2,2’-iminodiethanol was administered
to groups of 30 male and 30 female healthy young Wistar rats as addition
to the drinking water in concentrations of 100, 300 and 1000 ppm. The
vehicle control group was given plain drinking water. Analyses confirmed
the correct concentration and the stability of the test substance in
drinking water.
The overall mean doses of
2,2’-iminodiethanol throughout all study sections and across all cohorts
were 12.75 mg/kg body weight/day (mg/kg bw/d) in the 100 ppm group,
37.68 mg/kg bw/d in the 300 ppm group and 128.35 mg/kg bw/d in the 1000
ppm group. There were no test substance-related mortalities or adverse
clinical observations noted in the F0 generation parental animals at any
dose level as well as in the F1 adolescents at the low- and mid-dose
level (100 and 300 ppm).
The high-dose of the test substance
(1000 ppm) produced mortalities or adverse clinical observations in the
F1 adolescents. Three high-dose offspring (2 males, 1 female) in two
rearing cohorts (1A, 1B) were either sacrificed moribund or found dead
at different timepoints during the study. These casualties went along
with adverse clinical observations such as highstepping gait and
piloerection recurring across all F1 cohorts in several study sections.
Altogether 11 high-dose animals of both sexes in cohorts 1A, 1B, 2A and
3 were affected. Some of these affected high-dose male F1 offspring had,
in addition, small testes for which a histopathological correlate was
found. As these animals had a severe reduction in terminal body weight,
this size reduction was regarded to be secondary to the body weight
decrease. Treatment with 2,2’-iminodiethanol produced no effect on water
consumption in the F0 males at all dose levels, while F0 females at 300
and 1000 ppm had decreased water consumption beginning during gestation
and distinct to severe during lactation. The F1 adolescents showed this
effect consistently and distinctly across all cohorts at the 1000 ppm
dose level and less pronounced and consistent, but still noticeable, at
the 300 ppm dose level. No such effect was noted at 100 ppm. In the
affected groups reduced water consumption was associated with reduced
urine volume and histopathological findings in the kidneys.
In the 1000 ppm F0 parental males and
females food consumption was consistently reduced, at the 300 ppm level
reduced food consumption was only noted in females during lactation. In
the 1000 ppm F1 males and females food consumption was consistently
reduced postweaning, although there was some variability in the extent
of the reduction across the 1A, 1B, 2A and 3 cohorts. At the 300 ppm
level slightly and non-statistically significant reduced food
consumption was only noted in the cohort 1A males. In contrast to this,
food consumption of all males and females at the 100 ppm level remained
unchanged. In the 1000 ppm F0 parental males and females body weights
were consistently reduced throughout all study sections beginning on
study day 7. At the 300 ppm level reduced body weights were noted in F0
parental males from study day 28 and in females during lactation only.
This was caused by a similarly affected body weight gain, though the
course of body weight changes was variable in the different study
sections, generally for the females being more severe during the
gestation/lactation period. Body weights/body weight gain of the
high-dose (1000 ppm) Cohort 1A, Cohort 1B, Cohort 2A and Cohort 3
animals were similarly reduced as of the F0 parental animals, however
showing some variability across cohorts. At 300 ppm changes of body
weights/body weight gain look rather nonuniform and mild, however, there
appears to be evidence that this dose group was affected as well.
Regarding clinical pathology in F0
generation male and female rats of test group 3 (1000 ppm) as well as in
F1 generation male and female rats at PND92 of test group 13 (1000 ppm)
a microcytic anemia was present indicated by decreased red blood cell
(RBC) counts hematocrit and hemoglobin values as well as decreased mean
corpuscular volume (MCV). Increased urea values in male and female rats
of the same test groups in the F0 and F1 generation at PND 92 were due
to an increased protein metabolism. This is confirmed by higher albumin
levels in the rats of both sexes of test group 3 (1000 ppm) in the F0
generation and most probably also by a reduced prothrombin time in
females of test group 13 (1000 ppm) of the F1 generation at PND 92;
indicating higher synthesis of coagulation factors. Higher activities of
alkaline phosphatase in males of the F0 generation of test group 3 and
of the F1 generation in test group 13 may have been caused by a reduced
food consumption of these animals. Higher aspartate aminotransferase
(AST) activities in F0 males of test group 3 and in male and female rats
of the F1 generation of test group 13 may be due to a liver cell effect,
although other tissues could also have been involved because AST is not
a liver-specific enzyme in rats.
