Registration Dossier

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:
2013-11-25 to 2014-05-27
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
Adopted 2001-01-22
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: In-house bred animals
- Age at study initiation: 10-12 weeks
- Weight at study initiation: 195.62 - 198.01 g
- Housing:
i. Pre mating - Two rats of same sex per cage were housed in sterilized standard polypropylene cage (Size: L 430 x B 285 x H 150 mm). Steam sterilized clean paddy husk was provided as bedding material.
ii. Mating - During mating, three animals (one male and two females) were housed in standard polypropylene cages.
iii. Post mating - After confirming presence of sperm in the vaginal smear (Day 0 of pregnancy), the mated pairs were separated. Males were housed with their former cage mates while females were housed individually in polypropylene cages.
- Diet:Nutrilab rodent powder feed (Manufactured by Provimi Animal Nutrition India Pvt. Ltd., with Batch No. 0001149139) was provided ad libitum throughout the experimental period.
- Water Clean and potable drinking water was provided ad libitum throughout the acclimatization and experimental period. Deep bore well water passed through activated charcoal filter and exposed to UV rays in Aquaguard water filter cum purifier was provided
- Acclimation period: 5 to 21 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.1 to 23.7
- Humidity (%): 50 to 68
- Air changes (per hr): 12-15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From:2013-11-20 To: 2013-12-23

Administration / exposure

Route of administration:
oral: feed
Vehicle:
other: Acetone was used as vehicle for formulation preparation
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Test diets of the Test Article in the vehicle were prepared and used for 5 to 7 days with in the stability period. The Test Article was weighed as per the dose, dissolved in acetone and hand mixed with a small amount of feed in stainless steel container for 2 minutes. This premix was then added to appropriate amount of feed to obtain the desired dietary concentration and mixed in a double cone blender for 10 minutes. The test diets were stored in polyethylene bags which were kept in a stainless steel drums in study room at temperature 22 ± 3ºC and used within the stability period. Prior to the study, stability was verified at 10 and 300 ppm concentrations of dietary formulation, using acetone as a vehicle and followed storage for up to 8 days at ambient temperature (24 ± 5ºC).

VEHICLE
- Justification for use and choice of vehicle: The test item DOTC was soluble in Acetone. Acetone is routinely used vehicle in oral (dietary) toxicity studies.
- Lot/batch no.: 80160201D13
- Purity: pure (analytical grade)
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:

Formulation analysis for homogeneity and dose concentration verification was conducted by the Analytical Department of BIONEEDS during first week of treatment period via Gas Chromatography and FID detection. For homogeneity and concentration verification analyses, feed sample was collected in duplicate from top, middle and bottom layers of the container whereas vehicle control feed sample (in duplicate) was collected only from the middle layer of the container.
The collected samples were transferred to the Analytical Department of BIONEEDS for dose formulation analysis. One set of each aliquot of each formulation was analyzed. The second aliquot of each formulation was stored at 25 ± 4ºC. The second set of samples was discarded at the discretion of Study Director and analyst based the confirmation of the results.
Homogeneity was evaluated from the top, middle and bottom layers of the collected test formulations. The mean concentrations from all three sampled layers were used for the dose concentration verification. The acceptance criteria for results of homogeneity test and dose concentration verification was within the acceptance limits of ±20% to the nominal concentrations.
Details on mating procedure:
After minimum five days of acclimatization period, males and females were cohabitated at 1:2 ratio (one male and two females) until evidence of copulation is observed to obtain the required number of pregnant rats for each group or for two weeks. Every morning, the vaginal smear of each female was examined for presence of sperm in the vaginal smear and/or vaginal plug. The day of confirmation of mating was designated as day ‘0’ of gestation. Each day, the body weight of mated rats (day 0 pregnant females) was recorded and arranged in the ascending order of their body weight. These mated females were evenly distributed to all the groups based on their body weights so as to maintain comparable mean body weight for all groups and permanent identification numbers were assigned. Animals were kept for mating in seven batches to regulate the number of animals sacrificed on a particular day. Females not mated within 14 days of pairing with the first male were placed with a second proven male.
After obtaining required number of pregnant females for each group, the extra mated, non-mated females and all males were sacrificed without recording any observations.
Duration of treatment / exposure:
The test item was administered by admixture with the diet to the mated females from gestation day 5 to 19. The control animals were received diet mixed with acetone only.
Frequency of treatment:
daily via food
Doses / concentrationsopen allclose all
Dose / conc.:
0 ppm
Remarks:
Relates to 0 mg/kg bw/day (Quantitiy of test item per 5 kg feed: 0 mg)
Dose / conc.:
10 ppm
Remarks:
Relates to 0.8 mg/kg bw/day (Quantitiy of test item per 5 kg feed: 50 mg)
Dose / conc.:
100 ppm
Remarks:
Relates to 7.2 mg/kg bw/day (Quantitiy of test item per 5 kg feed: 500 mg)
Dose / conc.:
300 ppm
Remarks:
Relates to 22.4 mg/kg bw/day (Quantitiy of test item per 5 kg feed: 1500 mg)
No. of animals per sex per dose:
25 females per dose group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on the available literature, the doses of 0, 10, 100 and 300 ppm in feed for low, mid and high dose were selected by the sponsor.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were observed once daily for clinical signs of toxicity and twice daily for mortality/morbidity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily for clinical signs

