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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:
12 Aug 2018 to 30 Aug 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2019

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
22 January 2001
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Version / remarks:
30 May 2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
August 1998
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Wistar Han
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Deutschland, Sulzfeld, Germany
- Age at study initiation: 10-14 weeks
- Weight at study initiation: 186 – 266 g
- 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) provided ad libitum throughout the study
- Water: Municipal tap water was freely available to each animal via water bottles.
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 21
- Humidity (%): 49 to 78
- Air changes (per hr): ≥ 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES:
12 Aug 2018 to 30 Aug 2018

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
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 suspension and dosed within 6 hours after adding the 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. No correction was made for the purity/composition of the test item.

VEHICLE
- Justification for use and choice of vehicle: The use of corn oil was required due to the physicochemical properties of the test substance (low water solubility and stability)
- Concentration in vehicle: 25, 75 and 250 mg/mL
- Amount of vehicle: 4 mL/kg bw/day
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
CONCENTRATION ANALYSIS
Concentration was determined based on Zinc content since there was no better, feasible analytical method available for this type of molecule.
Duplicate sets of samples (approximately 500 mg) were sent to the analytical laboratory. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 15% for suspensions of target concentration.

HOMOGENEITY ANALYSIS
Homogeneity was determined based on Zinc content since there was no better, feasible analytical method available for this type of molecule.
Duplicate sets of samples (approximately 500 mg) 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%.

STABILITY ANALYSIS
As requested by the Sponsor, stability analysis of the test item in the vehicle was not determined since the available analytical method was only capable of measuring the zinc content in the test substance and, therefore, unable to measure the test item itself.
Details on mating procedure:
The females arrived on Day 0 or Day 1 post-coitum (Day 0 post-coitum is defined as the day of successful mating).
Duration of treatment / exposure:
The test item and vehicle were administered 7 days a week from day 6 to day 20 post-coitum, inclusive.
Frequency of treatment:
Once daily
Duration of test:
Animals surviving until scheduled euthanasia were euthanised on day 21 post-coitum.
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
Group 2
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Remarks:
Group 3
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
Group 4
No. of animals per sex per dose:
Group 1 to 4: 22 females per group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected based on the results of the dose range finder. In this dose range finder, doses up to 1000 mg/kg bw/day were tolerated by pregnant rats. Only transient test item-related clinical signs were noted during the first two days of treatment in all treated groups. No toxicologically relevant changes in mean body weight and food consumption were observed.
Based on the results of the dose range finder, selected dose levels for the main study were 100, 300 and 1000 mg/kg bw/day.
- Animal assignment: random

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS
- Time schedule: twice daily, in the morning and at the end of the working day
- Cage side observations included general health/mortality and moribundity.

DETAILED CLINICAL OBSERVATIONS
- Time schedule: once daily, beginning on Day 2 post-coitum and lasting up to the day prior to necropsy.
The time of onset, grade and duration of any observed sign was recorded. Signs were graded for severity and the maximum grade was predefined at 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) was scored.
Cage debris was examined to detect premature birth.

BODY WEIGHT
- Time schedule for examinations: Days 2, 6, 9, 12, 15, 18 and 21 post-coitum

FOOD CONSUMPTION
- Time schedule for examinations: Days 2-6, 6-9, 9-12, 12-15, 15-18 and 18-21 post-coitum
- Food consumption was determined for each animal and mean daily diet consumption calculated as g food/kg body weight/day.