In males of test groups 2 and 3 (300
and 1000 ppm) urine specific gravity was lower and urine volume was
higher (not statistically significantly) compared to controls. In
conjunction with histopathological alterations in the kidneys, this
change was regarded as treatment-related and adverse.
The increase of platelet counts in F0
males of test groups 2 and 3 (300 and 1000 ppm) as well as males of the
F1 generation in test groups 12 and 13 (300 and 1000 ppm) as well as the
shortened prothrombin time (Hepatoquick’s test) in males and females of
test groups 2 and 3 of the F0 generation and in F1 females of test group
13, indicated a dysregulation of the coagulation homeostasis. This
correlates with decreased serum platelet activating factor (PAF) values
in F0 females of test group 3 (1000 ppm).
Regarding pathology, target organs
were the kidneys, liver and glandular stomach.
F0 generation parental animals
The terminal body weight in the F0
generation parental males and females of test group 03 (1000 ppm) was
decreased to below historical control values and for test group 02
animals (300 ppm) it was within historical control values (see PART
III). This reflects the different extent of body weight effects at 300
and 1000 ppm in-life. In the kidneys of males of test group 02 and test
group 03 and in the kidneys of females of test group 03,
degeneration/regeneration of the proximal tubules was observed. This was
also regarded as treatment-related. The decrease in terminal body weight
in males of test group 12 and 13 (300 and 1000 ppm) and females of test
group 13 (1000 ppm) reflects the body weight effect at 300 and 1000 ppm
in life.
Similar to the findings in the kidneys
of the F0 generation, males and females of test group 12 and 13 (300 and
1000 ppm) revealed tubular degeneration and regeneration. This was also
reflected by the increased kidney weight. In test group 11 (100 ppm)
animals the kidney weight was still increased, but was regarded to be
non-adverse due to a missing histopathologic correlate. Comparable to
the F0 generation, animals of the F1 generation revealed also
mineralization: male animals at the transition between inner and outer
medulla and females in the papilla. As for the F0 generation this
finding was regarded to be treatment-related but not as adverse.
Males and females of test group 13
(1000 ppm) revealed centrilobular hypertrophy in the liver. Male animals
also had a peripheral hypertrophy. This correlated with the increased
liver weights. Furthermore, in both sexes an increase of fatty change
was observed in the peripheral area. These findings in combination with
clinical pathology findings were regarded to be adverse. In males of
test group 12 (300 ppm), the relative liver weight was increased, three
animals showed centrilobular hypertrophy and fatty change. Due to the
additional fatty change in combination with the hypertrophy, it was
regarded as adverse. Females of the same test group revealed increased
liver weights and one animal a centrilobular hypertrophy, but no
increase in fatty change when compared to control and no relevant
findings in clinical pathology. It was therefore regarded to be
treatment-related but not as adverse. The same comes true for the
increased liver weight in test group 11 males and females (100 ppm), in
which no relevant histopathologic findings were observed.
The mammary gland of males of test
group 13 (1000 ppm) revealed a female phenotype (feminization) in four
out of 16 animals. One male animal showed in addition a diffuse
hyperplasia of the mammary gland. Females of this test group showed an
increase in secretion. These findings were regarded to be adverse.
In the left testis, three males of
test group 13 (1000 ppm) revealed immature testicular tubules which
corresponded to the macroscopic finding “size reduced”. In one male,
there was focal degeneration in addition. As a consequence, the
secondary sexual glands showed also a reduction in size. In the
corresponding epididymis of the affected animals aspermia was found. In
the ductus deferens size reduction and an increase in macrovesicular
vacuolation was observed. The vacuolation in the ductus deferens of test
group 12 animals (300 ppm) might be still treatment-related but due to
the absence of findings in all other sexual organs this was regarded to
be non-adverse. These three males revealed a severe decrease in terminal
body weight. Therefore, this size decrease in the above-mentioned organs
was most likely due to the body weight reduction and was assessed to be
treatment-related but a secondary effect. A delayed maturation of the
testicular epithelium is known to occur in animals which have reduced
body weights (McInnes, 2012). But as in cohort 1A animals (and in cohort
1B animals) degeneration of the testicular epithelium was observed in
addition to the delay in maturation as well as in animals without a
delay in maturation in these organs, the degeneration of tubular
epithelium in the testis was assumed to be an adverse but secondary
effect.
