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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:
18 April - 16 August 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 guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
adopted 22 January 2001
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Health and Youth Care Inspectorate, Ministry of Health, Welfare and Sport, Utrecht, The Netherlands

Test material

1
Chemical structure
Reference substance name:
Bis(2-(2-butoxyethoxy)ethyl) adipate
EC Number:
205-465-5
EC Name:
Bis(2-(2-butoxyethoxy)ethyl) adipate
Cas Number:
141-17-3
Molecular formula:
C22H42O8
IUPAC Name:
bis(2-(2-butoxyethoxy)ethyl) adipate

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Crl: WI(Han)
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: approximately 10 -14 weeks
- Weight at study initiation: approximately 185 to 297 g (females)
- Housing: individually in Macrolon plastic cages (MIII type, height 18 cm) containing appropriate bedding (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) equipped with water bottles.
- Diet: pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany), ad libitum. Except during designated procedures.
- Water: municipal tap water, ad libitum
- Acclimation period: 5 days

DETAILS OF FOOD AND WATER QUALITY: analysis were performed

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 21
- Humidity (%): 40 - 68
- Air changes (per hr): 10 or more
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 01 July 2018 To: 19 July 2018

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Remarks:
400
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared daily as a solution and dosed within 5 h after adding vehicle to the test item. Test item dosing formulations were kept at room temperature until dosing. The dosing formulations and vehicle were continuously stirred until and during dosing. Adjustment was made for specific gravity of the vehicle (1.125) and test item (1.1 at 25 °C). No correction was made for the purity/composition of the test item.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Trial preparations were performed at the Test Facility to select the suitable vehicle and to establish a suitable formulation procedure. Polyethylene glycol 400 (Merck, Darmstadt, Germany) was identified as suitable vehicle.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dose formulation samples were collected for analysis in week 1 for homogeneity and concentration analysis. For homogeneity analysis, duplicate sets of samples (approximately 500 mg, accurately weighed) for each sampling time point were sent to the analytical laboratory. Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was ± 10%. In addition, stability was tested in samples collected in week 1.
For concentration analysis, duplicate sets of samples (approximately 500 mg, accurately weighed) for each sampling time point were sent to the analytical laboratory.Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 10% for solutions of target concentration.
Stability analysis performed previously in the repeated dose 90-day oral toxicity study were considered (Study No. 20148924) for the develomental toxicity study. Stability of the test item in the vehicle was determined over 5 h at room temperature under normal laboratory light conditions (lowest and highest concentration). Duplicate sets of each sample (approximately 500 mg, accurately weighed) were sent to the analytical laboratory. Stability results were considered acceptable if the sample analysis results were within or equal to ± 10% of the concentration determined by the initial analysis of each formulation.

