1,2-dihydro-5-nitro-3H-1,2,4-triazol-3-one

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

In a OECD 422 Screening for reproduction/developmental toxicity study doses of 31.25, 125, and 500 mg/kg bw/day NTO in corn oil were administered to Sprague-Dawley rats for four weeks.

Although the dose of 500 mg/kg bw/day resulted in testes degeneration, reduced sperm counts with no motile sperm observed, this dose did not affect reproduction or development. Therefore the NOAELs for reproduction and developmental toxicity are 125 and 500 mg/kg/day, respectively.

In a OECD 443 Extended one generation reproductive study, rats were given ad libitum access to NTO in drinking water at four concentrations (0, 144, 720 and 3600 mg/L NTO) for two (females) to four (males) weeks pre-mating and continued until weaning of litters. Direct dosing of F1 animals occurred from weaning through puberty. NTO did not affect measures of fertility including, mating index, pre-coital interval, gestation index, litter size, number of live and stillborn pups, and sex ratio. In the parent generation, NTO had an effect on the male reproductive system at 3600 mg/L(eq. 157 mg/kg/day) as previously seen in the subchronic and the OECD 422 study. The lowest BMDL10 value for the P generation (2335 mg/L eq. to 140 mg/kg bw/day) is based on the effect on epididymal mass.

Link to relevant study records

Reference

Reference 1

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

7 May 2012 to 2 July 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

- Analytical purity: 99.48%
- Lot No.: BAE 11L375-061
- Batch No.: 10NTO11-5

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

Born in-house

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: born in-house from a stock of timed-pregnant animals obtained from a Charles River Laboratories
- Age at study initiation: 8 weeks
- Weight at study initiation: Males: 339.8 ± 21.78 g; Females: 215.5 ± 12.82 g
- Housing: housed individually in suspended polycarbonate boxes except during the mating period when the animals were housed on elevated wire racks in the polycarbonate boxes to allow for the observation of sperm plugs.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 4 weeks
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.6 ± 0.4°C
- Humidity (%):51.0 ± 8.71%
- Photoperiod (hrs dark / hrs light): yes
IN-LIFE DATES: 7 May 2012 to 2 July 2012

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Dosing solutions/suspensions were prepared in volumes sufficient for approximately two-three weeks of dosing, resulting in preparation of two sets of dosing solutions. A third batch of 100 mg/ml dosing suspension was also required due to the additional volume needed for dosing of the recovery male animals. For each dosing solution/suspension, the calculated amount of NTO was weighed and placed in a ceramic mortar. The NTO was then wetted with a measured amount of corn oil and ground with a mortar and pestle to a fine consistency. The slurry was transferred to a volumetric flask and the mortar was rinsed with a measured amount of corn oil to remove any remaining slurry. The remaining corn oil was then added to the suspension to achieve the calculated concentration.

VEHICLE
- Concentration in vehicle: 6.25, 25 and 100mg/ml
- Amount of vehicle (if gavage): 5ml dosing solution per kg bw

Details on mating procedure:

- M/F ratio per cage:1/1
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- The female rats remained pair­ housed with the same male rat until a sperm or vaginal plug was observed or a period of two weeks elapsed.
- After successful mating each pregnant female was caged individually in suspended polycarbonate boxes

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

A separate dosing solution/suspension was prepared for each dose group at targeted concentrations of 6.25, 25, and 100 milligrams/milliliter (mg/ml).
A one milliliter (ml) sample was taken from each dosing solution/suspension prepared for the study and analyzed using an HPLC with ultraviolet detection to verify the concentration. In addition, the homogeneity of the solutions/suspensions was verified by determining the concentration of samples taken from the top, middle, and bottom of the highest concentration (100 mg/ml) suspension. Samples were collected from a representative suspension (6 mg/ml) at weekly intervals for an eight-week period to determine the stability of NTO in corn oil.

Duration of treatment / exposure:

Male rats were dosed for a total of four weeks encompassing the two-week pre-mating period and the two-week mating period.
Female rats were dosed throughout the study including the two-week pre-mating period, the variable time to conception, the duration of pregnancy, and up to and including postpartum day four.
In addition to the main study animals, male rats were used as a satellite group dosed concurrently with main study animals seven days/week for a period of four weeks and were then held, but not dosed, for an additional four-week period prior to necropsy.

Frequency of treatment:

Animals were dosed daily (7 days/week)

Dose / conc.:

31.25 mg/kg bw/day (nominal)

Remarks:

Main study

Dose / conc.:

125 mg/kg bw/day (nominal)

Remarks:

Main study

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

Main study

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

Satellite animals

No. of animals per sex per dose:

10 for main study
20 males for satellite study

Control animals:

yes

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose selection was based on the findings obtained from previously-performed 14- and 90-day oral toxicity studies with the expectation of producing reproductive effects at the highest dose.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Observations for mortality and signs of toxic effects were made twice daily, once in the morning following dosing and once in the afternoon, except on weekends when observations were only performed in the morning.
- Cage side observations checked included, but were not limited to, evaluation of the skin and fur, eyes and mucous membranes, respiratory and circulatory effects, autonomic effects (e.g., salivation), central nervous system effects (e.g., tremors and convulsions), changes in the level of activity, gait, and posture, reactivity to handling or sensory stimuli, altered strength, and stereotypes or changes in behavior (e.g., self­ mutilation). Pregnant females approaching gestation day 21 were observed more frequently to allow for an accurate determination of gestation duration.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
A thorough physical examination of each animal, including the male satellite group, was performed each day concurrently with the dosing procedure. Neurobehavioral observations were omitted from this study due to the negative results obtained during the performance of a subchronic oral toxicity study on NTO previously performed by this Institute (USAPHC (Provisional), 2010).

BODY WEIGHT: Yes
- Time schedule for examinations:
Male and female rats were weighed several times during the acclimation period, on the first day of dosing, and weekly thereafter. During pregnancy, female rats were weighed on gestational days 0, 7, 14, 20, within 24 hours of parturition, and on day 4 postpartum. Female rats showlng no evidence of copulation resumed their normal weekly weigh schedule following the 2-week mating period. Weekly body mass was obtained from satellite male animals during both the exposure and recovery periods. Terminal body mass was obtained the morning of necropsy following overnight fasting for all animals. Litters were weighed within 24 hours of parturition (day 0 or 1 postpartum) and on day 4 postpartum.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Feeder bins were reweighed on the same days body mass was obtained. Grams of food consumption for each period were calculated by subtracting the mass of the empty feeder from the mass of the full feeder. Food consumption was not monitored during the mating period due to pair-housing.