BODY WEIGHT: Yes
- Time schedule for examinations:Gestation Days 0, 3, 5, 8, 11, 14, 17, 19 and 20 (day of cesarean section).

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
Individual animal feed intake of mated females was recorded for days 0 to 3, 3 to 5, 5 to 8, 8 to 11, 11 to 14, 14 to 17, 17 to 19 and 19 to 20 of gestation.
- 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: Yes


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day #20
- Organs examined: Uterus of pregnant and non-pregnant animals
Ovaries and uterine content:
On the 20th day of gestation, the uteri of non-pregnant females were immersed in 10% ammonium sulphide and there was no evidence of implantation sites. The weight of the gravid uterus including cervix was recorded for each pregnant female at hysterectomy. The following counts/observations were performed for all pregnant animals.
-No. of corpora lutea
- No. of implantations
- No. of live and dead fetuses
- No. of early and late resorptions
Fetal examinations:
All fetuses were examined for
- Sex, number and weight of live fetuses
- External appearance of live fetuses (including oral cavity)
- External anomalies

Live fetuses were killed by keeping them on cool packs and allocated to either skeletal or visceral examinations, independent of sex. Approximately one-half of live fetuses from each litter were examined for skeletal alterations. The remaining fetuses were examined for soft tissue alterations (visceral examinations).

Visceral Examination
A detailed soft tissue examination was performed on the live fetuses with even numbers using micro dissection technique for body and a free-hand serial sectioning technique (Wilson technique) for head.After examination, the fetuses along with organs were preserved in a solution of glycerine. Observations of visceral abnormalities and variations were recorded.

Skeletal Examination
The live fetuses with odd numbers were skinned and eviscerated, fixed in 95% ethanol, subjected to preparation of Alcian blue staining for cartilage and Alizarin red S staining for bones and the specimens were examined under stereomicroscope for the presence or absence of skeletal malformation or variations. After examination, the fetuses along with were preserved in a solution of glycerine. Fetuses with abnormalities were photographed.
Statistics:
The raw data was subjected to computer statistical processing. The computer printout of the data (in the form of appendix) was verified with the raw data. After verification, the data was subjected to statistical analyses. The litter or litter mean was used as the experimental unit. One-way ANOVA with Dunnett’s post test was performed for the data (body weight, food consumption, number of pregnant corpora lutea, uterus weight, number of implantations, number of live fetuses, body weight and crown-rump length of fetuses) and Kruskal-Wallis test followed by Dunnett’s post test for the percentage of visceral or skeletal malformations (or variations) for each litter, percentage of individual malformation (or variation) for each litter using GraphPad Prism version 5.00, GraphPad Software. All analyses and comparisons were evaluated at the 95% or 99% or 99.9% level of confidence (P<0.05 or P<0.01 or P<0.001), indicated by the aforementioned tests were designated by the superscripts throughout the report as stated below:
* Statistically significant (P<0.05) change than the control group.
** Statistically significant (P<0.01) change than the control group.
*** Statistically significant (P<0.001) change than the control group.
Note: Data of non-pregnant females were not included in mean calculation and statistical analysis.
Indices:
Corrected body weight (g)
Pre-implantation loss (%)
Post-implantation loss (%)
Male/Female Sex Ratio
Male/Female fetuses (%)
Group/Litter Fetal Observation for External, Visceral and Skeletal Examinations:
Fetal incidence (%)