Six out of 20 females of test group 13
(1000 ppm) showed an increased incidence of luteal cysts in the ovary.
One female revealed a diffuse atrophy of the ovaries. Luteal cysts might
develop in case the follicle fails to ovulate. These findings were
regarded to be treatment-related and adverse.
Males and females of all treated
groups revealed cysts in the pars distalis of the pituitary gland which
were filled with a homogenous eosinophilic material and differed from
the spontaneously observed cysts (e.g. remnants of the Rathke’s pouch).
It could not be determined what material was present in these cysts and
whether they were functionally active or not. Thus this finding could
not be assessed with regard to potential adversity.
The results of the differential
ovarian follicle count (DOFC) – comprising the numbers of primordial and
growing follicles, as well as the combined incidence of primordial and
growing follicles – showed significant differences between the control
group 10 and animals of test group 13. The DOFC was performed in cohort
1A and cohort 1B animals together in a fully blinded manner. This
statistically significant decrease in primordial and growing follicles
was regarded to be adverse.
F1 rearing animals, cohort 1B
Target organs were the ovaries and
testes. No other organs were examined histopathologically.
The decrease in terminal body weight
in males and females of test group 13 (1000 ppm) reflects the body
weight effect at 1000 ppm in life.
In males and females of all treated
groups the liver weight was significantly increased which was regarded
to be treatment-related. Although a conclusive interpretation of
adversity cannot be made without a histopathological or clinical
pathological examination, similar effects as have been detected in
cohort 1A livers are the most likely explanation for this finding. The
reduced absolute and relative prostate weight in test group 13 (1000
ppm) males could be related to treatment. But as the terminal body
weight was also significantly decreased and the cohort 1B animals were
treated the same way as the cohort 1A animals and there were no
histopathologic findings in the prostate that could explain the weight
decrease beside the three affected animals, it was regarded to be
secondary to the body weight reduction. Three males of test group 13
(1000 ppm) had reduced testes sizes and corresponding reduced size of
the prostate and seminal vesicle that was thought to be secondary to the
testes findings. Microscopically, the gross lesion was related to
immaturity as described for cohort 1A. Two of these males had in
addition tubular degeneration and two males showed minimal tubular
degeneration without other findings. In test group 11 (100 ppm), one
male also revealed immature testicular epithelium and degeneration. As
there was no male affected in test group 12 (300 ppm) this finding was
regarded to be incidental. The immaturity and degeneration in test group
13 animals (1000 ppm) was regarded to be treatment-related and adverse,
however, as in the cohort 1A animals are most likely a secondary effect
of the body weight reduction, a hypothesis supported when the individual
animal data is taken into account.
Four females of test group 13 (1000
ppm) had macroscopically reduced ovaries that could be related
microscopically to a diffuse atrophy of the ovary. As described for
cohort 1A animals here were six females showing luteal cysts in the same
test group and in two females of test group 13 (1000 ppm) no corpora
lutea could be observed. These findings were regarded treatment-related
and adverse. The single animal in test group 12 (300 ppm) showing luteal
cysts was regarded to be incidental.
F1 rearing animals, cohort 3
(Immunotoxicity)
There was a reduction in terminal body
weight in test group 13 (1000 ppm) in males and females which reflects
the body weight effect at 1000 ppm in life.Macroscopically there was one
male showing reduced size of testes, epididymides, prostate and seminal
vesicle. As no microscopic investigation was performed a detailed
diagnosis could not be made. But considering the testis effects in the
other cohorts it is most likely the same finding here.
