17.0

16.7

17.9

21.0

number of animals

23

23

23

23

23

23

23

23

250

mean

252.0

253.6

254.3

260.1*

268.7**

290.5**

316.1**

358.9*

sd

18.6

18.2

17.5

18.6

18.9

19.1

20.6

26.5

number of animals

22

22

22

22

22

22

22

22

Historical data  range  204 - 291 / °  214 - 306 225 - 321  243 - 341  258 - 361  283 - 396  322 - 447 
 mean 247  / ° 260  273  292  310  340  385 
 sd  22 / ° 23  24  25  26  28  31 
 number of animals  288 / ° 290  290  289  290  289  290 

° For Day 4 no historical data is available

° *p<0.05; ** p<0.01

Table 4: Group Mean Body Weight Change Values

Dose Level (mg/kg bw/day)

 

Body Weight Change (g) during Days of Gestation

3 to 4

4 to 5

5 to 8

8 to 11

11 to 14

14 to 17

17 to 20

0 (Control)

mean

4.4

5.8

13.1

16.8

19.1

27.2

43.4

sd

4.1

3.7

3.4

4.1

3.5

4.9

5.9

number of animals

24

24

24

24

24

24

24

25

mean

6.6

5.3

13.1

16.5

19.6

25.2

45.3

sd

3.7

3.0

4.4

3.4

3.8

3.5

6.8

number of animals

23

23

23

23

23

23

23

100

mean

4.9

4.3

11.3

14.0*

19.3

24.4

43.9

sd

3.9

2.9

5.0

5.6

4.7

4.8

5.8

number of animals

23

23

23

23

23

23

23

250

mean

1.6

0.6***

5.8***

8.6**

21.9

25.6

42.7

sd

3.9

4.9

5.2

16.4

17.3

6.5

10.6

number of animals

22

22

22

22

22

22

22

° * p<0.05; ** p<0.01; *** p<0.001

Table 4a: Group Mean Body Weight Change Values - Historical Data

   

 Days 3 -5

 Days 5 - 6

Days 6 - 7 

 Days 7 - 8

 Days 8 - 11

 Days 11 - 14

 Days 14 - 18

 Days 17 - 20

Historical data: body weight change (g)

 range

 3 - 22

-5 - 10 

-2 - 13 

-2 - 11 

 10 - 29

10 - 27 

20 - 40 

32 - 53 

 mean

 12

19 

18 

30 

44 

 sd

 5

 number of animals

 286

128 

205 

298 

293 

288 

288 

289 

Table 5: Group Mean Gravid Uterus Weight and Adjusted Body Weight and Body Weight Change Values

Dose Level (mg/kg bw/day)

 

Body Weight (g) on Days of Gestation

Body Weight Change (g) during Days of Gestation

Gravid Uterus Weight
(g)

Adjusted
Body Weight (g)
 Day 20

Adjusted
Body Weight Change (g)
3-20

3

20

3-20

0 (Control)

mean

252.8

382.5

129.7

82.880

299.6

46.8

sd

23.1

33.6

12.9

8.641

29.4

10.1

number of animals

24

24

24

24

24

24

25

mean

251.8

383.3

131.4

85.789

297.5

45.6

sd

16.4

24.4

10.7

9.994

22.1

10.7

number of animals

23

23

23

23

23

23

100

mean

248.6

370.7

122.1

82.770

287.9

39.4

sd

13.2

21.0

14.0

7.550

17.4

10.7

number of animals

23

23

23

23

23

23

250

mean

252.0

358.9

106.9

78.508

280.4*

28.4***

sd

18.6

26.5

12.1

8.685

24.4

12.4

number of animals

22

22

22

22

22

22

°* p<0.05; ** p<0.01; *** p<0.001

° Available Historical Data:

- Gravid Uterus Weight (g):

~ Range: 63.56 - 106.59 ~ Mean: 85.08 +/- 10.76 ~ Number of animals: 286

- Adjusted Body Weight Day 20 (g):

