Registration Dossier

Administrative data

Endpoint:
reproductive toxicity, other
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24 June 2016 - 10 December 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose:
reference to same study
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24 June 2016 - 10 December 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Specific details on test material used for the study:
DBTE > 95 %
Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
In-house bred animals (healthy, yound adult animals)
4 Groups 25 pregnant females per group

110 female and 55 males were received. Males were used for cohabitation with females. After cohabitation, all males, extra mated and non mated females were euthanized under CO2 anesthesia.

Age at initiation of mating: 10 to 12 weeks

Animal Identification: Acclimatisation period: Cage cards and tail marking by marker pen. Treatment period: cage cards and body marking by tumeric solution.

Animals were housed under standard laboratory conditions, air-conditioned with adequate fresh air supply (12-15 Air changes per hour), room temperature 19.8 to 22.6 oC and relative humidity 49 to 68%, with 12 hours fluorescent light and 12 hours dark cycle. The temperature and relative humidity were recorded once daily.
Route of administration:
oral: gavage
Vehicle:
peanut oil
Details on exposure:
Test Item formulation was prepared daily before administration. The required quantity of test item was weighted into a clean beaker and there by adding little volume of peanut oil (vehicle) in to the beaker and was mixed well with glass rod and transferred into a measuring cylinder. The beaker was rinsed with peanut oil and the volume was transferred to measuring cylinder. This procedure was repeated until to ensure entire quantity of test item formulation was transferred into measuring cylinder. Finally the volume was made up to required quantity with peanut oil to get a desired concentration of different dose levels
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Formulation analysis for dose concentration verification was was performed for all dose formulations during week 1 and week 4 of dosing period. The dose formulation samples were collected in duplicates (2 x 5 ml ) for each dose formulation including vehicle control and transferred at ambient conditions for dose confirmation analysis at Auriga Research ltd, Unit-III, No 136, 6th Cross, 2nd stage, Yeshwanthpur industrial suburb, Bangalore-560022.
The samples are analyzed for tin content by ICP-OESand the results found to be within the acceptance range of +/- 10 % of the nominal conjcentration.
Results in Appendix 14 of the report
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:
GD 5 to 19
Frequency of treatment:
daily
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
G1 Vehicle control
Dose / conc.:
2.5 mg/kg bw/day (nominal)
Remarks:
G2 Low Dose
Dose / conc.:
8.5 mg/kg bw/day (nominal)
Remarks:
G3 Mid Dose
Dose / conc.:
25 mg/kg bw/day (nominal)
Remarks:
G4 High Dose
No. of animals per sex per dose:
25 mated females
Control animals:
yes, concurrent vehicle
Details on study design:

Treatment Group Description Dose (mg/kg bw) Concentration (mg/ml) No of preg. fem.

Vehicle G1 Control 0 0 25

DBTE G2 Low Dose 2.5 0.5 25

DBTE G3 Mid Dose 8.5 1.7 25

DBTE G4 High Dose 25.0 5.0 25

Maternal examinations:
Clinical Signs of Toxicity and Mortality/Morbidity
All animals were observed once daily for clinical signs of toxicity and twice daily for mortality/morbidity.

Body Weight
Individual animal body weight was weighed on Gestation Days (GD) 0, 3, and daily from DG 5 to 20 (day of caesarian section)

Feed Consumption
Individual animal feed intake of mated females was recorded for days 0 to 3, 3 to 5 and daily from day 5 to 20 of gestation.

Necropsy
All surviving animals were anaesthetized by exposing to CO2 and subjected to detailed necropsy on the day (GD 20) of cesarean section. The Thymus gland of each rat was weighed and recorded. The Thymus gland, ovary and uterus were collected and preserved in 10% neutral buffered formalin solution for microscopic examination.

Histopathology
Histopathological examination was conducted on thymus from the vehicle control and high dose group animals euthanized at termination.

Thymus samples were processed, embedded in paraffin, sectioned at a thickness of 3 to 5 micrometers and stained with hematoxylin and eosin.

The investigations were extended to the lower dose groups as there were treatment related effects at the ghigh dose level (thymus) were encountered.
Ovaries and uterine content:

Uteri Observations
On the 20th day of gestation, fetuses were taken out by cesarean section and females were subjected to macroscopic examination. 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
• Crown-rump length

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 fresh fetuses with even numbers using micro dissection technique (Staples 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 fresh 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 (variations).
After examination, the fetuses were preserved in a solution of glycerine.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
There was a treatment related reduction in thymus size observed in the high dose animals at necropsy. Total 14/25 females were noted as having a reduction in the size of thymus in gross examination
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
The treatment related histopathological change of decreased cellularity in the thymic cortex was reported for 15/25 females of the high dose group. The observed decreased cell population in the cortex was described as multifocal to diffuse and severity varied from minimal to marked. The histopathological evaluation of the thymus was extended to the lower dose groups and there was no treatment related changes observed in these animals.
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
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
Key result
Dose descriptor:
NOAEL
Effect level:
>= 8.5 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
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
External malformations:
no effects observed
Skeletal malformations:
effects observed, non-treatment-related
Visceral malformations:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
> 25 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: The NOAEL of the test item for develomental toxicity was set to > 25 mg/kg bw, the high dose, because there were no adverse effects on fetal developmentals or incidences for external, soft tissueu or skeletal anomalies.
Key result
Developmental effects observed:
no

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: OECD TG 414
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
DBTE > 95 %

