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EC number: 915-730-3
CAS number: -
Formulation Analysis: The mean
concentrations of OTNE in test formulations were within 8% of the
nominal concentration, confirming the accuracy of formulation. The
difference from mean remained within 4%, confirming precise analysis.
See Annex 2 of the study report.
The test substance was investigated, in a study performed
according to OECD TG 443, on systemic and reproductive toxicity when
administered continuously by oral gavage to Han Wistar rats. In the F0
generation, 24 Han Wistar rats per sex received OTNE at dose levels of
30, 100 or 300 mg/kg/day. The dose was based on all repeated dose
toxicity studies showing increase relative liver weights with
hypertrophy at 500 mg/kg bw of ca. 50% indicative for overloading the
metabolic pathway. The NOAEL for this effect was set at 120 mg/kg bw and
therefore 300 mg/kg bw was anticipated to show some liver toxicity,
which could be well tolerated. Males were treated for ten weeks before
pairing, up to necropsy after litters were weaned. Females were treated
for ten weeks before pairing, throughout pairing up to necropsy on Day
28 of lactation (resulting in exposure up ca 120 days. In the F1
generation, 40 rats per sex were treated from weaning to their scheduled
termination (relevant to each cohort) at the same dose levels and as the
F0 generation resulting in approximately 70 exposure days for the F1A
cohort and 77 exposure days for the F1B cohort. A similarly constituted
Control group received the vehicle, corn oil.
For the F0 generation data were recorded on clinical condition,
body weight, food consumption, estrous cycles, mating performance and
fertility, gestation length and parturition observations and
reproductive performance. Clinical pathology (haematology, blood
chemistry and thyroid-related hormones), sperm assessment, organ weight,
macroscopic pathology and microscopic pathology investigations were
For F1 offspring, clinical condition, litter size and survival,
sex ratio, body weight, ano-genital distance, organ weights and
macropathology were assessed. Nipple counts were performed on male
offspring on Day 13 and 20 of age. Blood samples were collected from
selected offspring on Day 4 and 22 of age for biomarker investigations.
For F1 generation - Cohort A, data were recorded on clinical
condition, body weight, food consumption, sexual maturation, vaginal
opening and estrous cycles. Clinical pathology (haematology, blood
chemistry, thyroid hormones and urinalysis), sperm assessment, ovarian
follicle counts, organ weight, macroscopic pathology, full microscopic
pathology and spleen cell immunophenotyping investigations were
For F1 generation - Cohort B, data was recorded on clinical
condition, body weight, food consumption, estrous cycles, sexual
maturation, organ weight, and a targeted set of macroscopic pathology
investigations were performed.
Results – systemic toxicity:
F0 adults (and F1 offspring up to weaning)
Mortality: There were two deaths throughout
the duration of study; Control male No. 12 and Female No. 267 that
received 100 mg/kg/day were euthanized prematurely on animal welfare
grounds during the 10-week pre-pairing treatment period and on Day 1 of
lactation respectively. Female No. 267 had shown signs comprising
decreased activity, irregular breathing and was abnormally cold to
touch, macropathological examination revealed abnormal contents of the
gastrointestinal tract and thickened uterus horns. When compared to the
animals killed at scheduled termination, these findings were atypical
and therefore the death of female No. 267 was not considered related to
Clinical signs: There were no clinical signs
considered to be related to treatment in F0 males or in the F0 females
which reared their litters to weaning.
Body weight gain of males and females before pairing and of
females during gestation and lactation was unaffected by treatment.
Food consumptionof both sexes before pairing,
males after pairing and females during gestation and lactation was
generally similar to that of the Controls. From Days 14 to 21 of
lactation, food intake was marginally high for females that received 100
or 300 mg/kg/day.
Haematologyinvestigations revealed slightly
low haematocrit and haemoglobin concentrations for males that received
300 mg/kg/day and an increase in mean corpuscular haemoglobin, mean
corpuscular haemoglobin concentration and mean corpuscular volume for
females that received 300 mg/kg/day. For females that received 30, 100
or 300 mg/kg/day, a slight increase in haemoglobin levels was evident.
All these effects were <6% and considered incidental and not adverse.
