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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
July 2019 to August 2019 (end of experiment), final report April 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report date:
2020

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
foetal gender determination via anogenital appearance rather than anogenital distance; method of euthanasia different from method in study plan; temperature (21.2-26.2C) fell outside expected range. No deviations adversely affect integrity of the study
GLP compliance:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
Diisodecyl sebacate
EC Number:
249-047-0
EC Name:
Diisodecyl sebacate
Cas Number:
28473-19-0
Molecular formula:
C26H50O4
IUPAC Name:
1,10-bis(2-methylnonyl) decanedioate
Test material form:
liquid
Specific details on test material used for the study:
Batch/lot number: ES18-02097
Description: Colourless liquid
Purity: 99.6%
Expiry date: 17 July 2020
Storage conditions: Controlled room temperature (15-25 C, <70% relative humidity)
Vehicle:

Test animals

Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
Age of animals: Young adult female rats, nulliparous and non-pregnant,
13 weeks old at mating.
Starting body weight: 194 - 258 g (the variation did not exceed ± 20% of the
mean weight)
Acclimation period: at least 14 days

Animal health: Only healthy animals were used for the test, as certified
by the staff Veterinarian.
Cage type: Type II polycarbonate cages were used during mating
and gestation period and Type III polycarbonate cages
were used during the acclimatisation period.
Bedding: SAFE Hygienic Animal Bedding (Batch number:
03027181126, Expiry date 26 November 2021),
produced by J. Rettenmaier & Söhne GmbH+Co.KG
(Holzmühle 1, D-73494 Rosenberg, Germany was used
in the study. Details of bedding quality are reported in
Appendix 8.
Nesting: Arbocel crinklets natural nesting material produced by
J. Rettenmaier & Söhne GmbH+Co.KG (Holzmühle 1,
D-73494 Rosenberg, Germany, Batch number:
05072181025, Expiry date: 12 December 2021) and
SAFE crinklets natural nesting material produced by J.
Rettenmaier & Söhne GmbH+Co.KG (Holzmühle 1, D-
73494 Rosenberg, Germany) were used in the study
(Batch number: 05072190726, Expiry date: 26 July
2022). Details of nesting quality are reported in
Appendix 8.
Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 21.2-26.2°C (target: 22 ± 3°C)
Relative humidity: 34-69% (target: 30-70%)
Ventilation: 15-20 air exchanges/hour
Housing/Enrichment: Successfully mated animals were housed individually.
Deep wood sawdust was use as bedding to allow digging
and other normal rodent activities. Nest building
material and hiding tunnels were also added to the cages.
The temperature and relative humidity were monitored continuously and recorded twice
on each day during the study. The bedding and nest building material were considered
not to contain any contaminants that could reasonably be expected to affect the purpose
or integrity of the study.