Analysis showed that concentrations analyzed were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%) and that no test item was detected in the vehicle formulation. Furthermore, the dose formulations were homogeneous (i.e. coefficient of variation ≤ 10%) and stable when stored at room temperature under normal laboratory light conditions for at least 5 h (i.e. differences between mean concentrations before and after storage ≤ 10%).
Details on mating procedure:
- Impregnation procedure: purchased timed pregnant
Duration of treatment / exposure:
days 6 to 20 post-coitum, inclusive
Frequency of treatment:
once daily
Duration of test:
until day 21 day post coitum
Doses / concentrationsopen allclose all
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
600 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
22
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on the results of a dose range finder study (Study No. 20148926). In this study 6 time-mated female Wistar Han rats per dose groups were administered the test substance at 300, 600 and 1000 mg/kg bw/day once daily oral gavage from day 6 to day 20 post-coitum, inclusive. No mortality occurred during the study period and treatment-related findings were noted in the high dose group only. The most sensitive was female no. 21 which had rales (up to a moderate degree) and a slightly shallow respiration on day 2 and days 9-11 (slight rales only), a hunched posture and piloerection (treatment Days 2-6), diarrhoea and/or slightly reduced faeces production (treatment Days 3-6), and a flat gait (treatment Days 3-6). Flat gait was also noted for another two high dose females (nos. 22 and 23) from treatment days 2-6. Salivation seen after dosing among animals of all test item treated groups on several days during the treatment period was not considered toxicologically relevant, taking into account the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign was considered to be a physiological response rather than a sign of systemic toxicity. Incidental findings included general erythema (at a slight degree), alopecia and scales. As these findings occurred in control animals treated with the vehicle only, a relation to treatment with the test item could be excluded.
The slight body weight loss (2%) observed in the high dose group from days 6-9 post-coitum was mainly caused by female no. 21 which lost 12% of her weight in this period. An additional measurement performed on day 8 post-coitum indicated 15% body weight loss from days 6-8 post-coitum. In the subsequent treatment period, mean body weight gain recovered to control levels again. After correction for the gravid uterus mean body weight on day 21 post-coitum was lower in the high dose group as compared to the control group (2.6% versus 8.4%; not statistically significant). This was again caused largely by female no. 21 which had a body weight loss of 7.1% after correction for the gravid uterus weight. Body weight gain before and after correction for (gravid) uterus weight of low and mid dose group females was unaffected by the treatment.
Absolute and relative food consumption was reduced in the high dose group between days 6-9 postcoitum. Again this was largely caused by the markedly lower food consumption of female no. 21 which was about 70% lower as compared to group 4 average (absolute and relative to body weight). In the subsequent period, it recovered to normal values again. The statistically significantly higher mean value for relative food consumption observed in the mid dose group from post-coitum days 9-12 was not considered to be toxicologically relevant as changes were transient and mean value remained within the range of available historical control data.

Necropsy did not reveal any toxicologically relevant alterations. One low dose female was observed with enlarged parathymic lymphnodes. As this was an isolated finding at the low dose only, it was considered unrelated to treatment with the test item.

All females were pregnant, except for one female of the low dose. All pregnant females had viable fetuses, except for one control female which had resorptions only. A relation to treatment with the test item could be excluded, since females of the control group were treated with the vehicle alone.
There were no females that aborted or delivered before scheduled necropsy. The number of implantation sites, pre- and post-implantation loss, sex ratio and fetal body weights were unaffected by treatment. Mean numbers of corpora lutea were statistically significantly increased in the 300 and 1000 mg/kg groups, but not in the 600 mg/kg group. As treatment started after implantation of the conceptus, a relation to the test item could be excluded.

Litter sizes were within normal limits for all groups, the male:female ratios were equal in litters of all groups and fetal body weights were unaffected by treatment. The only external malformation noted was an inwards rotated right fore limb in a single control fetus (A003-01). As this malformation occurred in the control treated with the vehicle alone, a relation to treatment with the test-item could be excluded. No external variations were observed.

Based on the results of the dose range finder, selected dose levels for the main study were 300, 600 and 1000 mg/kg.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations checked: mortality, moribundity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily, beginning on day 2 post-coitum - up to the day prior to necropsy.

BODY WEIGHT: Yes
- Time schedule for examinations: on days 2, 6, 9, 12, 15, 18 and 21 post-coitum.

FOOD CONSUMPTION AND COMPOUND INTAKE : Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No (gavage study)

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: Water consumption was monitored on regular basis throughout the study by visual inspection of the water bottles/containers.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21


Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: the number and distribution of live and dead fetuses, the number and distribution of embryo-fetal deaths, the sex of each fetus based on the ano-genital distance.
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter

Other: As skeletal malformations were suspected for one animal of the low and one animal of the mid dose group,which were selected for visceral examination, these fetuses were also subjected to skeletal examination
Statistics:
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% or 5% levels. Descriptive statistics number, meanand standard deviation (or %CV or SE when deemed appropriate) were reported whenever possible.
Inferential statistics were performed when possible, but excluded semi-quantitative data, and any group with less than 3 observations.

The following pairwise comparisons were made:
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1

Parametric:
Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).