Sperm parameters (parental animals):

Parameters examined in male parental generations:
testis weight, epididymis weight, sperm count in epididymides, sperm motility, sperm morphology
Cauda epididymal sperm counts were determined on all male rats using a computer assisted sperm analyzer (TOX IVOS-CASA). The number of sperm, number of motile sperm, and number of progressive sperm were determined in duplicate for each animal.

Litter observations:

PARAMETERS EXAMINED
The following parameters were examined in offspring:
Each litter was examined within 24 hours of parturition to establish the number and sex of live pups, litter mass, stillbirths, runts, and the presence of gross abnormalities. Litters were again examined on postpartum day 4 to establish the number and sex of live pups, litter mass, bizarre behavior, and the presence of gross external abnormalities.

Postmortem examinations (parental animals):

SACRIFICE
- All surviving males were euthanized following four weeks of treatment (including satellite group)
- All surviving females were euthanized on postpartum day 5

- Female rats that did not become pregnant were euthanized 24-25 days following the last day of the mating period.

GROSS NECROPSY
- A macroscopic examination was conducted on all terminal animals and animals that died during study, noting all lesions and abnormal observations. Tissues that were not grossly autolytic were submitted for histopathological evaluation.

HISTOPATHOLOGY / ORGAN WEIGHTS
- The following organs and tissues, or representative samples, were preserved in a suitable medium (in 10% buffered formalin, Testes and epididymides were preserved in modified Davidson's fixative for a period not exceeding 24 hours and were then transferred to 70% ethyl alcohol. Tissues were routinely processed and paraffin embedded. ): all gross lesions; brain (including sections of medulla/pons, cerebellar cortex and cerebral cortex}; pituitary; thyroid/parathyroid; thymus; lungs and trachea; pharynx; larynx; nose; heart; femur bone marrow; salivary glands; liver; spleen; kidney; adrenals; pancreas; testes; uterus; aorta; esophagus; stomach; duodenum; jejunum;ileum; caecum; colon; rectum; urinary bladder; representative lymph node; peripheral nerve; sternum with bone marrow; mammary gland; thigh musculature; eyes; femur (including articular surface}; spinal cord at three levels (cervical, midthoracic, and lumbar} and exorbital lachrymal glands. In addition, the following organs were removed, trimmed in a uniform manner, and weighed: liver; kidneys; adrenals; gonads; spleen; brain; epididymides, uterus; thymus, and heart.

Postmortem examinations (offspring):

SACRIFICE
- Litters were examined and euthanized on post-natal day 4.

Statistics:

Analyses conducted for males and females separately, SPSS 16.0 used to perform all analyses. For one-time measurement variables in adult animals (hematology, clinical chemistry, organ to body/brain mass ratios, sperm counts, litter/pup parameters), the dose groups compared using a one-factor analysis of variance (ANOVA). If the dose group effect significant, a Tukey post hoc test used to compare pairs of dose groups. The Levene's test was used to determine variance of groups. The Tukey post hoc test used because group variances were equal. Data was checked for normality by plotting residuals. If data was not normal it was natural log transformed. if transformation still did not satisfy ANOVA assumptions, a non-parametric Kruskal-Wallis (K-W) test was used to analyze dose group differences.

For absolute organ mass, comparison of the dose groups was made using analysis of covariance (ANCOVA), with body mass at the end of the study as the covariate. Even though the dose groups were assigned at Day 0 to keep the average starting mass for each dose group similar, the average mass could have changed during the study dependent on the dose group. ANCOVA adjusted for any differences in terminal body mass among the dose groups because heavier animals would tend to have heavier organs. If dose group effect was significant a Sidak post hoc test was used to compare pairs of dose groups and dose groups to the control group.

Repeated-measure variables for adult animals (body mass and food consumption) compared using repeated measures ANOVA. If dose effect in the ANOVA was significant, a Tukey post hoc test was used to compare pairs of dose groups. If interaction effect of week and dose group was statistically significant, weekly means were compared but overall dose group means were not because results would have been inconclusive. Verification of normally distributed data (residual plots) and equal variances among dose groups (Levene's test) assumptions performed.

Reproductive indices:

Length of Gestation (Days), Implants/Female, % Postimplantation Loss, Live Pups/Litter, Stillborn/Litter

Offspring viability indices:

Dead Pups, PND 1-4, Sex Ratio(% Male), Birth Mass

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Bright yellow-colored urine at dosages of 125 mg/kg bw/day and above

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One male rat in the 31.25 mg/kg bw/day dose group was found dead in its cage on day 18 of the study.This pre-term mortality was not considered test material related since the animal exhibited no clinical signs of toxicity prior to death.

Body weight and weight changes:

effects observed, non-treatment-related

Food efficiency:

not specified

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Clinical biochemistry findings:

effects observed, non-treatment-related

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

NTO-related lesions were limited to the reproductive system in male rats. Severe tubular degeneration and atrophy was observed in the testes of 10/10 male rats given 500 mg/kg bw/day NTO. The majority of the testicular seminiferous tubules in these animals were shrunken, retaining only Sertoli cells, spermatogonla, and early stage spermatocytes (leptotene and zygotene). Few tubules retained pachytene spermatocytes and most were degenerate. One male control animal was noted to have a severely degenerative/atrophied left testis however the finding was considered to be a random congenital underdeveloped testis and not related to the study. Moderate hypospermia was observed in the epididymides of 3/10 500 mg/kg bw/day males while severe hypospermia/aspermia was observed in 7/10 500 mg/kg bw/day males. Moderate hypospermia was defined as absence of mature spermatids in the head and body of the epididymis with mature spermatids evident in the tail section. In animals with severe hypospermia/aspermia, mature spermatids were not observed in any epididymal segment. Minimal to mild cribiform change of the epididymis, defined as an infolding and bridging of the epithelium in segments of ducts that have undergone contraction due to decreased/absent sperm, was observed in 10/10 500 mg/kg bw/day male rats. Severe hypospermia/aspermia as well as cribiform change of the epididymis was also observed in the one male control animal with an underdeveloped testis.
Following a 4-week recovery period, complete recovery from observed testicular and epidldymal lesions was not evident in male rats given 500 mg/kg bw/day NTO for 4 weeks. For recovery males, incidence and severity of testicular tubular degeneration/atrophy was reported separately for the left and right testes. Incidence and severity of tubular degeneration/atrophy for 500 mg/kg bw/day recovery males was as follows: 4/10 left testes and 4/8 right testes with mild degeneration, 4/10 left testes and 3/8 right testes with moderate degeneration, and 2/10 left testes and 1/8 right testes with severe degeneration. Two of the right testes in the 500 mg/kg bw/day recovery group were not available for microscopic evaluation. All spermatogonia and spermatocytes were present through all stages for recovery males. Mild degeneration was defined as variable mature 13-18 and 19 spermatids missing while moderate degeneration indicated that spermatids 1-11/12 were generally present with some 7-10 spermatid loss and 13-18 variably present. No mature 19 spermatids were present with moderate degeneration. Severe degeneration was defined as stages I-V completely intact , stages VII-VIII missing mature 19 spermatids, and stages IX-XIV missing spermatids 9-14 with more completely atrophic tubules. Moderate hypospermia was observed in the epididymides of 7/9 500 mg/kg bw/day recovery males with 2/9 exhibiting severe hypospermfa/aspermia.

