Registration Dossier

Administrative data

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

Referenceopen allclose all

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:
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 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
Description (incidence and severity):
Testes and epididymides mass, organ to body mass, and organ to brain mass ratios were decreased in the 500 mg/kg bw/day dose group compared to all other dose groups, including controls (p=0.00 for all). Testes and epididymides mass, organ to body mass. and organ to brain mass ratios remained decreased in the 500 mg/kg bw/day recovery male dose group compared to recovery controls following a 4-week recovery period (p=0.00 for all).
In female rats, differences between NTO treated and control animals were observed for brain, spleen, and uterus mass and mass ratios. Absolute brain mass was reduced in the 31.25 mg/kg bw/day dose group relative to controls (p=0.020). Spleen mass and spleen to body mass ratios were reduced in the 31.25 (p=0.035 and p=0.031) and 500 mg/kg bw/day (p=0.008 and p=0.005) dose groups relative to controls. Following the removal of one outlier in the 500 mg/kg bw/day dose group, absolute uterus mass and uterus to brain mass ratios were reduced in the 125 mg/kg bw/day dose group relative to the 500 mg/kg bw/day group (p=0.027 and p=.016, respectively)
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
All male animals in the 500 mg/kg bw/day group were noted as having small testes.
In male rats, liver findings included 3 with a pale liver (one control and two 31.25 mg/kg bw/day animals) and 18 with a dark/mottled liver (three control, four 31.25 mg/kg-d, four 125 mg/kg bw/day, and seven 500 mg/kg bw/day animals). Three male rats per dose group were reported to have dark kidneys with the exception of the 31.25 mg/kg bw/day group with two.
A number of male rats throughout all dose groups were noted as having pale intestines either with or without yellow fluid present (five controls, five 31.25 mg/kg bw/day, eight 125 mg/kg bw/day, and eight 500mg/kg bw/day animals). Three male controls, two 31.25 mg/kg bw/day, one 125 mg/kg bw/day, and two 500 mg/kg bw/day animals were observed with dark and/or enlarged spleens. One additional male in the 500 mg/kg bw/day group had a pale spleen and one in the 125 mg/kg bw/day group had a small spleen.
Additional male gross pathology findings included one control with a small left testes and one 31.25 mg/kg bw/day animal with 2 pale brown 1 millimeter areas on the left lobe of the thymus.
In recovery male rats, six rats in both the control and 500 mg/kg bw/day groups were noted to have dark livers and five animals had dark spleens (three control and two 500mg/kg bw/day animals). One control and three 500 mg/kg bw/day recovery males were observed with dark/mottled kidneys. Eight out of ten males in the 500 mg/kg bw/day recovery group were noted as having small testes. Additional recovery male gross pathology findings included one control with enlarged submandibular lymph nodes, one control with hydronephrosis of right kidney, and one 500 mg/kg bw/day male with hydronephrosis of right kidney.

In female rats, liver findings included 7 with a dark liver (three control, one 31.25 In female rats, liver findings included 7 with a dark liver (three control, one 31.25 mg/kg bw/day, two 125 mg/kg bw/day, and one 500 mg/kg bw/day animals}, one 125 mg/kg bw/day animal with a pale brown liver, and one control with a pale liver. Kidney findings included 4 with dark kidneys (two control, one 125 mg/kg bw/day, and one 500 mg/kg bw/day animals) and one 125 mg/kg bw/day female with left and right cystic kidneys. A number of female rats throughout all dose groups were noted as having pale intestines either with or without yellow fluid present (five controls, nine .31.25 mg/kg bw/day, eight 125 mg/kg bw/day, and seven 500 mg/kg bw/day animals). One female control and one 500 mg/kg bw/day female were observed with dark spleens. One female control had a 9 x 4 millimeter diverticulum on serosal side of the greater curvature of the stomach, 2 females had distended stomachs full of bedding (one 125 mg/kg bw/day and one 500 mg/kg bw/day animals), and one 500 mg/kg bw/day female had yellow fluid present in the stomach.
Female lung findings included one control with red spots on the lung and two 125 mg/kg bw/day animals with mottled lungs. Additional female gross pathology findings included a scab on ventral chin, a mass in the fat near the ovaries, yellow-tinged mesenteric lymph nodes, enlarged submandibular lymph nodes, thin appearance, yellow-tinged subcutaneous fat, fluid-filled uterus, dilated uterus, yellow-stained fur, and a gas distended cecum. , two 125 mg/kg bw/day, and one 500 mg/kg bw/day animals}, one 125 mg/kg bw/day animal with a pale brown liver, and one control with a pale liver. Kidney findings included 4 with dark kidneys (two control, one 125 mg/kg bw/day, and one 500 mg/kg bw/day animals) and one 125 mg/kg bw/day female with left and right cystic kidneys. A number of female rats throughout all dose groups were noted as having pale intestines either with or without yellow fluid present (five controls, nine .31.25 mg/kg bw/day, eight 125 mg/kg bw/day, and seven 500 mg/kg bw/day animals). One female control and one 500 mg/kg bw/day female were observed with dark spleens. One female control had a 9 x 4 millimeter diverticulum on serosal side of the greater curvature of the stomach, 2 females had distended stomachs full of bedding (one 125 mg/kg bw/day and one 500 mg/kg bw/day animals), and one 500 mg/kg bw/day female had yellow fluid present in the stomach.
Female lung findings included one control with red spots on the lung and two 125 mg/kg bw/day animals with mottled lungs. Additional female gross pathology findings included a scab on ventral chin, a mass in the fat near the ovaries, yellow-tinged mesenteric lymph nodes, enlarged submandibular lymph nodes, thin appearance, yellow-tinged subcutaneous fat, fluid-filled uterus, dilated uterus, yellow-stained fur, and a gas distended cecum.
Neuropathological findings:
no effects observed
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:
testes
seminiferous tubules
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.

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:
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 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
Description (incidence and severity):
Mass of the left and right epididymides were reduced (11%) in the 3600 mg/l males (p=0.009 and p=0.001, respectively) relative to both the control and other NTO treatment groups.There were no significant treatment-related effects on other organ masses in P animals.
In recovery males, mass of the epididymides (left and right) was reduced by 11% and 12%, respectively; however, this reduction was only statistically significant for the right epididymis (p=0.008). Mass of the SVCG with fluid was reduced by 13% in the 3600 mg/l recovery group
compared to the control recovery group (p=0.008). No other treatment-related effects on organ mass were observed for the recovery males.
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

In the Extended One-Generation Reproductive Toxicity study, NTO did not affect mean litter size (14.41–14.9 pups litter), number of live births (13.63-14.44 per litter), still births (0.25-0.68 per litter), or the percentage of pups in each litter that were male (45.3%-53.5%). Pre- and post-implantation loss and corpora lutea number were unaffected by NTO. In the reproductive and developmental toxicity screening study,  gross external abnormalities were not observed  in the litter examined  after either 24 hours of parturition or on postpartum day 4. Moreover,  Amitrole, RDX and TNT, which are structurally similar substances to NTO, are not teratogens at non toxic doses.

Reproductive development of male, but not female, offspring was altered by exposure to NTO in the EOGRTS.

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 F1 generation (1048 mg/L eq. to 120 mg/kg bw/day) is based on nipple retention effect.
Link to relevant study records
Reference
Endpoint:
developmental toxicity
Data waiving:
other justification
Justification for data waiving:
other:
Justification for type of information:
JUSTIFICATION FOR DATA WAIVING:
This information will be submitted later based on ECHA communication/decision number CCH-D-2114407685-46-01/F.
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.

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