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EC number: 200-854-6
CAS number: 75-25-2
Effect of the test substance on reproductive
function in Swiss D-1 Mice.
The effect of the test substance on
reproduction and fertility in Swiss CD-1 mice was assessed using a
United States National Toxicology Program Reproductive Assessment by
Continuous Breeding Protocol (RACB).
Male and female Swiss CD-1 mice were used
for the study.
The RACB protocol is divided into four
tasks. Task 1 is 14-day toxicity range-finder, Task 2 is a continuous
breading phase, Task 3 is a 1-week mating trial and task 4 is an
assessment of offspring. As the test substance had no
effect on fertility Task 3 was not conducted as part of this study.
Following dose range-finder study (Task 1)
the dose levels selected for Task 2 were 50, 100 and 200 mg/kg bw/day. For
task 2 there were 20 male and female breeding pairs per treatment group
and 40 male and female breeding pairs for control animals). Animals
were dosed daily by gavage using corn oil as the vehicle for a 7-day
pre-cohabitation and a 98-day cohabitation period. The
last litter born during the holding period following the continuous
breeding phase is reared by the dam until weaning, after which treatment
is initiated by the same route and at the same concentration as during
Task 2. These animals are used for assessment of
second generation fertility (Task 4).
Endpoints assessed were clinical signs,
parental body weight and average consumption of drinking water during
representative weeks, fertility (number producing a litter/number of
breeding pairs), litters per pair, live pups per
litter, proportion of pups born alive, sex of live pups and the pup body
weights immediately after birth. For Task 4 the last and generally the
fifth litter was reared and weaned from the control and 200 mg/kg and
kept to sexual maturity (74 ± 10 days) while housed by sex two per cage
(maximum) and receiving the same treatment as their parents. At sexual
maturity a male and female from different litters within the same
treatment group are cohabited for 7 days and then housed singly until
delivery. The endpoints assessed for this mating trial are the same as
Task 2 with the addition of checking for the presence of a copulatory
Six mice died or were sacrificed during this
task. For two mice the cause of death could not be
determined. The deaths of the other four mice were not
considered related to treatment.
Fertility index was 100% for control and
test substance groups (breeding pair was designated fertile if they
produced at least one live or dead pup. The control and bromoform groups
did not significantly differ in number of litters per pair, litter size,
proportion of live pups and proportion of male pups, female pup weight
or combined pup weight at birth. Male absolute pup weights in the 200
mg/kg test substance group were significantly less than control values
but this difference was not significant after pup weights were adjusted
for litter size. The cumulative days to litter were
essentially identical across groups.
Body weights at delivery of dams receiving
200 mg/kg of test substance were consistently less than control values;
these reduced body weights were statistically significant after
delivering the first, second, fourth and fifth litters. During the Task
2 holding period, body weights of lactating dams did not differ among
control and treatment groups.
Male and female adults were weighed on
representative weeks during Task 2 and simultaneously, the water
consumption per cage, or per pair, was monitored. Test substance
treatment had no significant effect on body weight of males or females.
The average daily consumption of drinking water was significantly
increased in males and females treated with 50 mg/kg of test substance
during weeks 2 and 10. In the 100 mg/kg dose group, male and female
consumption was significantly increased during week 10. Consumption was
also significantly increased in the 200 mg/kg dose group for males and
females during weeks 2, 10, and 14.
Eleven F1 mice died during this task. The
cause of death for four mice was not determined. The
remaining deaths were not considered treatment related.
The postnatal survival among F1 pups in the
200 mg/kg bw/day test substance group was significantly lower compared
to the control group. This decline was primarily attributed to three
dams, which lost all their pups by postnatal day 4. One dam in the
control group also lost her litter by postnatal day 4. Although these
incidences are too low to evaluate statistically, they are consistent
with a treatment effect on early maternal behaviour, early lactational
failure, or postnatal developmental processes.
