Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental start date (animal arrival) 22 July 2020 Experimental completion date (TSH analysis) 15 September 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report date:
2020

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: • Japanese Ministry of Agriculture, Forestry and Fisheries, Test Data for Registration of Agricultural Chemicals, 12 Nohsan No. 8147, Agricultural Production Bureau, November 24, 2000
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

1
Reference substance name:
NTO
IUPAC Name:
NTO
Test material form:
solid
Details on test material:
Appearance: White to off white (slightly yellow) solid.
Purity: 99.9%
Specific details on test material used for the study:
Test item: Nitrotriazolone.
Test item identity (including alternative names): 1,2-dihydro-5-nitro-3H-1,2,4-triazol-3-one, NTO.
Intended use: Explosive compound (Industrial Chemical).
Appearance: White to off white (slightly yellow) solid.
Storage conditions: At ambient temperature (15 to 25C). Locked and secured from fire.
Supplier: Sponsor
Batch number: 180004
Stability/expiry date: 05 February 2028
Purity: 99.9%
Supplier’s responsibilities: Characterization of the test item and the documentation of the methods of synthesis, fabrication or derivation and stability.
Archive sample: A 1.0 g representative sample was taken from the batch of test item. This sample was placed in a well closed glass container and stored in Pharmacy (Covance, Eye) in a secure metal cabinet under the same conditions as the bulk material.

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
Strain/Species: Crl:CD(SD) rat.
Supplier Charles River (UK) Ltd.

Number of animals ordered 90 females.
Spare animals were removed from the study room after treatment commenced.

Duration of acclimatization Six days before commencement of pairing.

Age of the animals at the start of the study (Day 0 of gestation) Approximately 72 days old.

Weight range of the animals at the start of the study (Day 0 of gestation) 232 to 277 g.

Animal Care and Husbandry
Environmental Control
Animal facility
Limited access- to minimize entry of external biological and chemical agents and to minimize the transference of such agents between rooms.

Air supply Filtered fresh air which was passed to atmosphere and not recirculated.

Temperature and relative humidity Monitored and maintained within the range of 20-24C and 40-70%.

There were no deviations from these ranges.

Lighting Artificial lighting, 12 hours light: 12 hours dark.

Electricity supply Public supply with automatic stand-by generators.

Animal Accommodation
Cages Cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals.

Solid (polycarbonate) bottom cages were used during the acclimatization and gestation periods.

Grid bottomed cages were used during pairing. Cages were suspended above absorbent paper which was changed daily during pairing.

Cage distribution The cages constituting each group were blocked by group and mounted in batteries.

Bedding Solid bottom cages contained softwood based bark-free fiber bedding, which was changed at appropriate intervals each week.

Number of animals per cage
Acclimatization up to four animals
During pairing one (stock) male and one female
Gestation one female

Environmental Enrichment
Aspen chew block A soft white untreated wood block; provided to each cage throughout the study and replaced when necessary.
Plastic shelter Provided to each cage throughout the study (except during pairing) and replaced at the same time as the cages.

Diet Supply
Diet SDS VRF1 Certified pelleted diet.
The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.
Availability Non-restricted.

Water Supply
Supply Potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals.
Availability Non-restricted.

Supplier Certificates of Analysis
Certificates of analysis for the diet are scrutinized and approved before any batch of diet was released for use. Certificates of analysis were routinely provided by the water supplier.
Certificates of analysis were also received from the suppliers of the softwood based bark-free fiber bedding and Aspen chew blocks.
No specific contaminants were known that may have interfered with or prejudiced the outcome of the study and therefore no special assays were performed.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 1% Methylcellulose.
Details on exposure:
Method of preparation
The required amount of test item was transferred to a mortar, wetted with vehicle (0.5 mL vehicle per gram test item), ground using a pestle and mixed with further vehicle to form a paste. (The test item was not dry ground). Further amounts of vehicle were gradually added and mixed to produce a smooth, pourable suspension. The suspension was quantitatively transferred and diluted to volume and finally mixed by magnetic stirring. (A high shear homogenizer was not used for this test item).

A series of suspensions, at the required concentrations were prepared by dilution of individual weighings of the test item.

Frequency of preparation
Weekly.

Storage of formulation
Refrigerated (2 to 8C).

