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EC number: 213-254-4
CAS number: 932-64-9
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,
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.
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.
and Homogeneity Results
6 (day 0 stability)
6 (day 7 stability)
6(day 14 stability)
6 (day 21 stability)
6(day 28 stability)
6 (day 35 stability)
6 (day 42 stability)
6(day 49 stability)
100 (homogeneity top)
100 (homogeneity middle)
100 (homogeneity bottom)
5.9, 5.7, 5.7 (repeats)
22, 22, 23(repeats)
87, 85, 90(repeats)
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
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
Historical Controls *
Length of Gestation (Days)
22.0 ± 0.00
22.1 ± 0.35
22.0 ± 0.47
22.42 ± 0.53
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
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
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
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.
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
Oral administration of Nitrotriazolone at dose levels of 100, 300 or
1000 mg/kg/day to pregnant Sprague Dawley rats throughout
organogenesis and the fetal growth phases was well tolerated, with no
adverse findings at any dose level. Consequently, the no observed adverse
effect level (NOAEL) for maternal toxicity and embryo-fetal survival
and development was considered to be 1000 mg/kg/day.
Mean recovery results obtained
on each analytical occasion during the study were within ±10% of nominal
showing the continued accuracy of the method.
The mean concentrations were
within – 15%/+10% of nominal concentrations confirming the accuracy of
formulation, with the exception of Group 4 prepared for the last week of
study that was – 18.5% of nominal. The Group 4 contingency samples were
analyzed confirming the original result and therefore this formulation
was discarded and a new Group 4 formulation prepared. The Group 4
reformulation was within limits.
Mean serum T3
Female Dams (pg/mL)
No statistically significant
difference was identified between the control group and treatment groups
using a Williams test.
Mean serum T4
The purpose of this study was to assessof the influence of
Nitrotriazolone (an industrial chemical) on embryo-fetal survival and
development when administered during the organogenesis and fetal growth
phases of pregnancy in the Sprague Dawley rat.
Three groups of 20 females receivedNitrotriazoloneat doses of 100,
300 or 1000 mg/kg/day by oral gavage administration, from Day 6 to 19
after mating. A similarly constituted Control group received the
vehicle, 1% Methylcellulose at the same volume dose as the treated
groups. Animals were killed on Day 20 after mating for reproductive
assessment and fetal examination.
Clinical observations, body weight and food consumption were
recorded. Adult females were examined macroscopically at necropsy on Day
20 after mating, blood samples were taken for thyroid hormone analysis
and the gravid uterus weight and thyroid weight were recorded.
Microscopic pathology investigations were also undertaken on the
thyroids. Ano-genital distance was measured for fetuses and all fetuses
were examined macroscopically at necropsy and subsequently by detailed
internal visceral examination or skeletal examination.
The mean concentrations were within – 15%/+10% of nominal
concentrations confirming the accuracy of formulation, with the
exception of Group 4 prepared for the last week of study that was
– 18.5% of nominal. The Group 4 contingency samples were analyzed
confirming the original result and therefore this formulation was
discarded and a new Group 4 formulation prepared. The Group 4
reformulation was within limits.
There were no premature deaths attributable to treatment.
There was no effect of treatment on maternal clinical condition in
females receiving 100 or 300 mg/kg/day where two females receiving
1000 mg/kg/day showed signs of piloerection and one of which also had
shallow breathing and was observed as underactive.
There was no effect of treatment on body weight, thyroid weight,
macropathology or histopathology of the maternal thyroid. Maternal body
weight change (adjusted for the gravid uterine) was slightly high in
females receiving 300 or 1000 mg/kg/day. In addition, food intake for
females receiving 1000 mg/kg/day was slightly low at the start of
treatment but increased towards the end of gestation and therefore was
The analysis of serum TSH, T3 and T4 concentrations performed at
scheduled termination on Day 20 of gestation revealed that serum thyroid
hormone concentrations were comparable with endogenous levels at all
dose levels when compared with the control.
Embryo-fetal survival, fetal ano-genital distance and development
were unaffected by maternal treatment. The fetal weights were slightly
low from maternal females treated at 1000 mg/kg/day. There was an
increase in incidence of short supernumerary 14thribs in the
litters of females receiving 300 or 1000 mg/kg/day and were outside the
historical control data range. These findings were considered
Oral administration of Nitrotriazolone at dose levels of 100, 300
or 1000 mg/kg/day to pregnant Sprague Dawley rats throughout
organogenesis and the fetal growth phases was well tolerated, with no
adverse findings at any dose level. Consequently, the no observed
adverse effect level (NOAEL) for maternal toxicity and embryo-fetal
survival and development was considered to be 1000 mg/kg/day.
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
In a OECD 414 study for developmental toxicity, doses of 100, 300 and
1000 mg/kg/day NTO in 1% methylcellulose were administered to female
Sprauge-Dawley rats from Day 6 to 19 after mating. Oral administration
of NTO was well tolerated, with no adverse findings at any dose level.
Consequently, the no observed adverse effect level (NOAEL) for maternal
toxicity and embryo-fetal survival and development was considered to be
1000 mg/kg/day, the highest dose tested.
Therefore NTO is not classified as a reproductive or developmental
toxicant in accordance with Regulation (EC) No 1272/2008
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