~ Range: 247 - 353 ~ Mean 300 +/- 26 ~ Number of animals: 286

- Adjusted Body Weight Change Days 5 - 20 (g):

~ Range: 20 - 64 ~ Mean 45 +/- 11 ~ Number of animals: 126

Table 6: Mean Number and Percent of Live Offspring

    Dose Level (mg/kg bw/day)             Number of Live Implants  % Male Fetuses  total live implants / number of implants x 100
 Male Female  Total     
    0 (Control)  mean  6.0 7.9  13.9  43.1  99.3 
 sd 1.8  2.0  1.5  12.7 
    25  mean  6.7 7.6  14.3  46.8  99.3 
 sd  1.7 1.9  1.9  11.2   /
    100  mean 7.3  6.7  14.0  51.9  98.6 
 sd 1.9  1.7  1.5  11.7 
    250  mean 6.0  7.5  13.5  43.9  98.5 
 sd 2.6  2.2  1.9  17.1 
    Historical data  mean  6 13  48.5   /
 sd  2 31.1 

Table 7: Mean Foetal / Pup Body Weight by Sex and Sexes Combined

 Dose Level (mg/kg bw/day   Mean Male Fetal Weight (g)   Mean Female Fetal Weight (g)  Mean Fetal Weight (g)  Mean Placental Weight (g)  Litter Weight (g)  Total Placental Weight (g)
0 (Control)  mean 3.959  3.747  3.840  0.541  53.382  7.537 
 sd 0.201  0.152  0.168  0.046  5.739  1.081 
number of animals  24  24  24  24  24  24 
25 mean  3.923  3.733  3.825  0.548  54.584  7.803 
sd  0.314  0.270  0.287  0.053  7.193  1.117 
 number of animals 23  23  23  23  23  23 
100  mean 3.922  3.686  3.805  0.566  52.922  7.883 
 sd 0.282  0.313  0.290  0.071  5.116  1.234 
number of animals  23  23  23  23  23  23 
250  mean 3.778  3.618  3.691  0.560  49.832  7.475 
 sd 0.345  0.320  0.319  0.106  6.747  1.038 
 number of animals 22  22  22  22  22  22 
Historical data  range 3.64 - 4.59  3.36 - 4.45  3.46 - 4.54  0.4 - 0.73   35.43 - 71.14 5.24 - 9.71 
 mean 4.11   3.9 0.57  53.28  7.47 
 sd  0.24  0.27 0.27  0.08  8.93  1.12 
 number of animals 200  203   203 203  202   202

° range = mean + / - 2 standard deviations

Table 8: Summary Incidence of Fetal External Findings

External Findings

Dose level (mg/kg bw/day)

0 (Control)

25

100

250

Number of fetuses (litters) examined

334 (24)

329 (23)

321 (23)

298 (22)

NF

NL

%†

NF

NL

%†

NF

NL

%†

NF

NL

%†

Small fetus

5

4

1.5

6

5

1.8

7

5

2.0

13

4

5.6

Small placenta

3

2

0.9

4

3

1.2

1

1

0.3

1

1

0.3

Pale fetus

1

1

0.3

0

0

0.0

3

3

0.9

1

1

0.3

Large placenta

0

0

0.0

1

1

0.3

5

2

1.5

12

2

5.4

Pale placenta

0

0

0.0

0

0

0.0

0

0

0.0

1

1

0.3

Total

8

5

2.4

8

6

2.3

14

8

4.1

15

5

6.4

 %†: Group mean percent

NF: Number of Fetuses

NL: Number of litters

 Table 9: Summary Incidence of Fetal Visceral Findings

Visceral Findings

Dose Level (mg/kg bw/day)

0 (Control)

25

100

250

Number of Fetuses (litters) Examined

172 (24)

169 (23)

166 (23)

156 (22)

NF

NL

%†

NF

NL

%†

NF

NL

%†

NF

NL

%†

External

 

 

 

 

 

 

 

 

 

 

 

 

Hemorrhage

1

1

0.6

1

1

0.5

0

0

0.0

0

0

0.0

Situs inversus

1

1

0.6

0

0

0.0

0

0

0.0

0

0

0.0

Head

 