Test item accounting
Detailed records of compound usage were maintained. The amount of test item necessary to prepare the formulations and the amount actually used were determined on each occasion. The difference between these amounts was checked before the formulations were dispensed.

Formulation Analysis
Stability and homogeneity
The homogeneity and stability of formulations during storage were confirmed as part of another study, Covance Study Number JK52QD. In that study, formulations in the range 1 to 200 mg/mL were determined to be stable for:

• One day at ambient temperature (15 to 25°C).
• 15 days when stored refrigerated (2 to 8°C).

Achieved concentration
Samples of each formulation prepared for administration in the first and last weeks of treatment were analyzed for achieved concentration of the test item.

Analysis of the samples taken during the last week of treatment demonstrated that the Group 4 formulation samples (200 mg/mL) were outside the acceptable standard variance limits (-15 to +10% of nominal concentration). A contingency sample was analyzed and confirmed the original results. Group 4 formulations prepared on 07 August 2020 were discarded and re-formulated. Samples were taken from the re-prepared Group 4 formulations on the last formulation occasion.

Administration
Route Oral gavage using a suitably graduated syringe and a semi-ridged cannula inserted via the mouth.

Treated at Constant doses in mg/kg/day.

Volume dose 5 mL/kg body weight.

Individual dose volume Calculated from the most recently recorded scheduled body weight.

Control (Group 1) Vehicle at the same volume dose as treated groups.

Frequency Females were treated from Day 6 to Day 19 (inclusive) after mating, once daily at approximately the same time each day.

Formulation A daily record of the usage of formulation was maintained based on weights. This balance was compared with the expected usage as a check of correct administration. No significant discrepancy was found.

The pH of the formulations were low (acidic). Therefore, following filling of the dose equipment for each animal, the semi-ridged cannula was wiped dry, dipped in tap water, wiped dry and dipped in tap water again, before administration, to prevent any unwanted animal response during the dosing procedure. This procedure was followed from the 4 to 6 August 2020.

From 7 August 2020 (which equated to Day 7, 8 or 9 of gestation depending on the animals allocation date), following filling of the dose equipment for each animal, the semi-ridged cannula was wiped dry, dipped in tap water, wiped dry and dipped in sugar solution before administration, to prevent any unwanted animal response during the dosing procedure.

Following completion of dosing, all remaining dose formulations were retained and returned to pharmacy for storage until disposal.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analytical method involved dissolution of a representative sample in methanol/water (99/1 v/v), followed by dilution in methanol and finally water/formic acid (100/0.1 v/v). The diluted sample was quantified by liquid chromatographic analysis with tandem mass spectrometric detection (LC-MS/MS). Sample concentrations were determined with reference to external calibration standards prepared in the concentration range 5 ng/ml to 25 ng/mL. The accuracy of the analytical procedure on the occasion of analysis was confirmed by preparation and concurrent analysis of procedural recovery samples.

Concentration of Dose Formulations
The formulations for the first and last week of the study and the repeat formulation for Group 4 last week of study, were sampled, Group 1, (2 × 3 mL) and Groups 2, 3, 4, (4 × 1 mL), from the middle of the formulation by Pharmacy personnel. Two aliquots from the first Group 1 sample and two samples from the Group 2, 3, 4 samples were analyzed in accordance with the analytical procedure. The remaining samples were retained for contingency. Samples were disposed of once satisfactory results were achieved.
Details on mating procedure:
3.3.2 Mating
Male/female ratio 1:1 with identified stock males.
Daily checks for evidence of mating Ejected copulation plugs in cage tray and vaginal smears were checked for the presence of sperm.
Day 0 of gestation When positive evidence of mating was detected.
A colony of stud males was maintained specifically for the purpose of mating; these animals were not part of the study and were maintained as stock animals.
Duration of treatment / exposure:
Females were treated from Day 6 to Day 19 (inclusive) after mating, once daily at approximately the same time each day.
Frequency of treatment:
Once per day
Duration of test:
Day 0-6: Mating
Day 6-28: Treatment
Day 29: Necropsy
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Vehicle only
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
20 females
Control animals:
yes, concurrent vehicle
Details on study design:
Animal Model
The rat was chosen as the test species because of the requirement for a rodent species by regulatory agencies. The Crl:CD(SD) strain was used because of the historical control data available at this laboratory.