POST-MORTEM EXAMINATIONS
- Sacrifice on gestation day 21 post-coitum
- Organs examined: All animals were subjected to an external, thoracic and abdominal examination, with special attention being paid to the reproductive organs. All macroscopic abnormalities were recorded, collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution). No organs (except for the uterus) were weighed.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination
Examinations included:
- Gravid uterus weight
- Number of corpora lutea
- Number of implantations
- Number of early resorptions
- Number of late resorptions
- Other: the number and distribution of live and dead fetuses; the sex of each fetus based on the ano-genital distance
Fetal examinations:
- External examinations: all viable fetuses per litter. Each viable foetus was sexed, examined in detail to detect macroscopic visible abnormalities and their weight was determined. For late resorptions, a gross external examination was performed.
- Soft tissue examinations: The sex of all fetuses was confirmed by internal examination and approximately one-half of the fetuses in each litter (all groups) were examined for visceral anomalies by dissection in the fresh (non-fixed) state. This examination included the heart and major vessels. Fetal kidneys were examined and graded for renal papillae development.
- Skeletal examinations: half per litter. In order to further examine an externally noted malformation, one fetus selected for visceral examination, was also subjected to skeletal examination.
- Head examinations: half per litter. This examination included serial sectioning of the head in at least seven even slices after removal of the lower jaw. For each slice, the front- and back side was examined. Multiple structures were distinguished, special attention was paid to nose/throat, eyes and brains.
Statistics:
STATISTICAL ANALYSIS
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.
Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation (or %CV or SE when deemed appropriate) were reported whenever possible. Inferential statistics were performed according to the matrix below 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:
MATERNAL VARIABLES
Body Weight Gains: Calculated against the body weight on Day 6 post-coitum
Corrected Body Weight Gains: Terminal body weight minus the body weight on Day 6 postcoitum and the weight of gravid uterus.
Relative Food Consumption: Calculated against the body weight for scheduled intervals.

REPRODUCTION AND DEVELOPMENTAL VARIABLES
For each group, the following calculations were performed:

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

Viable fetuses affected per 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, non-treatment-related
Description (incidence and severity):
Piloerection was observed in one control female for 3 consecutive days during the pre-treatment period and in one animal at 1000 mg/kg bw/day during the last 3 days of treatment. This clinical sign was considered to be unrelated to treatment at the incidence observed (in one high dose female only) and as it was also noted in one control animal.
Another clinical sign noted during the treatment period was alopecia which 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, this was considered to be unrelated to treatment.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
No mortality occurred during the study period.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Mean body weight and body weight gain of animals treated up to 1000 mg/kg remained in the same range as controls over the treatment period.
Body weight gain corrected for gravid uterus was considered to be unaffected by treatment up to 1000 mg/kg.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
No toxicologically relevant changes in food consumption before or after correction for body weight were observed over the study period.
Over Days 6-9 post-coitum, mean food consumption (before or after allowance for body weight) was significantly increased at 1000 mg/kg bw/day (relative food consumption 11% higher vs concurrent control mean). This significant increase in food consumption was considered to be due to a slightly low mean value of the concurrent control group. Mean values of all treated groups were well within the range of the historical control data.
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
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Macroscopic observations at necropsy did not reveal any alterations that were considered to have arisen as a result of treatment.
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
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
The numbers of pre-implantation loss in the control and test groups were similar and/or in the range of normal biological variation.
No toxicologically relevant changes in post-implantation loss were observed by treatment up to 1000 mg/kg.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Description (incidence and severity):
No toxicologically relevant changes in the litter incidence of early and late resorptions were observed by treatment up to 1000 mg/kg.
Dead fetuses:
no effects observed
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
no effects observed
Other effects:
not examined

Effect levels (maternal animals)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
Maternal
Effect level:
> 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Dose descriptor:
NOAEL
Remarks:
Maternal developmental
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Remarks on result:
not determinable due to absence of adverse toxic effects

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
There were no treatment-related effects on fetal body weights (both sexes) noted by treatment up to 1000 mg/kg.
Mean combined (male and female) fetal body weights were 5.3, 5.4, 5.4 and 5.3 gram for the control, 100, 300 and 1000 mg/kg groups, respectively.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Description (incidence and severity):
Sex ratios were unaffected by treatment up to 1000 mg/kg.
Mean sex ratios (males:females) were 47:53, 48:52, 49:51 and 57:43 for the control, 100, 300 and 1000 mg/kg groups, respectively.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
There were no treatment-related effects on litter size by treatment up to 1000 mg/kg.
Mean litter sizes were 10.8, 10.7, 11.2 and 11.1 fetuses/litter for the control, 100, 300 and 1000 mg/kg groups, respectively.
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment-related effects on external morphology following treatment up to 1000 mg/kg.
Two externally malformed fetuses were observed in this study, each in the control and 100 mg/kg bw/day groups: the low dose fetus was noted with a small lower jaw and cleft palate and the control fetus had a malrotated hindlimb. Skeletal examination substantiated the jaw and palate findings, but there was no apparent skeletal origin for the limb finding. Due to the single occurrence of these external malformations and their occurrence only in the low dose and control groups, these malformations were considered to be of spontaneous origin.
No external variations were noted in any of the groups.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment-related effects on skeletal morphology following treatment up to 1000 mg/kg.
Aside from the underlying skull malformations of the low dose fetus that presented with a small lower jaw and cleft palate, bent limb bones were observed in another low dose fetus and a vertebral anomaly was found in a mid dose fetus. The low incidence and group distribution of these malformations did not suggest a treatment relationship and litter incidences were well within the historical control range. Therefore, the above malformations were considered to be spontaneous in origin and not test item related.
Skeletal variations occurred at an incidence of 70.6%, 82.5%, 75.4% and 70.0% per litter in the control, 100, 300 and 1000 mg/kg groups, respectively. All skeletal variations noted 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. Therefore, they were not considered treatment related.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment-related effects on visceral morphology following treatment up to 1000 mg/kg. No remarkable observations were noted in the heads which were removed from one-half of the fetuses in each litter.
Only one visceral malformation (situs inversus) was observed and as it occurred in a single control fetus, it was as such considered to be of spontaneous origin.
Only one visceral variation (small supernumerary liver lobes) was observed in one and four fetuses at 100 and 300 mg/kg, respectively. This finding was considered to be unrelated to treatment in the absence of a dose-response relationship and at the low incidence observed (litter incidences were well within the range of the historical control data).
Other effects:
not examined