Non-Parametric:
Datasets with at least 3 groups were compared using a Steel-test (many-to-one rank test). Mean litter proportions (percent of litter) of the number of viable and dead fetuses, early and late resorptions, total resorptions, pre- and post-implantation loss, and sex distribution were compared using the Mann Whitney test. Mean litter proportions (percent per litter) of total fetal malformations and developmental variations (external, visceral and skeletal), and each particular external, visceral and skeletal malformation or variation were subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences.

Incidence
An overall Fisher’s exact test was used to compare all groups at the 5% significance level. The above pairwise comparisons were conducted using a two-sided Fisher’s exact test at the 5% significance level if the overall test was significant. No statistics were applied for data on maternal survival, pregnancy status, group mean numbers of dead fetuses, early and late resorptions, and pre- and post-implantation loss.
Indices:
Pre-implantation loss (%): ((number of corpora lutea - number of implantation sites) / number of corpora lutea) x 100
Post-implantation loss (%): ((number of implantation sites - number of live fetuses) / number of implantation sites) x 100
Number of viable fetuses affected/litter (%): (number of viable fetuses affected/litter / number of viable fetuses/litter) x 100

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
300 and 1000 mg/kg bw/day: slight salivation was observed in high dose animals from day 4 of treatment onwards, and in a single female of the low dose group on the last day of treatment. Salivation was considered to be a physiological response rather than a sign of systemic toxicity, taking into account the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). (non-adverse)

All other clinical signs noted occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered to be non-treatment-related.
(Please refer to table 1 in the "Any other information on results incl. tables" section.)

Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
300 and 1000 mg/kg bw/day: slightly (not statistically significantly) lower mean body weight gain after correction for gravid uterus was observed in high dose group females compared to concurrent control mean (19.0 vs 24.0 g) and in one female of the low dose group. The finding in the high dose group was mainly attributed to one female that appeared to have 0.3% body weight loss during the study period. In the absence of a dose-related trend, this observation was not considered toxicological relevant. (Please refer to table 2 and 3 in the "Any other information on results incl. tables" section.)
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
1000 mg/kg bw/day: absolute and relative food consumption between treatment days 6-9 was decreased by 16 and 12 %, respectively, when compared to controls, reaching statistical significance for relative values only. Food consumption, normalized over the next treatment days and remained within the normal range of biological variation over the rest of the treatment period and was therefore not considered toxicologically relevant (non-adverse). (Please refer to table 4 and 5 in the "Any other information on results incl. tables" section.)
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:
not examined
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
control and 600 mg/kg bw/day: one female of each group had an early delivery on the morning of scheduled necropsy (day 21 post-coitum). In the absence of a dose-related trend this finding was considered to be unrelated to treatment.

Other incidental findings among control and treated animals included uterus with a wateryclear cyst (one control female) and alopecia. These findings are occasionally seen among rats used in these types of studies and therefore were considered changes of no toxicological significance. (Please refer to table 6 in the "Any other information on results incl. tables" section.)
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:
no effects observed
Description (incidence and severity):
(Please refer to table 7 in the "Any other information on results incl. tables" section.)
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
control: relatively high mean values for post-implantation loss in the control group was caused by one female which had only one implantation that died early during gestation. Since this female was treated with the vehicle alone, a relation to the test item could be excluded. (Please refer to table 8 and 9 in the "Any other information on results incl. tables" section.)
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
control: relatively high mean values for total resorptions loss in the control group was caused by one female which had only one implantation that died early during gestation. Since this female was treated with the vehicle alone, a relation to the test item could be excluded. (Please refer to table 7, 8 and 9 in the "Any other information on results incl. tables" section.)
Early or late resorptions:
no effects observed
Description (incidence and severity):
control: one female had an early resorption (vehicle group, non-treatment related) (Please refer to table 7, 8 and 9 in the "Any other information on results incl. tables" section.)
Dead fetuses:
no effects observed
Description (incidence and severity):
(Please refer to table 7, 8 and 9 in the "Any other information on results incl. tables" section.)
Changes in pregnancy duration:
effects observed, non-treatment-related
Description (incidence and severity):
control and 600 mg/kg bw/day: one female of each group delivered their offspring on the day of scheduled necropsy (day 21 post-coitum) and delivered 5 (control) and 8 (600 mg/kg bw/day) viable pups, all without developmental malformations. This early delivery was
considered non-treatment-related as it occurred at the same incidence in the 600 mg/kg group as in the vehicle control group. (Please refer to table 7 in the "Any other information on results incl. tables" section.)
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
control: one female was not pregnant (vehicle group, non-treatment related)
(Please refer to table 7 and 8 in the "Any other information on results incl. tables" section.)
Other effects:
not examined