Histopathological findings: neoplastic:

no effects observed

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

effects observed, treatment-related

Description (incidence and severity):

The number of sperm per gram in the cauda epididymis in male rats in the 500 mg/kg bw/day group was reduced to 6.8% of the number of sperm per gram found in the controls (p=0.00). No motile sperm were found in any of the animals in the 500 mg/kg bw/day group. Average numbers of sperm per gram were only reduced to 91.7 and 87.2% of control averages in the 31.25 and 125 mg/kg bw/day dose groups, respectively. Following the 4-week recovery period, the number of sperm per gram in the cauda epididymis of male rats in the recovery 500 mg/kg bw/day group was reduced to 28.6% of the number of sperm per gram found in the recovery controls (p=0.00). No motile sperm were found in any of the animals in the recovery 500 mg/kg bw/day group

Reproductive performance:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

125 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

reproductive function (sperm measures)

Critical effects observed:

yes

Lowest effective dose / conc.:

500 mg/kg bw/day (nominal)

System:

male reproductive system

Organ:

seminiferous tubules

testes

Treatment related:

yes

Dose response relationship:

yes

Clinical signs:

no effects observed

Mortality / viability:

mortality observed, non-treatment-related

Body weight and weight changes:

no effects observed

Organ weight findings including organ / body weight ratios:

not examined

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

500 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

mortality

body weight and weight gain

Reproductive effects observed:

yes

Lowest effective dose / conc.:

500 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to other toxic effects:

not specified

Dose response relationship:

yes

Relevant for humans:

not specified

Analytical results

The analytical chemistry results are summarized in Tables below. The results of the 7-week stability study indicated that the NTO concentration in corn oil remained within acceptable ranges. Weekly recovery percentages ranged from 100-107% throughout the sampling period.

Homogeneity testing of the most concentrated NTO/corn oil suspension (100 mg/ml) yielded 92% recovery at the top and 89% recovery at the middle and bottom of the container. Verification of the dosing solution/suspension concentrations prior to use yielded recovery percentages ranging from 85-102% of the nominal concentrations for all batches mixed. Given the concentrated nature of these mixtures and the acceptable limits of the analytical laboratory control samples for this method, these analytical results were considered acceptable. All of the dosage levels are reported using the nominal concentrations.

Table Stability and Homogeneity Results

Nominal Concentration(mg/ml)	AnalyticalConcentration(mg/ml)
6 (day 0 stability)	6.3
6 (day 7 stability)	6.1
6(day 14 stability)	6.0
6 (day 21 stability)	6.3
6(day 28 stability)	6.3
6 (day 35 stability)	6.3
6 (day 42 stability)	6.4
6(day 49 stability)	6.3
100 (homogeneity top)	92
100 (homogeneity middle)	89
100 (homogeneity bottom)	89

Table Dosing solution/suspension concentration Resuts

NominalConcentration (mg/ml)	AnalyticalConcentration(mg/ml)
	Batch 1	Batch2	Batch 3
6.25	6.4	5.9, 5.7, 5.7 (repeats)
25	23	22, 22, 23(repeats)
100	96	87, 85, 90(repeats)	86

Reproductive/Developmental Parameters

No discernible differences were recognized among the dose groups, including controls, for the reproductive endpoints expressed as proportions. These endpoints included number of females showing evidence of copulation, number of females achieving pregnancy, number of dams with live young born, and number of dams with live young at postpartum day 4. NTO-treated animals in this study did not exhibit a reduction in pregnancy rates and were within historical averages.

Dose group averages for the number of days pair-housed prior to finding evidence of copulation, gestational length, pre-implantation loss, pre-natal loss, and post-natal loss were analysed using a non-parametric Kruskal-Wallis test and were not statistically different. Statistical analysis of the number of live pups at birth and at postpartum day 4, the pup sex ratio at birth and at postpartum day 4, the average litter weight at birth and at postpartum day 4, and the number of pup abnormalities (including stillbirths) at birth did not reveal any differences among dose group averages. A summary of the litter parameters which had historical control data are presented in Table below

Table Summary of Litter parameters

Mean ± S.D.	Corn Oil Control	31.25 mg/kg bw/day	125 mg/kg bw/day	500 mg/kg bw/day	Historical Controls *
Length of Gestation (Days)	22.0 ± 0.00	22.1 ± 0.35	22.0 ± 0.47	22.0 ± 0.00	22.42 ± 0.53
# Implants/Female	15.3 ± 3.01	15.6 ± 2.60	15.5 ± 2.07	15.3 ± 2.25	15.51 ± 1.85
% Postimplantation Loss	14.8 ± 16 .88	12.7 ± 8.82	12.5 ± 16 .19	8.1 ± 7.89	8.13 ± 3. 35
# Live Pups/Litter	12.8 ± 3.28	13.4 ± 2.35	13.4 ± 2.95	13.4 ± 2.13	14.14 ± 1.42
# Stillborn/Litter	1.8 ± 2.71	0.2 ± 0.44	0.4 ± 0.70	0.4 ± 0.74	0.24 ± 0.26
Dead Pups, PND 1-4	3.3 ± 6.04	0.3 ± 0.71	3.2 ± 6.23	2.4 ± 5.13	0.41 ± 0.39+
Sex Ratio(% Male)	45.3 ± 12.16	54.8 ± 13.80	54.4 ± 13.52	50.5 ± 15.32	49.68 ± 3.90
Birth weight	5.9 ± 0.65	6.4 ± 0.61	6.2 ± 0.70	6.1 ± 0.89	6.33 ± 0.29

* Charles River Laboratories, 2006

+ Historical controls reported as dead pups, PND 1 -21

# Includes pups humanely euthanized due to total neglect by dam

PND: Post-Natal Day

Conclusions:

In a OECD 422 Screening for reproduction/developmental toxicity study doses of 31.25, 125, and 500 mg/kg bw/day NTO in corn oil were administered to Sprague-Dawley rats for four weeks.
Although the dose of 500 mg/kg bw/day resulted in testes degeneration, reduced sperm counts with no motile sperm observed, this dose did not affect reproduction or development.Therefore the NOAELs for reproduction and developmental toxicity are 125 and 500 mg/kg/day, respectively.