Diet and water supply-
The animals were provided with ssniff® SM Autoclavable Complete Diet for Rats/Mice
– Breeding and Maintenance (Ssniff Spezialdiäten GmbH, D-59494 Soest, Germany)
and tap water (in water bottles) as for human consumption, ad libitum. The diet and
drinking water were routinely analysed and are considered not to contain any
contaminants that could reasonably be expected to affect the purpose or integrity of the
study.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 1% Tween 80 in Propylene glycol
Details on exposure:
A constant volume of 5 mL/kg body weight was administered to all dose groups,
including the controls. The individual volume of the treatment was based on the most
recent individual body weight of the animals.
The control or test item dose formulations were administered to mated, sperm positive
(assumed pregnant) female rats daily by oral gavage on a 7-days/week basis,
approximately at similar times, from gestation day 6 (GD 6) to gestation day 19
(GD 19).
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis of test item formulations for concentration and homogeneity was performed at the Laboratory of Charles River Laboratories Hungary Kft. using a validated HPLC-UV method. Top, middle and bottom samples were taken from the test item formulations two times during the study (during the first week and on the last week of treatment).
Samples were taken in duplicate, one set to analyse and one set as a back-up, if required for any confirmatory analyses. Similarly, duplicate samples were taken from the middle of the vehicle control formulation for concentration measurement. Acceptance criterion of the concentration analysis was set according to the analytical method validation, expected to be at 100 ± 15% of the nominal concentration.
Acceptance criteria of the homogeneity was that the CV of replicates (top, middle and bottom of test item formulations) to be less than 10%.
Details on mating procedure:
The oestrus cycle of female animals was examined a day before start of pairing. After acclimation, the females were paired according to their oestrus cycle with males in the
morning for approximately 2-3 hours (1 male : 1 female) until 24 sperm positive females/group were attained. After the daily mating period, a vaginal smear was prepared and stained with 1% aqueous methylene blue solution. The smear was examined with a light microscope; the presence of a vaginal plug or sperm in the vaginal smear was considered as evidence of copulation (gestation day 0, GD 0). Sperm positive females were separated and caged individually.
Duration of treatment / exposure:
Treatment was administered from gestation days 6 to 19
Frequency of treatment:
Treatment was administered on a 7-days a week basis
Duration of test:
Start of experiment: 25 July 2019
End of experiment: 23 August 2019
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
24 mated females per group
Control animals:
yes, concurrent vehicle
Details on study design:
The dose levels were set by the Study Director in consultation with the Sponsor, based on the available data and information from previous experimental work, including the results of a 28-Day Oral (gavage) Dose Range Finding Toxicity Study in Wistar Rats (Charles River Laboratories Hungary study code: 18/296-101PE). In that study, no adverse effect was observed in the test item treated group (1000 mg/kg bw/day). Increase of the liver and kidney weights in the test item treated group were recorded, with no correlating adverse liver histopathology findings.
Based on the results from the Dose Range Finding study, doses of 1000, 300 and 100 mg/kg bw/day were selected for the main study (designated High, Mid and Low dose
respectively). The aim is to use the highest dose of 1000 mg/kg bw/day to induce toxic effects, but ideally no death or suffering, and to obtain a NOAEL at the lowest dose
level. The oral route was selected since it is a route of administration recommended by the OECD guideline No. 414 and it was considered suitable to provide the exposure
required for this developmental toxicology study.

Examinations

Maternal examinations:
Animals were inspected for signs of morbidity and mortality twice daily (at the beginning and end of each working day).
Cage-side (general) clinical observations were made twice daily (at the beginning and end of each working day). Only one general clinical observation was made on the first
day (in the afternoon), on the afternoon on those days when detailed clinical observation was made in the morning. Furthermore, clinical observation (detailed) was made only
once on necropsy days (in the morning). Detailed clinical observations were made on all animals at the onset of treatment (GD 6) then weekly.
The animals were monitored for any changes including pertinent behavioural changes and signs of toxicity including mortality, changes in skin, fur, eyes, mucous membranes,
occurrence of secretions and excretions, and autonomic activity (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern), changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling), bizarre behaviour (e.g. self-mutilation, walking backwards), tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma [4].
On GD 13 and/or 14, the sperm positive females were examined for the presence of vaginal bleeding or “placental sign” (intra-uterine extravasation of blood as an early sign of pregnancy in rat, which is considered to confirm implantation).

The body weight of each animal was recorded with precision of ±1 g on GD 0, 3, 6, 8,10, 12, 14, 16, 18 and 20.
Food was measured with precision of ± 1 g on GD 0, 3, 6, 8, 10, 12, 14, 16, 18 and 20.
Food consumption was also calculated for each interval, including GD 0-6, GD6-10, GD 6-20 and GD 0-20.