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

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 1.6 BODY WEIGHTS (GRAM) SUMMARY

F0-GENERATION

 

 

GROUP 1

CONTROL

GROUP 2

100 MG/KG

GROUP 3

300 MG/KG

GROUP 4

1000 MG/KG

POST COITUM

 

 

 

 

 

DAY 2

MEAN

209

209

209

210

 

ST.DEV.

19.6

19.7

17.0

18.3

 

N

22

22

22

22

DAY 6

MEAN

224

225

225

227

 

ST.DEV.

20.0

19.9

17.7

18.5

 

N

22

22

22

22

DAY 9

MEAN

232

234

234

235

 

ST.DEV.

20.2

23.1

18.8

20.0

 

N

22

22

22

22

DAY 12

MEAN

246

248

249

251

 

ST.DEV.

21.3

24.5

18.1

21.0

 

N

22

22

22

22

DAY 15

MEAN

261

262

263

264

 

ST.DEV.

23.1

26.2

19.9

23.7

 

N

22

22

22

22

DAY 18

MEAN

292

294

296

296

 

ST.DEV.

25.0

31.1

23.6

26.0

 

N

22

22

22

22

DAY 21

MEAN

329

332

335

333

 

ST.DEV.

30.8

36.8

28.0

29.6

 

N

22

22

22

22

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

 

TABLE 1.7 BODY WEIGHT GAIN (%) SUMMARY

F0-GENERATION

 

 

GROUP 1

CONTROL

GROUP 2

100 MG/KG

GROUP 3

300 MG/KG

GROUP 4

1000 MG/KG

POST COITUM

 

 

 

 

 

DAY 2

MEAN

-7

-7

-7

-8

 

ST.DEV.

2.3

2.0

2.4

2.3

 

N

22

22

22

22

DAY 6

MEAN

0

0

0

0

 

ST.DEV.

0.0

0.0

0.0

0.0

 

N

22

22

22

22

DAY 9

MEAN

4

4

4

4

 

ST.DEV.

1.8

1.9

2.0

2.3

 

N

22

22

22

22

DAY 12

MEAN

10

10

11

10

 

ST.DEV.

2.3

3.0

2.8

3.1

 

N

22

22

22

22

DAY 15

MEAN

16

16

17

16

 

ST.DEV.

2.7

3.8

3.8

4.9

 

N

22

22

22

22

DAY 18

MEAN

30

30

32

31

 

ST.DEV.

3.7

5.2

4.4

6.0

 

N

22

22

22

22

DAY 21

MEAN

47

47

49

47

 

ST.DEV.

7.2

7.1

6.4

6.8

 

N

22

22

22

22

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

 

TABLE 1.8 FOOD CONSUMPTION (G/ANIMAL/DAY) SUMMARY

F0-GENERATION

 

 

GROUP 1

CONTROL

GROUP 2

100 MG/KG

GROUP 3

300 MG/KG

GROUP 4

1000 MG/KG

POST COITUM

 

 

 

 

 

DAYS 2-6

MEAN

22

22

21

21

 

ST.DEV.

