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EC number: 947-977-8
CAS number: -
The aim of this study was to assess the
possible effects of Naphthalenesulfonic acid, bis(1-methylethyl)-,methyl
derivs., sodium salt on male and female fertility and embryofoetal
development after repeated dose administration in Wistar rats.
The test item was administered daily in
graduated doses to 3 groups of test animals, one dose level per group
for a treatment period of 63 days, i.e. during 14 days of pre-mating and
maximum 14 days of mating in both males and females, during the
gestation period and up to post-natal day 12 in females. Males were
dosed after the mating period until the minimum total dosing period of
28 days were completed. Animals of an additional control group were
handled identically as the dose groups but received aqua ad iniectabilia
(sterile water), the vehicle used in this study. The 4 groups comprised
10 male and 10 female Wistar rats. Before dosing all females were
screened for two weeks for regular estrous cyclicity and animals (10
females/ group) with regular estrous cycle (4-5 day cycle) were used in
The following doses were evaluated:
Control: 0 mg/kg bw/day
Low Dose: 30 mg/kg bw/day
Medium Dose: 200 mg/kg bw/day
High Dose: 700 mg/kg bw day /
500 mg/kg bw/day*
* = the high dose level was reduced from 700
mg/kg bw to 500 mg/kg bw after 6 or 7 days of dose application
(staggered start of dosing) due to overt toxicity.
The test item formulation was prepared once
in 10 days based on stability data. The test item was dissolved in aqua
ad iniectabilia and administered daily during 14 days of pre-mating and
14 days of mating in both male and female animals, during the gestation
period and up to post-natal day 12 in females. Males were dosed for 28
days. Dose volumes were adjusted individually based on weekly body
weight measurements. The administration volume was 5 mL/kg body weight.
During the period of administration, the
animals were observed each day for signs of toxicity. Animals that died
were examined macroscopically and at the conclusion of the test,
surviving animals were sacrificed and observed macroscopically.
Body weight and food consumption were
measured weekly, except for food consumption measurements which were not
taken during the mating period in female animals and the mating and
post-mating period in male animals.
Haematological and clinical biochemistry
evaluations were performed on blood samples collected at terminal
sacrifice from five randomly selected males and females from each group.
Urinalysis was performed on samples collected at terminal sacrifice from
five randomly selected males from each group.
Functional observations including sensory
reactivity to different stimuli, grip strength, motor activity
assessments and other behavior observations were performed in the week
before the treatment from all animals and in the last week of treatment
in five randomly selected males and females of each group.
After 14 days of treatment to both male and
female, animals were mated (1:1) for a maximum of 14 days. The
subsequent morning onwards the vaginal smears of females were checked to
confirm the evidence of mating. After the confirmation of the mating,
females were separated and housed individually. Each litter was examined
as soon as possible after delivery of the dam to establish the number
and sex of pups, stillbirths, live births, runts and the presence of
Live pups were counted, sexed and litters
weighed within 24 hours of parturition, on day 4 and day 13 post-partum.
The anogenital distance (AGD) of each pup was measured on PND 0. The
number of nipples/areolae in male pups was counted on PND 12.
From 2 pups/litter on day 4 after birth;
from all dams and 2 pups /litter at termination on day 13 and from all
adults males at termination, blood samples were collected from the
defined site. All blood samples were stored under appropriate
conditions. Blood samples from the day 13 pups and from the adult males
were assessed for serum levels for thyroid hormones (T4). Further
assessment of T4 in blood samples from adult females and day 4 pups were
not deemed necessary. Pup blood was pooled by litter for thyroid hormone
The males were sacrificed after completion
of the mating period on treatment day 29 and the females along with
their pups were sacrificed on post natal day 13. Non-pregnant females
were sacrificed on day 26.
The number of implantation sites and corpora
lutea was recorded for each parental female at necropsy.
Pups sacrificed on post-natal day 4 or 13
and those found dead, were carefully examined for gross external
A full histopathological evaluation of the
preserved tissues was performed on high dose and control animals, in non
pregnant female animals and male mating partners of the LD and MD
animals. These examinations were extended to animals of all other dosage
groups as treatment-related changes were observed in the high dose group
for stomach, liver and kidney. For the testes, a detailed qualitative
examination was made taking into account the tubular stages of the
spermatogenic cycle at evaluation of additional hematoxylin-PAS
(Periodic Acid Schiff) stained slides. All gross lesions macroscopically
identified were examined microscopically in all animals.