There were no indications from
clinical examinations as well as gross and histopathology, that
2,2’-iminodiethanol adversely affected the fertility or reproductive
performance of the F0 parental animals up to and including the
administered dose of 300 ppm. Estrous cycle data, mating behaviour,
conception, gestation, parturition, lactation and weaning as well as
sexual organ weights and gross and histopathological findings of these
organs (specifically the differential ovarian follicle count) were
comparable between the rats of these groups including control and ranged
within the historical control data of the test facility. The high dose
of the test item (1000 ppm) exerted effects on a number of parameters
such as number of implants and duration of gestation in the F0 parental
animals as well as estrous cyclicity and morphology of pituitary,
ovaries, testes (subsequently accessory sexual glands) and mammary
glands in the F1 offspring.
The high-dose F0 generation animals
were successfully paired, fertility and gestation indices were
comparable to the concurrent control. There was, however, a
significantly lower number of implants and subsequently lower litter
size noted along with a small but significant increase in duration of
gestation. There were no morphological changes detected in tissues
related to reproduction which could have explained these effects.
The high dose F1 generation females
had a prolonged estrous cycle and the cycle was irregular in a higher
number of females. There was no obvious pattern of change in the
affected females, the irregularities consisted likewise of prolonged
diestrous, estrous or metestrous. These irregularities corresponded with
ovarian atrophy, pituitary and luteal cysts as well as a decrease in
primordial and growing ovarian follicles, as described in the
pathology/neuropathology sections.
Some high-dose F1 males had smaller
and lightweight testes which were immature and, in some cases, showed
degeneration of tubular epithelia. Cysts in the pars distalis of
pituitary were also present. Most of the affected males exhibited
distinct clinical symptoms of systemic toxicity as well. In addition,
some high-dose F1 males had feminized and/or hyperplastic mammary
glands, while high-dose females showed an increase in secretion, as
described in the pathology section.
For all liveborn male and female pups
of the F0 parents, no test substance-induced signs of developmental
toxicity were noted at dose levels as high as 100 ppm. Postnatal
survival, pup body weight gain as well as post-weaning development of
the offspring of this test group until puberty remained unaffected by
the test substance. Furthermore, clinical and/or gross necropsy
examinations of the F1 pups revealed no adverse findings. The high dose
of the test substance (1000 ppm) caused effects on pup survival during
early lactation. While the lower litter size in this dose group was a
consequence of a lower number of implants and not due to prenatal or
perinatal mortality, the slightly lower viability index came from a
higher number of dead and cannibalized pups which were distributed
across 8 litters. In 2 of those 8 litters none of the pups survived.
Altogether, the lower viability index in this group was only slightly
below the historical control range. Postnatal survival after PND 4 of
the offspring of all test groups until weaning remained unaffected by
the test substance. Furthermore, clinical and/or gross necropsy
examinations of the weaned F1 pups revealed no adverse findings.
Pup body weight development of the mid
and high-dose F1 offspring (300 and 1000 ppm) was affected by the
treatment. These offspring had similar weights as the control right
after birth but gained significantly less weight than control offspring
from PND 4 (high-dose) or PND 14 (mid-dose) onwards. The decrease in
terminal body weight in the 300 and 1000 ppm male and 1000 ppm female
weanlings not selected for cohorts reflects the body weight effect in
life. The impairment of body weight gain in the F1 offspring continued
after weaning in the offspring selected for cohorts.
Measurement of thyroid hormones
revealed higher T4 values in F0 males of test group 3 (1000 ppm) as well
as in F1 rats of both sexes in test group 13 (1000 ppm) at PND 92 as
well as in F1 females of the same test group at PND 22 and in females of
test groups 11 and 12 at PND 4. However, no effects were observed on THS
levels.
Anogenital distance of all test
substance treated F1 pups (100, 300 and 1000 ppm) was comparable to the
concurrent control values, as were anogenital indices. In addition, the
check for the presence of nipples/areolas, also a very sensitive marker
of potential endocrine-mediated imbalances, revealed no test
substance-related effects at all. Vaginal opening and preputial
separation are commonly used developmental markers for onset of puberty
in laboratory rats. A statistically significant delay in vaginal opening
of about 1-2 days beyond the control was observed in the female F1
offspring of the mid- and high-dose groups (300 and 1000 ppm). The
values for pubertal age and weight of mid-dose females were both at the
lower end of the historical control range; thus the apparent statistical
increase is considered to be due to the very low concurrent control
values in this study and not treatment-related.