Route of Administration
The oral gavage route of administration was chosen because it is a common route of exposure used in studies of this type.

Rationale for Dose Level Selection
Dose levels of 100, 300 and 1000 mg/kg/day were selected in conjunction with the Sponsor based on the results from a preliminary embryo-fetal development study conducted at these laboratories (Covance Study No. 8437213).

In the preliminary study, administration of NTO at dose levels of 250, 500 and 1000 mg/kg/day was generally well tolerated. There were no premature deaths, no signs observed in relation to dose administration, no test item-related changes in clinical condition, no adverse effects on body weight or embryo-fetal survival and development and no treatment related macroscopic findings at any dose level investigated. From Day 6 to 10 of gestation food consumption was marginally lower than control at 1000 mg/kg/day, however, food consumption was unaffected for the remainder of the study.

A high dose level of 1000 mg/kg/day was therefore considered suitable for use on this OECD 414 study, with intermediate and low dose levels of 300 and 100 mg/kg/day selected to fulfill the 2-fold to 4-fold dosing interval as specified in the test guideline.

Examinations

Maternal examinations:
Serial Observations
Mortality
A viability check was performed near the start and end of each working day. One female was found dead.

A complete necropsy was performed.

Clinical Observations
Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupant(s). Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate.

During the acclimatization period, observations of the animals and their cages were recorded at least once per day.

Signs Associated with Dosing
Detailed observations were recorded daily during the treatment period at the following times in relation to dose administration:
• Pre-dose observation
• One to two hours after completion of dosing
• As late as possible in the working day.

Clinical Signs
A detailed physical examination was performed on each animal on Days 0, 5, 12, 18 and 20 after mating to monitor general health.

Body Weight
The weight of each adult was recorded on Days 0, 3 and 6-20 after mating.

Food Consumption
The weight of food supplied to each adult, that remaining and an estimate of any spilled was recorded for the periods Days 0-3, 3-6, 6-10, 10-14, 14-18 and 18-20 after mating inclusive.

Thyroid Hormone Analysis
Blood samples were collected at the following occasion:

Occasion Animals
At termination All adults

Sequence of blood sampling on each occasion To minimize any potential confounding effect of the time of day of blood sampling, the time of blood sampling was controlled to allow satisfactory inter-group comparisons.

Conditions No overnight deprivation of food.
Blood sample site Sublingual vein.
Anesthetic Isoflurane.
Anticoagulant None.
Tubes Greiner Minicollect tubes with clotting activator.
Blood volume 1.0 mL.
Treatment of samples Samples were kept at ambient temperature (15 to 25°C) for a minimum of 30 minutes prior to centrifugation.
Centrifugation conditions At 2000g for ten minutes at 4°C.
Number of aliquots Two per animal. Aliquot 1: 0.2 mL serum for T3/T4
Aliquot 2: residual serum for TSH
Final storage conditions Deep frozen (approximately -60°C to -90C) pending analysis.
Fate of samples Aliquot 1 (T3 and T4): dispatched to the Department of Bioanalysis (LC-MS/MS), Covance.
Aliquot 2 (TSH): dispatched to the Department of Immunology & Immunotoxicology, Covance.
T3 and T4 Performed by the Department of Bioanalysis (LC-MS/MS), Covance.


TSH Performed by the Department of Immunology & Immunotoxicology, Covance.
Ovaries and uterine content:
Reproductive Assessment
For females surviving to Day 20 after mating only, the following was recorded:
Uterus Gravid uterine weight (including cervix and ovaries).

The following were recorded for all animals:
For each ovary/uterine horn
Number of: Corpora lutea. Implantation sites. Resorption sites (classified as early or late). Fetuses (live and dead).
Fetal examinations:
Fetal Examination and Processing
Examination of all viable fetuses and placentae
Dissected from the uterus, individually weighed and identified within the litter using a coding system based on their position in the uterus. Fetuses were examined externally with abnormalities recorded. The sex and ano-genital distance of each fetus was recorded.

Examination of nominally 50% of fetuses in each litter
Sexed internally and eviscerated.

Fixation
Fetuses eviscerated were fixed in Industrial Methylated Spirit (IMS).
Remaining fetuses were fixed whole in Bouin’s fluid.