Executive summary:

Daily oral exposure to male and female rats at dosages of 31.25, 125, and 500 mg/kg bw/day NTO in corn oil for four weeks did not induce compound-related pre-term mortality. Clinical signs of toxicity were mainly limited to bright yellow-colored urine at higher dosages with no changes in body mass, body mass gain, and food consumption compared to controls observed throughout the study period.

Treatment with NTO resulted in reductions in testes and epididymides mass and mass ratios in male rats given 500 mg/kg bw/day. Microscopic evaluation of these tissues revealed severe degeneration/atrophy of the testicular seminiferous tubules along with moderate to severe hypospermia and cribiform change of the epididymides. Sperm counts were reduced in the high dose group resulted in reductions in testes  Complete recovery was not evident in the high dose satellite group following a 4-week recovery period. Reductions in sperm counts with no motile sperm were also observed in the male satellite group.

However, under the stated study conditions, oral dosages of up to and including 500 mg/kg bw/day NTO did not appear to affect reproduction or development in Sprague-Dawley rats. Gross external examinations of the offspring on theday of birth and on day 4 postpartum did not indicate that NTO presents a developmental hazard.

Therefore the NOAELs for reproduction and developmental toxicity are 125 and 500 mg/kg/day, respectively.

The authors of the report discussed the rat testes tubular degeneration/atrophy and male rat fertility and pointed out that although the spermatogenic cycle repeats approximately every 12.9 days in the Sprague-Dawley rat, the complete process of spermatogenesis requires approximately 56 days or 4.5 cycles (Creasy, 1997). Since the premating treatment duration was only 2 weeks, this could explain why no reduction was observed in the number of females becoming pregnant between NTO-treated and control animals.

Reference 2

Endpoint:

extended one-generation reproductive toxicity - with developmental immunotoxicity (Cohorts 1A, 1B without extension, and 3)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

4 February 2013 - 19 April 2016 (in life 28 October 2013 - 11 August 2014)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)

GLP compliance:

yes

Limit test:

no

Justification for study design:

SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS
- Premating exposure duration for parental (P0) animals: 4 weeks for males and 2 weeks for females
- Basis for dose level selection:In subacute and subchronic toxicity studies with NTO, the limit dose (1000 mg/kg-day) produced minimal systemic toxicity. Testicular toxicity was the primary effect, occurring at doses as low as 315 mg/kg-day in the 90-day study and 500 mg/kg-day in the 14-day study. Reproductive toxicity was not, however, observed at 500 mg/kg-day in the reproductive toxicity and developmental toxicity screening study. Reduced sperm counts were observed in the reproductive screen after four weeks of dosing at 500 mg/kg-day. As such, this study was conducted with the same nominal high dose of 500 mg/kg-day, but with the pre-mating dosing period extended to four weeks to induce testicular toxicity prior to mating. Subsequent dose groups were set at five-fold intervals (i.e., 100 and 20 mg/kg-day). To determine approximately equivalent doses via drinking water, a default water intake of 0.037 L/day and a default body weight of 0.267 kg were used to arrive at a rate of 0.139 L/kg-day in young adult male rats. This resulted in a drinking water concentration of 3597 mg/l. The selected doses were therefore 3600, 720, 144, and 0 mg/l.
- Inclusion/exclusion of developmental neurotoxicity Cohorts 2A and 2B: no signs of developmental neurotoxicity in the reproductive toxicity and developmental toxicity screening study.
- Inclusion/exclusion of extension of Cohort 1B: no reproductive toxicity observed in P1
- Inclusion/exclusion of developmental immunotoxicity Cohort 3: A potential sensitive endpoint is the development of the nascent immune system in animals that have been exposed in utero and as pups to NTO in drinking water.
- Route of administration: drinking water

Specific details on test material used for the study:

- Source:BAE systems, Inc
- lot/batch No.of test material: 11C305-009
- purity:100%

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

(Crl:CD(SD))

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Wilmington, MA
- Females (if applicable) nulliparous and non-pregnant: [yes/no]
- Age at study initiation: (P) 10 wks for the female rats and 8 weeks for the male rats; (F1) x wks
- Weight at study initiation: (P) Males: 290.5 ± 10.6 g; Females: 240.7 ± 9.0 g; (F1) Males: x-x g; Females: x-x g
- Fasting period before study: no
- Housing: suspended polycarbonate cages with Diamond Dry® bedding.
- Diet: ad libitum
- Water: Control animals were provided filtered tap water ad libitum whereas treated rats were provided solutions of NTO in filtered tap water ad libitum.
- Acclimation period: 5 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-22.2 C
- Humidity (%): 30-70%
- Photoperiod (hrs dark / hrs light): 12/12
IN-LIFE DATES: From: 28 October 2013 To: 24 February 2014

Route of administration:

oral: drinking water

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

Dosing solutions were prepared by weighing the required amount of NTO, transferring to a 4L volumetric flask, adding approximately 3.5L of water from the animal room, stirring using a magnetic stir bar and stir plate until dissolved, and adding water to 4L. Three drinking water dosing solutions, 144, 720, and 3600 milligrams NTO per liter (mg/l) of water, were used throughout the study. Solutions were prepared as needed according to the consumption rate of the rats. Drinking water/dosing reservoirs were refilled as needed and reservoirs were changed and solutions replaced completely every 2 weeks.
NTO was previously determined to be stable in water for at least three weeks (Houpt et al, 2010); therefore a stability study was not conducted.

Houpt, J.T. and M. Hable, Determination of the Water Solubility, Octanol-Water Partition
Coefficient and Biodegradation Potential of 3-Nitro-1,2,4-Triazol-5-One (NTO). 2010, U.S. Army
Public Health Command: Aberdeen Proving Ground, MD.