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Remarks:
general and developmental toxicity
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: no adverse effect observed at this dose level

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
Mean combined (male and female) fetal body weights were 5.3, 5.3, 5.2 and 5.1 gram for the control, 300, 600 and 1000 mg/kg groups, respectively. These values remained well within the available historical control range. (Please refer to table 8 in the "Any other information on results incl. tables" section.)
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
1000 mg/kg bw/day: one dead fetus observed (1/260 fetuses, incidental finding, non-treatment-related) (Please refer to table 7, 8 and 9 in the "Any other information on results incl. tables" section.)
Changes in sex ratio:
no effects observed
Description (incidence and severity):
(Please refer to table 8 and 9 in the "Any other information on results incl. tables" section.)
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
control: slightly lower mean litter size in the control group was caused by one female which had one early resorption only. As this female was treated with the vehicle only, a relation to the test item could be excluded.
Mean litter sizes were 10.5, 11.7, 11.5 and 11.8 fetuses/litter for the control, 300, 600 and 1000 mg/kg groups, respectively. (Please refer to table 9 in the "Any other information on results incl. tables" section.)
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
300 and 600 mg/kg bw/day: one fetus of the low dose group had gastroschisis and missed one digit of a forepaw and one fetus of the mid dose group had a filamentous tail. (Additional skeletal examination revealed that the fetus of the low dose group appeared to have sternoschisis and vertebral anomaly with associated rib anomaly as well.) At the isolated incidence and in the absence of a dose-related trend, these malformations were considered to be of spontaneous origin. (Please refer to table 10 and 11 in the "Any other information on results incl. tables" section.)
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
300 mg/kg bw/day: the externally malformed fetus of the low dose group also had vertebral anomaly and sternoschisis, skeletal examination revealed another vertebral anomaly in a littermate and bent limb bones (humerus and scapula) in a furtherlow dose fetus. At the isolated incidence and in the absence of a dose-related trend, these finidngs were considered to be of spontaneous origin, since all the malformations that were noted were observed previously in historical control fetuses.

Skeletal variations: Skeletal variations occurred at an incidence of 73.1%, 70.8%, 88.6% and 73.5% per litter in the control, 300, 600 and 1000 mg/kg groups, respectively.
600 mg/kg bw/day: statistically significantly higher incidence of total skeletal variations compared to the control. The reason for these increased incidences in this group is unknown, but as none single finding reached statistical significance or showed a dose relationship, they were not considered toxicologically relevant. Therefore, also the statistically significant higher incidence of total variations in this group was considered to be a chance finding.

All other variations occurred in the absence of a dose-related incidence trend, infrequently and/or at frequencies that were within the range of available historical control data.
(Please refer to table 10 and 11 in the "Any other information on results incl. tables" section.)
Visceral malformations:
no effects observed
Description (incidence and severity):
Visceral variations that were noted occurred in the absence of a dose-related incidence trend, occurred infrequently, and/or at frequencies that were within the range of available historical control data (non-treatment-related). (Please refer to table 10 and 11 in the "Any other information on results incl. tables" section.)
Other effects:
not examined

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effect observed at this dose level

Fetal abnormalities

Key result
Abnormalities:
effects observed, non-treatment-related
Localisation:
external: paw
external: tail
skeletal: sternum
skeletal: vertebra
other: External: gastroschisis
Description (incidence and severity):
At the isolated incidence and in the absence of a dose-related trend, these findings were considered to be of spontaneous origin, since all the malformations that were noted were observed previously in historical control fetuses.