Blood chemistryinvestigations revealed
marginally low alkaline phosphatase levels for males that received 100
or 300 mg/kg/day and females that received 30, 100 or 300 mg/kg/day (<
20%). Low aspartate aminotransferase and phosphate levels were evident
for males that received 300 mg/kg/day. Gamma-glutamyl transpeptidase
increased from zero to 1. Total protein and albumin levels increased
minimally for males that received 300 mg/kg/day and protein levels at
this dose for females but <=6%. Bilirubin and bile acid levels were low
and for females that received 30, 100 or 300 mg/kg/day. For females,
cholesterol was slightly increased at the 300 mg/kg/day (+16%). The
toxicological relevance decreases of alkaline phosphate levels, low
aspartate transferase and bile acid levels is unclear, because increase
is considered to be related to liver effects. Others liver related
parameters such as, increase in gamma-glutamyl transferase and some
increase in cholesterol are considered to be due to increased liver
Macropathology: There were no test item
related macropathology findings in any of the organs.
Organ weights and Histopathology:Liver:
Relative Liver weights were dose dependently statistically significantly
high among males and females that received 100 or 300 mg/kg/day (9.5 and
29.5%, respectively). For females only, relative liver weights were dose
dependently increased up to 26.5%. Treatment-related changes in the
liver were present in both males and females of F0 generation. In some
males and approximately half of the females treated with OTNE at 300
mg/kg/day, the hepatic changes were consistent with minimal
centrilobular or general hepatocellular hypertrophy (males only). Some
changes in blood chemistry values such as the increase in gamma glutamyl
peptide transferase, increase in total protein, increase in cholesterol,
increase in liver weight and slight liver hypertrophy is anticipated to
be related to an adaptive increase of liver function and not considered
adverse.Thyroid: Relative thyroid weights were also increased in
males but less so in females. No histopathological findings were seen
and therefore their increases were not considered adverse.Spleen:
The higher mean weights of the spleen and in F0 males that received 300
mg/kg/day were not supported by any microscopic pathology changes and
thus biological significance and any relationship to treatment is not
evident.Kidneys: Treatment-related changes in the kidneys were
present in males only. Increased amounts of hyaline droplets within the
renal proximal tubular epithelium were considered to be treatment
related in kidneys of males treated with OTNE at 300 mg/kg/day and at
100 mg/kg/day examined: 15/25 and 23/24 animals, respectively.
Additionally, higher incidence of minimal focal to multifocal, tubular
basophilia was observed in the kidneys of males treated with OTNE at 300
mg/kg/day: 11/24 animals. Hyaline droplets and the increase in tubular
basophilia are considered to be related to alpha-2u-globulin hydrocarbon
nephropathy, an adaptive response, male sex specific and not considered
adverse for humans; but no immune staining was done.
Results Reproductive toxicity (F0)
Fertility P0: Irregular, extended, and acyclic
estrous cycles were observed in both control and treatment groups. The
cause was thought to be related due to a light cycle error in the animal
facility room and therefore these abnormalities were not considered
related to treatment. There was no effect of treatment on pre-coital
interval, fertility, gestation length and gestation index. Sperm
motility, counts and morphology were unaffected by treatment.
Developmental toxicity: At 30, 100 or 300
mg/kg/day, there was no effect of treatment on post implantation
survival, the mean number of offspring born and the number of live
offspring on Day 1. Offspring survival after birth was unaffected by
treatment and there was no clear effect on offspring sex ratio. There
was no effect of treatment on offspring ano-genital distance, nipple
counts in males, organ weights or macropathological findings. Male and
female reproductive organs were not affected. There was no effect of
treatment on serum T4 levels in adults or offspring, and no conclusive
effect on TSH levels.
Results: Selected F1 offspring - Cohorts 1A and 1B
Clinical signs: Treatment of the F1A or F1B
males and females was generally well tolerated and there were no
test-item related changes in clinical condition observed from weaning on
Day 21 of age up until scheduled termination at approximately Week 13 or
14 of age.
Body weight: At 30, 100 or 300 mg/kg/day, body
weight gain of both sexes between Days 21 to 25 of age was generally
similar to that of the Controls. For males that received 300 mg/kg/day,
group mean body weight gain from Day 1 to 57 after the formal
commencement of the F1 generation was marginally low when compared to
the controls (<=-5%). Food consumption was not affected.
Haematology investigationof the F1A cohort did
not reveal any treatment related changes. Two significant finding in
females were not considered toxicologically relevant: Hemoglobulin
concentrations decrease of 5% and platelet count increase of 17% in
absence of other related findings.