Details on mating procedure:

Each P female was co-housed in a solid bottom cage with a wire bottom insert with a single, randomly selected, unrelated male from the same dose group (1:1 pairing). Cages were examined for the presence of sperm plugs each morning during the co-housing period. Animals were paired
until a sperm plug was found or 2 weeks elapsed, whichever occurred first. The day on which a sperm plug was found was defined as GD 0. For each pairing date of pairing, date of mating, and number of sperm plugs observed were recorded.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

A one milliliter sample was taken from each batch of dosing solution prepared and analyzed by Army Institute of Public Health (AIPH) Laboratory Sciences Portfolio via high performance liquid chromatography with ultra violet detection to verify the concentration.

Duration of treatment / exposure:

Administration of NTO via drinking water was continued for both males and females during pregnancy and lactation until termination of the P generation after weaning of the litters (i.e., total of 10 and 12 weeks of treatment for females and males, respectively). Males in the recovery groups (control and high dose) were dosed until termination of the P generation, at which time treatment was stopped and they began receiving untreated (control) water for 10 weeks.
F1 males and females were given NTO in drinking water beginning at weaning (post-natal day (PND) 22±1). NTO in drinking water provided to the P females was also available to nursing/weanling pups during the weaning period; therefore, direct dosing of the F1 generation likely began prior to PND 22±1. Selected F1 offspring were maintained on the NTO treatment through puberty (PND 42±1 and 53±1 for females and males, respectively).

Frequency of treatment:

daily

Dose / conc.:

0 mg/L drinking water

Dose / conc.:

144 mg/L drinking water

Dose / conc.:

720 mg/L drinking water

Dose / conc.:

3 600 mg/L drinking water

No. of animals per sex per dose:

Main study: 25 animals/sex/dose
Satellite (recovery): 10 males for control and 10 males for high dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on subacute and subchronic studies and the dose level at which testicular toxicity occurs (ca 315-500 mg/kg bw/day.
As such, this study was conducted with the same nominal high dose of 500 mg/kg-day, but with the pre-mating dosing period extended to four weeks to induce testicular toxicity prior to mating. Subsequent dose groups were set at five-fold intervals (i.e., 100 and 20 mg/kg-day). To determine approximately equivalent doses via drinking water, a default water intake of 0.037 L/day and a default body weight of 0.267 kg were used to arrive at a rate of 0.139 L/kg-day in young adult male rats. This resulted in a drinking water concentration of 3597 mg/l.

Parental animals: Observations and examinations:

All animals were observed twice daily for signs of toxicity, morbidity, and mortality. All animals were given handheld physical examinations at least once per week. In addition, P females were carefully examined at the time of expected parturition for signs of dystocia, while dams were
observed for abnormalities in nesting behavior, nursing, or failure to care for litters.

P animals were weighed on the first day of dosing, weekly thereafter, and at termination (pre-fasted and fasted). During pregnancy, female rats were weighed on the day on which a sperm plug was found (gestational day (GD) 0), every two days thereafter, and on GD 21.
During lactation, females were weighed on the same days as pups in their litters (i.e., PND 0 or 1, 4, 7, 14, and 21).

Food consumption was monitored weekly during pre-mating, pregnancy, and lactation. Food and water consumption were not monitored during the 2-week co-housing period. Food consumption was monitored weekly for all recovery . Frequency of water consumption monitoring varied based on consumption rate. Water consumption was determined every 3-4 days in P1 males, 2-3 days in P1 females.

Clinical Pathology and Thyroid Hormone Assessment
Fasted blood samples were taken from P1 at scheduled necropsy and subjected to clinical chemistry, hematology, and thyroid hormone analyses.

Sperm parameters (parental animals):

Cauda epididymal sperm counts were determined using a computer assisted sperm analyzer (TOX IVOS-CASA; Hamilton-Thorne Research, Beverly, Massachusetts). The cauda was weighed. The number of sperm, number of motile sperm, and number of progressive sperm was determined in duplicate for each animal.

Litter observations:

Litters were examined as soon after delivery as possible to determine the number of stillbirths, live births, runts, and the presence of gross abnormalities in each litter. The number of live and dead pups and the sex and body weight of each pup were determined on PND 0/1, 4, 7, 14, and 21. On PND 4, litter size was standardized to 10. Culled pups were selected randomly within sex and, where possible, litters were culled to five males and five females.
At weaning (PND 21±1), 20 litters per dose group and control group were selected for further use. One male and one female per litter per dose group were randomly selected (20/sex/group) for continuation on treatment. Obvious runts (animals with a body weight more than two standard deviations below the mean pup weight of the respective litter) were not included. Additionally, one PND 22 weanling/sex/litter (10/sex/group) was randomly selected for necropsy, thyroid hormone analyses, and measurement of organ weights.

Reproductive Development: Endocrine Sensitive Endpoints
The ano-genital distance (AGD) of each pup was measured PND 4 using digital calipers and was analyzed as absolute AGD and relative to the cube root of body weight. Male pups were examined for the presence of nipples/areolae on PND 13. All selected F1 animals were evaluated, at approximately the same time daily, for vaginal opening (VO) beginning on PND 22 or for completion of preputial separation PPS beginning on PND 30. Age and body weight were recorded on the day these markers of puberty were observed.

Clinical Pathology and Thyroid Hormone Assessment
Fasted blood samples were taken from PND 22 weanlings (thyroid hormones only), and F1 animals (10 randomly selected/sex/group) at scheduled necropsy and subjected to clinical chemistry, hematology, and thyroid hormone analyses.

Thymic and Splenic Lymphocyte Subpopulation Analysis
At necropsy of the F1 generation, a portion of the spleen and ½ the thymus from selected animals (10 randomly selected per sex dose group) were

Postmortem examinations (parental animals):

A complete necropsy was performed on all P1. A complete necropsy was performed on three animals that died prior to scheduled termination; one male from the 3600 mg/l group found dead on study day 4, one female control found dead on PPD 0, and one female from the 3600 mg/l group found dead on PND 14. No tissues were collected from the male or the control female due to autolysis.
Adrenals, brain, heart, kidneys, epididymides, liver, ovaries (without oviducts), prostate, seminal vesicles with coagulating glands (weighed with and without fluid), spleen, testes, thymus, and thyroid (trimmed and weighed post-fixation) were collected, weighed, and preserved for all groups.

All tissues were preserved in10% buffered formalin except the testes and epididymides which were placed in Davidson’s fixative no longer than 24 hours. After fixation, all tissues were rinsed and stored in 70% ethanol.