No histopathology evaluation was performed in the study (as it was not required for interpretation).
Ovaries and uterine content:
Before expected delivery, on GD 20, Caesarean section was performed on each treated dam. Sodium pentobarbital (Euthanimal 40%, 400 mg/mL sodium pentobarbital
solution; Supplier: Alfasan Nederland BV, Batch number: 1811347-03, Expiry date:
31 December 2021) administered by intraperitoneal injection and followed by exsanguination was used for euthanasia.
The dams’ viscera were examined macroscopically for any structural abnormalities or pathological changes; all the gross findings were retained in 10% buffered formalin solution (or modified Davidson fixative, in case of the eyes, if any abnormalities noted) for possible future evaluation.
Furthermore, the livers of the dams were weighed and retained in 10% buffered formalin solution for any possible histological examination.
The ovaries and uterus were removed and the pregnancy status ascertained. The uterus including the cervix was weighed and examined for early and late embryonic or foetal
deaths and for the number of live foetuses.
The number of corpora lutea in each ovary and implantation sites in each uterine horn, the number of live foetuses, early and late embryonic death and foetal death were counted, the number and percentage of pre- and post-implantation losses were calculated. The degree of resorption was described in order to estimate the relative time of death of the conceptus. The placentas were examined macroscopically.
Fetal examinations:
Each foetus was weighed individually (accuracy ±0.01 g) and subjected to external examination, plus an additional examination of the great arteries. The gender of foetuses
was determined according to the appearance of the anogenital distance. Thereafter the foetuses were individually identified; approximately half of each litter was subjected to visceral examination, and the other half was processed for skeletal examination.
For the foetuses subjected to visceral examination, the abdominal and thoracic region was opened and the thymus and great arteries were freshly examined by means of a
dissecting microscope. The rest of the body was fixed in Sannomiya mixture (a mixture of 920 mL concentrated isopropanol, 30 g sulfosalicylic acid, and 50 mL acetic acid),
then, after fixation, the body was micro-dissected by means of a dissecting microscope. The heads were examined by Wilson's free-hand razor blade method.
For the foetuses subjected to skeletal examination, the abdominal region was opened, and the viscera and skin of foetuses were removed and the cadaver was fixed in alcianblue- acetic acid – isopropanol mixture. After fixation in isopropanol the skeletons were stained by KOH-Alizarin red-S method and examined by means of a dissecting
microscope. All abnormalities (external, soft tissue and skeletal malformations, and variations) found during the foetal examinations were recorded; photographic records were made additionally.

No histopathology evaluation was performed in the study (as it was not required for interpretation).
Statistics:
Data were collected using the software PROVANTIS v.9 or recorded on appropriate forms from the relevant SOPs of Charles River Laboratories Hungary Kft. (foetal evaluation data) and tabulated with PROVANTIS v.9/Microsoft Office Excel 2010. Statistical evaluation of data was performed with the program package SAS v9.2 in case of Provantis v.9 or SPSS PC+4.0 (SPSS Hungary Kft, Budapest) for data tabulated in Excel by an appropriate statistical method. For the SAS v9.2 software package (within the validated Provantis system) the normality and heterogeneity of variance between groups were checked by Shapiro-Wilk and Levene tests using the most appropriate data format (log-transformed when justified). Where both tests showed no significant heterogeneity, an Anova / Ancova (one-way analysis of variance) test was carried out. If the obtained result was positive, Dunnett’s (Multiple Range) test was used to assess the significance of intergroup differences. If either of the Shapiro-Wilk or Levene tests showed significance a non-parametric analysis was used. A Kruskal-Wallis analysis of variance was used after Rank Transformation. If there was a positive result, the inter-group comparisons were performed using Dunn test.
For the SPSS PC+4.0 program package, the heterogeneity of variance between groups was checked by Bartlett's test. Where no significant heterogeneity was detected, a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant, then Duncan's Multiple Range test assessed the significance of inter-group differences. Where significant heterogeneity was found, the normal distribution of data was examined by Kolmogorow-Smirnow test. In the case of not normal distribution, the non-parametric method of Kruskal-Wallis ANOVA was applied. If a positive result was detected, the inter-group comparisons were performed using Mann-Whitney U-test. The Chi squared test was used for comparing group incidences against the controls.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Thin fur was observed in 2 out of 24 animals in the Control group, in 3 out of 24 animals in the Low dose group, and in 1 out of 23 animals in the High dose group. These changes were most probably incidental, and not considered to be test item related effects.
Piloerection was present in 4 out of 22 animals in the Mid dose group, and 19 out of 23 animals in the High dose group. The finding appeared one day after the start of the treatment (Day7), and peaked for both dose groups at Day 10, the fifth treatment day.
While the distribution of the symptoms indicates that the animals adapted to the treatment, these findings correlate with the observed food intake and body weight loss during the peak period (first week of treatment). Noisy respiration was observed only in the Low dose group (1 out of 24 animals).

Based on the relatively high proportion of this otherwise non-specific finding, piloerection is considered to be a test item related, maternal effect. It is not known if the effect was a systemic effect, or a digestive or other non-systemic cause.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
No mortality was observed in the study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
A test item related body weight decrease was observed after the onset of treatment in animals in the High dose group.

Subsequently, significantly lower (p < 0.01) body weight gain was observed in animals in the High dose group from Day 6 (the first day of treatment) up to Day 12, and the whole treatment and experimental period. Furthermore, significantly lower corrected body weight, corrected body weight gain, and net body weight gain was observed in animals in the High dose group.
A similar, more transient change, not reaching statistical significance, was observed in animals in the Mid dose group.