Historical control data:
Availbale on request

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
There were no statistically significant decreases in maternal body weight for dams in the 10 or 100 ppm dose groups on any gestation day (GD) when compared to control dams. There was a statistically significantly decrease in maternal body weight for dams in the 300 ppm group from GD 17 to termination at GD 20.
Maternal body weight change for several periods during gestation was not statistically different than control dams for the 10 and 100 ppm groups. There was a statistically significantly decrease in maternal body weight change among the 300 ppm dams for the periods GD 5-8, GD 11-14, GD 14-17, and GD 17-19.
There was no statistically significantly decrease in body weight change GD 5-20 for dams in the 10 ppm group compared to controls. There was a statistically significantly decrease in body weight change GD 5-20 for dams in the 100 ppm (11.9%) and in 300 ppm (30.8%) groups as compared with controls.
The corrected body weight change and the percent change were similar to controls in the 10 and 100 ppm groups. In the 300 ppm group the corrected body weight change and the percent change were both statistically significantly decreased compared to control. Gravid uterine weight was similar to controls at all doses.
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
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, treatment-related
Description (incidence and severity):
A macroscopic observation of reduced size of the thymus was recorded for 7 of 25 females at 100 ppm and all females (25 of 25) at 300 ppm. These observations were judged to be treatment-related. No gross pathological observations were noted in any 10 ppm animals
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):
Pre-Implantation Loss
No treatment-related pre-implantation loss was noted across all doses when compared to the vehicle control. The statistically significant difference in this parameter for the 300 ppm group was attributed to two dams [Ra6262 and Ra6270] which had pre-implantation losses of 44.4% and 60.4%, respectively. In the 100 ppm group two dams [Ra6248 and Ra6249] were noted with a pre-implantation loss of 45.5% and 62.5% respectively. These occurrences were judged as incidental and not treatment related.

Post-Implantation Loss
No statistically significant difference in the percentage of post-implantation loss was observed for any dose group when compared to vehicle controls.
The observed post-implantation losses were 6.9%, 4.9% and 6.9% in the 10, 100, and 300 ppm groups, respectively versus 0.8% in the vehicle control. This outcome was judged as incidental, due to lack of dose dependency and the absence of an effect on the number of live fetuses in treated groups as compared to vehicle controls.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Description (incidence and severity):
Early Resorptions
The incidence of early resorptions was statistically significantly increased at the mid dose compared to the vehicle control. This was judged to be an incidental occurrence. One dam (Animal No. Ra6232) in this group had 4 early resorptions compared to a maximum of 2 early resorptions in any dam. In addition the incidence of early resorptions did not demonstrate a dose-response.

Late Resorptions
No statistically significant differences in the number of late resorptions per dam were observed across groups. There was no treatment related effect on the occurrence of late resorptions.


Dead fetuses:
no effects observed
Description (incidence and severity):
No dead fetuses were observed in the 100 or 300 ppm groups or in the controls. In the 10 ppm group, 2 dead fetuses were observed in a single litter. This was judged as an incidental finding and not the result of treatment with the Test Article
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
no effects observed

Effect levels (maternal animals)

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
0.8 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
other: decreased thymus size
Dose descriptor:
LOAEL
Effect level:
7.2 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
other: decreased thymus weight

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
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
Changes in postnatal survival:
no effects observed
External malformations:
no effects observed
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
Malformations:
During skeletal examination of fetuses, the malformations were noted:

- Control group: 1 incidence in 132 examined fetuses, which relates to 1 of 22 litters affected.
- 10 ppm group: 11 incidences in 115 examined fetuses, which relates to 8 of 21 litters affected.
- 100 ppm group 22 incidences in 105 examined fetuses, which relates to 11 of 20 litters affected.
- 300 ppm group: 47 incidences of 107 examined fetuses, which relates to 19 of 20 litters affected.