Processing
Bouin’s fixed fetuses were subject to free-hand serial sectioning.
IMS fixed fetuses were processed and stained with Alizarin Red.

Fetal Pathology Examination
Bouin’s fixed fetuses Serial sections were examined for visceral abnormalities.
Alizarin Red stained fetuses Assessed for skeletal development and abnormalities.
Statistics:
Please refer to "Any other comments on materials and methods"
Indices:
Reproductive Assessment
Prenatal losses are separated into pre- and post-implantation phases. Pre-implantation loss was considered to reflect losses due to non-fertilization of ova and failure to implant. It was calculated from the formula:

Pre-implantation loss (%) = ((Number of corpora lutea - Number of implantations) / Number of corpora lutea) x 100

Where the number of implantations exceeded the number of corpora lutea observed, pre implantation loss was assumed to be zero (i.e. no pre-implantation loss was considered to have occurred).

Post-implantation loss was calculated from the formula:

Post-implantation loss (%) = ((Number of implantations - Number of live fetuses) / Number of implantations) x 100

All group values and SD were calculated from the individual litter values.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
At routine physical examination one female (4F No. 68) receiving 1000 mg/kg/day had piloerection and slow breathing on Day 8 of gestation and this was observed during Days 10 to 12 of gestation. This female was also observed as underactive on Days 8 and 10 of gestation. A kinked tail was the only observation recorded for this female from Day 13 of gestation onwards.

One female receiving 1000 mg/kg/day (4F No. 74) had piloerection after dosing on Days 8 and 9 of gestation, there were no other observations associated with dosing in this group.

There were no clinical signs or signs associated with dosing that were related to treatment with Nitrotriazolone in females receiving 100 or 300 mg/kg/day.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One female (4F No. 79) receiving 1000 mg/kg/day was found dead 1 to 2 hours after dosing on Day 10 of gestation. Macroscopic examination of this animal revealed a perforated esophagus, clear fluid was found in the thoracic cavity affecting numerous organs and the animal was pregnant with 14 live embryos. This female had no signs observed related to dosing but was observed as having hair loss on its forelimbs. The findings observed at necropsy were indicative of a dosing trauma and therefore this death was considered not related to treatment.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There was no effect of treatment, at any dose level, on body weight or body weight gain.

The gravid uterine weight and terminal body weight were unaffected by treatment with Nitrotriazolone. Maternal body weight gain when adjusted for the gravid uterine weight was statistically significantly higher for females receiving 300 or 1000 mg/kg/day when compared to controls during Days 6 to 20 of gestation (1.33X and 1.27X control, respectively).
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption was marginally low over Days 6-10 of gestation (start of treatment) and marginally high over Days 14-20 of gestation in females receiving 1000 mg/kg/day when compared with controls. Food consumption was unaffected in females receiving 100 or 300 mg/kg/day Nitrotriazolone.

PLEASE REFER TO ATTACHED TABLE
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
Description (incidence and severity):
The group mean absolute and terminal body weight adjusted thyroids weights in females receiving 100, 300 or 1000 mg/kg/day were similar to control females.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No Nitrotriazolone-related macroscopic findings were found at terminal sacrifice. At macroscopic examination small thyroids were observed for one control female, one female receiving 100 mg/kg/day and one female receiving 1000 mg/kg/day. There were no other macroscopic findings.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Histopathological evaluation was limited to the thyroids, and no Nitrotriazolone-related microscopic findings were found at terminal sacrifice.
Histopathological findings: neoplastic:
not examined
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Thyroid Hormone Analysis
No statistically significant differences in mean serum TSH levels collected at scheduled termination on Day 20 of gestation were observed when comparing dosed groups to the Control animals. Mean female TSH concentrations in groups 2, 3 and 4 (dosed with Nitrotriazolone at 100, 300 and 1000 mg/kg/day, respectively) were similar to the mean Control Group concentration of 1040 pg/mL.

Variation in TSH levels was observed within the groups, including the control. The extent of this variation in Groups 1, 3 and 4 was similar, but was greater in group 2. This was due to animal 36 having a higher individual concentration of TSH than that observed in the other groups: (3960 pg/mL, compared to 2290, 2470 and 2370 from Groups 1, 3 and 4 respectively).