Test substance treatment had no apparent
effect on fertility and reproduction in F1 mice. Some symptoms of
general toxicity were noted in treated F1 mice. This included decreased
male terminal body weights, increased adjusted liver weights in both
sexes with accompanying hepatocellular cell degeneration and decreased
adjusted kidney weights in both sexes. Epididymal ductal epithelium
degeneration was noted in both control and bromoform-treated F1 males,
and was not considered treatment-related.
Treatment with the test substance at up to
200 mg/kg bw/day had no effect reproduction or fertility in either the
parental or F1generation. Treatment
with test substance at 200 mg/kg treatment caused minimal to moderate
histopathologic changes in the liver of second generation CD-1 mice.
The No Observed Effect Level (NOEL) for the
study is considered to be 100 mg/kg bw/day.
Teratogenic effect of the test susbtance in
The teratogenic potential of the test
ratswas assessed using a study design that pre-dated the OCED 414
guideline, but which nevertheless covered many of the endpoints included
in the guideline.
Test substance was administered in corn oil
by gavage to pregnant Sprague-Dawley rats from day 6 to day 15 of
gestation at doses of 50, 100 or 200 mg/kg/day. A
control group was administered corn oil only. Each
group consisted of 15 rats and each rat was housed individually with
free access to food and water.
On day 22 of gestation rates sacrificed and
their viscera including the uteri were examined for pathological
changes. The foetuses were removed, weighed individually and examined
for viability and external malformations. Two pups from each dam were
fixed in formalin for histological evaluation. Approximately two-thirds
of the remaining live foetuses from each litter were placed in absolute
ethanol for future staining of the skeleton with Alizarin red and
subsequent examination for skeleton abnormalities. The rest of the
foetuses were fixed in Bouin's fluid and studied for visceral changes.
Maternal rats were subject to gross
pathological assessment and the liver, hear, brain spleen and one kidney
were removed and weighed. The following tissue samples
were taken from each animal and fixed: brain, heart, pituitary, thyroid,
parathyroid, thymus, lungs, trachea and bronchi, bronchial node, liver,
kidney, adrenal gland, spleen, skeleton muscle, peripheral nerve,
salivary gland, skin, bone marrow, ovaries, uterus and bladder.
Maternal blood samples were taken for
haematological and clinical chemistry assessments.
Following gross pathological examination a
liver sample was taken from maternal animals for liver protein
aniline hydroxylase (AH) and aminopyrine demethylase (APDM) activity.
Body and Organ Weights: There were no
effects on maternal weight, liver, kidney or spleen weights resulting
from treatment with test substance.
Foetal effects: There were no effects or
litter size, incidence of resorptions, foetal weight or incidence of
visceral anomalies resulting from treatment with test substance.
Histopathology: There were
no dose related histological, changes in mother or foetuses resulting
from treatment with test substance.
haematological parameters assessed showed no affects from treatment with
Clinical Chemistry: Maternal
clinical chemistry parameters assessed showed no affects from treatment
with test substance.
Liver Enzymes: Maternal liver enzyme
parameters assessed showed no affects from treatment with test substance.
Foetal toxicity: There was some of evidence
of dose related increase in sternebra aberrations and observations of
interparietal anomalies in foetuses at 100 and 200 mg/kg bw/day. These
were considered to be the result of a foetal toxicity effect and not
The test substance was not considered
teratogenic to Sprague-Dawley rats. The study No Observed Effect Level
(NOEL) for teratogenicity was 200 mg/kg bw/day (highest dose
There was some evidence of a foetal toxicity
caused by the test substance. The study No Observed Adverse Effect Level
(NOAEL) for foetal toxicity was considered to be 100 mg/kg bw/day.
There was some of evidence of dose related
increase in sternebra aberrations and observations of interparietal
anomalies in foetuses at 100 and 200 mg/kg bw/day. These
were considered to be the result of a foetal toxicity and not
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