 

 

 

 

 

 

 

 

 

 

 

Tongue - short

0

0

0.0

0

0

0.0

1

1

0.7

0

0

0.0

Rugae - non-uniform patterning

5

5

2.8

11

8

6.6

7

7

4.3

13

9

7.9

Brain - olfactory ventricle - enlarged

0

0

0.0

0

0

0.0

1

1

0.5

0

0

0.0

Brain - cerebral aqueduct - enlarged

0

0

0.0

0

0

0.0

2

1

1.1

0

0

0.0

Abdomen

 

 

 

 

 

 

 

 

 

 

 

 

Liver - additional lobe between right and left median

2

2

1.2

1

1

0.7

0

0

0.0

0

0

0.0

Liver - papillary process - reduced in size

1

1

0.6

0

0

0.0

0

0

0.0

0

0

0.0

Umbilical artery - left-sided

1

1

0.5

0

0

0.0

0

0

0.0

0

0

0.0

Testis - partially undescended

1

1

0.5

1

1

0.6

0

0

0.0

1

1

0.6

Ureter - kinked

28

13

16.3

17

11

10.2

10

7

5.7

12

8

7.3

Ureter - dilated

23

11

13.4

12

8

7.4

7

4

4.0*

3

3

1.9**

Renal pelvic cavitation - increased

9

5

5.1

10

7

5.9

4

4

2.3

11

6

6.4

Renal papilla - absent

2

2

1.2

1

1

0.6

1

1

0.5

4

2

2.5

Renal medulla - reduced in size

0

0

0.0

0

0

0.0

1

1

0.5

0

0

0.0

Renal medulla - absent

0

0

0.0

0

0

0.0

0

0

0.0

1

1

0.6

Thorax

 

 

 

 

 

 

 

 

 

 

 

 

Thyroid - Isthmus thickened

0

0

0.0

0

0

0.0

2

1

1.1

0

0

0.0

Thyroid - parathyroid - enlarged

0

0

0.0

0

0

0.0

1

1

0.5

0

0

0.0

Thymus - lobe partially undescended

9

5

4.9

10

6

6.0

4

4

2.4

13

9

8.0

Lungs - right lobe - single lobe present

1

1

0.6

0

0

0.0

0

0

0.0

0

0

0.0

%†: Group mean percent

NF: Number of Fetuses

NL: Number of litters

* p<0.05; ** p<0.01

Table 9a: Summary Incidence of Fetal Visceral Findings Continued

Visceral Findings

Dose Level (mg/kg bw/day)

0 (Control)

25

100

250

Number of Fetuses (litters) Examined

172 (24)

169 (23)

166 (23)

156 (22)

NF

NL

%†

NF

NL

%†

NF

NL

%†

NF

NL

%†

Thorax (continued)

 

 

 

 

 

 

 

 

 

 

 

 

Atrium - enlarged

1

1

0.6

0

0

0.0

1

1

0.6

0

0

0.0

Single atrioventricular valve

1

1

0.6

0

0

0.0

0

0

0.0

0

0

0.0

Atrial septal defect

1

1

0.6

0

0

0.0

0

0

0.0

0

0

0.0

Ventricle - enlarged

1

1

0.6

0

0

0.0

0

0

0.0

0

0

0.0

Ventricular septal defect

1

1

0.6

0

0

0.0

0

0

0.0

0

0

0.0

Total

45

17

25.7

37

18

22.2

25

16

14.8

39

15

23.7

%†: Group mean percent

NF: Number of Fetuses

NL: Number of litters

Table 10: Summary Incidence of Fetal Skeletal Findings

Skeletal Findings

Dose Level (mg/kg bw/day)

0 (Control)

25

100

250

Number of Fetuses (litters) Examined

162 (24)

160 (23)

155 (23)

142 (22)

NF

NL

%†

NF

NL

%†

NF

NL

%†

NF

NL

%†

Skull

 

 

 

 

 

 

 

 

 

 

 

 