During the treatment period of this study,
few mortalities observed were male nos. 31, 35 (HD) found dead on
premating day (PMD) 6, male no. 38 (HD) was found dead on premating day
(PMD) 5, female no. 75 (HD) was found dead on premating day (PMD) 3 and
female no. 77 (HD) was found dead on premating day (PMD) 5.
Histopathologically, in females, gastric changes (ulceration in female
No. 75 and erosion in female No. 77) contributed to both morbidity and
mortality. In males, based on the histopathology investigation, the
cause of death was not evident although, there were slight to moderate
gastric changes were observed which might have contributed to the
In terminally sacrificed males, predominant
clinical signs observed during the treatment period (premating day 1 to
mating/post mating day 14) were moderately increased salivation in one
animal of MD and moving the bedding in two animals of MD group during
very few days of mating and postmating period. In HD group, major
clinical signs observed were slight to moderately reduced spontaneous
activity, half eyelid closure, moving the bedding, slight to moderate
salivation and piloerection in all animals during majority of premating
and mating/postmating period.
In terminally sacrificed females, major
clinical signs observed during the treatment period (Premating day 1 to
PND 12) were moving the bedding on one day during lactation period in
one female of LD group, moving the bedding, slightly to moderately
increased salivation in few animals on few days of MD group and moving
the bedding, slightly to severely increased salivation, slightly to
moderately increased piloerection, reduced spontaneous activity, half
eyelid closure in all animals on majority of days during treatment
period in HD group.
The clinical signs salivation and moving the
bedding were observed immediately after the dose administration and
therefore were considered to be a sign of discomfort due to a local
reaction to the test item rather than a systemic adverse effect and has
no toxicological relevance. However, clinical signs like slightly to
moderately increased piloerection, reduced spontaneous activity and half
eyelid closure in HD group could be attributed to treatment with the
None of the females showed signs of abortion
or premature delivery.
During the weekly detailed clinical
observation, no relevant differences between the groups were found.
In males and females, no relevant effects
were observed in any of the parameters of the functional observation
battery before and at the end of the treatment period except incidental
statistically significantly higher/lower count for few parameters before
the initiation of treatment or at the end of treatment when compared
with the controls. There were no biologically relevant differences
observed in body temperature between the groups.
Body Weight Development:
In males, there was no statistically
significant difference observed on body weight between the LD and the
control group during the entire study period. However, lower group mean
body weights from premating day 14 to terminal sacrifice were observed
in MD group without achieving statistical significance when compared
with the controls. A statistically significantly lower group mean body
weight was also observed in HD group from day 7 during entire study
period although statistical significance was achieved only on premating
day 7, 14 and mating/postmating day 7 when compared with the controls.
In correlation to body weight, group mean
body weight gain was statistically significantly lower during
mating/post mating day 7-14, premating day 1-mating/postmating day 14
and premating day 1 to terminal sacrifice in MD group males when
compared with the controls. There was also statistically significantly
lower group mean body weight gain observed during premating day 1-7,
premating day 1 to mating/postmating day 14, premating day 1 to terminal
sacrifice and statistically significantly higher group mean body weight
gain during premating day 7-14 and mating/postmating day 7-14 in HD
group when compared with the controls.
In females, no statistically significant
difference in group mean body weight was observed in treatment groups
during entire study period when compared with controls. There was
statistically significantly higher group mean body weight gain observed
during premating day 7-14 in HD group when compared with the controls.
This statistically and biologically
significant effect on body weight and body weight gain in MD and HD
group male was considered as test item related and toxicologically
relevant. However, statistically significant effect on female group mean
body weight gain in HD group was attributed to possible compensatory
recovery after the reduction of dose from second week of the study.
In males, the food consumption during
treatment period tended to increase with the progress of the study in
the control, the LD, the MD and the HD group. However, In HD group,
during premating day 1-7, food consumption was lower without achieving
statistical significance compared to the control group and this effect
on food consumption in males was considered to be test item related.
In females, no statistically significant
effect on food consumption was observed during premating period,
gestation and lactation period in treatment groups when compared with
the controls. However, in correlation to body weight development,
marginally lower food consumption was observed in HD females during
premating day 1-7 when compared with the controls. As this effect on
female food consumption was marginal and in the light of no significant
effect on body weight development, this effect on food consumption in
females was not considered to be adverse.