3.5

2.6

2.6

2.2

 

N

22

22

22

22

DAYS 6-9

MEAN

16

18

18

18 *

 

ST.DEV.

3.0

2.8

2.1

2.9

 

N

22

22

22

22

DAYS 9-12

MEAN

20

20

20

21

 

ST.DEV.

2.7

2.8

2.0

2.7

 

N

22

22

22

22

DAYS 12-15

MEAN

19

19

19

20

 

ST.DEV.

2.4

3.1

2.4

2.1

 

N

22

22

22

22

DAYS 15-18

MEAN

24

24

24

25

 

ST.DEV.

2.4

2.8

2.5

3.1

 

N

22

22

22

22

DAYS 18-21

MEAN

23

23

23

24

 

ST.DEV.

2.8

3.5

2.8

2.4

 

N

22

22

22

22

MEAN OF MEANS

 

21

21

21

22

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

 

TABLE1.9 RELATIVE FOOD CONSUMPTION (G/KG BODY WEIGHT/DAY) SUMMARY

F0-GENERATION

 

 

GROUP 1

CONTROL

GROUP 2

100 MG/KG

GROUP 3

300 MG/KG

GROUP 4

1000 MG/KG

POST COITUM

 

 

 

 

 

DAYS 2-6

MEAN

98

97

95

93

 

ST.DEV.

14.8

9.3

11.6

7.8

 

N

22

22

22

22

DAYS 6-9

MEAN

70

76

76

78 *

 

ST.DEV.

12.5

7.5

7.0

9.7

 

N

22

22

22

22

DAYS 9-12

MEAN

79

82

81

84

 

ST.DEV.

8.7

8.4

7.5

7.5

 

N

22

22

22

22

DAYS 12-15

MEAN

74

74

73

77

 

ST.DEV.

6.3

7.1

7.4

6.0

 

N

22

22

22

22

DAYS 15-18

MEAN

83

83

82

85

 

ST.DEV.

7.2

5.7

7.4

7.9

 

N

22

22

22

22

DAYS 18-21

MEAN

71

70

70

73

 

ST.DEV.

4.4

8.1

7.2

8.0

 

N

22

22

22

22

MEAN OF MEANS

 

79

80

80

82

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

 

TABLE 1.10 MACROSCOPIC FINDINGS SUMMARY

 

GROUP 1

CONTROL

GROUP 2

100 MG/KG

GROUP 3

300 MG/KG

GROUP 4

1000 MG/KG

POST COITUM

 

 

 

 

Animals examined

22

22

22

22

Animals without findings

22

21

22

20

Animals affected

0

1

0

2

Liver
Focus/foci

0

0

0

1

Skin

 

 

 

 

Alopecia

0

1

0

1

# / ## Fisher's Exact test significant at 5% (#) or 1% (##) level

TABLE 1.11 SUMMARY OF MATERNAL SURVIVAL AND PREGNANCY STATUS

DOSE GROUP :

1

 

2

 

3

 

4

 

 

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

0

0.0

0

0.0

0

0.0

0

0.0

  GRAVID

22

100.0

22

100.0

22

100.0

22

100.0

     WITH RESORPTIONS ONLY

0

0.0

0

0.0

0

0.0

0

0.0

     WITH VIABLE FETUSES

22

100.0

22

100.0

22

100.0

22

100.0

 

 

 

 

 

 

 

 

 

TOTAL FEMALES GRAVID

22

100.0

22

100.0

22

100.0

22

100.0

 1-  0 MG/KG; 2-  100 MG/KG; 3 -  300 MG/KG; 4- 1000 MG/KG

1.12 SUMMARY OF FETAL DATA AT SCHEDULED NECROPSY

 

 

 

 

 

 

 

 

POST

 

 

PRE

FETAL

NO. OF

SEX

VIABLE

DEAD

RESORPTIONS

IMPLANTATION

IMPLANTATION

CORPORA

IMPLANTATION

WEIGHTS

GRAVID

GROUP

 

M

F

FETUSES

FETUSES

EARLY

LATE

LOSS

SITES

LUTEA

LOSS

IN GRAMS

FEMALES

1

TOTAL

112

125

237

0

11

0

11

248

274

26

NA

22

MEAN

5.1

5.7

10.8

0

0.5

0

0.5

11.3

12.5

1.2

5.3

S.D.