Fontanelle (anterior) - large

2

1

1.0

0

0

0.0

4

4

2.5

1

1

0.9

Nasal - incomplete ossification

15

8

8.9

23

13

14.3

14

8

8.7

32

14

21.1

Nasal/Frontal - sutural bone

1

1

0.6

1

1

0.5

0

0

0.0

0

0

0.0

Frontal - incomplete ossification

3

3

1.7

4

4

2.5

4

4

2.4

12

9

7.8

Frontal - unossified area

3

3

1.8

1

1

0.7

5

4

3.1

14

7

9.9

Frontal - fissure

0

0

0.0

1

1

0.7

0

0

0.0

0

0

0.0

Parietal - incomplete ossification

9

6

5.3

14

10

8.6

12

7

7.4

27

12

17.9*

Parietal/Interparietal - sutural bone

0

0

0.0

0

0

0.0

0

0

0.0

2

2

1.3

Interparietal - incomplete ossification

32

13

19.9

27

12

16.9

26

13

16.6

54

17

37.2*

Occipital (Supra-occipital) - incomplete ossification

26

12

16.1

20

12

12.6

22

13

14.2

46

16

30.7

Occipital (Supra-occipital) - unossified area(s)

11

9

7.1

10

8

6.3

9

6

5.7

6

6

4.0

Squamosal - incomplete ossification

18

9

11.3

22

11

13.6

18

9

11.5

33

13

22.1

Squamosal - unossified area(s)

0

0

0.0

1

1

0.5

2

2

1.2

4

3

2.5

Jugal - incomplete ossification

5

4

2.9

11

8

7.3

6

6

4.2

17

9

11.7

Zygomatic process of maxilla - incomplete ossification

9

7

5.5

19

11

12.4

18

10

11.7

25

11

17.4

Zygomatic process of maxilla -elongated

1

1

0.6

0

0

0.0

0

0

0.0

0

0

0.0

Zygomatic process of squamosal - incomplete ossification

0

0

0.0

5

4

3.0*

1

1

0.6

9

5

6.2*

Zygomatic process of squamosal - fused to jugal

1

1

0.6

0

0

0.0

0

0

0.0

0

0

0.0

Premaxilla - incomplete ossification

4

3

2.3

1

1

0.6

4

4

2.3

9

6

6.2

Hyoid - incomplete ossification

10

9

5.8

16

11

9.7

24

15

15.7*

17

8

11.1*

Hyoid - not ossified

21

12

12.5

17

12

9.7

26

13

16.6

30

14

19.2

Tympanic annulus - incomplete ossification

0

0

0.0

0

0

0.0

1

1

0.6

0

0

0.0

%†: Group mean percent

NF: Number of Fetuses

NL: Number of litters

* p<0.05

Table 10a: Summary Incidence of Fetal Skeletal Findings - Continued

Skeletal Findings

Dose Level (mg/kg bw/day)

0 (Control)

25

100

250

Number of Fetuses (litters) Examined

162 (24)

160 (23)

155 (23)

142 (22)

NF

NL

%†

NF

NL

%†

NF

NL

%†

NF

NL

%†

Skull (continued)

 

 

 

 

 

 

 

 

 

 

 

 

Presphenoid - incomplete ossification

1

1

0.7

5

2

2.6

2

2

1.2

3

2

1.9

Presphenoid - not ossified

0

0

0.0

2

2

1.1

0

0

0.0

3

3

1.9

Vertebral column

 

 

 

 

 

 

 

 

 

 

 

 

