Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04 April 2018 - 04 June 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report Date:
2019

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 guidelines for Toxicology studies, 12 NohSan No 8147
Version / remarks:
24 November 2000
Deviations:
no
Qualifier:
according to
Guideline:
other: Commission Regulation (EC) No 440/2008 of 30 May 2008 test methods pursuant to Regulations (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH)
Deviations:
no
GLP compliance:
yes (incl. certificate)
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: TE767F05G
- Expiration date of the lot/batch: 05 June 2019
- Purity: 99.7%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark
- Solubility and stability of the test substance in the solvent/vehicle: stable for 4 hours
- Samples were taken of test item formulations and were analyzed on two occasions for
concentration of Methoxypropylamine (MOPA) at Envigo Analytical Laboratory, Shardlow. The results indicate that the prepared formulations were within 7% of the nominal concentration.

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: 199 to 285g
- Fasting period before study: no data
- Housing: The animals were housed in a single air-conditioned room within the Envigo Research Limited, Shardlow, UK Barrier Maintained Rodent Facility.
- Diet: ad libitum; A pelleted diet (Rodent 2018C Teklad Global Certified Diet, Envigo RMS (UK) Limited, Oxon, UK)
- Water: ad libitum; Mains drinking water was supplied from polycarbonate bottles attached to the cage.
- Acclimation period: no data

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

IN-LIFE DATES: From: 07 April 2018 (first day of treatment) and 26 April 2018 (final day of necropsy).

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Formulations were shown to be stable for four hours. Formulations were therefore prepared daily and dosed within four hours of preparation.

VEHICLE
- Concentration in vehicle: 7.5, 18.75 and 37.5 mg/mL
- Treatment volume: 4 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 Arachis Oil BP formulations at nominal concentrations of 2.5 mg/mL and 100 mg/mL when stored for 4 hours during study number WD30CK.
The mean concentrations of test item in test formulations analyzed for the study were within ± 10% of nominal concentrations, confirming accurate formulation.
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 19 of gestation
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Control group
Dose / conc.:
30 mg/kg bw/day (nominal)
Remarks:
low treatment group
Dose / conc.:
75 mg/kg bw/day (nominal)
Remarks:
Intermediate treatment group
Dose / conc.:
150 mg/kg bw/day (nominal)
Remarks:
high treatment group
No. of animals per sex per dose:
24 females per group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were chosen in collaboration with the Sponsor Representative and were based on available toxicity data including a Preliminary Oral (Gavage) Pre-Natal Development Toxicity Study in the Rat (Covance CRS Study Number VF78GT) and a 90 Day Oral (Gavage) Toxicity Study in the Rat (Covance CRS Study Number WD30CK).
- 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 4, 5, 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:
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:
Data was assessed using the R Environment for Statistical Computing. Initially, the distribution of the data was assessed by the Shapiro-Wilk normality test, followed by assessment of the homogeneity of the data using Bartlett’s test. Where considered appropriate, parametric analysis of the data was applied incorporating analysis of variance (ANOVA), which if significant, was followed by pair-wise comparisons using Dunnett’s test. Where parametric analysis of the data was considered to be unsuitable, non-parametric analysis of the data was performed incorporating the Kruskal-Wallis test which if significant was followed by the Mann-Whitney "U" test. Dose response relationships were also investigated by linear regression.
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 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):
Instances of increased salivation were evident in all females treated with 150 mg/kg bw/day between Days 13 and 19 and in two females treated with 75 mg/kg bw/day on Day 19 only.
No such effects were detected in females treated with 30 mg/kg bw/day.

The following observations were noted in a small number of control or treated females and in isolation, these were considered to be incidental and unrelated to the test item. One control female had scattered scabs on one occasion, another control female had red/brown staining around the eyes on one occasion, one female treated with 75 mg/kg bw/day had red/brown staining around the mouth on one occasion, and three females treated with 150 mg/kg bw/day had generalized fur loss between Days 16 and 20, with one of these females also having red/brown staining around the mouth on one occasion.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
There were no unscheduled deaths.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weight and body weight gain, including adjustment for the contribution of the gravid uterus, was unaffected by treatment at 30, 75 or 150 mg/kg bw/day.

Females from all treatment groups showed a statistically significant increase (p<0.05-0.001) in body weight gain between Days 3 and 4 of gestation. An increase in body weight gain is considered not to reflect an adverse effect of treatment and the increase was not dose related and was therefore considered not to be of toxicological significance.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No adverse effect on food consumption was evident in females from any treatment group.