Overall developmental toxicity

Key result
Developmental effects observed:
no

Any other information on results incl. tables

Table 1: Summary of clinical signs

 

 

Pre-treatment

Treatment

Dose group (mg/kg bw/day)

Week:

 

1xxx

1xxxxxxxxxxxxxx

Sign (Max grade, Location)

Day:

1234

123456712345671

Control

 

 

 

Gait / motility

G:

xxxx

xxx1xxxxxxxxxxx

Flat gait (1)

%:

xxxx

xxx0xxxxxxxxxxx

Skin / fur

G:

xxxx

xx1xxx111111111

Piloerection (1)

%:

xxxx

xx0xxx110000000

Fissures (3)

G:

xxxx

xxxx11111111111

(Flewes)

%:

xxxx

xxxx00000000000

300

 

 

 

Breathing

G:

xxxx

xxxxxxxxxxxxxx1

Rales (3)

%:

xxxx

xxxxxxxxxxxxxx0

Skin / fur

G:

xxxx

xx111xxxxxxxxxx

Piloerection (1)

%:

xxxx

xx000xxxxxxxxxx

Alopecia (3)

G:

xxxx

xxxxxxxxxxxxx11

 

%:

xxxx

xxxxxxxxxxxxx00

Secretion / excretion

G:

xxxx

xxxxxxxxxxxxxx1

Salivation (3)

%:

xxxx

xxxxxxxxxxxxxx0

600

 

 

 

Skin / fur

G:

xxxx

xx1xx11111xxxxx

Piloerection (1)

%:

xxxx

xx0xx00000xxxxx

Secretion / excretion

G:

xxxx

xxxxxxxx1xxxxxx

Chromodacryorrhoea (3) (snout)

%:

xxxx

xxxxxxxx0xxxxxx

1000

 

 

 

Skin / fur

G:

xxxx

xxx111111111111

Piloerection (1)

%:

xxxx

xxx011122221000

Alopecia (3)

G:

xxxx

xx1111111111111

 

%:

xxxx

xx0000000011111

Secretion / excretion

G:

xxxx

xxx111111111111

Salivation (3)

%:

xxxx

xxx379999AAAAAA

G: Median value of the highest individual daily grades

%: Percent of affected animals (0=less than 5%, 1=between 5% and 15%,..., A=more than 95%)

x: Observation performed, sign not present

 

Table 2: Summary of body weights (g)

Dose group (mg/kg bw/day)

 

Control

300

600

1000

Post Coitum

Day 2

 

Mean

 

210

 

211

 

212

 

210

 

ST.DEV.

22.5

20.1

15.2

16.3

 

N

21

22

22

22

Day 6

Mean

224

225

225

226

 

ST.DEV.

23.6

22.0

19.0

15.8

 

N

21

22

22

22

Day 9

Mean

231

234

235

230

 

ST.DEV.

24.5

22.6

17.9

16.6

 

N

21

22

22

22

Day 12

Mean

246

249

250

245

 

ST.DEV.

27.1

24.8

19.5

17.0

 

N

21

22

22

22

Day 15

Mean

260

262

265

259

 

ST.DEV.

29.1

25.8

20.4

19.2

 

N

21

22

22

22

Day 18

Mean

288

293

297

289

 

ST.DEV.

36.7

27.3

23.5

20.3

 

N

21

22

22

22

Day 21

Mean

325

329

334

325

 

ST.DEV.

44.4

30.8

24.6

22.9

 

N

20

22

21

22

 

Table 3: Summary of body weight gain (%)

Dose group (mg/kg bw/day)

 

Control

300

600

1000

Post Coitum

Day 2

 

Mean

 

-6

 

-6

 

-6

 

-7

 

ST.DEV.

