Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
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

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 (incl. certificate)
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
Sex:
female
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.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
peanut oil
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
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
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

Observations and examinations performed and frequency:
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 so
lution 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 micr
ometers 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
Other examinations:
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 (visceralexaminations).
Visceral Examination
A detailed soft tissue examination was performed on the fresh fetuses with even numbers using microdissection 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.

Results and discussion

Results of examinations

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 treatment related changes observed in these animals.

Effect levels

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

Target system / organ toxicity

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
25 mg/kg bw/day (nominal)
System:
immune system
Organ:
thymus
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

Applicant's summary and conclusion