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EC number: 695-748-3
CAS number: 65286-55-7
In a key, K1 combined repeated dose toxicity study with the
reproduction/developmental toxicity screening test, the test substance
was administered daily to rats at dose levels up to 100 mg/kg
bodyweight/day (OECD guideline 422; Edwards, 2018). The NOAEL was
considered to be 100 mg/kg bw/day for reproductive toxicity.
Discussion of the results
The administration of the test substance to rats at 20, 60 and 100 mg/kg
bw/day resulted in statistically significant reductions in body weight
development throughout the treatment period for both males and females
treated with 60 or 100 mg/kg bw/day compared to controls. A reduction in
food consumption for these animals was also apparent throughout the
treatment period and with no visible effects on water intake. No such
effects were detected in animals of either sex treated with 20 mg/kg
bw/day. There were no clinical observations of true systemic toxicity
and only sporadic instances of noisy respiration from isolated
Statistically significant differences in males and females treated with
60 or 100 mg/kg bw/day were noted in comparison to controls for the
hematological and blood chemical parameters measured. Due to the effects
apparent at histological examination an association with treatment
cannot be discounted for the majority of these differences.
The evaluation of Thyroxine (T4) in adults and male/female offspring
(Day 13 of age) did not identify any treatment-related findings in the
offspring. However, the adults did show a dose related reduction
attaining statistical significance for adult males treated with 60 and
100 mg/kg bw/day. This could be an associated effect due to the
histopathological changes to the thyroid gland.
Mating performance and fertility were unaffected by treatment. However,
offspring from 60 or 100 mg/kg bw/day litters showed a reduction (p<0.05
- p<0.001) in body weight, body weight gain and cumulative gains which
resulted in statistically significantly lower litter weights at 100
mg/kg bw/day (p<0.05 - p<0.01). No such effects were noted for
offspring from 20 mg/kg bw/day litters.
Microscopic examination of tissues revealed extensive and unusual
changes within this study which were considered to be a direct effect of
treatment with the test item. Vacuolation/vacuolated macrophages were
present in numerous tissues. Males treated with 20 mg/kg bw/day and
females treated with 60 and 100 mg/kg bw/day also had
vacuolation/vacuolated macrophages present in the choroid plexus.
Vacuolation is seen as part of a degeneration process, degeneration of
hepatocytes was seen in the liver of males at all dose levels and
females at 60 or 100 mg/kg bw/day. Although the exact cause of the
vacuolation cannot be determined, it may be related to the uptake and/or
clearance of the test item or its metabolite(s) or due to
phospholipidosis although this is not clear. It may also be related to
a change in, or disruption of, the storage process within the cells.
Special staining with Oil-Red-O and PAS did not give any indication of
the identity of the contents of the vacuoles.
There were also reduced amounts of splenic hematopoiesis present in all
groups treated with the test item. There were no notable correlating
changes were apparent in the hematology parameters therefore the
significance is unclear but unlikely to be significant.
Based on these findings, a No Observed Adverse Effect Level (NOAEL) for
systemic toxicity can be established for females only at 20 mg/kg bw/day
within the confines of this study, due to the absence of vacuolation or
any further changes in the tissues. This could not be established for
males. A No Observed Effect Level (NOEL) for reproductive toxicity was
considered to be at least 20 mg/kg bw/day.
A key, K1 reliable combined repeated dose toxicity study with
reproductive/developmental screening was performed according to OECD
guideline 422 (Edwards, 2018). 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 eleven weeks (males) and
twelve weeks (females) (including a two week pre-pairing phase, pairing,
gestation and early lactation for females), at dose levels of 20, 60 and
100 mg/kg bw/day. A control group of twelve males and
twelve females was dosed with vehicle alone (distilled water) 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 offspring only).
Extensive 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
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 offspring and
adult females on Days 13 and 14 post-partum, respectively. Any female
which did not produce a pregnancy 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.
There was no mortality observed during the study. There were no clinical
signs apparent that were considered to be related to systemic toxicity
of the test item.