Blood chemistry investigationof the F1A cohort
revealed low alanine amino transferase and glucose levels for males that
received 300 mg/kg/day. For males and females that received 30, 100 or
300 mg/kg/day, bile acids were low when compared to the controls. Low
alkaline phosphatase values were evident in females that received 300
mg/kg/day. Calcium was minimally but significantly increased (+3%). For
females that received 100 or 300 mg/kg/day, cholesterol was high (+17
and +30%, respectively) when compared to the controls. Except for
calcium most parameters are liver related.
Urinalysis investigationof the F1A cohort
revealed low pH in males and females that received 300 mg/kg/day: from
ca 7 to 6.1. For males that received 100 or 300 mg/kg/day, total protein
and protein was high when compared to the controls 92 and 275%,
respectively. Additionally, total sodium levels for males that received
300 mg/kg/day was low and for males that received 30 or 100 mg/kg/day,
Females that received 300 mg/kg/day, urine volume, total protein and
total sodium was low whereas, protein, urine potassium and urine
chloride was high when compared to the controls. Urine sodium levels
were high. These effects are considered minor and not considered adverse
in absence of related finding in kidney.
Macropathology: There were no test item related macropathology
findings on all organs.
Organ weight: Relativeliverweights were
high for cohort 1A males and females that received 30, 100 or 300
mg/kg/day (<=28% for the high dose). For cohort 1A and 1B males that
received 300 mg/kg/day and cohort 1A and 1B females that received 300
mg/kg/day.Thyroid and parathyroidweights were high in F1A and 1B
males when compared to the controls (<=37%); in females it was also
increased but not significantly (<11%), which are anticipated to be
related to increase in liver function. RelativeKidneyweights were
statistically significantly high among cohort 1A males and females that
received 300 mg/kg/day (<=16%) and slightly high for males that received
100 mg/kg/day (+7%).
Histopathology, Liver: Treatment-related
changes in the liver were present in both males and females of the F1
generation. In some males and approximately half of the females treated
with OTNE at 300 mg/kg/day, minimal centrilobular hypertrophy was
apparent with one male showing minimal general hypertrophy. These are
similar to what is seen in the F0 generation.
Microscopic pathology changes related to treatment with OTNE were
seen in the kidneys and the liver but not in any other organs. Increases
in the weight of some organs in some cohorts e.g. thyroid, prostate and
oviduct were therefore not considered adverse.Kidney:
Treatment-related changes in the kidneys were present in males only. In
males of the F1 generation, increased amounts of hyaline droplets within
the renal proximal tubular epithelium was considered to be treatment
related in kidneys of males treated with OTNE at 300 and 100 mg/kg/day.
Additionally, higher incidence of minimal to slight, focal to
multifocal, tubular basophilia was observed in the kidneys of males
treated with OTNE at 100 or 300 mg/kg/day (number of animals). These
effects are considered to be related to alpha-2u-globulin hydrocarbon
Reproductive toxicity (F1A and F1B):
Reproductive parameters for the F1 group, there was no adverse effect of
treatment on sperm motility, counts or morphology. At 300 mg/kg/day
there was a slight increase in cauda epididymal weight and sperm
concentration, which is not considered toxicological relevant. Age at
sexual maturation, time between vaginal opening and first estrous and
oestrus cycles and ovarian follicle counts were unaffected by treatment.
For cohort 1A females that received 100 or 300 mg/kg/day and cohort 1B
females that received 300 mg/kg/day, ovaries and oviduct weights were
high when compared to the controls just <=11%. No histopathological
findings were seen and therefore biological significance or any
relationship to treatment is uncertain. Prostate weights of cohort 1B
males that received 300 mg/kg/day was high when compared to the controls
(+19%). No histopathological findings were seen.
Conclusion: Considering systemic toxicity for
F0 and F1 animals, minor effects were seen on haematology and blood
chemistry of which the latter can be related to increased liver
function. In the liver the increased relative liver weights and
increased minimal hypertrophy is considered adaptive. Relative kidney
weights and histopathological changes in the kidneys of F0 and F1 Cohort
1A males, and changes in the liver of F0 and F1 Cohort 1A males and
females were not considered adverse within the context of this study and
for males these were related to alpha 2u-globulin hydrocarbon
nephropathy. The NOAEL for systemic toxicity in the F0 and F1 A adult
animals was concluded to be the high dose of 300 mg/kg/day. Based on the
results and absence of effects obtained in this study it was concluded
that the No-Observed-Adverse-Effect-Level (NOAEL) for reproductive
performance of the F0 and F1 animals was the high dose of 300 mg/kg/day
for males and females.
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