2.8

2.9

3.9

2.5

 

N

21

22

22

22

Day 6

Mean

0

0

0

0

 

ST.DEV.

0.0

0.0

0.0

0.0

 

N

21

22

22

22

Day 9

Mean

3

4

4

2

 

ST.DEV.

2.8

2.7

3.4

2.4

 

N

21

22

22

22

Day 12

Mean

10

11

11

8

 

ST.DEV.

2.9

3.1

3.6

2.6

 

N

21

22

22

22

Day 15

Mean

16

16

18

15

 

ST.DEV.

4.3

3.2

3.3

3.6

 

N

21

22

22

22

Day 18

Mean

28

30

32

28

 

ST.DEV.

7.9

4.5

3.9

4.2

 

N

21

22

22

22

Day 21

Mean

44

46

48

44

 

ST.DEV.

11.1

7.3

6.2

5.7

 

N

20

22

21

22

 

 

Table 4: Summary of food consumption (g/animal/day)

Dose group (mg/kg bw/day)

 

Control

300

600

1000

Post coitum

Days 2-6

 

Mean

 

22

 

22

 

22

 

23

 

St.dev.

2.7

3.2

3.0

2.5

 

N

21

22

22

22

Days 6-9

Mean

19

19

19

16

 

St.dev.

2.8

3.7

3.3

4.4

 

N

21

22

22

22

Days 9-12

Mean

21

22

21

20

 

St.dev.

2.7

4.0

3.9

3.4

 

N

21

22

22

22

Days 12-15

Mean

22

21

22

21

 

St.dev.

3.6

3.5

3.3

2.6

 

N

21

22

22

22

Days 15-18

Mean

23

23

24

22

 

St.dev.

3.3

3.2

2.4

2.6

 

N

21

22

22

22

Days 18-21

Mean

24

24

24

23

 

St.dev.

3.9

3.5

2.6

2.9

 

N

21

22

22

22

 

Mean of means

 

22

22

22

21

 

Table 5: Summary relative food consumption (g/kg body weight/day)

Dose group (mg/kg bw/day)

 

Control

300

600

1000

Post coitum

Days 2-6

 

Mean

 

98

 

100

 

96

 

103

 

ST.DEV.

9.6

9.6

10.9

10.4

 

N

21

22

22

22

Days 6-9

Mean

80

81

83

70 *

 

ST.DEV.

10.1

11.9

13.7

17.1

 

N

21

22

22

22

Days 9-12

Mean

85

86

82

82

 

ST.DEV.

6.8

9.4

13.9

11.8

 

N

21

22

22

22

Days 12-15

Mean

85

82

84

82

 

ST.DEV.

9.0

8.4

8.9

6.8

 

N

21

22

22

22

Days 15-18

Mean

79

80

81

77

 

ST.DEV.

4.3

6.2

4.6

6.5

 

N

21

22

22

22

Days 18-21

Mean

75

72

74

71

 

ST.DEV.

6.4

6.7

7.7

6.6

 

N

21

22

22

22

Mean of means

 

84

83

83

81

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

 

Table 6: Macroscopic findings

Dose group (mg/kg bw/day)

Control

300

600

1000

Post coitum

Animals examined

22

22

22

 

22

Animals without findings

20

21

21

20

Animals affected

2

1

1

2

General observations Early delivery

 

1

 

0

 

1

 

0

Uterus

Cyst(s)

1

0

0

0

Skin

Alopecia

0

1

0

2

 

Table 7: Summary of maternal survival and pregancy status

Dose group (mg/kg bw/day)

control

300

600

1000

No.

%

No.

%

No.

%

No.