Females treated with 150 mg/kg bw/day exhibited a statistically significant reduction (p<0.01) in food consumption between Days 8 and 11 of gestation. As no such effects were evident before or after this period and all but two individual values for food consumption at this treatment group were within the historical control data range, this reduction was considered not to be of any toxicological significance.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Daily visual inspection of water bottles did not reveal any overt intergroup differences.
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, treatment-related
Description (incidence and severity):
Sixteen females treated with 150 mg/kg bw/day had ulceration on the non-glandular region of the stomach. One of these females also had a pale stomach. A further five females from this treatment group had ulceration on both the non-glandular and glandular regions of the stomach. Eleven females treated with 75 mg/kg bw/day had ulceration on the non-glandular region of the stomach and one female treated with 30 mg/kg bw/day had white ridges on the non-glandular region of the stomach.
One female treated with 150 mg/kg bw/day had a mass on the right side of the abdominal cavity. In isolation this was considered to be incidental and unrelated to treatment.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined

Maternal developmental toxicity

Number of abortions:
no effects observed
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: 15.6%, 18.1%, 17.1%, 18.7% at 0, 30, 75 and 150 mg/kg bw/day
- post-implantation loss: 2.3%, 1.3%, 1.5%, 2.7% at 0, 30, 75 and 150 mg/kg bw/day
All data was within the historical control range available for this species and strain.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
There were no total litter losses by resorption observed.
Early or late resorptions:
no effects observed
Description (incidence and severity):
There were no treatment-related effects on early or late resorptions observed.
Dead fetuses:
no effects observed
Description (incidence and severity):
There were no treatment-related effects on the number of dead fetuses observed.
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
Cumulative Body Weight Changes were recorded until day 20 of pregnancy, suggesting a duration of 20 days for all females.
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, 24/24, 24/24 and 24/24 at 0,25, 100 and 250 mg/kg bw/day, resp. No historical control data is available for this parameter.
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 and pre and post-implantation losses at 30, 75 or 150 mg/kg bw/day.
Intergroup differences for mean fetal, litter or placental weights did not indicate any adverse effects of maternal treatment at 30, 75 or 150 mg/kg bw/day.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
clinical signs
gross pathology
Remarks on result:
other: at a lesser extent, also evident in females treated with 75 mg/kg bw/day

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Intergroup differences for mean fetal, litter or placental weights did not indicate any adverse effects of maternal treatment at 30, 75 or 150 mg/kg bw/day.
Male fetal weight and combined fetal weights for litters from females treated with 30 mg/kg bw/day showed a statistically significant increase (p<0.05-0.01) when compared to controls. An increase in fetal weight is generally not considered to represent an adverse effect of treatment and in the absence of a similar effect at the higher dosages, the intergroup differences are considered not to be of toxicological significance.
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.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
There was no treatment-related effect observed on sex ratio.
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.
Changes in postnatal survival:
no effects observed
Description (incidence and severity):
There was no treatment-related effects observed on postnatal survival.
External malformations:
no effects observed
Description (incidence and severity):
Neither the type, incidence nor distribution of external finding apparent for fetuses at Day 20 of gestation indicated an adverse effect of maternal treatment on fetal development at 30, 75 or 150 mg/kg bw/day.

A statistically significant increase (p<0.05) in the number of fetuses/litters showing a large placenta was evident at 150 mg/kg bw/day. The group mean value (4.7%) was slightly above the historical control range (0.0 - 4.2%), however, this was considered to be the result of one litter which had five fetuses that had this finding. With this litter excluded, the group mean value was within the historical control range. In the absence of any associated effects, the intergroup difference was considered not to be of toxicological significance.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Skeletal examinations of fetuses on Day 20 of gestation did not indicate any obvious effect of maternal treatment on fetal development at 30, 75 or 150 mg/kg bw/day.
Statistically significant increases (p<0.01) in the number of fetuses/litters showing a ossification centre associated with the first lumbar vertebra, incomplete ossification of the squamosal of the skull and zygomatic process of maxilla of the skull were noted in females treated with 150 mg/kg bw/day. The observations of one variant at a higher incidence compared with controls is not significant when evaluated in isolation. In the absence of a particular pattern of abnormal skeletal development of skeletal structures affecting treated fetuses, the observation of one affected skeletal structure can be considered unlikely to represent true developmental abnormality. This also takes account of these findings being seen regularly on this study type amongst control group fetuses.
A statistically significant increase (p<0.05) in the number of fetuses/litter showing incomplete ossification of the sternebra was evident at 75 mg/kg bw/day. Statistically significant reductions (p<0.05-0.01) in incomplete ossification of the cervical (neural) arch and incomplete ossification of the occipital (supra-occipital) was evident at 30 mg/kg bw/day. In the absence of similar findings at higher dosages or any particular pattern of abnormal skeletal development of skeletal structures affecting treated fetuses, the observation of one affected skeletal structure can be considered unlikely to represent true developmental abnormality.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Visceral examinations of fetuses on Day 20 of gestation did not indicate any obvious effect of maternal treatment on fetal development at 30, 75 or 150 mg/kg bw/day.