These changes were considered to be test item related, adverse effects.

No test item related effect on body weight or body weight parameters were observed in animals in the Low dose group when compared to control.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
A transient, test item related reduction of the food consumption was observed at the start of the treatment in the test item treated dose groups, reaching statistical significance (p < 0.01) in the periods between Day 6 (the first day of treatment) and Day 12 in the Mid and High dose groups and, while the reduction was compensated during the last week of the treatment, a statistically significant decrease was present when the whole treatment or experimental period was considered, both in the Mid (p < 0.05) and the High (p < 0.01) dose groups.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
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:
no effects observed
Description (incidence and severity):
No effect on gravid uterine weight, or liver weight
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No test item related macroscopic findings were present at necropsy in any of the animals in the study.
Thin fur, correlating with the clinical signs, was observed in one animal in the Low and the High dose group, respectively, but it was considered to be an incidental finding, not a test item related change.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Maternal developmental toxicity

Number of abortions:
not examined
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
No effect on implantation loss
Total litter losses by resorption:
not examined
Early or late resorptions:
no effects observed
Description (incidence and severity):
No effect on resorption.
Dead fetuses:
no effects observed
Description (incidence and severity):
No effect on intra-uterine mortality
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
Ninety-six females (24 each for the Control, the Low, Mid, and High dose group, respectively) were mated in the study. The number of confirmed pregnant, evaluated dams was 24 in the Control and the Low dose group, 22 in the Mid dose group, and 23 in the High dose group.
Other effects:
no effects observed
Description (incidence and severity):
Corpora lutea and implantation sites
There were no statistically significant differences in the intra-uterine parameters in the test item treated animals when compared to the control.

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

Results (fetuses)

Fetal body weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Significantly lower body weights/litter were observed in animals in the Mid (p<0.05), and High dose groups (p<0.01).
While the body weight difference reached significance in the Mid and High dose group, the actual values are within (Mid dose group) or slightly below (High dose group) the expected historical control range (mean ± SD, 3.407 ± 0.225), and therefore not considered to be an adverse test item related effect.

Significantly higher number of foetuses with retarded body weight were observed in the High dose group (p<0.05). These test item related changes were considered to be a secondary effect to the observed maternal toxicity.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
Significantly lower body weights/litter were observed in animals in the Mid (p<0.05), and High dose groups (p<0.01).
While the body weight difference reached significance in the Mid and High dose group, the actual values are within (Mid dose group) or slightly below (High dose group) the expected historical control range (mean ± SD, 3.407 ± 0.225), and therefore not considered to be an adverse test item related effect.
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no external varieties or malformations in the test item treated groups.
The malformation observed in the Control group (Absent mandible) was considered to be an incidental finding, not related to the treatment.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
The observed skeletal abnormalities are summarized in Table 9. Most of the skeletal findings correspond with the current historical control (HC) or the concurrent study control data, or were considered to be incidental findings without dose response (as shown in Table 10). In some cases, skeletal variations (without toxicological significance) shows higher incidence in the test item treated groups, indicating a secondary effect due to maternal toxicity.

Based on the skeletal findings the number of malformed / intact foetuses were comparable with the control in the Low, Mid and High dose groups. The number of variations were higher in all test item treated groups, reaching significance in the Mid and High dose groups.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
All of the visceral findings (variations) were consistent in general nature and incidence with the concurrent study control data / existing historical control data or showed incidental occurrence, therefore considered as incidental findings. Based on the visceral findings, the number of malformed / variant / intact foetuses were comparable with the control in the Low, Mid and High dose groups.
Other effects:
not examined

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes

Overall developmental toxicity

Key result
Developmental effects observed:
not specified

Any other information on results incl. tables

Group no./ Designation

Dose Level (mg/kg bw/day)

Concentration (mg/mL)

Dose volume (mL/kg)

Number of mated females

Evaluation No.

1/ Control

0

0

5

24

1501-1524

2/ Low dose

100

20

24

2501-2524

3/ Mid dose

300

60

24

3501-3524

4/ High dose

1000

200

24

4501-4524

Table 1 - Experimental design

 

Table 2 – Summary of the dose formulation analysis

Formulation

30 July 2019

19 August 2019

Mean measured concentration (mg/mL)

Relative to the nominal concentration (%)

Measured concentration (mg/mL)

Relative to the nominal concentration (%)

Control

Not detected

-

Not detected

-

20 mg/mL

19.5 ± 0.56

97

19.9 ± 0.43

99

60 mg/mL

57.8 ± 0.91

96

60.3 ± 0.36

100

200 mg/mL

199 ± 3.7

100

206 ± 4.12

103

Note: Samples collected freshly on the days indicated in the header of the table.