Preserved tissues from the high dose and control groups from each generation were prepared. Tissues from lower dose groups were examined if exposure-related effects were seen in the high-dose group, gross lesions were present, or other signs of organ toxicity were noted in
the dose group (e.g., changes in organ mass). Male reproductive tissues were examined in all dose groups in the P generation.

Postmortem examinations (offspring):

A complete necropsy was performed on all F1 animals and a subset of PND 22 weanlings. Adrenals, brain, heart, kidneys, epididymides, liver,
ovaries (without oviducts), prostate, seminal vesicles with coagulating glands (weighed with and without fluid), spleen, testes, thymus, and thyroid (trimmed and weighed post-fixation) were collected, weighed, and preserved for all groups with the exception of male secondary sex organs
in the PND 22 weanlings.
All tissues were preserved in10% buffered formalin except the testes and epididymides which were placed in Davidson’s fixative no longer than 24 hours. After fixation, all tissues were rinsed and stored in 70% ethanol.

Preserved tissues from the high dose and control groups from each generation were prepared. Tissues from lower dose groups were examined if exposure-related effects were seen in the high-dose group, gross lesions were present, or other signs of organ toxicity were noted in
the dose group (e.g., changes in organ mass). Male reproductive tissues were examined in all dose groups in the F1 generation.

Statistics:

Reproductive indices calculated as described in the Guideline. Organ mass ratios calculated relative to final body mass and brain mass. AGD normalized to the cubed root of body weight. Nipple retention data converted to % of pups per litter with retained nipples for analysis. All analyses were performed separately by sex. Continuous data were analyzed using a one-way ANOVA with dose group as the main effect. Age and body weight at VO and PPS were analyzed by ANCOVA using body weight at PND 21±1 as the covariate. Absolute organ mass was analyzed by Analysis of Covariance (ANCOVA), with dose group as the main effect and body mass at the end of the study as the covariate. Interpretation of changes in absolute organ mass, organ-tobody mass ratio, and organ-to-brain mass ratio in the evaluation of compound related effects was based on published analysis of control animal data. Weekly body weight and food consumption data were analyzed using repeated measures ANOVA to determine dose effect. If the interaction between within and between subject factors was significant, the effect of dose group on the parameter was determined within each sampling day using a one-factor ANOVA. When significant main effects were observed (p<0.05), appropriate post hoc tests were used to compare dose groups to the control group [e.g., Tukey’s multiple comparison test, Dunnett’s T3 (if variances were unequal), or Sidak (for ANCOVA)]. Variance equality was determined by Levene’s test. If the
data were not normally distributed, the data were either log transformed or arcsine transformed prior to ANOVA/ANCOVA. Some hematology, clinical chemistry, and hormone parameters were analyzed using nonparametric tests (Kruskal-Wallis test).
Fisher exact test was used to determine significant differences between treated and control groups for nominal or count data (e.g., mating, conception, fertility, gestation indices, histology, etc.).

Reproductive indices:

Male Mating Index: No. of males with confirmed mating/ Total No. of males cohabited X 100
Female Mating Index: No. of sperm-positive females/Total No. of females cohabited x 100
Male Fertility Index: No. of males impregnating a female/Total No. of males cohabited x 100
Female Fertility Index: No. of pregnant females/No. of sperm-positive females x 100
Gestation Index: No. of females with live born pups/No. of pregnant females x 100

Offspring viability indices:

Survival Index: No. of live pups (at designated time)/No. of pups born x 100
Pre-Implantation Loss: No. of corpora lutea – No. of implantation sites
Post-Implantation Loss: No. of implantation sites – (No. of live + No. of dead pups)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Yellow stained fur on nose/face and feet/stomach was noted only in NTO treated rats.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One 3600 mg/l recovery male was found dead on day 6 of dosing and one 3600 mg/l female was found dead on PPD 14 (dosing day 54) with no prior clinical signs. One control female was found dead with a partially delivered litter.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body mass did not differ among treated and control groups at any time for P1 generation males. In P1 females, the effect of NTO on body mass differed with time (interaction effect p=0.001). Body mass was generally unaffected by NTO treatment until the post-partum/lactation phase. During the lactation phase, body mass was reduced in females in the 3600 mg/l group compared to the remaining groups. This reduction in body mass was statistically significant at PPD 14 (p<0.001)
and PPD 21 (p<0.001) (3% and 4%, respectively).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption for P1 generation females was unaffected by NTO treatment. In P1 males, the effect of NTO treatment on food consumption differed with time (interaction effect p<0.001). The only differences between control and treated groups were increases in food consumption in the 144 mg/l group during days 18-21 (7.4%, p=0.027) and 21-24 (8.8%, p=0.031). All remaining differences were due to consistently lower food consumption in the 3600 mg/l compared to the 114 mg/l group, resulting in an overall 8% lower food consumption rate (p=0.027).

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

Food conversion efficiency (FER) was not affected by NTO treatment in P1 generation males, but was reduced in the P1 generation females in the 3600 mg/l group compared to the 144 mg/l group (p=0.031).

Water consumption and compound intake (if drinking water study):

effects observed, treatment-related

Description (incidence and severity):

Reduced water consumption in higher dose groups likely associated with taste aversion.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Red blood cell counts (RBC) were reduced (8%) in P1 males in the 3600 mg/l group compared to those from the control (p=0.042), but were within published normal ranges. Mean cell hemoglobin (MCH) was elevated (6%) in P1 males in the 3600 mg/l group; both compared to control (p=0.002) and normal ranges. Mean cell volume (MCV) was slightly increased (6% and 4%, respectively) in both P1 males and females (p=0.002 and p=0.044, respectively).

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Glucose levels were elevated in P1 males exposed to NTO compared to controls (20 - 40%) and reported normal values. This increase was statistically significant only for the 144 mg/l group compared to the controls (p=0.020).

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

NTO treated P generation males exhibited histologic changes consistent with seminiferous tubule degeneration or atrophy. Vacuoles within Sertoli cell cytoplasm were observed in 44% and 68% of animals in the 144 and 3600 mg/l groups (p =0.003 and p <0.001, respectively). Germ cell-free gaps were observed in 24% and 88% of animals in the 144 and 3600 mg/l groups (p = 0.022 and p<0 001, respectively). Animals in the 3600 mg/l group also exhibited retained spermatids in Stage IX-X (28%; p = 0.048), apoptotic cells (36% ; p=0.016), Sertoli-only tubules (28%), multinucleategiant cells (12%), sloughed germ cells (16%), and lack of elongating spermatids (8%). Animals in the 720 mg/l group did not exhibit similar signs of seminiferous tubule degeneration and the incidence of testicular interstitial proteinaceous fluid was lower in this group than in controls (p = 0.021). Reduction in sperm count and inappropriate cell types in the lumen of the epididymides
were also noted for 20% of males in the 3600 mg/l group; however, the frequency of these lesions was not statistically different from control. No changes were noted in the epididymides in lower dose groups.
Only reproductive tissues were evaluated for recovery males. Animals in the 3600 mg/l recovery group exhibited protein between tubules (44%), Sertoli-only tubules (22%), vacuoles within Sertoli cell cytoplasm (22%), and germ cell-free gaps (11%). The incidence of these findings was not significantly different between treated recovery males and control recovery males.

