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EC number: 500-263-6
CAS number: 77138-45-5
The study was designed to investigate the systemic toxicity and
potential adverse effects of the test item on reproduction (including
offspring development), to evaluate some endocrine disruptor relevant
endpoints and is designed to be compatible with the requirements of the
OECD Guidelines for Testing of Chemicals No. 422 “Combined Repeated Dose
Toxicity Study with the Reproduction/ Developmental Toxicity Screening
Test” (adopted 29 July 2016).
This study was also designed to be compatible with Commission Regulation
(EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to
Regulation (EC) No 1907/2006 of the European Parliament and of the
Council on the Registration, Evaluation, Authorisation and Restriction
of Chemicals (REACH).
The test item was administered by gavage to three groups, each of twelve
male and twelve female Wistar Han™:RccHan™:WIST strain rats, for
approximately 6 weeks (males) and up to eight weeks (females) (including
a two week pre-pairing phase, pairing, gestation and early lactation for
females), at dose levels of 100, 300 and 500 mg/kg bw/day. A control
group of twelve males and twelve females was dosed with vehicle alone
(Polyethylene glycol 400) over the same period.
Clinical signs, behavioral assessments, body weight change and food and
water consumption were monitored during the study.
Pairing of animals within each dose group was undertaken on a one male:
one female basis within each treatment group on Day 15 of the study,
with females subsequently being allowed to litter and rear their
offspring to Day 13 of lactation.
During the lactation phase, daily clinical observations were performed
on all surviving offspring, together with litter size and offspring
weights and ano-genital distance and visible nipple count (male
Functional observations were performed on five selected males from each
dose group after the completion of the pairing phase, and for five
selected parental females from each dose group on Day 12 post partum.
Hematology and blood chemistry were evaluated prior to
termination on five selected males and females from each dose group.
Additionally, blood samples were taken at termination from all adult
animals and from one male and one female offspring per litter (where
possible) on Days 4 and 13 post partum, for thyroid hormone
analysis; samples from adult males and Day 13 offspring were analyzed
for Thyroxine (T4).
Vaginal smears were performed for all females from the day after arrival
(enabling the exclusion of females not showing appropriate estrous
cycling from dosing) and for all treated females including controls
through pre-pairing, pairing and up to confirmation of mating.
Vaginal smears were also performed in the morning on the day of
termination for all treated females.
Adult males were terminated on Day 44 or 45, followed by the termination
of all surviving females and offspring on Days 14 and 13 post partum
respectively. Any female which did not produce a pregnancy was
terminated around the same time as littering females. Any
female which did not show positive evidence of mating was terminated
around the same time as littering females. All animals were subjected
to a gross necropsy examination and histopathological evaluation of
selected tissues was performed. All offspring were examined
externally; where external observations were detected an internal
necropsy was performed.
One male and one female animal treated with 500 mg/kg bw/day were found
dead on Day 17 or Day 40 respectively.
There were no clinical signs specifically related to systemic toxicity
elicited by the test item for any treated males or females.
All animals of either sex treated with 500 mg/kg bw/day showed
incidences of increased salivation throughout the treatment period.
Noisy respiration was also noted in all of these animals throughout the
majority of the treatment period.
All males and eleven female animals treated with 300 mg/kg bw/day showed
incidences of increased salivation from Day 2 (males) or Day 17
throughout the treatment period. Noisy respiration was also noted in
all male animals and in nine females throughout the majority of
the treatment period.
An isolated occurrence of increased salivation was noted in one male
animal treated with 100 mg/kg bw/day and occasional instances were noted
in five females towards the end of the treatment period. Three female
animals also exhibited noisy respiration towards the end of the
The male animal which was found dead on Day 17 did not show any clinical
signs of toxicity the day prior to death. The female animal which was
found dead on Day 40 exhibited signs of noisy respiration on the day
prior to death.
Noisy respiration was apparent in one male animal treated with 500 mg/kg
bw/day on Days 26 and 33. There were no further changes in the
behavioral parameters noted in any treated male animals throughout the
Two female animals treated with 500 mg/kg bw/day exhibited noisy
respiration on Day 18 of gestation. Two female animals treated with 300
mg/kg bw/day exhibited noisy respiration on Day 4 or Day 12 of lactation.
One female animal treated with 100 mg/kg bw/day exhibited hunched
posture, pilo-erection and tip-toe gait on Day 18 of gestation. A
further female animal from this group exhibited signs of noisy
respiration on Day 12 of lactation. One control female also exhibited
respiration on Day 12 of lactation.
Functional Performance Tests
There were no inter-group differences in functional performance that
were considered to be related to treatment at 100, 300 or 500 mg/kg
Sensory Reactivity Assessments
There were no inter-group differences in sensory reactivity scores at
100, 300 or 500 mg/kg bw/day.