There were no treatment-related changes in the behavioral parameters in
animals of either sex treated with 20, 60 and 100 mg/kg bw/day. Some
isolated incidences of noisy respiration from one male each from 60 and
100 mg/kg bw/day dose groups, three males and three females at 100 mg/kg
bw/day were noted. However, these observations this finding was
considered to be of no toxicological significance due to the sporadic
There were considered to be no treatment related changes in functional
performance considered to be related to treatment at 20, 60 and 100
mg/kg bw/day. Males treated with 20 mg/kg bw/day showed a statistically
significant increase (p<0.05) in hind limb grip strength but in the
absence of any similar effects at higher dosages, this finding was
considered to be incidental and of no toxicological importance.
In terms of body weight, the males treated with 60 or 100 mg/kg bw/day
had statistically significantly lower body weight gains (p<0.01 -
p<0.05) throughout the treatment period in a dose related manner. No
differences, compared to control, were observed in males treated at 20
mg/kg bw/day throughout the study.
Females at 100 mg/kg bw/day showed a mean body weight loss during the
first week of treatment, and a reduced body weight gain during the
second week. At 60 mg/kg bw/day, females showed a
statistically significant (p<0.05) group mean body weight loss during
the second week of treatment. There was no effect of treatment in
females treated with 20 mg/kg bw/day.
During the gestation phase, females treated with 60 or 100 mg/kg bw/day
showed lower body weight gains in relation to controls during the first
week of gestation. These females attained statistical significance on
Days 0 to 7 (p<0.05 and p<0.001, respectively). Signs of recovery were
noted during the second and third weeks of gestation in animals treated
with 100 mg/kg bw/day and after the first week of gestation for animals
treated with 60 mg/kg bw/day.
Females treated with 60 or 100 mg/kg bw/day showed body weights on Day
14 of lactation which were statistically significantly lower (p<0.01)
compared to controls.
Body weights and body weight gains were unaffected by treatment at 20
mg/kg bw/day during the gestation and lactation phases. Male food
consumption at 60 or 100 mg/kg bw/day was generally lower in relation to
controls from Week 2 of treatment onwards. The food conversion
efficiency showed a similar trend to the male body weight changes and
food intake, as males treated with 60 or 100 mg/kg bw/day values were
generally lower than controls. No such effects were detected for males
treated with 20 mg/kg bw/day. There were no adverse effects noted on
food consumption during the pre-pairing phase, however, food conversion
efficiency showed a similar trend to the female body weight changes, as
females treated with 60 or 100 mg/kg bw/day were generally lower than
controls. Throughout gestation statistically significantly (p<0.01 –
p<0.001) lower food consumption was apparent. This
trend continued during lactation in females treated with 100 mg/kg
bw/day but, females treated with 60 mg/kg bw/day showed signs of
recovery during lactation.
No effect on food consumption was detected for 20 mg/kg bw/day females
during gestation or lactation.
Mating performance as assessed by the number of paired animals that
mated was unaffected by treatment at 20, 60 or 100 mg/kg bw/day.
1, 2, 2 and 2 females from control, 20, 60 and 100 mg/kg bw/day dose
groups were nonpregnant. For the pairings which did not result in
pregnancy, no findings were noted in the tissues examined which could
account unequivocally for the lack of pregnancy. However, the uterus of
two animals treated at 100 mg/kg bw/day showed mild atrophy and one had
anestrus morphology in the vagina and the changes in the uterus caused
by the test item cannot be discounted.
There was considered to be no adverse effect of treatment with the test
item on the mean number of implantations, post-implantation loss, litter
size, sex ratio and subsequent offspring survival to Day 13 of age at
20, 60 and 100 mg/kg bw/day.
Live birth index and offspring viability in treated females was
comparable to controls.
There was no effect of treatment with the test item on offspring
development, as indicated by ano-genital distance on Day 1 post partum
and visible nipple count in male offspring on Day 13 post partum at 20,
60 or 100 mg/kg bw/day.
Offspring weights of both sexes with maternal treatment of 60 and 100
mg/kg bw/day were initially comparable to controls. However, body weight
gains were lower throughout which achieved statistical significance from
Day 4 until termination (p<0.01 - p<0.001). The offspring of females
treated with 20 mg/kg bw/day had litter weights that were comparable to
or exceeded controls throughout lactation.