 

Table 3 – selected body weight parameters

Parameters

Historical control

Dose (mg/kg bw/day)

 

 

0

100

300

1000

 

Number of evaluated dams

507

24

24

22

23

 

Body weight on GD20 (g)

316.4±23.1

324.2

321.9

319.0

307.0#

DN

Body weight gain GD6-20 (g)

83.1±14.1

82.1

81.8

78.3

63.8##

DN

Body weight gain GD0-20 (g)

104.3±16.2

104.2

102.2

97.5

84.5##

DN

Corrected body weight on GD20 (g)

258.2±18.7

267.0

265.5

263.9

251.6#

DN

Corrected body weight gain GD0-20 (g)

46.1±11.8

47.0

45.8

42.3

29.0**

U

Net body weight gain GD6-20 (g)

24.9±9.8

24.9

25.5

23.1

8.3**

U

Notes: Historical control data is mean ± 1SD. Body weight data were rounded to one decimal place. Corrected and net weight/weight gains refer to body weight values minus the weight of the gravid uterus.

U: *=p<0.05; **=p<0.01; Dunn two sided test

DN: #=p<0.05; ##=p<0.01; Dunnett two sided test

 

Table 4 – Summary of pregnancy data

Parameters

Dose (mg/kg bw/day)

0

100

300

1000

Number of mated females

24

24

24

24

Pre-terminal death or euthanasia

0

0

0

0

Number of non-pregnant females

0

0

2

1

Number of evaluated females with5 implantation sites

0

0

0

0

Number of evaluated females on GD20 (Caesarean section)

24

24

22

23

 

Table 5 – Summary of the intra-uterine evaluation

Parameters

Dose (mg/kg bw/day)

 

0

100

300

1000

 

Number of evaluated dams

24

24

22

23

 

Mean number of corpora lutea

11.63

11.33

10.46

10.76

NS

Pre-implantation loss, mean

1.67

1.71

1.82

0.91

NS

Pre-implantation loss (%), mean

13.10

14.17

15.18

7.53

NS

Mean number of implantations

9.96

9.63

9.59

10.35

NS

Early embryonic loss, mean

0.21

0.00

0.05

0.13

NS

Early embryonic loss (%), mean

2.27

0.00

0.41

1.16

NS

Late embryonic loss, mean

0.08

0.00

0.14

0.22

NS

Late embryonic loss (%), mean

1.11

0.00

1.17

2.15

NS

Dead foetuses, mean

0.00

0.00

0.00

0.00

NS

Dead foetuses (%), mean

0.00

0.00

0.00

0.00

NS

Post-implantation loss, mean

0.29

0.00

0.18

0.35

NS

Post-implantation loss (%), mean

3.38

0.00

1.58

3.31

NS

Total intra-uterine mortality, mean

1.96

1.71

2.00

1.26

NS

Total intra-uterine mortality (%), mean

16.28

14.17

16.73

10.56

NS

Viable foetuses, mean

9.67

9.63

9.41

10.00

NS

Notes: Most important parameters are shown in bold.

NS: Statistically not significant when compared to the vehicle control.

NA: Not applicable

 

Table 6 – Examination of viable foetuses

Parameters

Dose (mg/kg bw/day)

 

0

100

300

1000

 

Number of examined litters

24

24

22

23

 

Viable foetuses, mean

9.67

9.63

9.41

10.00

NS

Male foetuses, mean

4.67

4.75

5.05

5.04

NS

Female foetuses, mean

5.00

4.88

4.36

4.96

NS

Total number of foetuses

232

231**

207

230

CH

Total number of male foetuses

112

114

111

116

NS

Total number of female foetuses

120

117

96

114

NS

Sex distribution (% of males/females)

48/52

49/51

54/46

50/50

NS

Mean foetal weight/litter (g)

3.435

3.404

3.367*

3.135**

U

Number of foetuses with retarded body weight

9

6

10

58**

CH

Number of affected litters (with runts)

7

4

7

14*

CH

 

Table 7 – Summary table of the external abnormalities

Parameter

 