A statistically significant increase (p<0.05) in the number of fetuses/litter showing a nonuniform patterning of the rugae was evident at 75 and 30 mg/kg bw/day. In the absence of a similar finding at the highest dosage, the intergroup differences were considered unrelated to treatment.
Details on embryotoxic / teratogenic effects:
No treatment-related changes were detected in the offspring parameters measured or on embryofetal development. The ‘No Observed Effect Level’ (NOEL) for developmental toxicity was therefore considered to be 150 mg/kg bw/day.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: developmental toxicity

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Any other information on results incl. tables

Table 1   Summary of Female Performance

Category

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

0 (Control)

30

75

150

Initial Group Size

24

24

24

24

Pregnant

24

24

24

24

 

Table 2   Summary Incidence of Daily Clinical Observations

Dose Level

(mg/kg bw/day)

Number of Animals

Clinical Observations

Number Showing Effect (Days post coitum affected)

0 (Control)

24

No Abnormalities Detected

Scattered Scabs

22 (0-20)

1 (5)

 

 

Red/Brown Staining around the Eyes

1 (9)

30

24

No Abnormalities Detected

24 (0-20)

75

24

No Abnormalities Detected

Increased Salivation

21 (0-20)

2 (19)

 

 

Red/Brown Staining Around the Mouth

1 (19)

150

24

No Abnormalities Detected

Fur Loss (Hind Quarters)

3 (0-20)

3 (16-20)

 

 

Red/Brown Staining Around the Mouth

1 (13)

 

 

Increased Salivation

20 (13-19)

 

Table 3   Group Mean Body Weight Values

Dose Level

(mg/kg bw/day)

 

3

4

Body Weight (g) on Day of Gestation

5          8          11       14

17

20

0 (Control)

mean

sd

260.7

21.6

261.6

20.9

267.0

20.2

280.6

22.9

300.8

23.1

320.0

25.0

347.4

27.4

395.8

33.6

 

n

24

24

24

24

24

24

24

24

30

mean

sd

258.6

16.2

261.3

17.4

266.4

17.9

280.0

19.5

300.1

21.0

318.7

23.2

349.5

23.8

393.9

30.6

 

n

24

24

24

24

24

24

24

24

75

mean

sd

256.8

15.8

261.7

15.5

264.7

16.2

276.6

16.5

295.9

17.3

314.8

18.6

342.8

22.1

388.9

28.7

 

n

24

24

24

24

24

24

24

24

150

mean

sd

255.8

16.3

260.5

16.4

264.2

16.9

276.1

15.6

293.9

17.3

312.6

17.7

339.6

21.5

384.5

26.7

 

n

24

24

24

24

24

24

24

24

 

Table 4   Group Mean Body Weight Change Values 

Dose

Level

 

(mg/kg bw/day)

3 to 4

 

Body Weight Change (g) during Days of Gestation

4 to 5   5 to 8   8 to 11    11 to 14   14 to 17

17 to 20

0

(Control)

mean

sd

0.9

5.6

 

5.4

3.7

13.6

5.3

20.2

4.8

19.2

6.1

27.4

5.3

48.4

8.1

 

n

24

 

24

24

24

24

24

24

30

mean

sd

2.8*

3.8

 

5.1 3.9

13.6

4.6

20.1

4.4

18.6

6.5

30.8

5.3

44.4

9.2

 

n

24

 

24

24

24

24

24

24

75

mean

sd

4.9***

2.9

 

3.0 3.2

11.9

4.4

19.3

4.7

18.9

5.5

28.0

6.6

46.1

8.3

 

n

24

 

24

24

24

24

24

24

150

mean

sd

4.7*

7.8

 