Histopathological findings: neoplastic:

no effects observed

Reproductive function: sperm measures:

effects observed, treatment-related

Description (incidence and severity):

All measures of sperm count were reduced in P1 males in the 3600 mg/l group compared to controls. Total sperm concentration was reduced by 20% (p=0.024) while motile (p=0.009) and progressively motile sperm concentrations (p=0.016) were reduced by 27% and 28%, respectively. The percent motile sperm did not differ between NTO treated groups and the control. Although total (44%) and motile sperm (27%) concentrations were also reduced in the 3600 mg/l recovery males compared to control recovery males, these reductions were not statistically significant.

Reproductive performance:

no effects observed

Description (incidence and severity):

NTO had no effect on male and female mating indices (96-100%) or mean pre-coital interval (2.5-3.3 days). Male and female fertility indices were lowest in the 3600 mg/l group (88%), but did not differ from the control group. All females determined to be pregnant gave birth to at least one live pup, resulting in gestation indices of 100% for all dose groups. The mean gestation interval was approximately 22 days for all dose groups.

Dose descriptor:

BMDL10

Effect level:

2 335 mg/L drinking water

Based on:

test mat.

Sex:

male

Basis for effect level:

organ weights and organ / body weight ratios

Dose descriptor:

BMDL10

Effect level:

2 775 mg/L drinking water

Based on:

test mat.

Sex:

male

Basis for effect level:

reproductive function (sperm measures)

Critical effects observed:

yes

Lowest effective dose / conc.:

3 600 mg/L drinking water

System:

male reproductive system

Organ:

cauda epididymis

Treatment related:

yes

Dose response relationship:

yes

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

One male in the 144 mg/l group was determined to have an undescended testis based on the presence of an abdominal mass and a single testis in the scrotum. Yellow stained fur was noted in all NTO treatment groups.

Mortality / viability:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In F1 pups, the effect of NTO on body mass varied over time, with no effects being evident until PND 21 (interaction effect p<0.001). On PND 21, body mass of pups in the 3600 mg/l group (46.4 grams) was reduced relative to the other NTO treatment groups (50.7 g and 49.8 g for 144 and 720 mg/l groups, respectively) but not the control group (49.3 g) (p=0.004). Body mass of the F1 females was unaffected by NTO treatment. In the F1 males, body mass did not differ between control and NTO treated groups at PND 21, but was reduced by approximately 8% in the 3600 mg/l group from PND 28 through 52

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption for F1 females was unaffected by NTO treatment. In F1 males, total food consumption was 10% lower in the 3600 mg/l group compared to the control and 144 mg/l group (p= 0.014).

Food efficiency:

no effects observed

Water consumption and compound intake (if drinking water study):

no effects observed

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related effects on hematology parameters in F1 pubertal animals.

Sexual maturation:

effects observed, treatment-related

Description (incidence and severity):

Anogenital Distance and Nipple Retention: Absolute anogenital distance and the ratio of anogenital distance to the cube root of body weight
were not affected by NTO exposure for either males or females. See Appendix H for details. Treatment with NTO increased both the percentage of pups per litter with retained nipples (>1 nipple) and the number of nipples retained per pup (p=0.012 and p=0.028, respectively). Percent of male pups with retained nipples at PND 13 was increased in all NTO dose groups (35%, 24%, and 30%) compared to controls (8%) (p=0.027, p=0.035, and p=0.017, respectively). Pups in the 144, 720 and 3600 mg/l NTO groups retained 1.1, 0.9, and 1.0 nipples per pup, respectively, compared to 0.4 nipples retained per pup in the control group. This difference was only statistically significant for the 144 and 3600 mg/l groups (p=0.041 and p=0.049, respectively).

Vaginal Opening and Preputial Separation
Age at VO and body mass at VO were not affected by treatment with NTO. Preputial separation was delayed (p<0.001) in the 3600 mg/l dose group by 2.6, 2.6, and 2.4 days relative to the control, 144 and 720 mg/l dose groups, respectively. Body mass at PPS was slightly higher in the 3600 mg/l group (218.3 g compared to 214.0 g in the controls), but did not differ between NTO treated rats and the control group.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

In F1 pubertal males, epididymides (left and right), testis (left and right), prostate, and seminal vesicles with coagulating glands (SVCG) (with and without fluid) masses were decreased in the 3600 mg/l NTO group. Mass of the epididymides was reduced by 24-27% in the 3600 mg/l group compared to the control and other NTO groups (p<0.001). Testis mass was similarly reduced by 30-31% in the 3600 mg/l group compared to the control and other NTO groups (p<0.001).Mass of the SVCG with fluid was reduced by approximately 32% in the 3600 mg/l group compared to all other groups (p<0.001). Mass of the SVCG without fluid was reduced in both the 720 (22%) and 3600 (25%) mg/l NTO groups (p=0.022 and p=0.008, respectively).
There were no treatment-related effects on the mass of organs in female F1 weanlings. In weanling males, left and right testis masses were reduced (15% and 13%, respectively) in the 3600 mg/l group compared to the control and other NTO groups (p=0.006 and p=0.023). However, the reduction in the 3600 mg/l group was statistically significant compared only to the 144 mg/l (p=0.012, p=0.013, respectively) and 720 mg/l groups (p=0.020). Thymus mass differed by 24% between the 144 mg/l group and the 3600 mg/l group (p=0.016), however, neither group differed from the control nor was a dose response evident. There were no other significant treatmentrelated effects on organ mass in male weanlings.