0

100

300

1000

Total number of examined litters

24

24

22

23

Total number of examined foetuses

118

114

104

113

Total number of intact (normal) foetuses

115

112

103

113

Total number of foetuses / litters with malformation

0 / 0

0 / 0

0 / 0

0 / 0

Total number of foetuses / litters with variation

3 / 3

2 / 2

1 / 1

7 / 7

Notes: Numbers represent the number of abnormalities / number of affected litters

 

Table 9 – Details of the visceral abnormalities

Parameter

Dose (mg/kg bw/day_

HC data

0

100

300

1000

Total number of examined litters

24

24

22

23

507

Total number of examined foetuses

118

114

104

113

2608

Visceral malformations

Visceral variations

Brachiocephalic trunk, short

Litter incidence

n

0

0

0

1

23

%

0.0

0.0

0.0

4.3

4.5

Foetal incidence

n

0

0

0

1

24

%

0.000

0.000

0.000

0.885

0.920

Liver lobe, fused

Litter incidence

n

0

1

0

0

--

%

0.0

4.2

0.0

0.0

--

Foetal incidence

n

0

1

0

0

--

%

0.000

0.877

0.000

0.000

--

Renal papilla, small

Litter incidence

n

1

0

0

1

16

%

4.2

0.0

0.0

4.3

3.2

Foetal incidence

n

1

0

0

1

17

%

0.847

0.000

0.000

0.885

0.652

Ureter convoluted

Litter incidence

n

1

0

0

0

7

%

4.2

0.0

0.0

0.0

1.4

Foetal incidence

n

1

0

0

0

7

%

0.847

0.000

0.000

0.000

0.268

Thymic cord

Litter incidence

n

2

1

1

5

51

%

8.3

4.2

4.5

21.7

10.1

Foetal incidence

n

2

1

1

5

60

%

1.695

0.877

0.962

4.425

2.31

Notes: Numbers represent the number (n) or ratio (%) of abnormalities.

HC: historical control (data provided where considered useful)

No statistically significant differences were noted when compared to the control group.

--: No data

 

Table 10 – summary table of the skeletal abnormalities

Parameter

Dose (mg/kg bw/day)

0

100

300

1000

Total number of examined litters

24

24

22

23

Total number of examined foetuses

114

117

103

117

Total number of intact (normal) foetuses

95

93

71CH*

64CH**

Total number of foetuses / litters with malformation

4 / 4

2 / 2

7 / 5

4 / 3

Total number of foetuses / litters with variation

15 / 12

22 / 12

25CH*/ 12

49CH**/ 19

Notes: Numbers represent the number of abnormalities / number of affected litters

CH: Chi2test; * = p < 0.05; ** = p < 0.01

 

Table 11 – Details of the skeletal abnormalities

Parameter

Dose (mg/kg bw/day)

HC data

0

100

300

1000

Total number of examined litters

24

24

22

23

507

Total number of examined foetuses

114

117

103

117

2599

Skeletal malformations

Transverse process fused; Pelvic girdle, malpositioned

Litter incidence

n

2

1

5

2

7

%

8.3

4.2

22.7

8.7

1.4

Foetal incidence

n

2

1

7

3

7

%

1.754

0.855

6.796

2.564

0.269

Multiple malformed vertebrae (hemicentric, hemivertebrae, misshapen)

Litter incidence

n

1

1

0

0

--

%

4.2

4.2

0.0

0.0

--

Foetal incidence

n

1

1

0

0

--

%

0.877

0.855

0.000

0.000

--

Mandible, short, fused

Litter incidence

n

1

0

0

0

1

%

4.2

0.0

0.0

0.0

0.2

Foetal incidence

n

1

0

0

0

1

%

0.877

0.000

0.000

0.000

0.038

Rib, branched

Litter incidence

n

1

0

0

0

--

%

4.2

0.0

0.0

0.0

--

Foetal incidence

n

1

0

0

0

--

%

0.877

0.000

0.000

0.000

--

Humerus short

Litter incidence

n

1

0

0

0

--

%

4.2

0.0

0.0

0.0

--

Foetal incidence

n

1

0

0

0

--

%

0.877

0.000

0.000

0.000

--

Scapula short, bent

Litter incidence

n

1

0

0

1

--

%

4.2

0.0

0.0

4.3

--

Foetal incidence

n

1

0

0

1

--

%

0.877

0.000

0.000

0.855

--

Skull: Incomplete ossification (More than 3)