3.7 3.6

11.9

4.1

17.8

5.5

18.7

5.2

27.0

8.2

44.9

7.7

 

n

24

 

24

24

24

24

24

24

 

Dose Level

(mg/kg bw/day)

 

4

Cumulative Body Weight Change (g) from Day 3 of Gestation

           5                8               11              14              17

20

0 (Control)

mean

sd

0.9 5.6

6.3

5.1

19.9

7.9

40.1

8.6

59.3

10.7

86.7

13.2

135.1

19.3

 

n

24

24

24

24

24

24

24

30

mean

sd

2.8*

3.8

7.8

4.5

21.4

6.7

41.5

8.4

60.1

11.5

90.9

12.3

135.3

19.8

 

n

24

24

24

24

24

24

24

75

mean

sd

4.9***

2.9

7.9

3.2

19.8

5.6

39.1

6.2

58.0

7.9

86.0 10.1

132.1

16.6

 

n

24

24

24

24

24

24

24

150

mean

sd

4.7*

7.8

8.4

8.1

20.3

9.1

38.1

10.9

56.8

12.2

83.8

15.8

128.7

21.4

 

n

24

24

24

24

24

24

24

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

 3            20

Body Weight

Change (g) during Days of Gestation

3-20

Gravid

Uterus

Weight

(g)

Adjusted

Body

Weight (g) Day 20

Adjusted

Body Weight

Change (g)

3-20

0 (Control)

mean

sd

260.7

21.6

395.8

33.6

135.1

19.3

80.985 13.696

314.8

27.1

54.1 11.5

 

n

24

24

24

24

24

24

30

mean

sd

258.6

16.2

393.8

30.4

135.2

19.5

78.509 14.164

315.3

25.8

56.7 15.9

 

n

24

24

24

24

24

24

75

mean

sd

256.8

15.8

388.9

28.7

132.1

16.6

80.375 12.871

308.5

23.0

51.8 11.4

 

n

24

24

24

24

24

24

150

mean

sd

255.8

16.3

384.5

26.7

128.7

21.4

77.238 19.951

307.3

19.3

51.5 12.2

 

n

24

24

24

24

24

24

 

Table 6   Group Mean Food Consumption Values

Dose Level

(mg/kg bw/day)

 

Food Consumption (g/rat/day) between Days of Gestation

3 - 5        5 - 8       8 - 11     11 - 14    14-17      17 - 20

0 (Control)

mean

sd

21.4

1.8

23.0

2.5

24.2

2.2

24.3

2.4

25.3

4.8

27.3

4.5

 

n

24

23

24

24

24

24

30

mean

sd

21.0

2.8

23.2

3.2

23.7

3.0

24.4

6.0

26.5

5.1

26.3

3.4

 

n

24

24

24

24

24

24

75

mean

sd

21.5

3.3

24.2

10.0

24.1

6.4

23.2

2.7

26.0

4.9

26.2

3.6

 

n

24

24+

23#

24

24

24

150

mean

sd

20.6

4.6

21.2

2.0

21.7**

2.5

22.6

1.9

24.1

4.9

26.0

5.1

 

n

24

24

24

24

24

24

   + = Food consumption for Female 61 calculated for Days 5-7 only due to female being present in incorrect cage on Day 8. 

# = Data unavailable for Female No. 63, due to erroneous value recorded for food allocated on Day 8. 

Table 7   Summary Incidence of Necropsy Findings

 

 

Dose Level (mg/kg bw/day)

 

0 (Control)

30

75

150

TERMINAL DEATH

 

Number of Animals Examined

 

 

24

 

 

24

 

 

24

 

 

24

 

External:

Generalized Fur Loss

 

 

0

 

 

0

 

 

0

 

 

1

Mass on Right Side

0

0

0

1

 

Internal:

Stomach – Non-Glandular Region: White Ridges

 

 

0

 

 

1

 

 

0

 

 

0

Stomach – Non-Glandular Region: Ulcerated

0

0

11

16

Stomach: Ulcerated

0

0

0

5

Stomach: Pale

0

0

0

1

Outer Right Side of Abdominal Cavity: Mass

0

0

0

1

 

No Abnormalities Detected

 

 

24

 

 

23 

 

13

 

 

3

 

 


Table 8   Group Mean Litter Data Values

Dose Level

(mg/kg bw/day)

 

Number of

Corpora Lutea

Number of

Implants

Number of

Embryonic/Fetal Deaths

Implantation

Loss 

%

Number of Live Implants

%

Male

Fetuses

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)