A statistically significant and treatment-related increase in the percentage of malformations was observed. These were described as:
- Missing metacarpal No. 5 bilateral (11.4% at 100 ppm; 34.6% at 300 ppm; versus 0.8% in controls).
- Missing proximal phalanx No. 3 bilateral (14.3% at 100 ppm; 28.0% at 300 ppm; versus 0.8% in controls).
- Missing proximal phalanx No. 4 bilateral (13.3% at 100 ppm; 27.1% at 300 ppm; versus 0.8% in controls).
- Missing of metacarpal No. 5 on both sides was noted in a single litter (one fetus) in control, 3 litters (3 fetuses) at 10 ppm, 6 litters (12 fetuses) at 100 ppm and 18 litters (37 fetuses) at 300 ppm.
- Missing of proximal phalanx No. 3 was noted in single litter (one fetus) in control, 7 litters (9 fetuses) at 10 ppm, 10 litters (15 fetuses) at 100 ppm and 17 litters (30 fetuses) at 300 ppm.
- Missing of proximal phalanx No. 4 was noted in single litter (one fetus) in control, 6 litters (8 fetuses) at 10 ppm, 9 litters (15 fetuses) at 100 ppm and 16 litters (29 fetuses) in high dose.
- Split thoracic vertebra centrum No. 12 was noted as a singular occurrence in a single litter at 10 ppm. This observation was judged to be incidental occurrence.
- Missing caudal vertebral arch No. 2 on both sides was noted in 2 litters (2 fetuses) at 10 ppm and 2 litters (3 fetuses) at 300 ppm. This observation was judged to be incidental occurrence.

Variations:
During skeletal examination of fetuses, the Variations were noted:
- Control group: 6 incidences in 132 examined fetuses (5 of 22 litters affected).
- 10 ppm group: 11 of 115 examined fetuses (7 of 21 litters affected).
- 100 ppm group: 10 of 105 examined fetuses (4 of 20 litters affected).
- 300 ppm group: 26 of 107 examined fetuses (12 of 20 litters affected).
A statistically significant and treatment-related increase in the percentage of poor ossification of sternum No.5 and No.6 (6.5% and 14.0% as compared with 0% in controls) was noted in the 300 ppm group.
Poor or incomplete ossification of sternum No.5 was noted in a single litter (one fetus) at 10 ppm, 4 litters (7 fetuses) in high dose. Poor or incomplete ossification of sternum No.6 was noted in single litter (2 fetuses) at 100 ppm, 8 litters (16 fetuses) in the 300 ppm group.
A dose dependent and treatment related increase in poor ossification of metacarpal No. 5 was observed at 100 ppm (1.0%) and at 300 ppm (3.7%).
Poor or incomplete ossification of metacarpal No. 5 was noted in a single litter (one fetus) at 100 ppm, 3 litters (4 fetuses) in high dose.

The following occasional findings occurred across groups treated with the Test Article and vehicle control. These observations showed no dose dependency and therefore considered not to be treatment related:
- 10 ppm group:
Poor or incomplete ossification of skull bones (parietal, inter-parietal and supra-occipital bones) was noted in a single litter (one fetus) in control, 3 litters (4 fetuses).
Dumbbell shaped thoracic vertebrae centrum No. 10 and bipartite thoracic vertebrae centrum No. 13 in a single litter (two fetuses) and dumbbell shaped thoracic vertebrae centrum No. 11 in single litter (one fetus).
Poor or incomplete ossification of lumbar vertebrae arch No. 4 on left side was noted in a single litter (two fetuses).

Moreover, poor or incomplete ossification of proximal phalanx No. 3 was noted in 3 litters (4 fetuses) in control, 2 litters (2 fetuses) at 10 ppm, 4 litters (8 fetuses) at 100 ppm and 3 litters (3 fetuses) in high dose.

Poor or incomplete ossification of proximal phalanx No. 4 was noted in 3 litters (4 fetuses) in control, 2 litters (2 fetuses) at 10 ppm, 3 litters (7 fetuses) at 100 ppm and 3 litters (3 fetuses) in high dose.
Poor ossification of caudal vertebrae arch No. 2 was noted in a single litter (one fetus) in control and 1 litter (1fetus) at 10 ppm.


Visceral malformations:
no effects observed
Description (incidence and severity):
Malformations:
During visceral examination of fetuses, the following malformations were noted:
Control group: 0 incidences in 119 examined fetuses, which relates to 0 of 22 litters affected.
10 ppm group:3 incidences in 105 examined fetuses, which relates to 2 of 21 litters affected.
100 ppm group: 1 incidence in 97 examined fetuses, which relates to 1 of 20 litters affected.
300 ppm group: 1 incidence in 95 examined fetuses, which relates to 1 of 20 litters affected.

Lateral ventricular dilation of the 3rd ventricle of the brain was the malformation noted in each case. Since these occurrences did not show any dose dependency they were judged as incidental occurrences.