Male animals across all treatment groups exhibited reductions in body
weight gain during the first week of treatment with animals treated with
500 and 300 mg/kg bw/day respectively exhibiting actual body weight
Male animals treated with 500 mg/kg bw/day generally showed body weight
gains which were lower than control during the remainder of the
treatment period, this resulted in an overall reduction in body weight
gain of 52% when compared to control animals.
Male animals treated with 300 mg/kg bw/day showed signs of recovery
during the next three weeks, however, reductions in body weight gains
were again apparent during the final two weeks of treatment. An overall
reduction in body weight gain of 26.9% when compared to
control animals was noted in these animals.
Fluctuations in body weight gains were noted in male animals treated
with 100 mg/kg bw/day during the remainder of the treatment period,
however, mean body weight gains generally remained closer to control
than the other two treatment groups. An overall reduction in body
weight gain of 16.9% was still noted in this group when compared to
During maturation, female animals treated with 500 mg/kg bw/day
exhibited reductions in body weight gains during the first week of
treatment. An improvement was noted during the second week as body
weight gains were comparable to control. Body weight gains in the other
two treatment groups were comparable to control during Week 1, however,
both groups showed reductions in body weight gains during the second
week of treatment.
There were no effects on body weight gain during the gestation or
lactation periods for any of the treated female animals.
Male animals treated with 500 mg/kg bw/day showed reductions in food
consumption when compared to control during the first week of the
treatment period. Recovery was evident thereafter, however, group means
still remained slightly lower than control throughout the
remainder of the treatment period. A similar effect was noted in the
male animals treated with 300 mg/kg bw/day, however, the reductions were
not as marked as those seen at 500 mg/kg bw/day.
Male animals treated with 100 mg/kg bw/day exhibited reductions in food
consumption when compared to control during the first two weeks of the
treatment period, improvements were noted during the final two weeks of
treatment where food consumptions were comparable to control.
There were no adverse effects noted in food consumption in any treated
female during maturation, gestation or lactation.
Food Conversion Efficiency
At 500 mg/kg bw/day, food conversion efficiency (where calculated) for
males appeared inferior to control during the treatment period. A
similar effect was noted in male animals treated with 300 mg/kg bw/day
(with the exception of Week 2), however, the effect was less
marked. At 100 mg/kg bw/day, food conversion efficiency for males
appeared generally unaffected by treatment.
Food conversion efficiency for females during the pre-pairing phase of
the study was reduced in animals treated with 500 mg/kg bw/day during
the first week and in animals treated with 100 or 300 mg/kg bw/day
during the second week.
There were no effects in water consumption for any treated animal.
There was no effect of treatment with the test item at any dose level on
the nature of estrous cycle. All females generally showed regular
cycles over the pre-pairing phase of the study. There were also no
intergroup differences in the stage of estrus on the day of necropsy.
No treatment-related effects were detected in mating performance.
No treatment-related effects were detected in fertility..
Gestation lengths for controls and treated females were between 22 and
23½ days. Overall, the distribution of gestation lengths for treated
females was considered to be essentially similar to control.
Offspring Litter Size, Sex Ratio and Viability
There was no adverse effect on litter size and offspring survival from
implantation to birth and subsequently to termination on Day 13 of age
in any treatment group. Sex ratios across all treatment groups were
comparable to controls and did not indicate any selective effect of
maternal treatment on survival for either sex. Live birth index and
offspring viability in treated females was also comparable to controls.
Offspring Growth and Development
There was no detrimental effect of treatment with the test item
indicated by offspring body weight or body weight gain and litter
weights, ano-genital distance on Day 1 post partum or visible nipple
count in male offspring on Day 13 post partum at 100, 300 or 500 mg/kg
Male animals treated with 500 mg/kg bw/day and 300 mg/kg bw/day
exhibited increases in neutrophils. Due to the effects noted in the
stomach at histopathological examination an association with treatment
cannot be discounted.
There were no further effects which could be associated with treatment
on the hematological parameters measured.
An increase in bile acids was noted in female animals treated with 500
There were no adverse effects of treatment on the blood chemical
parameters measured in any treated male animal or in females treated
with 100 or 300 mg/kg bw/day.
Necropsy findings apparent for offspring on the study were typical for
the age observed. Neither the incidence nor distribution of these
observations indicated any adverse effect of maternal treatment on
offspring development in any treatment group.