Early

Late

Total

Pre

Post

Male

Female

Total

0 (Control)

mean

sd

16.3

2.3

13.8

2.6

0.2

0.5

0.1

0.3

0.3 0.6

15.6 12.3

2.3 3.8

6.7

2.2

6.7

2.0

13.4

2.5

50.3

13.0

3.917

0.315

3.742

0.306

3.831

0.286

0.537

0.065

51.054

9.067

7.137

1.459

 

n

23#

24

24

24

24

23

24

24

24

24

24

24

24

24

23+

24

23+

30

mean

sd

15.4

2.4

12.8

2.8

0.1 0.3

0.0 0.2

0.2 0.4

18.1 13.4

1.3 3.0

6.3 1.8

6.3 2.2

12.6

2.9

52.1

15.2

4.253**

0.546

3.906

0.271

4.140*

0.569

0.571 0.132

50.878

9.780

6.834

1.202

 

n

24

24

24

24

24

24

24

24

24

24

24

24

23

24

24+

24

24+

75

mean

sd

16.2

2.3

13.5

2.5

0.2

0.4

0.0 0.2

0.2 0.4

17.1

7.4

1.5 3.0

7.1 2.7

6.2 2.1

13.3

2.5

53.0

17.2

3.961

0.291

3.771

0.305

3.877 0.279

0.540 0.045

51.140

8.766

7.138

1.334

 

n

24

24

24

24

24

24

24

24

24

24

24

24

24

24

24

24

24

150

mean

sd

15.3

2.2

12.6

3.2

0.1

0.4

0.0

0.2

0.2

0.5

18.7

14.7

2.7

10.3

6.5

2.5

5.9

2.5

12.4

3.4

52.9

14.3

4.029

0.264

3.781

0.309

3.913

0.289

0.608

0.165

48.735

13.796

7.201

1.968

 

n

24

24

24

24

24

24

24

24

24

24

24

24

24

24

24

24

24

+ = Placental weights not recorded in error for Female No. 21’s litter and for fetus No. 8 from Female No. 25

# = Data unavailable for Female No. 16

Table 9   Summary Incidence of Fetal External Findings

 

External Findings

 

Dose level (mg/kg bw/day)

 

 

0 (Control)

30

75

 

150

 

Number of fetuses (litters) examined

 

 

322 (24)

303 (24)

318 (24)

 

298 (24)

NF

NL

%†

NF

NL

%†

NF

NL

%†

NF

NL

%†

Total Number Affected

 

4

1

1.3

8

5

5.9

7

6

2.2

15

6

6.6

Small Fetus

 

4

1

1.3

6

4

1.8

5

4

1.6

8

4

2.8

Pale Fetus

 

1

1

0.3

0

0

0.0

0

0

0.0

0

0

0.0

Large Fetus

 

0

0

0.0

2

1

4.2

0

0

0.0

0

0

0.0

Large Placenta

 

0

0

0.0

2

1

4.2

1

1

0.3

9

5

4.7*

Small Placenta

 

2

1

0.6

1

1

0.2

2

2

0.7

1

1

0.3

 

            

Table 10 Summary Incidence of Fetal Visceral Findings

Visceral Findings

 

 

 

 

Dose Level (mg/kg bw/day)

 

 

 

 

 

0 (Control)

 

30

75

 

 

150

 

 

 

 

 

Number of Fetuses (litters) Examined

 

 

 

 

 

168 (24)

 

 

156 (24)

164 (24)

 

 

157 (24)

 

NF

 

NL

%†

NF

NL

%†

NF

NL

%†

NF

NL

%†

External

Hemorrhage 

 

0

 

 

0

 

0.0

 

0

 

0

 

0.0

 

1

 

1

 

0.5

 

0

 

0

 

0.0

Head

Rugae - non-uniform patterning

 

2

 

 

2

 

1.0

 

11

 

8

 

6.8*

 

8

 

8

 

4.8*

 

5

 

2

 

3.9

Abdomen

Liver - additional lobe between right and left median

 

2

 

 

2

 

1.2

 

2

 

1

 

1.0

 

0

 

0

 

0.0

 

0

 

0

 

0.0

Umbilical artery - left-sided

0

 

0

0.0

0

0

0.0

1

1

0.8

2

2

1.2

Testis - partially undescended

4

 

3

2.4

4

4

2.5

1

1

0.6

2

2

1.2

Testis - malrotated

0

 

