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Toxicological information

Toxicity to reproduction

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Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guideline
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline
Details on test material:
- Name of test material : 3-NITRO-1 ,2,4-TRIAZOL-5-0NE (NTO)
- Substance type: energetic explosive
- Physical state: light green to white crystalline solid with no odor
- Purity ca.99%
Specific details on test material used for the study:
- Analytical purity: 99.48%
- Lot No.: BAE 11L375-061
- Batch No.: 10NTO11-5

Test animals

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

Administration / exposure

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)
Doses / concentrationsopen allclose all
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.

Examinations

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

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

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 / performance (P0)

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

Effect levels (P0)

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)

Target system / organ toxicity (P0)

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

Results: F1 generation

General toxicity (F1)

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

Effect levels (F1)

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

Overall reproductive toxicity

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

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.