2.07

2.06

2.65

0

0.8

0

0.8

2.83

2.42

1.76

0.25

2

TOTAL

109

126

235

0

11

0

11

246

265

19

NA

22

MEAN

5.0

5.7

10.7

0

0.5

0

0.5

11.2

12

0.9

5.4

S.D.

2.01

2.55

2.5

0

0.96

0

0.96

2.38

1.91

1.21

0.26

3

TOTAL

120

127

247

0

7

0

7

254

273

19

NA

22

MEAN

5.5

5.8

11.2

0

0.3

0

0.3

11.5

12.4

0.9

5.4

S.D.

2.18

2.11

2.02

0

0.48

0

0.48

2.11

1.87

1.25

0.22

4

TOTAL

141

103

244

0

6

1

7

251

264

13

NA

22

MEAN

6.4

4.7

11.1

0

0.3

0

0.3

11.4

12

0.6

5.3

 

S.D.

1.71

1.25

2.04

0

0.55

0.21

0.65

2.24

1.75

0.85

0.23

 

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

1- 0 MG/KG; 2- 100 MG/KG; 3- 300 MG/KG; 4- 1000 MG/KG

TABLE 1.13 SUMMARY OF FETAL DATA AT SCHEDULED NECROPSY [% PER LITTER]

GROUP

0 MG/KG

100 MG/KG

300 MG/KG

1000 MG/KG

CORPORA LUTEA

 

 

 

 

MEAN

12.5

12.0

12.4

12.0

S.D.

2.42

1.91

1.87

1.75

N

22

22

22

22

 

 

 

 

 

IMPLANTATION SITES

 

 

 

 

MEAN

11.3

11.2

11.5

11.4

S.D.

2.83

2.38

2.11

2.24

N

22

22

22

22

 

 

 

 

 

VIABLE FETUSES (%)

 

 

 

 

MEAN

96.0

95.3

97.4

97.6

S.D.

6.27

8.65

4.01

4.84

N

22

22

22

22

 

 

 

 

 

DEAD FETUSES (%)

 

 

 

 

MEAN

0.0

0.0

0.0

0.0

S.D.

0.00

0.00

0.00

0.00

N

22

22

22

22

 

 

 

 

 

EARLY RESORPTIONS (%)

 

 

 

 

MEAN

4.0

4.7

2.6

2.1

S.D.

6.27

8.65

4.01

4.08

N

22

22

22

22

 

 

 

 

 

LATE RESORPTIONS (%)

 

 

 

 

MEAN

0.0

0.0

0.0

0.4

S.D.

0.00

0.00

0.00

1.64

N

22

22

22

22

 

 

 

 

 

TOTAL RESORPTIONS (%)

 

 

 

 

MEAN

4.0

4.7

2.6

2.4

S.D.

6.27

8.65

4.01

4.84

N

22

22

22

22

 

 

 

 

 

PRE-IMPLANTATION LOSS (%)

 

 

 

 

MEAN

10.0

7.6

6.8

5.6

S.D.

14.92

11.30

10.27

9.10

N

22

22

22

22

 

 

 

 

 

POST-IMPLANTATION LOSS (%)

 

 

 

 

MEAN

4.0

4.7

2.6

2.4

S.D.

6.27

8.65

4.01

4.84

N

22

22

22

22

 

 

 

 

 

MALES (%)

 

 

 

 

MEAN

47.1

47.7

48.5

57.0

S.D.

14.89

18.12

16.55

11.94

N

22

22

22

22

 

 

 

 

 

FEMALES (%)

 

 

 

 

MEAN

52.9

52.3

51.5

43.0

S.D.

14.89

18.12

16.55

11.94

N

22

22

22

22

 

 

 

 

 

MALE FETAL WEIGHTS (g)

 

 

 

 

MEAN

5.5

5.5

5.5

5.4

S.D.

0.27

0.27

0.30

0.24

N

22

22

22

22

 

 

 

 

 

FEMALE FETAL WEIGHTS (g)

 

 

 

 

MEAN

5.2

5.2

5.2

5.1

S.D.

0.25

0.24

0.23

0.24

N

22

22

22

22

 

 

 

 

 

COMBINED FETAL WEIGHTS (g)

 

 

 

 

MEAN

5.3

5.4

5.4

5.3

S.D.