Variations:
During visceral examination of fetuses, the following variations were noted:
Control group: 0 incidences in 119 examined fetuses, which relates to 0 of 22 litters affected.
10 ppm group: 3 incidences in 105 examined fetuses, which relates to 2 of 21 litters affected.
100 ppm group: 1 incidence in 97 examined fetuses, which relates to 1 of 20 litters affected.
300 ppm group: 1 incidence in 95 examined fetuses, which relates to 1 of 20 litters affected.
Two variations were noted: abnormal liver lobation and renal pelvic dilation. The incidences of abnormal liver lobation were 0, 1, 0, 0, and 0 in the control, 10, 100, and 300 ppm groups, respectively. The incidences of renal pelvic dilation were 0, 2, 1, and 1 in the control, 10, 100, and 300 ppm groups, respectively. The noted observations, abnormal liver lobation and dilation of the renal pelvis, are common findings for rat fetuses and were judged as incidental occurrences
Other effects:
no effects observed

Effect levels (fetuses)

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
0.8 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
skeletal malformations
Dose descriptor:
LOAEL
Effect level:
7.2 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
skeletal malformations

Overall developmental toxicity

Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
7.2 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
yes
Relevant for humans:
not specified

Any other information on results incl. tables

TABLE 1.          SUMMARY OF THE STUDY

Parameters

Group

G1

G2

G3

G4

Dose (ppm)

0

10

100

300

Pregnancy Data

Initial animals per group

25

25

25

25

Confirmed pregnancy at necropsy

22

21

20

20

Pregnancy rate (%)

88

84

80

80

Maternal Data

Initial body weight (g)/gestation day 0

Mean

195.62

197.88

197.79

198.01

SD

±12.45

±11.99

±9.62

±9.52

Final body weight (g)/gestation day 20

Mean

305.34

300.90

296.62

278.54

SD

±18.98

±18.42

±18.08

±25.85

Body weight change (g) during GD 0 - 20

Mean

96.56

86.54

79.06

64.43

SD

±38.49

±40.15

±42.35

±40.15

Correct Body weight (g)

Mean

23.94

26.57

19.47

5.85

SD

±15.48

±10.57

±11.98

±18.22

Uterine Observation

Gravid uterus weight (g)

Mean

69.96

62.23

63.26

59.10

SD

±15.06

±14.46

±16.20

±19.67

Corpora lutea (no.)

Mean

11.7

11.2

11.4

11.6

SD

±2.1

±1.9

±1.8

±2.5

Total implantation per female (no.)

Mean

11.5

11.1

10.7

10.7

SD

±2.1

±1.9

±2.6

±3.3

Live fetuses (no.)

Mean

11.4

10.1

10.1

10.1

SD

±2.2

±2.8

±2.7

±3.8

Dead fetuses (no.)

Mean

0.0

0.1

0.0

0.0

SD

±0.0

±0.4

±0.0

±0.0

Early resorptions (no.)

Mean

0.0

0.4

0.6*

0.4

SD

±0.2

±0.6

±1.1

±0.6

Late resorptions (no.)

Mean

0.0

0.1

0.0

0.2

SD

±0.2

±0.4

±0.0

±0.5

Pre-implantation loss (%)

Mean

1.5

0.8

7.0

10.4*

SD

±3.3

±2.4

±16.8

±17.1

Post-implantation loss (%)

Mean

0.8

6.9

4.9

6.9

SD

±3.6

±10.2

±10.0

±13.8

TABLE 1 (Contd..,). SUMMARYOF THE STUDY

Parameters

Group

G1

G2

G3

G4

Dose (ppm)

0

10

100

300

Litter Data

 

 

 

 

 

Litter size (no.)

Mean

11.4

10.2

10.1

10.1

SD

±2.2

±2.8

±2.7

±3.8

Total live fetuses (no.)

Mean

11.4

10.1

10.1

10.1

SD

±2.2

±2.8

±2.7

±3.8

Live male fetuses (no.)

Mean

5.1

4.7

4.7

5.4

SD

±1.6

±1.7

±1.8

±2.6

Live female fetuses (no.)

Mean

6.3

5.8

5.5

4.8*

SD

±2.0

±1.8

±2.3

±2.1

Male/female sex ratio (no.)