The male animal treated with 500 mg/kg bw/day which was found dead on
Day 17 exhibited gaseous distension of the cecum, duodenum, ileum
(including peyers patches) jejunum and stomach. The stomach also
exhibited red patches on the glandular region and a raised limiting
ridge. The female animal treated with 500 mg/kg bw/day which was found
dead on Day 40 exhibited enlarged and red adrenals, gaseous distension
was noted in the cecum, colon, duodenum, ileum (including peyers
patches), jejunum and stomach. The stomach was
also enlarged and fluid filled and also had red patches on the glandular
region, the non-glandular region also appeared thin.
One female treated with 100 mg/kg bw/day, three females treated with 300
mg/kg bw/day and one male and two females treated with 500 mg/kg bw/day
exhibited pale livers.
Four females treated with 500 mg/kg bw/day exhibited pale and mottled
livers. One female treated with 100 mg/kg bw/day and one female treated
with 500 mg/kg bw/day exhibited a thickened glandular region of the
There were no further findings noted amongst surviving animals at
necropsy that were considered to be specifically related to treatment
with the test item.
There were no toxicologically significant effects detected in the organ
weights measured in any treated animal.
Significant erosion with hemorrhage was noted in the glandular stomach
of the male animal treated with 500 mg/kg bw/day which was found dead on
Day 17 and this was considered to be the cause of death for this animal.
No changes which could account for the death of the female animal which
was found dead on Day 40 were found at histopathology.
Inflammatory cell infiltration in the glandular region was present in
4/5 males treated with 500 mg/kg bw/day. This occurred mainly in the
submucosal area but spread into the mucosa and was of a moderate or mild
severity. It was also present in 4/5 males treated with
300 mg/kg bw/day at a minimal or mild severity. One of the males
treated with 300 mg/kg bw/day also had a mild erosion in the glandular
region. Spongiosis was present at the limiting ridge in one control
male along with 5/5 and 3/5 males treated with 300 or 500 mg/kg bw/day
Increased cytoplasmic rarefaction was present in one female from the
control and one from groups treated with 100 or 300 mg/kg bw/day (mild)
along with 5/7 in females treated with 500 mg/kg bw/day (mild or
moderate). This is likely to represent increased glycogen
deposition and represents a mild adaptive or metabolic response.
Pigment deposition was present at the involuting implantation sites of
all applicable females treated with 500 mg/kg bw/day (10) and 4/12
treated with 300 mg/kg bw/day. Whilst pigment is seen at a low level at
most involuting implantation site this was more intense and
orange in colour. There was no associated pathology. No other changes
were noted which could be related to the administration of the test item
and all are considered to be incidental.
Animal 24F (Control) paired with 12M had inflammatory changes in the
uterus that are likely to have resulted in failure to produce a litter.
Animal 44F (treated with 100 mg/kg bw/day) and 32M showed no changes to
account for the lack of pregnancy.
Animal 48F (treated with 300 mg/kg bw/day) suffered total litter loss.
This animal had significant kidney changes which were likely to have
had a functional effect on the animal and may have contributed to the
inability to raise a litter.
Animal 92F (treated with 500 mg/kg bw/day) and male 84 showed no changes
at histopathology to account for the lack of mating.
There were no test item-related microscopic findings noted in the
reproductive tracts following the qualitative examination of the stages
of spermatogenesis in the testes (no test item-related abnormalities in
the integrity of the various cell types present within the different
stages of the sperm cycle) or the evaluation of follicles and corpora
lutea in the ovaries.
Thyroid Hormone Analysis
Evaluation of Thyroxine (T4) in adult males and offspring at Day 13 of
age did not identify any obvious effect of treatment or indication of
endocrine disruption at 100, 300 or 500 mg/kg bw/day
The oral administration of Formaldehyde, oligomeric reaction products
with 4,4’-isopropylidenediphenol and diethylenetriamine (EK110) to
rats by gavage, at dose levels of100, 300 and 500 mg/kg bw/day, resulted
in the early deaths of one male and one female animal treated with 500
mg/kg bw/day. Significant erosion with hemorrhage was noted in the
glandular stomach of the male animal treated with 500 mg/kg bw/day which
was found dead on Day 17 and this was considered to be the cause of
death for this animal.
In general, it is considered that the effects noted in the animals at
500 mg/kg bw/day were considered to be the result of gastric irritancy
caused by the test item rather than attributable to true systemic
toxicity. However, due to the two deaths noted a No Observed Adverse
Effect Level (NOAEL) cannot be established at this dose level.
A No Observed Adverse Effect Level (NOAEL) can be established at 300
mg/kg bw/day for animals of either sex because the findings in general
do not reflect true systemic toxicity as the effects noted are
considered to be in relation to the irritant nature of the test item.
The No Observed Adverse Effect Level (NOAEL) for reproductive toxicity
was considered to be 500 mg/kg bw/day.
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.
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