There were no considerable differences in
the length or sequence of cycle stages between the LD and MD dose groups
and the control group. However, statistically significantly higher mean
cycle length and lower number of normal cycles were observed in HD group
females when compared with controls. Furthermore, 6/8 females were
observed with no single estrus cycles (acyclicity) and exhibited
persistent diestrus stage. In the light of no effect on pregnancy rate
and various reproductive indices in HD group, this effect on estrus
cyclicity in HD group was considered as a secondary effect due the
treatment with the test item.
There were no test item treatment related or
statistically significant effects observed in treatment groups on litter
data parameters like group mean total number of pups born, number of
male pups, number of female pups, sex ratio, number of live pups, still
birth, runt on PND 0 as well as number of live pups, male pups, number
of female pups and sex ratio on PND 4 and PND 13 when compared with the
Litter Weight Data:
There was no statistically significant
effect on pup mean weight, total litter weight, female litter weight on
PND 0, PND 4 and PND 13 observed in treatment groups when compared with
the controls. However, in correlation to low number of male pups and sex
ratio, lower group mean male litter weight values were observed on PND
0, 4 and 13 although statistical significance was achieved only on PND 0
and 4. This decrease in male litter weight in HD group was attributed to
low male pups in few females (71, 73 and 78) of the HD group. Therefore
this effect on male litter weight was not considered to be test item
related and assumed to be biological variation.
Precoital Interval and Duration of
There was no effects on the duration of
gestation. However, precoital interval was statistically significantly
higher in HD group females compared to the controls. As this difference
in precoital interval was marginal (1 day) and therefore considered as
biological variation and not related to treatment with test item.
Pre and Post-Natal Data:
There were no test item treatment related
effects observed on the number of corpora lutea, implantation sites,
live pups on PND 0, 4 and 13, percent preimplantation loss and post
implantation loss in treatment groups when compared with the control
There were no test item related effects on
the reproductive indices (copulation, fertility, viability and delivery
indices) in the dose groups when compared to the control group.
Pup Survival Data:
No effect on mean mortality of pups between
PND 0 and PND 4 and during PND 4-13 in treatment groups when compared to
the control group and all pups survived until terminal sacrifice on PND
Anogenital Distance and Nipple Retention:
In males, statistically significant lower
pup weight and cube root of pup weight on the day of anogenital
measurement was observed in male LD and HD group (not dose dependent)
when compared with the controls. There was also statistically
significantly higher absolute (not in HD group) and relative anogenital
distance in MD and HD group observed when compared to the controls.
In females, statistically significantly
higher absolute and relative anogenital distance was observed in all
treatment groups when compared to the controls.
In male and females, parameters like pup
body weight, litter size and sex ratio were not affected in LD and MD
groups and these parameters are correlated with anogenital distance
(AGD) although statistically significant group mean AGD value in MD
group males and LD and MD group females were observed. Therefore effect
on AGD in LD and MD group males and females cannot be considered as test
In HD males effect on absolute and relative
AGD was not consistent and dose dependent although statistically
significantly higher relative AGD in HD group was observed when compared
to the controls. In females also, as no effect on body weight, litter
size and sex ratio was observed, marginal higher but statistically
significant effect on female absolute and relative AGD was not
considered to be adverse.
All anogenital values in male and female
pups were within historical control data range. AGD is not the only
parameter to confirm the endocrine disruption and AGD itself needs to be
correlated with lot of other parameters in the study. No additional
finding in the study supporting a possible androgen-mediated activity
(agonistic) or any other endocrine disruption modality of the test item.
Thus, there is no conclusive evidence of an endocrine disrupting effect
of the test item and as a result of that, the findings on AGD cannot be
considered as adverse.
Additional comments indicating that
statistical relevenat efefcts on AGD are of no biological consequence:
The AGD of the females are still below the average historical control
for all does groups, and additionally the variability (SD) is
exceptionally low when compared variability in historical control data.
Besides, there is hardly any dose related increase visible. Also the
indicated AGD increase in males is not biologically relevant. Also here,
the increase is only minimal and only slightly above the average
historical control, also for the control group. Besides, studies with
testosterone indicated that specifically AGD did not increase:
“masculinization in males appears to operate at maximum capacity with
normal endogenous testosterone concentrations.” (Welsh M. et al., 2008,
Identification in rats of a programming window for reproductive tract
masculinization, disruption of which leads to hypospadias and
cryptorchidism). This suggests that an observed increase in AGD in males
is of no biological significance.
No statistically significant effect of
toxicological relevance was observed on nipple retention in the pups of
any of the groups when compared with the controls.