Mean

0.9

0.9

1.2

1.3

SD

±0.4

±0.5

±1.2

±0.7

Average fetal weight (g)

Mean

4.0

4.0

4.2

4.0

SD

±0.3

±0.5

±0.2

±0.2

Average male fetal weight (g)

Mean

4.2

4.1

4.3

4.1

SD

±0.3

±0.5

±0.2

±0.3

Average female fetal weight (g)

Mean

3.9

3.9

4.1

3.9

 SD

±0.3

±0.5

±0.3

±0.3

Fetal Data

 

 

 

 

 

Total No. of live fetuses

 

251

220

202

202

Total No. offetuses examined for Visceral examination

 

119

105

97

95

Total No. offetuses examined for Skeletal examination

 

132

115

105

107

Total fetuses available for gross external evaluation (no.)

Mean

11.4

10.5

10.1

10.1

SD

±2.2

±2.2

±2.7

±3.8

Fetuses available for visceral examination (no.)

Mean

5.4

5.0

4.8

4.8

SD

±1.2

±1.2

±1.4

±1.8

Fetuses available for skeletal examination (no.)

Mean

6.0

5.5

5.3

5.4

SD

±1.0

±1.1

±1.3

±2.0

TABLE 1 (Contd..,). SUMMARY OF THE STUDY

Parameters

Group

G1

G2

G3

G4

Dose (ppm)

0

10

100

300

External Examination

 

 

 

 

 

Malformations

No. of Fetuses

0

0

0

0

%

0.0

0.0

0.0

0.0

Variations

No. of Fetuses

0

0

0

0

%

0.0

0.0

0.0

0.0

Visceral Examination

 

 

 

 

 

Malformations

No. of Fetuses

0

3

1

1

%

0.0

2.9

1.0

1.1

Variations

No. of Fetuses

0

3

1

1       

%

0.0

2.9

1.0

1.1

Skeletal Examination

 

 

 

 

 

Malformations

No. of Fetuses

1

11

22**

47***

%

0.8

9.6

21.0

43.9

Variations

No. of Fetuses

6

11

10

26*

%

4.5

9.6

9.5

24.3

* Statistically significant (P < 0.05) change than the control group

** Statistically significant (P<0.01) change than the control group

*** Statistically significant (P<0.001) change than the control group


TABLE 2.             SUMMARY OF VISCERAL EXAMINATION OF FETUSES (MALFORMATIONS)

Refer Appendix - 13

Parameters

Group

G1

G2

G3

G4

Dose (ppm)

0

10

100

300

Number of Dams

22

21

20

20

Number of fetuses for visceral examination

119

105

97

95

 

 

 

 

 

 

BRAIN

 

 

 

 

 

Lateral ventricular dilation

No.

0

3

1

1

%

0

2.9

1.0

1.1

 


TABLE 3.             SUMMARY OF VISCERAL EXAMINATION OF FETUSES (VARIATIONS)

                                                                                                                                      Refer Appendix - 13

Parameters

Group

G1

G2

G3

G4

Dose (ppm)

0

10

100

300

Number of Dams

22

21

20

20

Number of fetuses for visceral examination

119

105

97

95

 

 

 

 

 

 

LIVER

 

 

 

 

 

Abnormal lobation

No.

0

1

0

0

%

0

1.0

0

0

 

 

 

 

 

 

KIDNEYS 

 

 

 

 

 

Renal pelvis dilation

No.

0

2

1

1

%

0

1.9

1.0

1.1


 

TABLE 4.             SUMMARY OF SKELETAL EXAMINATION OF FETUSES (MALFORMATIONS)

Refer Appendix - 14

Parameters

Group

G1

G2

G3

G4

Dose (ppm)

0

10

100

300

Number of Dams

22

21

20

20

Number of fetuses for skeletal examination

132

115

105

107

 

 

 

 

 

 

THORACIC VERTEBRAE

 

 

 

 

 

Centrum No. 12 - Split

No.

0

1

0

0

%

0

0.9

0

0

FORE LIMB

 

 

 

 

 

Metacarpal No. 5 - Absent

No.

1

3

12

37

%

0.8

2.6

11.4

34.6***

Proximal Phalanx No. 3 - Absent

No.

1

9

15

30

%

0.8

7.8

14.3*

28.0***

Proximal Phalanx No. 4 - Absent

No.

1

8

15

29

%

0.8

7.0

14.3*

27.1***

CAUDAL VERTEBRAE

 

 

 

 

 

Arch No.2 (bilateral) - Absent

No.