0

0.0

0

0

0.0

0

0

0.0

1

1

0.5

Ureter - kinked

15

 

10

10.0

11

7

7.0

12

6

8.3

9

7

7.3

Ureter - dilated 

11

 

7

7.9

3

3

1.9

8

4

5.9

5

4

4.9

Kidney - malpositioned 

0

 

0

0.0

0

0

0.0

1

1

0.8

0

0

0.0

Renal pelvic cavitation - increased 

8

 

6

4.4

8

7

5.0

7

5

4.5

10

6

7.5

Renal papilla - absent

1

 

1

0.5

2

2

1.3

3

2

2.3

1

1

1.4

Renal medulla - reduced in size

1

 

1

0.5

0

0

0.0

0

0

0.0

0

0

0.0

Thorax

Thymus - lobe partially undescended

 

5

 

 

4

 

2.8

 

5

 

4

 

2.9

 

3

 

3

 

1.8

 

4

 

4

 

2.2

Atrium - enlarged

4

 

4

2.3

1

1

0.7

2

2

1.1

3

3

1.6

Ventricle - reduced in size

0

 

0

0.0

0

0

0.0

1

1

0.5

0

0

0.0

Ventricle - vacuole

0

 

0

0.0

0

0

0.0

1

1

0.5

0

0

0.0

Total

30

 

18

18.5

31

14

18.9

27

14

17.0

30

15

20.3

 

Table 11 Summary Incidence of Fetal Skeletal Findings

Skeletal Findings

 

Dose Level (mg/kg bw/day)

 

 

0 (Control)

30

75

 

150

 

Number of Fetuses (litters) Examined

 

 

154 (24)

147 (24)

154 (24)

 

141 (24)

NF

NL

%†

NF

NL

%†

NF

NL

%†

NF

NL

%†

Skull

Fontanelle (anterior) - large

 

2

 

1

 

1.4

 

2

 

2

 

1.1

 

9

 

4

 

5.2

 

1

 

1

 

0.6

Nasal - incomplete ossification

10

5

6.2

6

5

3.9

18

9

10.5

19

9

12.5

Nasal/Frontal - sutural bone

1

1

0.7

0

0

0.0

0

0

0.0

0

0

0.0

Frontal - incomplete ossification

1

1

0.7

0

0

0.0

5

4

2.9

8

5

5.1

Frontal - unossified area

1

1

0.7

3

2

1.9

5

3

3.5

3

2

1.6

Parietal - incomplete ossification

6

4

3.7

3

2

2.1

8

6

5.0

20

10

14.2

Interparietal - incomplete ossification

29

11

18.4

14

8

9.1

30

12

18.5

38

14

25.7

Occipital (Supra-occipital) - incomplete ossification

17

12

11.1

6

4

4.1*

23

10

14.2

26

14

18.4

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

12

8

7.2

7

6

4.3

8

5

4.6

8

7

6.5

Squamosal - incomplete ossification

7

5

4.4

8

4

5.4

14

7

8.1

30

14

20.3**

Squamosal - unossified area(s)

1

1

0.8

0

0

0.0

1

1

0.5

1

1

1.4

Jugal - incomplete ossification

5

3

3.2

4

4

2.6

4

4

2.5

8

6

6.7

Zygomatic process of maxilla - incomplete ossification

4

3

2.6

7

6

4.3

13

9

7.6

18

11

12.5**

Zygomatic process of squamosal - incomplete ossification

1

1

0.7

0

0

0.0

2

2

1.1

6

3

4.2

Zygomatic process of squamosal - fused to jugal

1

1

0.7

0

0

0.0

0

0

0.0

0

0

0.0

Premaxilla - incomplete ossification

0

0

0.0

0

0

0.0

3

3

1.8

2

1

1.4

Hyoid - incomplete ossification

14

9

8.6

17

11

11.9

11

8

7.0

16

9

10.1

Hyoid - not ossified

14

10

9.1

5

5

3.6

11

6

6.4

14

7

8.3

Presphenoid - incomplete ossification

1

1

0.7

0

0

0.0

1

1

0.7

2

2

1.3

Presphenoid - not ossified

0

0

0.0

1

1

0.7

2

2

1.3

0

0

0.0

NOTE: a fetus may appear in more than one category


Skeletal Findings

Dose Level (mg/kg bw/day)

 

 

0 (Control)

30

75

 

150

 

Number of Fetuses (litters) Examined

 

 

154 (24)

147 (24)

154 (24)

