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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
fertility, other
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2004
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
2004

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
The effect of 2,5-Hexanedione on the fertility of male rats is examined.
GLP compliance:
not specified
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
not specified

Test animals

Species:
rat
Strain:
Wistar
Sex:
male
Details on test animals and environmental conditions:
MALE WISTAR RATS, approximately 10 weeks old,
were used in the current study. The health status
of the rats were checked and then acclimated to the
laboratory environment for 2 weeks before the experiment.
The room temperature and relative humidity were
set at 24°C and 54%, respectively. The light was controlled
to provide a 12 h light cycle and a 12 h dark cycle. All
the animals were kept in stainless-steel cages and given
pelleted commercial laboratory animal food and water
throughout the study.

Administration / exposure

Route of administration:
subcutaneous
Vehicle:
unchanged (no vehicle)
Details on exposure:
Male rats were randomly allocated to three treatment
groups. Each group had six rats and each group was
given 2,5-HD subcutaneously at dose levels of 100, 200
and 400 mg/kg per day for 5 days per week (no
injection given on the other 2 days i.e., resting days) for
12 weeks. The control group had 10 rats which were
given only food and water throughout the experiment.
The concentration of the doses were based on the most
recent bodyweights. The dose levels and period were
selected as most likely to cause toxicity based on preliminary
studies on both testicular and nerve toxicity.
Details on mating procedure:
not applicable
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
12 weeks
Frequency of treatment:
5 days/week
Doses / concentrations
Remarks:
Doses / Concentrations:
100, 200, 400 mg/kg body weight
Basis:
nominal conc.
No. of animals per sex per dose:
6 animals per group, 10 control animals
Control animals:
yes, concurrent no treatment
Details on study design:
Male rats were randomly allocated to three treatment
groups. Each group had six rats and each group was
given 2,5-HD subcutaneously at dose levels of 100, 200
and 400 mg/kg per day for 5 days per week (no
injection given on the other 2 days i.e., resting days) for
12 weeks. The control group had 10 rats which were
given only food and water throughout the experiment.
The concentration of the doses were based on the most
recent bodyweights. The dose levels and period were
selected as most likely to cause toxicity based on preliminary
studies on both testicular and nerve toxicity.
Positive control:
No

Examinations

Parental animals: Observations and examinations:
On day 90 of 2,5-HD treatment, all the rats were anesthetized
with diethyl ether, weighed and killed. The
testis and epididymides were collected for analysis of
sperm motility and count. The right-sided epididymis
was excised and placed in a pre-warmed Petri dish
containing 1 mL of calcium and magnesium free Hank’s
solution at 37°C. The tissue was minced with scaples
for approximately 60 s and then placed in a 37°C incubator
for approximately 20 min prior to determining
the sperm motility. The suspension was stirred and one
drop was placed on to a pre-warmed slide and a coverslip
added. A minimum of 10 microscopic fields were
observed for each slide at 400× magnification using a
standard optical microscope and the percentage of motile
sperm were counted. For the morphological examination,
another drop of the sperm suspension was added
to another slide and then a thin smear was made and
stained with Papanicolau and the morphology of 200
sperm cells was assessed for each slide.
The left-sided epididymis and testis were frozen immediately
until evaluation. After thawing at room temperature,
the whole epididymis and the testis specimens
were homogenized separately in 1 mL of a solution of
0.9% NaCl containing 0.01 mL Triton X-100. The testis
and epididymis homogenates were each diluted with
1.5 mL of the same solution and spermatozoa and
spermatids were counted 400× in a Neubauer hemocytometer
(Erma, Tokyo, Japan) and three counts per sample
were averaged.
Sperm parameters (parental animals):
Histologic examination of the testis was performed after
fixing the right-sided testis in a formalin solution. Sixmicron
thick paraffin sections were stained with hematoxylin
and eosin and examined by light microscopy.
Statistics:
Data were analyzed using the Levene’s test for equality
of variances. If P > 0.05, the variances were considered
equal and the Student’s t-test was calculated and P < 0.05
was considered as significant. If P < 0.05, then the variances
were considered unequal and a Wilcoxon signed-ranked
test was also performed and P < 0.05 was considered
significant.

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
not specified
Food consumption and compound intake (if feeding study):
not specified
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
testicles
Other effects:
not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
effects observed, treatment-related
Description (incidence and severity):
adverse effects observed
Reproductive performance:
not examined

Effect levels (P0)

open allclose all
Dose descriptor:
other: reduced sperm motility
Effect level:
ca. 100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Dose descriptor:
other: no sperm motility, maturation arrest
Effect level:
ca. 200 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Dose descriptor:
other: no sperm motility, reduced sperm concentration, testicular injury
Effect level:
ca. 400 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male

Results: F1 generation

General toxicity (F1)

Clinical signs:
not examined
Mortality / viability:
not examined
Body weight and weight changes:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings:
not examined

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
In conclusion, we have shown that 2,5-hexanedione
severely affected sperm motility even at low doses, whereas
high doses adversely affected all the sperm parameters as well
as causing testicular injury.
Executive summary:

No sperm motility was observed in the 200 mg and

400 mg/kg per day treatment groups and significantly reduced

motility was observed in the 100 mg/kg per day group. The

morphology were also significantly reduced in the 200 mg and

400 mg/kg per day groups compared to the control group,

but the sperm concentration was significantly reduced only in

the 400 mg/kg per day group. Histological examination of

the testes in the 400 mg/kg per day group revealed that twothirds

of the testes had Sertoli cell only syndrome, whereas in

the 200 mg/kg per day group half of the testes showed maturation

arrest and sperm as well as spermatids were observed in

83% of the testes.