Odontoid - ossification present

3

3

1.8

0

0

0.0

0

0

0.0

0

0

0.0

Ventral arch of vertebra 1 - ossification present

30

16

18.6

47

22

29.8

40

19

26.8

49

16

34.8

Cervical (neural) arch - incomplete ossification

13

7

7.5

10

7

6.7

9

8

5.7

8

6

5.4

Cervical (neural) arch - fused

0

0

0.0

0

0

0.0

0

0

0.0

1

1

0.6

Thoracic centrum - incomplete ossification

5

4

2.9

5

4

3.4

2

2

1.2

5

4

3.5

Thoracic centrum - not ossified

1

1

0.6

0

0

0.0

2

2

1.2

0

0

0.0

Thoracic centrum - bipartite ossification

2

2

1.1

3

3

2.1

1

1

0.6

2

2

1.7

Thoracic centrum - dumb-bell-shaped

18

12

11.4

15

11

9.2

12

9

8.1

20

12

14.0

Thoracic centrum - asymmetrically ossified

4

2

2.3

2

2

1.6

4

4

2.7

2

2

1.2

Lumbar centrum - incomplete ossification

1

1

0.6

1

1

0.5

0

0

0.0

0

0

0.0

Lumbar (neural) arch - incomplete ossification

0

0

0.0

0

0

0.0

0

0

0.0

1

1

0.6

Sacral centrum - incomplete ossification

0

0

0.0

0

0

0.0

1

1

0.5

0

0

0.0

Sacral (neural) arch - incomplete ossification

25

12

15.0

28

12

17.7

33

16

21.2

51

17

34.9*

Sacral (neural) arch - not ossified

0

0

0.0

0

0

0.0

1

1

0.5

1

1

0.8

Caudal vertebrae - less than 4 ossified

39

18

23.6

39

15

23.9

43

15

27.4

59

16

39.6

Number of pre-sacral vertebrae = 25/27

2

2

1.3

0

0

0.0

1

1

0.6

0

0

0.0

Ribs

 

 

 

 

 

 

 

 

 

 

 

 

Ossification centre - associated with 7th cervical vertebra

0

0

0.0

0

0

0.0

1

1

0.6

0

0

0.0

14th rib - extra - associated with 1st lumbar vertebra

0

0

0.0

0

0

0.0

1

1

0.6

0

0

0.0

Ossification centre - associated with 1st lumbar vertebra

5

5

3.2

9

3

5.8

6

5

4.0

2

2

1.3

%†: Group mean percent

NF: Number of Fetuses

NL: Number of litters

* p<0.05

Table 10b: Summary Incidence of Fetal Skeletal Findings - Continued

Skeletal Findings

Dose Level (mg/kg bw/day)

0 (Control)

25

100

250

Number of Fetuses (litters) Examined

162 (24)

160 (23)

155 (23)

142 (22)

NF

NL

%†

NF

NL

%†

NF

NL

%†

NF

NL

%†

Ribs (continued)

 

 

 

 

 

 

 

 

 

 

 

 

One or more ribs - wavy

1

1

0.7

1

1

0.6

0

0

0.0

1

1

0.6

One or more ribs - thickened

1

1

0.7

0

0

0.0

1

1

0.6

0

0

0.0

Rib - short

1

1

0.7

1

1

0.5

0

0

0.0

0

0

0.0

Rib - rudimentary

1

1

0.6

0

0

0.0

0

0

0.0

0

0

0.0

Rib - fused

1

1

0.6

0

0

0.0

0

0

0.0

0

0

0.0

Rib - no articulation point

1

1

0.6

1

1

0.5

0

0

0.0

0

0

0.0

Costal cartilage - misaligned

4

4

2.4

2

2

1.0

2

2

1.2

1

1

0.6

Costal cartilage - not fused to sternebra

28

12

17.9

16

8

10.3

14

9

9.2

13

10

9.6

Sternebrae

 

 

 

 

 

 

 

 

 

 

 

 

Sternebra - incomplete ossification

0

0

0.0

5

4

3.0*

5

5

3.1*

8

7

5.4**

Sternebra - not ossified

2

2

1.2

0

0

0.0

2

2

1.2

0

0

0.0

Sternebra - bipartite ossification

1

1

0.6

0

0

0.0

1

1

0.9

0

0

0.0

Sternebra - misaligned

2

2

1.2

3

3

1.7

6

6

4.0

5

4

3.6

Sternebra - misshapen

1

1

0.6

0

0

0.0

0

0

0.0

0

0

0.0

Xiphoid cartilage - partially split

4

4

2.5

6

6

3.8

11

7

8.0

6

4

4.4

Pectoral Girdle

 