0.25

0.26

0.22

0.23

N

22

22

22

22

PROPORTIONAL (%) DATA COMPARED USING THE MANN-WHITNEY TEST

CORPORA LUTEA, IMPLANTATION SITES AND FETAL WEIGHTS COMPARED USING DUNNETT'S TEST

None significantly different from control group

TABLE 1.14 SUMMARY OF FETUSES AND LITTERS WITH MALFORMATIONS [ABSOLUTE NO.]

 

DAY 21

F E T U S E S

L I T T E R S

DOSE GROUP:

1

2

3

4

1

2

3

4

NUMBER EXAMINED EXTERNALLY

237

235

247

244

22

22

22

22

LOWER JAW- ABSENT OR SMALL

0

1

0

0

0

1

0

0

LIMB(S)- MALROTATED

1

0

0

0

1

0

0

0

CLEFT PALATE

0

1

0

0

0

1

0

0

 

 

 

 

 

 

 

 

 

NUMBER EXAMINED VISCERALLY

120

118

122

123

22

22

22

22

SITUS INVERSUS

1

0

0

0

1

0

0

0

 

 

 

 

 

 

 

 

 

NUMBER EXAMINED SKELETALLY

118

117

125

121

22

22

22

22

VERTEBRAL ANOMALY WITH OR WITHOUT ASSOCIATED RIB ANOMALY

0

0

1

0

0

0

1

0

BENT LIMB BONE(S)

0

1

0

0

0

1

0

0

 

 

 

 

 

 

 

 

 

TOTAL NUMBER WITH MALFORMATIONS

 

 

 

 

 

 

 

 

EXTERNAL:

1

1

0

0

1

1

0

0

SOFT TISSUE :

1

0

0

0

1

0

0

0

SKELETAL :

0

1

1

0

0

1

1

0

 

 

 

 

 

 

 

 

 

COMBINED :

2

2

1

0

2

2

1

0

1-  0 MG/KG; 2-  100 MG/KG; 3-  300 MG/KG; 4-  1000 MG/KG

 

TABLE 1.15 SUMMARY OF LITTER PROPORTIONS OF MALFORMATIONS% PER LITTER

 

DAY 21

DOSE GROUP:

 

1

2

3

4

NUMBER OF LITTERS EXAMINED EXTERNALLY

 

22

22

22

22

LOWER JAW- ABSENT OR SMALL

MEAN

0.0

0.6

0.0

0.0

 

S.D.

0.00

2.67

0.00

0.00

LIMB(S)- MALROTATED

MEAN

0.4

0.0

0.0

0.0

 

S.D.

1.94

0.00

0.00

0.00

CLEFT PALATE

MEAN

0.0

0.6

0.0

0.0

 

S.D.

0.00

2.67

0.00

0.00

NUMBER OF LITTERS EXAMINED VISCERALLY

 

22

22

22

22

SITUS INVERSUS

MEAN

0.9

0.0

0.0

0.0

 

S.D.

4.26

0.00

0.00

0.00

NUMBER OF LITTERS EXAMINED SKELETALLY

 

22

22

22

22

VERTEBRAL ANOMALY WITH OR WITHOUT ASSOCIATED RIB ANOMALY

MEAN

0.0

0.0

0.8

0.0

 

S.D.

0.00

0.00

3.55

0.00

BENT LIMB BONE(S)

MEAN

0.0

0.9

0.0

0.0

 

S.D.

0.00

4.26

0.00

0.00

TOTAL MALFORMATIONS

 

 

 

 

 

PERCENT PER LITTER WITH EXTERNAL MALFORMATIONS

MEAN

0.4

0.6

0.0

0.0

 

S.D.

1.94

2.67

0.00

0.00

PERCENT PER LITTER WITH SOFT TISSUE MALFORMATIONS

MEAN

0.9

0.0

0.0

0.0

 

S.D.

4.26

0.00

0.00

0.00

PERCENT PER LITTER WITH SKELETAL MALFORMATIONS

MEAN

0.0

0.9

0.8

0.0

 

S.D.

0.00

4.26

3.55

0.00

TOTAL PERCENT PER LITTER WITH MALFORMATIONS

MEAN

1.3

1.5

0.8

0.0

 

S.D.

4.60

4.92

3.55

0.00

1-  0 MG/KG; 2-  100 MG/KG; 3-  300 MG/KG; 4-  1000 MG/KG

None significantly different from control group

TABLE 1.16 SUMMARY OF FETUSES AND LITTERS WITH VARIATIONS [ABSOLUTE NO.]