0

2

0

3

%

0

1.7

0

2.8

* Statistically significant (P < 0.05) change than the control group

*** Statistically significant (P<0.001) change than the control group

 


TABLE 5.             SUMMARY OF SKELETAL EXAMINATION OF FETUSES (VARIATIONS)

                                                                                                                                   Refer Appendix - 14


Parameters

Group

G1

G2

G3

G4

Dose (ppm)

0

10

100

300

Number of Dams

22

21

20

20

Number of fetuses for skeletal examination

132

115

105

107

 

 

 

 

 

 

SKULL

 

 

 

 

 

Parietal Bones - Poor / Incomplete ossification

No.

1

4

0

0

%

0.8

3.5

0

0

Interparietal Bones - Poor / Incomplete ossification

No.

1

4

0

0

%

0.8

3.5

0

0

Supraoccipital Bones - Poor / Incomplete ossification

No.

1

4

0

0

%

0.8

3.5

0

0

STERNUM

 

 

 

 

 

Sternum No. 5 - Poor / Incomplete ossification

No.

0

1

0

7

%

0

0.9

0

6.5*

Sternum No. 6 - Poor / Incomplete ossification

No.

0

1

2

16

%

0

0.9

1.9

15.0***

THORACIC VERTIBRAE

 

 

 

 

 

Centrum No. 10 - Dumbbell shaped

No.

0

1

0

0

%

0

0.9

0

0

Centrum No. 11- Dumbbell shaped

No.

0

1

0

0

%

0

0.9

0

0

Centrum No. 13 - Bipartite

No.

0

1

0

0

%

0

0.9

0

0

LUMBAR VERTEBRAE

 

 

 

 

 

Arch No. 4 (Left side) - Poor ossification

No.

0

2

0

0

%

0

1.7

0

0

 * Statistically significant (P < 0.05) change than the control group

 *** Statistically significant (P<0.001) change than the control group


 


 

Applicant's summary and conclusion

Conclusions:
The NOAEL and the LOAEL for maternal toxicity to the dams and was determined to be 10 ppm (0.8 mg/kg bw/day) and 100 ppm (7.2 mg/kg bw/day), respectively based on a statistically significant decrease in maternal body weight and reduced thymus size.
The NOAEL and the LOAEL for developmental toxicity was also determined to be 10 ppm (0.8 mg/kg bw/day) and 100 ppm (7.2 mg/kg bw/day), respectively, based on a statistically significant and treatment-related increase in the percentage of skeletal malformations associated with delayed fetal ossification. As the observed skeletal malformation associated with delayed fetal ossification was only noted at maternally toxic doses DOTC is regarded as not teratogenic in the rat but as fetotoxic at a maternally toxic doses.
Executive summary:

A prenatal developmental toxicity study with the registered substance Dioctyltin Dichloride (DOTC) was conducted in accordance with the OECD Guideline 414 and GLP. The purpose of this study was to assess the effects of prenatal exposure of Test Article (DOTC) on the pregnant females and in the developing organisms and assessment of maternal toxicity as well as death, structural abnormalities, or altered growth in the fetuses when administered by a mixture with the diet to the mated females from gestation day 5 to 19. A total of 100 mated female Sprague Dawley rats were allocated to four groups. Each group consisted of 25 mated female Sprague Dawley rats (Dams). DOTC was administered by a mixture with the diet (using insignificant amounts of acetone as suitable vehicle) at dose levels of: Vehicle control: 0 ppm, Low dose group 10 ppm (0.8 mg/kg bw/day), Mid dose group: 100 ppm (7.2 mg/kg bw/day), High dose group: 300 ppm (22.4 mg/kg bw/day). Both the Test Article and vehicle control were administered by a mixture with the diet. All the animals were sacrificed on gestation day 20 by carbon dioxide exposure and subjected to detailed gross pathology. The gravid uterus was collected by hysterectomy and fetuses were removed by caesarean section. Examination of dams and fetuses was performed and the following results were obtained.

PREGNANCY DATA

A total number of 22, 21, 20 and 20 mated females were confirmed with pregnancy at a pregnancy rate of 88%, 84%, 80% and 80% at the time of caesarean section at 0, 10, 100 and 300 ppm respectively.

MATERNAL DATA

General Tolerability

No deaths and abortions were noted during the experimental period.