Litter incidence

n

3

5

9

11

13

%

12.5

20.8

40.9

47.8

2.6

Foetal incidence

n

3

10

21CH**

20CH**

18

%

2.632

8.547

20.388

17.094

0.693

Skull: Hyoid Body Unossified

Litter incidence

n

5

6

5

8

3

%

20.8

25.0

22.7

34.8

0.6

Foetal incidence

n

5

9

6

16CH*

5

%

4.386

7.692

5.825

13.675

0.192

Sternum: Unossified Sternebra (3 or More)

Litter incidence

n

2

1

2

13

--

%

8.3

4.2

9.1

56.5

--

Foetal incidence

n

2

3

2

27CH**

--

%

1.754

2.564

1.942

23.077

--

Sternum: Misaligned

Litter incidence

n

0

0

2

0

1

%

0.0

0.0

9.1

0.0

0.2

Foetal incidence

n

0

0

2

0

1

%

0.000

0.000

1.942

0.000

0.038

Ribs: Wavy, Extreme

Litter incidence

n

3

0

1

2

132

%

12.5

0.0

4.5

8.7

26.0

Foetal incidence

n

3

0

1

3

202

%

2.632

0.000

0.971

2.564

7.772

Vertebrae: 2 or More Dumbbell or Asymmetric Ossification

Litter incidence

n

2

1

3

1

51

%

8.3

4.2

13.6

4.3

10.1

Foetal incidence

n

3

1

3

1

56

%

2.632

0.855

2.913

0.855

2.155

Vertebrae: Dumbbell Shaped

Litter incidence

n

0

5

3

0

6

%

0.0

20.8

13.6

0.0

1.2

Foetal incidence

n

0

6CH*

3

0

6

%

0.000

5.128

2.913

0.000

0.231

Vertebrae: Unossified centra or arch

Litter incidence

n

2

2

2

5

--

%

8.3

8.3

9.1

21.7

--

Foetal incidence

n

2

4

2

14CH**

--

%

1.753

3.419

1.942

11.966

--

Vertebrae: Bipartite Ossification

Litter incidence

n

1

3

1

0

11

%

4.2

12.5

4.5

0.0

2.2

Foetal incidence

n

1

3

1

0

11

%

0.877

2.564

0.971

0.000

0.423

Limbs (C/T): Unossified Carpal Bone (2 or More)

Litter incidence

n

2

1

2

2

--

%

8.3

4.2

9.1

8.7

--

Foetal incidence

n

2

3

2

6

--

%

1.754

2.564

1.942

5.128

--

Limbs (C/T): Unossified Tarsal Bone

Litter incidence

n

1

1

1

5

--

%

4.2

4.2

4.5

21.7

--

Foetal incidence

n

1

1

2

12CH**

--

%

0.877

0.855

1.942

10.256

--

Limbs (C/T): Unossified Pubis, Ischium

Litter incidence

n

0

1

0

1

--

%

0.0

4.2

0.0

4.3

--

Foetal incidence

n

0

1

0

1

--

%

0.000

0.855

0.000

0.855

--

Notes: Numbers represent the number (n) or ratio (%) of abnormalities.

HC: historical control (data provided where considered useful)

No statistically significant differences were noted compared to the control group

--: No data

CH: Chi2test; * = p <0.05; ** = p < 0.01

Applicant's summary and conclusion

Conclusions:
In conclusion, CEREPLAS DIDS, when administered daily by oral gavage to pregnant Hannover Wistar rats from gestation days GD6 to GD19 at 1000 mg/kg bw/day induced a slight maternal toxicity, with an initial body weight loss, reduced food consumption and clinical signs of piloerection. The Mid dose of 300 mg/kg bw/day had minimal signs with a less expressed effect on body weight, but there was evidence of a slight maternal toxicity. No embryotoxicity was observed in the study based on overall development. The lower mean foetal body weight and the increased number of runts and affected litters indicates foetotoxicity seen in animals in the High dose group should be considered as secondary to maternal toxicity. There were no malformations attributed to the test item at any dose level. Slight differences in the incidence of retarded ossification in the foetuses of animals of the High dose group were considered to be within the normal range, but may have been related to maternal toxicity.
Executive summary:

This developmental toxicity study (OECD 414, 2001 version as required by Sponsor) was performed to assess the effects of the test item CEREPLAS DIDS on the embryonic and foetal development (including the organogenesis period) of Hannover Wistar rats in their first pregnancy. The dams (one control and three test item treated groups) were treated daily by oral (gavage) administration, from gestation day 6 (GD 6) up to and including gestation day 19 (GD 19), where sperm positive day was counted as day 0 of pregnancy (GD 0). Control dams were treated with the vehicle (1% Tween 80 in propylene glycol) only. Caesarean sections, necropsy of dams and examination of uterine contents were performed on GD 20. The dose levels were set by the Study Director in consultation with the Sponsor, based on the available data and information from previous experimental work, including the

results of a 28-Day Oral (gavage) Dose Range Finding Toxicity Study in Wistar Rats

[8].

Based on the results from the Dose Range Finding study, doses of 1000, 300 and 100 mg/kg bw/day were selected for the main study (designated High, Mid and Low dose respectively). The aim is to use the highest dose of 1000 mg/kg bw/day to induce toxic effects, but ideally no death or suffering, and to obtain a NOAEL at the lowest dose level.

 

Test item formulations were analysed for concentration and homogeneity two times during the treatment period using a validated HPLC-UV method. Simultaneously, vehicle control formulations were analysed for the test item. Parameters monitored during the study included mortality and clinical observations, body weight, body weight gain and individual food consumption. Maternal reproductive parameters associated with uterine examination were evaluated, and the foetuses were weighed and examined for external, visceral and skeletal abnormalities. Placentas were examined macroscopically. The number of confirmed pregnant, evaluated dams was 24 in the Control and Low, 22 in the Mid dose group, and 23 in the High dose groups.

 

Results

All test item formulations were within the range of 96-103% of nominal concentration and were found to be homogenous. No test item was detected in the vehicle control samples. Based on these results, test item formulations were considered suitable for the study purposes.

No mortality was observed in the study.

Piloerection was present in 4 out of 22 animals in the Mid dose group, and 19 out of 23 animals in the High dose group. These adverse effects were considered to be test item related.

Test item related body weight decrease was observed after the onset of treatment in animals in the High dose group. Consequently, significantly lower body weight gain, and lower corrected body weight, corrected body weight gain, and net body weight gain was observed in animals in the High dose group. A similar, more transient change, not reaching statistical significance, was observed in animals in the Mid dose group. No test item related effect on the body weight parameters were observed in animals in the Low dose group when compared to controls. Transient, test item related reduction of the food consumption was observed at the start of the treatment in the test item treated groups, reaching statistical significance in the Mid and High dose group. No test item related macroscopic findings were present at necropsy in any of the animals in the study.

Taking the above maternal findings together, it is considered that were was slight/minimal maternal toxicity present in animals in the Mid dose group, and more significant maternal toxicity in animals in the High dose group. There were no statistically significant differences in the intra-uterine parameters in the test item treated animals when compared to the controls. There was no toxicologically significant difference in the sex distribution of foetuses between the control and treatment groups. Significantly lower foetal body weights were observed in the test item treated groups. The well-expressed reduction in body weight in animals in the High dose group correlated with the increased number of runts and affected litters in this dose group. There was no toxicologically significant difference in the number of runts and affected

litters between the control and the Low and Mid dose groups. There were no test item related effects on external or visceral development of foetuses in the study. The number of skeletal variations was higher in the test item treated groups when compared to the controls, reaching statistical significance in the Mid and High dose group due to maternal toxicity. Foetal malformations observed in the study were all considered to be incidental. They showed no dose dependency and thus were not regarded as a test item related effect.

 

In conclusion, CEREPLAS DIDS, when administered daily by oral gavage to pregnant Hannover Wistar rats from gestation days GD6 to GD19 at 1000 mg/kg bw/day induced a slight maternal toxicity, with an initial body weight loss, reduced food consumption and clinical signs of piloerection. The Mid dose of 300 mg/kg bw/day had minimal signs with a less expressed effect on body weight, but there was evidence of a slight maternal toxicity. No embryotoxicity was observed in the study based on overall development. The lower mean foetal body weight and the increased number of runts and affected litters indicates that foetotoxicity seen in animals in the High dose group should be considered as secondary to maternal toxicity. There were no malformations attributed to the test item at any dose level. Slight differences in the incidence of retarded ossification in the foetuses of animals in the High dose group were considered to be within the normal range, but may have been related to maternal toxicity.