The clinical signs apparent for offspring on the study were typical for
the age observed. There were no treatment-related abnormalities detected
An evaluation of Thyroxine (T4) in adults and male/female offspring (Day
13 of age) was performed; this did not identify any treatment-related
findings in the offspring. However, the adults did
show a dose related reduction attaining statistical significance
(p<0.001) for adult males treated with 60 and 100 mg/kg bw/day. This
could be an associated effect due to the histopathological changes to
the thyroid gland.
Histopathology results revealed extensive and unusual changes which were
considered to be a direct effect of treatment with the test item. These
changes included Vacuolation/vacuolated macrophages (at the adrenal
glands, aorta, brain, eyes, esophagus, heart, kidneys, liver, pancreas,
respiratory track, parathyroid glands, Pituitary Gland, prostate,
seminal vesicles, spleen, stomach, intestines, Thyroid Glands, Urinary
Bladder and uterus), reduced hematopoiesis in the spleen, atrophy of the
thymus. As described in the repeated dose section, these effects are
considered to be osmotic effects that are transient in nature and
There were a number of non-productive matings within the study,
generally spread through the groups. No findings were noted in the
tissues examined, of the animals involved, which could account
unequivocally for the lack of pregnancy. The uterus of 2 animals treated
with the test substance at 100 mg/kg bw/day showed mild atrophy and one
had anestrus morphology in the vagina and the changes in the uterus
caused by the test item cannot be discounted. The follicles and corpora
lutea in the ovaries indicated no test item related microscopic findings.
There were no test item-related microscopic findings 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).
The oral administration of the test substance to rats by gavage at dose
levels of 20, 60 and 100 mg/ kg bw/day resulted in treatment related,
non-adverse effects. The primary observation in the study was
vacuolisation/ vacuolated macrophages in numerous tissues from males
treated with 20 mg/kg bw/day and above and degeneration of hepatocytes
in the liver for males of all dose levels and females treated with 60 or
100 mg/kg bw/day. With regards to vacuolisation/vacuolated macrophages
in numerous tissues, the vacuoles observed are considered transient and
non-adverse based on scientific peer-reviewed literature regarding
weakly basic aliphatic amines (which can be primary, secondary or
tertiary amines) and supporting evidence from similar compounds within
the same chemical family of aliphatic amines. With regards to
degeneration of hepatocytes in the liver for males and females, there
are no other parameters within the study that supports this observation
as being adverse or progressive (i.e., clinical pathology or
hematology/clinical blood chemistry). Thus, with no obvious adverse
effects at 100 mg/kg bw/day, a NOAEL of 100 mg/kg bw/day is considered
acceptable (Lewis et al. 2012).
reproductive/developmental NOAEL based on various parameters for the
test substance from the OECD 422 study (OECD 422 - Combined Repeated
Dose Toxicity Study with the Reproduction/Developmental Toxicity
Screening Test), dosed at 20, 60 and 100 mg/kg/day, should be set at 100
primary reproductive/developmental observation in the study was a
decrease in fetal body weights (male, female and total litter weight)
for the various dose groups compared to the Control Group. However,
there was always a positive weight gain throughout the study in each
dose group at each body weight measurement timepoint. This observation
is considered to be a secondary effect due to lower maternal (female)
food consumption in the dose groups compared to Control Group, which is
also reflected in maternal (female) body weights. Even though food
consumption tended to be lower for dose group animals compared to the
Control Group, there was always a positive increase in food consumption
throughout the study for all groups.
with no obvious adverse effects at 100 mg/kg/day (i.e., mortalities) and
positive maternal and fetal weight gains, as well as, positive maternal
food consumption throughout the study, a NOAEL of 100 mg/kg/day is
considered acceptable (Lewis at al, 2012).
testing proposal for a prenatal development study in a first species is
the key, K1 combined repeated dose toxicity study with the
reproduction/developmental toxicity screening test
(OECD guideline 422; Edwards, 2018), an effect on offspring weight was
observed but considered to be non-adverse. The NOAEL for developmental
toxicity is considered to be 100 mg/kg bw/day.
Annex IX testing:
A testing proposal for a prenatal development study in a first species
Based on the results, the test substance is considered not to be
classified as reproductive toxicant, according to the criteria laid down
in the CLP regulation.
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