Body weight, Body Weight Change and Corrected body weight

There was a statistically significant decrease in maternal body weight for the period of gestation days (GD) 17-20 and maternal body weight change for the periods GD 5-8, GD 11-14, GD 14-17 and GD 17-19 for dams in the 300 ppm group was noted. There were no statistically significant decreases in maternal body weight and maternal body weight change for dams in the 10 or 100 ppm dose groups on any gestation day when compared to control dams.

No statistically significant decrease in body weight change GD 5-20 for dams in the 10 ppm and 100 ppm groups compared to controls was noted. A statistically significant decrease in body weight change GD 5-20 for dams in the 100 ppm (11.9%) and in 300 ppm (30.8%) groups as compared with controls was noted.

The corrected body weight change and the percent change were similar to controls in the 10 and 100 ppm groups. In the 300 ppm group the corrected body weight change and the percent change (75.6%) were both statistically significantly decreased compared to controls. Gravid uterine weight was similar to controls at all doses.

Feed Consumption and Test Article Consumption

There were no treatment related differences in average feed consumption at any of the tested dose.

Test Article consumption for each dose was calculated as 0.8, 7.2 and 22.4 mg/kg/day for the low, mid and high dose groups, respectively.

Gross Pathology

Macroscopic observations of reduced size of thymus in 7 of 25 females at 100 ppm and in all females (25 of 25) at 300 ppm were observed. These observations were judged to be treatment-related. No gross pathological observations were noted in 10 ppm animals.

Maternal Data (Uterine Observations)

No treatment related differences in mean gravid uterus weight, number of corpora lutea per dam, number of implantation sites per dam, incidence of early and late resorptions, number of dead and live fetuses, pre and post-implantation losses and male/female sex ratio were noted at all the doses.

The occurrence of early resorptions at 100 ppm, late resorptions at 10 and 300 ppm, pre-implantation loss at 300 ppm and post-implantation loss at 10, 100 and 300 ppm were judged as incidental and not treatment related.

FETAL DATA

Fetal Sex Ratio, Average Fetal Weight and Average Crown Rump Length

No treatment related effects on the fetal sex ratio, average fetal weight and average crown-rump length were noted at any of the dose.

External Examination

No gross external abnormalities were noted within any group after external examination of fetuses.

Visceral Examination

No treatment related malformations and variations were observed at all doses. The noted observations, abnormal liver lobation and dilation of the renal pelvis, are common findings for rat fetuses and were judged as incidental occurrences. 

Skeletal Examination

Statistically significant and treatment related increase in percentage of malformations of missing metacarpal No. 5 (11.4% at 100 ppm and 34.6% at 300 ppm as compared with 0.8% in control), proximal phalanx No. 3 bilateral (14.3% at 100 ppm and 28.0% at 300 ppm as compared with 0.8% in control) and proximal phalanx No. 4 (13.3% at 100 ppm and 27.1% at 300 ppm as compared with 0.8% in control) were noted.

Statistically significant and treatment related increase in percentage variations of poor ossification of sternum No. 5 and No. 6 (6.5% and 14.0% as compared with 0% in control) was noted at 300 ppm. A dose dependent and treatment related increase in poor ossification of metacarpal No. 5 was observed at 100 ppm (1.0%) and at 300 ppm (3.7%) as compared with 0% in control.

CONCLUSION

The results of the experiment support the conclusion that the NOAEL (No Observed Adverse Effect Level) of DOTC for the maternal toxicity endpoint was 10 ppm (0.8 mg/kg bw/day). The LOAEL (low observed adverse effect level) for the maternal toxicity endpoint was 100 ppm (7.2 mg/kg bw/day) based on a statistically significant decrease in corrected body weight change from gestation day 5-20 and a decrease in thymus size at 100 ppm. A statistically significant decrease in maternal body weight, maternal body weight gain, corrected maternal body weight change from gestation day 5-20, and corrected maternal weight occurred at the 300 ppm dose.

The results of the experiment support the conclusion that the NOAEL of the Test Article DOTC for the developmental toxicity endpoint was 10 ppm (0.8 mg/kg bw/day). The LOAEL (low observed adverse effect level) was 100 ppm (7.2 mg/kg bw/day)

The results of the experiment also support the conclusion that the NOAEL (No Observed Adverse Effect Level) and LOAEL (low observed adverse effect level) of the Test Article DOTC for the maternal and developmental toxicity endpoints are the same. Hence, DOTC is regarded as not teratogenic in the rat but is fetotoxic at a maternally toxic dose.