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
In-house bred animals (healthy, yound adult animals)
4 Groups 25 pregnant females per group
110 female and 55 males were received. Males were used for cohabitation with females. After cohab
itation, all males, extra mated and non mated females were euthanized under CO2 anesthesia.
Age at initiation of mating: 10 to 12 weeks
Animal Identification: Acclimatisation period: Cage cards and tail marking by marker pen. Treatment
period: cage cards and body marking by tumeric solution.
Animals were housed under standard laboratory conditions, air-conditioned with adequate fresh air su
pply (12-15 Air changes per hour), room temperature 19.8 to 22.6 oC and relative humidity 49 to 68%
, with 12 hours fluorescent light and 12 hours dark cycle. The temperature and relative humidity were
recorded once daily.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
peanut oil
Details on exposure:
Test Item formulation was prepared daily before administration. The required quantity of test item was
weighted into a clean beaker and there by adding little volume of peanut oil (vehicle) in to the beaker
and was mixed well with glass rod and transferred into a measuring cylinder. The beaker was rinsed
with peanut oil and the volume was transferred to measuring cylinder. This procedure was repeated
until to ensure entire quantity of test item formulation was transferred into measuring cylinder. Fin
ally the volume was made up to required quantity with peanut oil to get a desired concentration of
different dose levels
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 ofpregnant rats for each group or for two weeks. Every morning, the vaginal smear of each female wasexamined 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 matedfemales were evenly distributed to all the groups based on their body weights so as to maintaincomparable 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 provenmale.
After obtaining required number of pregnant females for each group, the extra mated, non-matedfemales and all males were sacrificed without recording any observations
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Formulation analysis for dose concentration verification was was performed for all dose formulations during week 1 and week 4 of dosing period. The dose formulation samples were collected in duplicates (2 x 5 ml ) for each dose formulation including vehicle control and transferred at ambient conditions for dose confirmation analysis at Auriga Research ltd, Unit-III, No 136, 6th Cross, 2nd stage,Yeshwanthpur industrial suburb, Bangalore-560022.

The samples are analyzed for tin content by ICP-OES and the results found to be within the acceptance range of +/- 10 % of the nominal conjcentration.

Results in Appendix 14 of the report
Duration of treatment / exposure:
GD 5 to 19
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
G1 Vehicle Control
Dose / conc.:
2.5 mg/kg bw/day (nominal)
Remarks:
G2 Low Dose
Dose / conc.:
8.5 mg/kg bw/day (nominal)
Remarks:
G3 Mid Dose
Dose / conc.:
25 mg/kg bw/day (nominal)
Remarks:
G4 High Dose
No. of animals per sex per dose:
25 mated females
Control animals:
yes, concurrent vehicle
Details on study design:
Treatment Group Description Dose (mg/kg bw) Concentration (mg/ml) No of preg. fem.
Vehicle G1 Control 0 0 25
DBTE G2 Low Dose 2.5 0.5 25
DBTE G3 Mid Dose 8.5 1.7 25
DBTE G4 High Dose 25.0 5.0 25

Examinations

Parental animals: Observations and examinations:

Clinical Signs of Toxicity and Mortality/Morbidity
All animals were observed once daily for clinical signs of toxicity and twice daily for mortality/morbidity.
Body Weight
Individual animal body weight was weighed on Gestation Days (GD) 0, 3, and daily from DG 5 to 20 (day of caesarian section)
Feed Consumption
Individual animal feed intake of mated females was recorded for days 0 to 3, 3 to 5 and daily from day5 to 20 of gestation.
Postmortem examinations (parental animals):
Necropsy
All surviving animals were anaesthetized by exposing to CO2 and subjected to detailed necropsy on
the day (GD 20) of cesarean section. The Thymus gland of each rat was weighed and recorded. The
Thymus gland, ovary and uterus were collected and preserved in 10% neutral buffered formalin so
lution for microscopic examination.
Reproductive indices:
Uteri Observations
On the 20th day of gestation, fetuses were taken out by cesarean section and females were subjecte
d to macroscopic examination. 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
Offspring viability indices:
• Sex, number and weight of live fetuses
• External appearance of live fetuses (including oral cavity)
• External anomalies
• Crown-rump length
Live fetuses were killed by keeping them on cool packs and allocated to either skeletal or visceral exa
minations, independent of sex. Approximately one-half of live fetuses from each litter were examine
d 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 fresh fetuses with even numbers using micro
dissection technique (Staples 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. Obser
vations of visceral abnormalities and variations were recorded.
Skeletal Examination
The fresh 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 specim
ens were examined under stereomicroscope for the presence or absence of skeletal malformation
(variations).
After examination, the fetuses were preserved in a solution of glycerine.

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
There was a treatment related reduction in thymus size observed in the high dose animals at necropsy. Total 14/25 females were noted as having a reduction in the size of thymus in gross examination
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
There was a treatment related reduction in thymus size observed in the high dose animals at necropsy. Total 14/25 females were noted as having a reduction in the size of thymus in gross examination
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
The treatment related histopathological change of decreased cellularity in the thymic cortex was reported for 15/25 females of the high dose group. The observed decreased cell population in the cortex was described as multifocal to diffuse and severity varied from minimal to marked. The histopathological evaluation of the thymus was extended to the lower dose groups and there was no treatmentrelated changes observed in these animals.

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
> 8.5 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic

Results: F1 generation

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
> 25 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: The NOAEL of the test item for develomental toxicity was set to > 25 mg/kg bw, the high dose, because there were no adverse effects on fetal developmentals or incidences for external, soft tissueu or skeletal anomalies

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Applicant's summary and conclusion