%

 

Females on study 22

 

22

 

22

 

22

 

 

Females that aborted or delivered

0

0.0

0

0.0

0

0.0

0

0.0

Females that died

0

0.0

0

0.0

0

0.0

0

0.0

Females that aborted

0

0.0

0

0.0

0

0.0

0

0.0

Nongravid

0

0.0

0

0.0

0

0.0

0

0.0

Gravid

0

0.0

0

0.0

0

0.0

0

0.0

Females that were euthanized

0

0.0

0

0.0

0

0.0

0

0.0

Nongravid

0

0.0

0

0.0

0

0.0

0

0.0

Gravid

0

0.0

0

0.0

0

0.0

0

0.0

Females examined at scheduled necropsy

22#

100.0

22

100.0

22#

100.0

22

100.0

Nongravid

1

4.5

0

0.0

0

0.0

0

0.0

Gravid

21

95.5

22

100.0

22

100.0

22

100.0

With resorptions only

1

4.8

0

0.0

0

0.0

0

0.0

With viable fetuses

20

95.2

22

100.0

22

100.0

22

100.0

# Including females that delivered day 21 (control and 600 mg/kg bw/day dose group)

 

Table 8: Summary of fetal data at scheduled necropsy

 

 

Sex

Viable Fetuses

Dead fetuses

Resorptions

Post-implantation

Corpora lutea

Pre-implantation loss

Fetal weight (g)

No. of gravid females

Dose group

(mg/kg bw/day)

 

Male

Female

Early

Late

Loss

Sites

Vehicle

Total

119

101

220

0

17

0

17

237

239

2

NA

21

 

Mean

5.7

4.8

10.5

0.0

0.8

0.0

0.8

11.3

11.4

0.1

5.3

 

 

S.D.

2.15

2.06

3.43

0.00

1.21

0.00

1.21

3.13

3.14

0.30

0.40

 

300

Total

122

136

258

0

8

0

8

266

276

10

NA

22

 

Mean

5.5

6.2

11.7

0.0

0.4

0.0

0.4

12.1

12.5

0.5

5.3

 

 

S.D.

2.22

1.65

1.83

0.00

0.58

0.00

0.58

1.69

1.60

0.96

0.30

 

600

Total

127

125

252

0

5

0

5

257

263

6

NA

22

 

Mean

5.8

5.7

11.5

0.0

0.2

0.0

0.2

11.7

12.0

0.3

5.2

 

 

S.D.

1.57

1.73

1.60

0.00

0.53

0.00

0.53

1.64

1.56

0.55

0.24

 

1000

Total

124

135

259

1

15

0

16

275

280

5

NA

22

 

Mean

5.6

6.1

11.8

0.0

0.7

0.0

0.7

12.5

12.7

0.2

5.1

 

 

S.D.

2.22

2.19

1.48

0.21

0.84

0.00

0.94

1.30

1.28

0.43

0.40

 

None significantly different from control group

NA = not applicable

Mean number of viable fetuses, mean number of implantation sites, mean number of corpora lutea, fetal weights compared using dunnett's test

SD standard deviation

 

Table 9: Summary of fetal data at scheduled necropsy (% per litter)

 

Dose group (mg/kg bw/day)

control

300

600

1000

Corpora Lutea

Mean

11.4

12.5

12.0

12.7

 

S.D.

3.14

1.60

1.56

1.28

 

N

21

22

22

22

Viable Fetuses (%)

Mean

88.4

96.9

98.2

94.2

 

S.D.

23.53

4.86

4.18

7.16

 

N

21

22

22

22

Dead Fetuses (%)

 

Mean

0.0

0.0

0.0

0.4

 

S.D.

0.00

0.00

0.00

1.77

 

N

21

22

22

22

Early Resorptions (%)

Mean

11.6

3.1

1.8

5.4

 

S.D.

23.53

4.86

4.18

6.27

 

N

21

22

22

22

Late Resorptions (%)

Mean

0.0

0.0

0.0

0.0

 

S.D.

0.00

0.00

0.00

0.00

 

N

21

22

22

22

Total Resorptions (%)

Mean

11.6

3.1

1.8

5.4

 

S.D.

23.53

4.86

4.18

6.27

 

N

21

22

22

22

Pre-Implantation Loss (%)

Mean

0.8

3.5

2.3

1.8

 

S.D.