Histopathological findings:

effects observed, treatment-related

Description (incidence and severity):

F1 Pubertal Animals
NTO treated F1 generation males exhibited histologic changes consistent with seminiferous tubule hypoplasia or degeneration/atrophy. Testes of males in the 3600 mg/l group demonstrated apoptotic cells (100%; p <0 001), sloughed germ cells (100%; p <0 001), multinucleate giant cells (95%; p <0 001), lack of elongating spermatids (100%; p <0.001), germ cell-free gaps (95%; p <0.001), Sertoli cell vacuoles (85%; p <0.001); dilatation of seminiferous tubules (55%; p=0.012),
Sertoli-only tubules (30%; p=0.031), and reduced diameter of the testis (100%; p<0.001). Males in the 720 mg/l group demonstrated reduction in testis diameter (p=0.028), but did not exhibit signs of seminiferous tubule degeneration.
Corresponding increases in the frequency of epididymal hypospermia (95, and 100%, respectively) were observed in the 720 and 3600 mg/l groups (p<0.001, and p<0.001, respectively). Inappropriate cell types in the lumen (90%) and cribriform change in cauda (85%) of the epididymides were also observed in males in the 3600 mg/l group (p<0.001 and p<0.001).
In somatic (i.e., non-reproductive) tissues, differences between F1 males in the 3600 mg/l group and control rats included a slight increase in pulmonary alveolar hemorrhage (60%; p=0.019), minimal pyknosis of the inner stripe of the kidneys (55%; p=0.001), and minimal hepatic congestion (40%; p=0.003).

Developmental immunotoxicity:

effects observed, non-treatment-related

Description (incidence and severity):

Thymocyte cellularity in F1 male and female rats did not differ between treatment groups. Thymus cellularity for one male in the 720 mg/l group (14-0276) was approximately double (14 X109) the average cellularity for that group and was dropped from further analysis. The distributions of DN/DP/CD4+/CD8+ thymocytes in female rats were not affected by treatment with NTO. In male rats, the percent of double negative cells (DN) was 33% lower in the 3600 mg/l group compared to
the control (p=0.036). There were no differences between the control and NTO treated males for the remaining thymic cell types.
NTO had no effect on splenic cellularity or the proportion of B, T, and NK cells in F1 male and female spleens.

Dose descriptor:

BMDL10

Generation:

F1

Effect level:

1 048 mg/L drinking water

Based on:

test mat.

Sex:

male

Basis for effect level:

sexual maturation

Dose descriptor:

BMDL10

Generation:

F1

Effect level:

1 149 mg/L drinking water

Based on:

test mat.

Sex:

male

Basis for effect level:

organ weights and organ / body weight ratios

Dose descriptor:

BMDL10

Generation:

F1

Effect level:

1 420 mg/L drinking water

Based on:

test mat.

Sex:

male

Basis for effect level:

sexual maturation

Dose descriptor:

BMDL10

Generation:

F1

Effect level:

2 443 mg/L drinking water

Based on:

test mat.

Sex:

male

Basis for effect level:

organ weights and organ / body weight ratios

Critical effects observed:

yes

Lowest effective dose / conc.:

144 mg/L drinking water

System:

male reproductive system

Organ:

mammary gland

Treatment related:

yes

Dose response relationship:

yes

Reproductive effects observed:

yes

Lowest effective dose / conc.:

1 048 mg/L drinking water

Treatment related:

yes

Relation to other toxic effects:

reproductive effects occurring together with other toxic effects, but not as a secondary non-specific consequence of other toxic effects

Dose response relationship:

yes

Analytical Chemistry

The analytical chemistry results are summarized in Appendix D. Mean analytical concentrations were 137 ± 4.9, 681 ± 15.5, and 3344 ± 52.7 mg/l for the 144, 720 and 3400 mg/l NTO solutions, respectively. All results were within the 70-130% recovery limits for the analysis. As such, all results were reported using the nominal concentrations.

Thyroid Hormone Analyses

There was no consistent pattern of effects on thyroid hormones between sexes or across study phases. There were no treatment-related effects on thyroid hormones in P1 or F1 females or weanlings of both sexes. In P1 males, TSH levels demonstrated a non-significant dose response and were reduced (35%) in the 3600 mg/l group. In F1 males, T4 levels had a non-significant dose response and were reduced (15%) in the 3600 mg/l group. All thyroid hormone values were within previously reported control values for the species

 

Conclusions:

Rats were given ad libitum access to NTO in drinking water at four concentrations (0, 144, 720 and 3600 mg/L NTO) for two (females) to four (males) weeks pre-mating and continued until weaning of litters. Direct dosing of F1 animals occurred from weaning through puberty. NTO did not affect measures of fertility including, mating index, pre-coital interval, gestation index, litter size, number of live and stillborn pups, and sex ratio. In the Parent generation, NTO had an effect on the male reproductive system at 3600 mg/L as previously seen in the subchronic and the reproductive and developmental screening study. Reproductive development of male, but not female, offspring was altered by exposure to NTO. Both the proportion of pups that had retained nipples and number of nipples retained were increased in NTO exposed males compared to controls. Attainment of puberty was delayed by 2.6 days in the 3600 mg/l NTO exposed males relative to controls. Pubertal males in the 3600 mg/l NTO group exhibited reduced mass of the testis, epididymides, and accessory sex organs and associated histologic changes consistent with seminiferous tubule hypoplasia or degeneration/atrophy. The lowest BMDL10 value for the P generation (2335 mg/L eq. to 140 mg/kg bw/day) is based on the effect on epididymal mass. The lowest BMDL10 value for the F1 generation (1048 mg/L eq. to 120 mg/kg bw/day) is based on nipple retention effect.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Effects on developmental toxicity

Description of key information

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.

Link to relevant study records

Reference

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

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

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.

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.

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

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.

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.

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.

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

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.

Key result

Abnormalities:

no effects observed

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.

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

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.

Key result

Developmental effects observed:

no

Lowest effective dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Treatment related:

no

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.

 

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 14^thribs 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.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Justification for classification or non-classification

In a OECD 422 Screening for reproduction/developmental toxicity study doses of 31.25, 125, and 500 mg/kg bw/day NTO in corn oil were administered to Sprague-Dawley rats for four weeks.

Although the dose of 150 and 500 mg/kg bw/day resulted in testes degeneration, reduced sperm counts with no or less motile sperm observed, this dose did not affect reproduction or development of the offsprings.

In a OECD 414 study for developmental toxicity, doses of 100, 300 and 1000 mg/kg/day NTO in 1% methylcellulose were administered to female Sprauge-Dawley rats from Day 6 to 19 after mating. Oral administration of NTO 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, the highest dose tested.

Therefore NTO is not classified as a reproductive or developmental toxicant in accordance with Regulation (EC) No 1272/2008