Thyroid Hormone (T4) Analysis:
No test item related effect of toxicological
relevance or statistical significance was observed on pup thyroid
weight, male and PND 13 pup thyroxine hormone (T4) in the treatment
groups when compared to the controls.
Pup External Findings:
No test item related gross external
abnormalities of toxicological relevance on PND 0-12 and death were
observed in the pups of any of the groups.
Haematology and Coagulation:
In males sacrificed at the end of treatment
period, no test item related adverse effects were observed for
haematological parameters. However, there was a statistically
significantly higher platelets (PLT) count observed in HD group compared
to the control group. All group mean and most of the individual values
were within the historical control data range.
As group mean value was within historical
control data limit, statistically significant effect on PLT in HD group
was considered to be incidental and not related to the treatment with
test item. No test item related effect was observed on coagulation
parameters when compared with the controls.
In females sacrificed at the end of
treatment period, no statistically significant or test item effect
observed on any of the haematology or blood coagulation parameters when
compared with the controls. All group mean and most of the individual
values were within the historical control data range.
In males and females sacrificed at the end
of treatment period, no test item related or statistically significant
effect on any clinical biochemistry parameter in treatment groups was
observed when compared with the control.
The urinalysis performed in selected male
and female animals sacrificed at the end of treatment period revealed no
test item treatment related effect and all urinary parameters were in
the normal range of variation. High protein levels were found in the
urine of few male and females of all groups including control group.
Therefore, this effect on urine parameters was not considered to be test
Few specific macroscopic changes were
recorded for the male and female animals, which based on microscopic
examination were not considered to be of test item treatment relevance.
However, In decedents, red discoloration observed in the thymus of males
no. 35 and No. 38 of HD group was correlated microscopically with
congestion and hemorrhage in male no. 35 and with hemorrhage in male no.
38. The above mentioned changes were considered incidental agonal
changes which are occasionally found in animals being subjected to
necropsy. In survivors, small testes and epididymides recorded in male
no. 14 (LD group) correlated microscopically with testis and epididymis
atrophy and aspermia. In male No. 21 (MD group) small epididymis on the
left side was correlated microscopically with unilateral epididymis
atrophy and aspermia. In addition, in the male no. 30, a dilated right
kidney was correlated microscopically with pelvic dilatation. In female
No. 59 (LD group), the observed diaphragmal herniation corresponded
histologically to a hepatic nodule.
The above mentioned changes were considered
to be most likely incidental in nature.
In males sacrificed at the end of treatment
period, there were statistically significantly higher absolute liver
weights in all treatment groups (except MD group), higher relative (to
body weight) liver weights in MD and HD group and higher kidney weights
in LD and HD group although statistical significance only achieved in LD
group when compared with the controls. Increased liver absolute weights
were histologically correlated with slight hepatocellular hypertrophy
only in one animal from the HD group, whereas in the MD and LD group
neither the absolute nor the relative increase in liver weight
correlated with underlying histological changes. Therefore, without a
dose dependent correlation between liver weight increase and the
observed histopathological hepatic changes and in absence of
hepatic-derived enzyme profiling, the toxicological relevance of the
liver weight increase could not be established.
In females sacrificed at the end of
treatment period, significantly higher absolute liver weights were
observed in HD group when compared with the controls although
statistical significance was not achieved.
There were also statistically significantly
higher relative (to body weight) kidney weights observed in HD group
when compared with the controls. In females, statistically significantly
higher relative kidney weights in HD group were considered of no
toxicological relevance in absence of correlating histological changes.
There were a number of early decedents. in
females, gastric changes (ulceration in female No. 75 and erosion in
female No. 77) contributed to both morbidity and mortality. In males
(31, 35 and 38) based on the histopathology investigation, the cause of
death was not evident although, there were slight to moderate gastric
changes were observed which might have contributed to the morbidity. The
above mentioned changes were associated with vacuolization of
forestomach epithelial cells (epithelial vacuolization), hyperkeratosis,
mixed cell infiltrates and squamous hyperplasia. In male stomach,
hyperkeratosis and squamous hyperplasia were observed. In liver minor
degree of hepatocytes vacuolation (fatty change) and centrilobular
hypertrophy was observed in few males and females.
In Survivors of LD, MD and HD groups, test
item related histopathology changes were observed in liver, kidney and
In liver, minor degree of centrilobular
hepatocytes hypertrophy was observed only in few males from the HD group
and was considered test item related. In kidney, minimal to slight
hyaline droplets accumulation in tubular epithelial cells were observed
in the majority of males from all dose groups and the control group.