 

141 (24)

 

NF

NL

%†

NF

NL

%†

NF

NL

%†

NF

NL

%†

Vertebral Column

Odontoid - ossification present

 

0

 

0

 

0.0

 

1

 

1

 

4.2

 

0

 

0

 

0.0

 

1

 

1

 

0.7

Ventral arch of vertebra 1 - ossification present

45

18

31.7

49

18

36.3

37

16

23.9

34

11

22.2

Cervical (neural) arch - incomplete ossification

8

7

5.1

0

0

0.0**

10

7

6.3

16

9

10.1

Thoracic centrum - incomplete ossification

12

9

8.1

3

2

1.8

10

5

6.3

7

6

5.0

Thoracic centrum - not ossified

3

1

2.1

0

0

0.0

1

1

0.8

2

2

1.3

Thoracic centrum - bipartite ossification

5

4

3.8

2

2

1.4

2

2

1.1

1

1

0.8

Thoracic centrum - dumb-bell-shaped

20

10

13.9

19

11

16.2

19

13

11.7

21

14

18.5

Thoracic centrum - asymmetrically ossified

4

4

3.0

1

1

0.6

2

2

1.1

3

3

2.2

Thoracic centrum - misaligned

1

1

0.8

0

0

0.0

0

0

0.0

0

0

0.0

Thoracic centrum - hemicentric 

2

2

1.5

0

0

0.0

0

0

0.0

0

0

0.0

Thoracic vertebra - absent (arch and centrum)

1

1

0.8

0

0

0.0

0

0

0.0

0

0

0.0

Lumbar centrum - incomplete ossification

3

2

2.1

0

0

0.0

0

0

0.0

0

0

0.0

Lumbar centrum - bipartite ossification

0

0

0.0

0

0

0.0

1

1

0.8

0

0

0.0

Lumbar centrum - dumb-bell-shaped

0

0

0.0

0

0

0.0

1

1

0.8

0

0

0.0

Lumbar centrum - asymmetrically ossified

0

0

0.0

0

0

0.0

1

1

0.8

0

0

0.0

Sacral centrum - incomplete ossification

0

0

0.0

0

0

0.0

1

1

0.8

0

0

0.0

Sacral centrum - bipartite ossification

0

0

0.0

0

0

0.0

1

1

0.8

0

0

0.0

Sacral (neural) arch - incomplete ossification

25

11

15.9

22

9

15.3

31

13

19.9

39

14

26.5

Sacral (neural) arch - not ossified

0

0

0.0

0

0

0.0

1

1

0.6

0

0

0.0

Caudal vertebrae - less than 4 ossified

28

14

18.0

24

12

14.8

47

13

28.0

51

15

32.8

Number of pre-sacral vertebrae = 25/27 

1

1

0.8

1

1

0.7

1

1

0.8

1

1

2.1

 

Skeletal Findings

Dose Level (mg/kg bw/day)

 

0 (Control)

30

75

 

150

Number of Fetuses (litters) Examined

 

154 (24)

147 (24)

154 (24)

 

141 (24)

NF

NL

%†

NF

NL

%†

NF

NL

%†

NF

NL

%†

Ribs

Ossification centre - associated with 7th cervical vertebra

 

0

 

0

 

0.0

 

1

 

1

 

0.8

 

0

 

0

 

0.0

 

2

 

2

 

1.4

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

0

0

0.0

1

1

0.7

0

0

0.0

0

0

0.0

Ossification centre - associated with 1st lumbar vertebra

2

2

1.5

8

4

5.5

4

3

2.5

18

11

14.1**

One or more ribs - wavy

0

0

0.0

0

0

0.0

0

0

0.0

2

1

1.0

One or more ribs - thickened

1

1

0.6

0

0

0.0

0

0

0.0

2

1

1.0

Rib - short

3

1

1.8

1

1

0.7

2

2

1.5

0

0

0.0

Rib - fused 

2

2

1.5

0

0

0.0

0

0

0.0

0

0

0.0

Rib - incomplete ossification

0

0

0.0

0

0

0.0

0

0

0.0

1

1

0.5

Rib - interrupted ossification

1

1

0.8

0

0

0.0

0

0

0.0

0

0

0.0

Rib - not ossified

1

1

0.8

0

0

0.0

0

0

0.0

0

0

0.0

Rib - absent

1

1

0.8

0

0

0.0

0

0

0.0

0

0

0.0

Rib - no articulation point

1

1

0.8

0

0

0.0

1

1

0.5

0

0

0.0

Rib - bifurcated

1

1

0.8

0

0

0.0

0

0

0.0

0

0

0.0

Costal cartilage - misaligned

3

2

2.0

2

2

1.2

3

3

1.6

1

1

0.7

Costal cartilage - not fused to sternebra

14

9

8.7

11

9

7.5

12

7

7.9

14

7

11.1

Sternebrae

Sternebra - incomplete ossification

 