In the high-dose group the pubertal
age is just above the upper limit of the historical range while the
weight at puberty is below the historical control range. This clearly
indicates that the later onset of puberty is a consequence of a general
developmental delay and not a specific effect on the timing of puberty.
A statistically significant delay in preputial separation of about 2
days beyond the control was observed in the male F1 offspring of the
high-dose group (1000 ppm). In the high-dose group the pubertal age is
well within of the historical range while the weight at puberty is
distinctly below the historical control range. This indicates that the
apparent later onset of puberty may be a spurious finding, and if at
all, is a consequence of a general developmental delay and not a
specific effect on the timing of puberty. No clinical signs of
developmental neurotoxicity were evident in male and female F1 offspring
at dose levels as high as 300 ppm. There were no compound related
effects on motor activity, auditory startle habituation, and in the
field observation battery following exposure to the test compound in
these animals.
Some findings which might be related
to an affection of the nervous system were observed in the F1 offspring
at 1000 ppm. Clinically, high-stepping gait and piloerection were
observed recurring across all F1 cohorts in several study sections. The
only notable finding in neurobehavioral testing, however, were lower
maximum amplitudes in the auditory startle response test of the
high-dose F1 males and females in Cohort 2A, while FOB and motor
activity testing remained without findings. In the auditory startle test
there was also no habituation to the test environment seen in these
animals, males slightly more affected by this than females. Notably, no
corresponding effects were recorded for startle response latency. In
addition, regarding neuropathology, treatment-related findings were seen
in the medulla oblongata, spinal cord and pituitary gland of Cohort 2A
animals (adults, PND 77) as well as in the pituitary gland of Cohort 2B
animals (weanlings, PND 22).
F1 rearing animals, cohort 2A (adults)
The terminal body weight was decreased
in male and female test group 13 animals, reflects the body weight
effect at 1000 ppm in life.
The medulla oblongata and the spinal
cord of male and female animals of test group 13 (1000 ppm) revealed a
minimal to marked, multifocal degeneration of nerve fibers. This finding
was characterized by disintegrated myelin sheaths, vacuolation of myelin
sheaths, pyknotic nuclei of oligodendroglia and spheroids as well as
gitter cells within the lesions. The lesions were visible especially in
longitudinal sections and less in cross sections of the spinal cord. As
also in several control animals minimal spontaneous degeneration was
seen, only degeneration with a higher severity grade was assessed as
treatment-related and adverse. In the pars distalis of the pituitary
gland of male and female animals (all test groups), multifocally
distributed very small eosinophilic cysts with a non-ciliated, irregular
border and an eosinophilic homogenous content were seen. As no
functional or mechanistic data are available, this finding could not be
assessed with regard to potential adversity. The brain weight
determination, brain length and width measurements as well as brain
morphometry and neuropathological examination by light microscopy of all
other tissues did not reveal further treatment-related findings.
F1 rearing animals, cohort 2B
(weanlings)
The terminal body weight was decreased
in male and female test group 13 animals and in females of test group
12, which reflects the body weight effect at 300 and 1000 ppm in life.
In the pars distalis of the pituitary gland of four male and five female
animals of test group 13 (1000 ppm), multifocally distributed, very
small eosinophilic cysts with a non-ciliated, irregular border and an
eosinophilic homogenous content were seen. As no functional or
mechanistic data are available, this finding could not be assessed with
regard to potential adversity. There was no evidence that the test
substance produced any developmental immunotoxicity up to and including
a dose of 300 ppm in both sexes and 1000 ppm in males. Neither T-cell
dependent anti-SRBC IgM antibody response, nor absolute and relative
lymphocyte subpopulation cell counts in the spleen tissue (B-,
T-lymphocytes, CD4-, CD8-T-lymphocytes and natural killer (NK) cells)
displayed any treatment-related changes. However, a test
compound-related effect on the T-helper cells and cytotoxic T-cells in
the spleen in the high-dose F1 females (1000 ppm) cannot be excluded.