The T3 and T4 concentrations in the pregnant females were comparable with endogenous levels in all treatment groups, and therefore, no effect of treatment was inferred.

Maternal developmental toxicity

Number of abortions:
no effects observed
Description (incidence and severity):
Rats do not abort
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
The number of implantations, the number of resorptions, implantation losses, the number of live male and female pups and the sex ratio of the young were unaffected by treatment with Nitrotriazolone.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
The number of implantations, the number of resorptions, implantation losses, the number of live male and female pups and the sex ratio of the young were unaffected by treatment with Nitrotriazolone.
Early or late resorptions:
no effects observed
Description (incidence and severity):
The number of implantations, the number of resorptions, implantation losses, the number of live male and female pups and the sex ratio of the young were unaffected by treatment with Nitrotriazolone.
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: Oral administration of Nitrotriazolone at dose levels of 100, 300 or 1000 mg/kg/day to pregnant Sprague Dawley rats throughout organogenesis and the fetal growth phases was well tolerated, with no adverse findings at any dose level.

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
Male, female and overall fetal weights in maternal females receiving 1000 mg/kg/day were slightly low attaining statistical significance when compared with controls. Fetal weights for females receiving 100 or 300 mg/kg/day were similar to controls.

Placental and litter weights were unaffected by maternal treatment with Nitrotriazolone.

PLEASE REFER TO ATTACHED TABLE
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
The number of implantations, the number of resorptions, implantation losses, the number of live male and female pups and the sex ratio of the young were unaffected by treatment with Nitrotriazolone.
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):
At 300 and 1000 mg/kg/day there was an increased incidence of short supernumerary 14th rib when compared with the concurrent controls; the fetal and litter incidences at 300 and 1000 mg/kg/day were outside the Historical Control data (HCD) range. Studies have shown that this finding does not persist in the rat (Chernoff et al, 1991) and therefore in isolation the increased incidence in this variant is considered not to be adverse.

In all groups receiving Nitroriazolone, there was an increase in incidence of unossified 5th and/or 6th sternebrae compared to concurrent control. Incomplete ossification is a transient stage in fetal development and is indicative of slight fetal immaturity however all incidences were unrelated to treatment and were within the HCD range and therefore it was concluded that these findings at all dose levels were not adverse.

PLEASE REFER TO ATTACHED TABLE
Visceral malformations:
no effects observed
Description (incidence and severity):
The incidence of major and minor visceral abnormalities showed no relationship to treatment.
Other effects:
no effects observed
Description (incidence and severity):
The ano-genital distance of male and female fetuses on gestation Day 20 was considered to be unaffected by maternal treatment with Nitrotriazolone.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Oral administration of Nitrotriazolone at dose levels of 100, 300 or 1000 mg/kg/day to pregnant Sprague Dawley rats throughout organogenesis and the fetal growth phases was well tolerated, with no adverse findings at any dose level

Fetal abnormalities

Key result
Abnormalities:
effects observed, non-treatment-related
Localisation:
skeletal: sternum
skeletal: rib
Description (incidence and severity):
At 300 and 1000 mg/kg/day there was an increased incidence of short supernumerary 14th rib when compared with the concurrent controls; the fetal and litter incidences at 300 and 1000 mg/kg/day were outside the Historical Control data (HCD) range.

Overall developmental toxicity

Key result
Developmental effects observed:
no
Lowest effective dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Treatment related:
no

Any other information on results incl. tables

Formulation Analysis

Mean recovery results obtained on each analytical occasion during the study were within ±10% of nominal showing the continued accuracy of the method.

The mean concentrations were within – 15%/+10% of nominal concentrations confirming the accuracy of formulation, with the exception of Group 4 prepared for the last week of study that was – 18.5% of nominal. The Group 4 contingency samples were analyzed confirming the original result and therefore this formulation was discarded and a new Group 4 formulation prepared. The Group 4 reformulation was within limits.

Mean serum T3 concentrations(pg/mL)

Group

Treatment

Dose

(mg/kg/day)

 T3 concentrations

Female Dams (pg/mL)

1

Control

(Vehicle)

0

Mean

515

SD

66.1

CV%

12.8

N

20

2

Nitrotriazolone

100

Mean

490

SD

74.0

CV%

15.1

N

20

3

Nitrotriazolone

300

Mean

474

SD

88.1

CV%

18.6

N

20

4

Nitrotriazolone

1000

Mean

484

SD

96.1

CV%

19.9

N

19

No statistically significant difference was identified between the control group and treatment groups using a Williams test.