4

 

2

 

2.7

 

0

 

0

 

0.0

 

11

 

8

 

6.2*

 

4

 

3

 

2.7

Sternebra - not ossified

3

1

2.1

0

0

0.0

1

1

0.7

0

0

0.0

Sternebra - bipartite ossification

0

0

0.0

0

0

0.0

0

0

0.0

1

1

0.7

Sternebra - misaligned

2

2

1.3

4

4

2.4

6

5

3.3

8

6

4.9

Sternum - split

0

0

0.0

0

0

0.0

1

1

0.7

0

0

0.0

 

Skeletal Findings

Dose Level (mg/kg bw/day)

 

 

0 (Control)

30

75

 

150

 

Number of Fetuses (litters) Examined

 

 

154 (24)

147 (24)

154 (24)

 

141 (24)

 

 

 

NF

NL

%†

NF

NL

%†

NF

NL

%†

NF

NL

%†

Sternebrae (continued)

Xiphoid cartilage - split

 

0

 

0

 

0.0

 

0

 

0

 

0.0

 

1

 

1

 

0.7

 

0

 

0

 

0.0

Xiphoid cartilage - partially split

14

11

9.9

9

8

5.7

8

8

5.1

14

11

10.1

Pectoral Girdle

Scapula - misshapen 

 

1

 

1

 

0.6

 

5

 

5

 

6.6

 

2

 

2

 

1.0

 

4

 

2

 

2.6

Pelvic Girdle

Ischium - not ossified

 

0

 

0

 

0.0

 

0

 

0

 

0.0

 

1

 

1

 

0.7

 

0

 

0

 

0.0

Ischium - incomplete ossification

1

1

0.7

1

1

0.7

4

2

2.6

2

2

1.2

Pubis - not ossified

0

0

0.0

0

0

0.0

3

3

1.8

1

1

0.7

Pubis - incomplete ossification

8

3

5.4

8

5

5.8

11

8

7.0

13

8

9.3

Forelimbs

Metacarpal - not ossified

 

51

 

20

 

31.8

 

41

 

15

 

27.0

 

65

 

19

 

42.2

 

57

 

17

 

37.7

Metacarpal - incomplete ossification

0

0

0.0

1

1

0.7

4

3

2.9

3

3

1.9

Forepaw phalanges - 1 or more - ossified

14

10

10.5

13

9

12.8

10

5

6.5

10

5

7.0

Humerus - incomplete ossification

2

2

1.3

1

1

0.7

2

2

1.7

7

5

5.6

Humerus - hole

1

1

0.6

0

0

0.0

1

1

0.6

0

0

0.0

Hindlimbs

Metatarsal - 1st - ossified

 

0

 

0

 

0.0

 

1

 

1

 

4.2

 

0

 

0

 

0.0

 

0

 

0

 

0.0

Metatarsal - not ossified

0

0

0.0

0

0

0.0

1

1

0.7

0

0

0.0

Metatarsal - incomplete ossification

3

2

2.2

0

0

0.0

5

4

2.9

2

2

1.3

Femur - incomplete ossification

12

7

7.7

2

2

1.4

9

6

5.9

7

6

4.6

Total

132

24

86.6

118

24

81.4

130

24

82.7

126

24

90.2

 

Applicant's summary and conclusion

Conclusions:
The oral administration of the test substance to pregnant rats by oral gavage during gestation at dose levels of 30, 75 and 150 mg/kg bw/day, resulted in treatment-related macroscopic abnormalities detected in females treated with 150 and 75 mg/kg bw/day. The macroscopic findings detected were considered to be the result of localized irritation from the test item formulations and are not considered to reflect systemic toxicity. The ‘No Observed Adverse Effect Level’ (NOAEL) for the pregnant female was therefore considered to be 150 mg/kg bw/day.
No treatment-related changes were detected in the offspring parameters measured or on embryofetal development. The ‘No Observed Effect Level’ (NOEL) for developmental toxicity was therefore considered to be 150 mg/kg bw/day.