2.50

7.54

4.60

3.34

 

N

21

22

22

22

Post-Implantation Loss (%)

Mean

11.6

3.1

1.8

5.8

 

S.D.

23.53

4.86

4.18

7.16

 

N

21

22

22

22

Males (%)

Mean

54.7

46.4

50.6

47.7

 

S.D.

12.55

15.90

12.26

17.26

 

N

20

22

22

22

Females (%)

Mean

45.3

53.6

49.4

52.3

 

S.D.

12.55

15.90

12.26

17.26

 

N

20

22

22

22

Male fetal weights (g)

Mean

5.5

5.4

5.4

5.2

 

S.D.

0.40

0.35

0.30

0.36

 

N

20

22

22

22

Female fetal weights (g)

Mean

5.2

5.2

5.1

5.0

 

S.D.

0.45

0.30

0.20

0.44

 

N

20

22

22

22

Combined fetal weights (g)

Mean

5.3

5.3

5.2

5.1

 

S.D.

0.40

0.30

0.24

0.40

 

N

20

22

22

22

proportional (%) data compared using the mann-whitney test

corpora lutea and implantation sites compared using dunnett's test

fetal weights compared using dunnett's test

none significantly different from control group

SD standard deviation

N number

 

Table 10: Summary of fetuses and litters with malformation (absolute number)

 

Fetuses

Litters

Dose group (mg/kg bw/day)

control

300

600

1000

control

300

600

1000

Number examined externally

220

258

252

259

20

22

22

22

Trunk- gastroschisis

0

1

0

0

0

1

0

0

Tail- absent, short or filamentous

0

0

1

0

0

0

1

0

Ectrodactyly

0

1

0

0

0

1

0

0

Number examined viscerally

110

130

128

129

20

22

22

22

Number with findings

0

0

0

0

0

0

0

0

Number examined skeletally

110

129

125

130

20

22

22

22

Vertebral anomaly with or without associated rib anomaly

0

2

0

0

0

1

0

0

Bent limb bone(s)

0

1

0

0

0

1

0

0

Sternoschisis

0

1

0

0

0

1

0

0

 

Total number with malformations

 

 

 

 

 

 

 

 

External :

0

1

1

0

0

1

1

0

Soft tissue :

0

0

0

0

0

0

0

0

Skeletal :

0

3

0

0

0

2

0

0

Combined :

0

3

1

0

0

2

1

0

 

Table 11: Summary of fetuses and litters with variations (absolute number)

 

Fetuses

Litters

Dose group (mg/kg bw/day)

control

300

600

1000

control

300

600

1000

Number examined externally

220

258

252

259

20

22

22

22

Number with findings

0

0

0

0

0

0

0

0

Number examined viscerally

110

130

128

129

20

22

22

22

Liver- small supernumerary lobe(s)

4

1

1

6

3

1

1

6

Ureter(s)- convoluted

0

3

1

0

0

3

1

0

Ureter(s)- dilated

0

1

0

0

0

1

0

0

Renal papilla(e)- absent and/or small

0

1

0

0

0

1

0

0

Number examined skeletally

110

129

125

130

20

22

22

22

14th rudimentary rib(s)

59

74

78

71

18

22

21

19

14th full rib(s)

7

7

20

6

6

5

11

4

Bent rib(s)

12

12

24

14

7

7

14

7

Pelvic girdle- caudal shift

5

9

13

4

5

6

8

3

Reduced ossification of the skull

9

8

16

10

7

6

10

5

Sternebra(e) malaligned

9

14

13

10

8

12

11

6

Scapula(e)- bent

0

0

2

0

0

0

2

0

Metacarpal(s) and/or metatarsal(s) unossified

1

1

0

7

1

1

0

5

7th cervical ossification site(s)

4

7

8

6

4

7

7

4

Sternebra(e)- branched

2

0

1

1

2

0

1

1

Vertebral centra- reduced ossification

0

1

0

1

0

1

0

1

7th cervical full rib(s)

0

2

0

0

0

2

0

0

Applicant's summary and conclusion

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