 

 

 

 

 

 

 

 

 

 

 

Scapula - bent

0

0

0.0

0

0

0.0

1

1

0.5

0

0

0.0

Scapula - misshapen

3

3

1.8

0

0

0.0

1

1

0.7

4

4

2.7

Pelvic Girdle

 

 

 

 

 

 

 

 

 

 

 

 

Ischium - incomplete ossification

1

1

0.6

2

2

1.3

1

1

0.6

6

5

4.0

Pubis - not ossified

1

1

0.6

2

2

1.1

1

1

0.6

3

3

2.2

Pubis - incomplete ossification

8

6

4.6

5

5

3.1

12

7

7.6

15

11

9.9

Table 10c: Summary Incidence of Fetal Skeletal Findings - Continued

Skeletal Findings

Dose Level (mg/kg bw/day)

0 (Control)

25

100

250

Number of Fetuses (litters) Examined

162 (24)

160 (23)

155 (23)

142 (22)

NF

NL

%†

NF

NL

%†

NF

NL

%†

NF

NL

%†

Forelimbs

 

 

 

 

 

 

 

 

 

 

 

 

Metacarpal - not ossified

56

19

34.3

46

16

28.4

63

19

39.5

83

18

56.4

Metacarpal - incomplete ossification

2

2

1.2

2

2

1.2

3

3

1.9

9

6

5.8

Forepaw phalanges - 1 or more - ossified

8

4

4.8

14

7

8.5

10

6

6.3

8

6

5.6

Humerus - incomplete ossification

1

1

0.6

3

3

1.8

2

2

1.2

7

5

4.8

Humerus - hole

3

3

1.8

0

0

0.0

1

1

0.6

0

0

0.0

Humerus - bent

0

0

0.0

0

0

0.0

1

1

0.5

0

0

0.0

Hindlimbs

 

 

 

 

 

 

 

 

 

 

 

 

Metatarsal - 1st - ossified

1

1

0.8

1

1

0.5

0

0

0.0

0

0

0.0

Metatarsal - not ossified

0

0

0.0

0

0

0.0

2

2

1.2

0

0

0.0

Metatarsal - incomplete ossification

1

1

0.6

0

0

0.0

2

2

1.2

2

2

1.6

Femur - incomplete ossification

11

7

6.9

6

4

3.8

17

8

10.1

30

14

20.6**

Total

136

24

84.3

132

23

82.3

134

23

86.8

135

22

95.2

%†: Group mean percent

NF: Number of Fetuses

NL: Number of litters

* p<0.05; ** p<0.01

Table 11 Caesarea necropsy findings

   number of females  V  ED  LD  DF  NP
 control  24  22  1  1    
 25 mg/kg bw/d  24  20  3      1
 100 mg/kg bw/d  24  20  3  1    1
 250 mg/kg bw/d  24  19  2  1  1  2

V: viable fetuses; ED: early death (no visible distinction between placental/decidual tissue and embryonic tissue); LD: late death: seperate embryonic/fetal and placental tissue visible; DF: dead fetus: a fetus that died shortly before necropsy; NP: non pregnant

Applicant's summary and conclusion

Conclusions:
The oral (gavage) administration of the test substance to pregnant rats during gestation at dose levels of 25, 100 and 250 mg/kg bw/day resulted in reductions in body weight gain and food consumption throughout the treatment period in females treated with 250 mg/kg bw/day. Although a slight reduction in body weight gain and cumulative body weight gain was noted for the 100 mg/kg bw/day dose group between Days 8 and 11 and Days 3 and 11 and 3 and 17 respectively, the effects were minimal and body weight gains for the remainder of the periods were comparable to controls. Consequently, 100 mg/kg bw/day was considered to represent the No Observed Adverse Effect Level (NOAEL) for the pregnant female.
No treatment-related changes were detected in the offspring parameters measured or on embryofetal development. The ‘No Observed Adverse Effect Level’ (NOAEL) for developmental toxicity was therefore considered to be 250 mg/kg bw/day.