 

Mean serum T4 concentrations(pg/mL)

Group

Treatment

Dose

(mg/kg/day)

T4 concentrations

Female Dams (pg/mL)

1

Control

(Vehicle)

0

Mean

14800

SD

2830

CV%

19.1

N

20

2

 

Nitrotriazolone

100

Mean

13600

SD

2780

CV%

20.4

N

20

3

Nitrotriazolone

300

Mean

13500

SD

2980

CV%

22.1

N

20

4

Nitrotriazolone

1000

 

Mean

13500

SD

3560

CV%

26.4

N

19

No statistically significant difference was identified between the control group and treatment groups using a Williams test.

 

Applicant's summary and conclusion

Conclusions:
Oral administration of Nitrotriazolone at dose levels of 100, 300 or 1000 mg/kg/day to
pregnant Sprague Dawley rats throughout organogenesis and the fetal growth phases was
well tolerated, with no adverse findings at any dose level. Consequently, the no observed adverse effect level (NOAEL) for maternal toxicity and embryo-fetal survival and development was considered to be 1000 mg/kg/day.

Executive summary:

The purpose of this study was to assessof the influence of Nitrotriazolone (an industrial chemical) on embryo-fetal survival and development when administered during the organogenesis and fetal growth phases of pregnancy in the Sprague Dawley rat.

Three groups of 20 females receivedNitrotriazoloneat doses of 100, 300 or 1000 mg/kg/day by oral gavage administration, from Day 6 to 19 after mating. A similarly constituted Control group received the vehicle, 1% Methylcellulose at the same volume dose as the treated groups. Animals were killed on Day 20 after mating for reproductive assessment and fetal examination.

Clinical observations, body weight and food consumption were recorded. Adult females were examined macroscopically at necropsy on Day 20 after mating, blood samples were taken for thyroid hormone analysis and the gravid uterus weight and thyroid weight were recorded. Microscopic pathology investigations were also undertaken on the thyroids. Ano-genital distance was measured for fetuses and all fetuses were examined macroscopically at necropsy and subsequently by detailed internal visceral examination or skeletal examination.

Results

The mean concentrations were within – 15%/+10% of nominal concentrations confirming the accuracy of formulation, with the exception of Group 4 prepared for the last week of study that was – 18.5% of nominal. The Group 4 contingency samples were analyzed confirming the original result and therefore this formulation was discarded and a new Group 4 formulation prepared. The Group 4 reformulation was within limits.

Maternal responses

There were no premature deaths attributable to treatment.

There was no effect of treatment on maternal clinical condition in females receiving 100 or 300 mg/kg/day where two females receiving 1000 mg/kg/day showed signs of piloerection and one of which also had shallow breathing and was observed as underactive.

 

There was no effect of treatment on body weight, thyroid weight, macropathology or histopathology of the maternal thyroid. Maternal body weight change (adjusted for the gravid uterine) was slightly high in females receiving 300 or 1000 mg/kg/day. In addition, food intake for females receiving 1000 mg/kg/day was slightly low at the start of treatment but increased towards the end of gestation and therefore was considered non-adverse. 

 

The analysis of serum TSH, T3 and T4 concentrations performed at scheduled termination on Day 20 of gestation revealed that serum thyroid hormone concentrations were comparable with endogenous levels at all dose levels when compared with the control.

Litter responses

Embryo-fetal survival, fetal ano-genital distance and development were unaffected by maternal treatment. The fetal weights were slightly low from maternal females treated at 1000 mg/kg/day. There was an increase in incidence of short supernumerary 14thribs in the litters of females receiving 300 or 1000 mg/kg/day and were outside the historical control data range. These findings were considered non-adverse.

Conclusion

Oral administration of Nitrotriazolone at dose levels of 100, 300 or 1000 mg/kg/day to pregnant Sprague Dawley rats throughout organogenesis and the fetal growth phases was well tolerated, with no adverse findings at any dose level. Consequently, the no observed adverse effect level (NOAEL) for maternal toxicity and embryo-fetal survival and development was considered to be 1000 mg/kg/day.