This renal change is a male rat specific phenomenon of no toxicological
relevance in humans. Minimal to slight tubular basophilia was observed
in few animals from the high dose group only. The tubular basophilia was
considered most likely related to the test item administration. In
stomach, moderate forestomach ulceration accompanied by severe
multifocal mixed cell infiltrates, moderate submucosal edema wall and
epithelial vacuolization was observed in one male from the MD group.
Minimal to moderate mixed cell infiltrates mainly located at the
limiting ridge and sometimes extending in the adjacent forestomach and
glandular stomach submucosa of males and females from the LD and MD
groups and only in males from the HD group. Minimal to moderate
epithelial vacuolization was noticed in few males from LD, MD and HD
dose groups only. Furthermore, a minor degree of hyperkeratosis was
observed in some males from the LD and HD dose groups, whereas slight to
moderate squamous hyperplasia was present only in few males from the HD
group. These changes in stomach were considered to be most likely
related to the test item administration.
The test item did not produced any
histological evidence of toxicity in the male and female reproductive
organs and tissues.
In conclusion, due to the early mortality
observed in males and females from the high dose group and the presence
of several histopathological adverse changes in different organs, a
histomorphological NOAEL (no observed adverse level) could be
established at 30 mg/kg bw/day for the male rats and 200 mg/kg bw/day
for female rats.
Dose Formulation Analysis:
Dose formulation analysis for nominal
concentration revealed that nominal concentrations for all formulations
were confirmed throughout the study period as measured concentrations
were within acceptance criterion of 10 %.
On the basis of this combined repeated dose
oral toxicity and reproduction/ developmental toxicity screening test
with Naphthalenesulfonic acid, bis(1-methylethyl)-,methyl derivs.,
sodium salt in male and female Wistar rats with dose levels of 30, 200,
and 500/700 mg/kg body weight day the following conclusions can be made:
- There were few mortalities observed in the
study (3 males and 2 females) during the early days of the study.
Histopathologically cause of the death could not be established for all
of them. In females, gastric changes (ulceration in female No. 75 and
erosion in female No. 77) contributed to both morbidity and mortality.
- No adverse effects of test item were found
on male and female clinical observations in LD and MD group, clinical
signs like slightly to moderately increased piloerection, reduced
spontaneous activity and half eyelid closure in HD group could be
attributed to treatment with the test item.
- No adverse effects of test item were found
on female body weight development and food consumption in any treatment
group. However, test item related effect on male body weight development
observed in MD and HD group and on food consumption in HD group.
- Functional observations, haematology and
coagulation, clinical biochemistry, urinalysis, gross pathological
findings at necropsy and organ weight remained unaffected in male and
females up to dose levels of 500/700 mg/kg bw/day.
- There were also no effects on litter data,
litter weight data, nipple retention, precoital interval and duration of
gestation, reproductive indices, pup thyroid weight and parental male
and pup thyroxine hormone, pre and post-natal data, pup survival and pup
external findings on PND 0 and at death observed up to dose levels of
500/700 mg/kg bw/day.
- There were test item related effects
observed on estrous cyclicity in female HD group (secondary effect
without effect on pregnancy rate) and anogenital distance in male and
female HD group when compared with the controls. However, all anogenital
values in male and female pups were within historical control data range
and no additional finding is supporting a possible androgen-mediated
activity (agonistic) or any other endocrine disruption modality of the
- Histopathologically, in Survivors of LD,
MD and HD groups, test item related histopathology changes were observed
in liver, kidney and stomach. However, the test item did not produced
any histological evidence of toxicity in the male and female
reproductive organs and tissues.
The report concluded that,
due to the early mortality observed in males and females from the high
dose group (at 700 mg/kg bw/day in first week) and the presence of
several histopathological adverse changes in different organs, a
histomorphological NOAEL could be established at 30 mg/kg bw/day for the
male rats and 200 mg/kg bw/day for female rats.
Close examination shows that the only
effects observed at MD (200 mg/kg) are a slight lower BW compared to
control (-6%) in males, and an increased combined effects in stomach
upon histopathological examinations in males and females. (See attached
All other effects are observed at HD at 700
mg/kg bw (mortality and effects on oestrous cycle first week study) and
500 mg/kg (Bw males, slight effects food consumption, clinical signs,
slight effects on liver and kidney weights, and increased platelets in
No adverse effects were observed on
reproduction an developmenatl parameters, and the NOAEL for reproduction
and development in this study established at 500 mg/kg bw/day.
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