DAY 21

 

F E T U S E S

L I T T E R S

DOSE GROUP:

1

2

3

4

1

2

3

4

NUMBER EXAMINED EXTERNALLY

237

235

247

244

22

22

22

22

NUMBER WITH FINDINGS

0

0

0

0

0

0

0

0

NUMBER EXAMINED VISCERALLY

120

118

122

123

22

22

22

22

LIVER- SMALL SUPERNUMERARY LOBE(S)

0

1

4

0

0

1

3

0

NUMBER EXAMINED SKELETALLY

118

117

125

121

22

22

22

22

14TH RUDIMENTARY RIB(S)

58

70

72

64

19

21

20

21

14TH FULL RIB(S)

13

10

7

9

9

6

6

7

PELVIC GIRDLE- CAUDAL SHIFT

8

7

4

7

7

5

3

4

BENT RIB(S)

12

11

11

7

6

9

7

3

STERNEBRA(E) MALALIGNED

4

12

6

7

4

10

5

5

REDUCED OSSIFICATION OF THE SKULL

13

12

10

8

8

7

3

5

METACARPAL(S) AND/OR METATARSAL(S) UNOSSIFIED

2

4

1

2

2

2

1

2

VERTEBRAL CENTRA- REDUCED OSSIFICATION

4

2

0

1

3

2

0

1

7TH CERVICAL OSSIFICATION SITE(S)

7

6

7

7

6

5

4

4

SKULL- SUPERNUMERARY SITE

0

0

0

1

0

0

0

1

7TH CERVICAL FULL RIB(S)

0

0

1

0

0

0

1

0

STERNEBRA(E)- BRANCHED

0

2

0

0

0

2

0

0

STERNEBRA(E) #5 AND/OR #6 UNOSSIFIED

0

1

0

0

0

1

0

0

1-  0 MG/KG; 2-  100 MG/KG; 3-  300 MG/KG; 4-  1000 MG/KG

TABLE 1.17 SUMMARY OF LITTER PROPORTIONS OF VARIATIONS % PER LITTER

 

 

DAY 21

DOSE GROUP:

1

2

3

4

NUMBER OF LITTERS EXAMINED EXTERNALLY

22

22

22

22

NUMBER OF LITTERS WITH FINDINGS

0

0

0

0

1-  0 MG/KG; 2-  100 MG/KG; 3-  300 MG/KG; 4-  1000 MG/KG

None significantly different from control group

Applicant's summary and conclusion

Conclusions:
In this GLP compliant OECD 414 study, time-mated female Wistar Han rats were treated with the test substance from day 6 to 20 post-coitum. Based on the results in this prenatal developmental toxicity study the maternal and developmental No Observed Adverse Effect Level (NOAEL) for the test substance was established as being at least 1000 mg/kg bw/day.
Executive summary:

In this GLP compliant OECD 414 study, the potential of the test substance to induce developmental toxicity after maternal exposure during the critical period of organogenesis was examined and maternal toxicity at the exposure levels tested when given orally by gavage to time-mated female Wistar Han rats from Day 6 to 20 post-coitum, inclusive was characterized. In addition, the No Observed Adverse Effect Levels (NOAELs) for maternal toxicity and developmental toxicity were evaluated. The dose levels in this study were selected to be 0, 100, 300, 1000 mg/kg bw/day, based on the results of the dose range finder. Chemical analyses of formulations were conducted once during the study to assess accuracy and homogeneity. The following parameters and endpoints were evaluated in this study for the F0-generation: mortality/moribundity, clinical signs, body weights, food consumption, gross necropsy findings, number of corpora lutea, (gravid) uterine weight and uterine contents. In addition, the following parameters were determined for the F1-generation:the number of live and dead fetuses, early and late resorptions, total implantations, fetal body weights, sex ratio, external, visceral and skeletal malformations and developmental variations.

Formulation analyses confirmed that formulations of test item in corn oil were prepared accurately and homogenously. No maternal and developmental toxicity was observed in the 100, 300 and 1000 mg/kg bw/day groups.

In conclusion, based on the results in this prenatal developmental toxicity study a maternal and developmental No Observed Adverse Effect Level (NOAEL) for the test substance of at least 1000 mg/kg bw/day was established.