  SEQ CHAPTER \h \r 1 ICF Consulting Review of Yamada et al. (2003)

ICF has been asked to review the journal article, “Exposure to
1-bromopropane (i.e., n-propylbromide) causes ovarian dysfunction in
rats” by Yamada and coworkers (2003).  Details regarding the study
design, results, and major conclusions are detailed in the following
text.  Further, ICF will discuss the relevance of this study to other
studies in rodents and will discuss whether this study allows for the
identification of an alternate NOAEL/LOAEL for the determination of an
Acceptable Exposure Limit for n-propylbromide.	

								

Study Design

Female Wistar rats at 10 weeks of age (and presumably nulliparous), were
exposed to target concentrations of 0 (fresh air), 200, 400, or 800 ppm
n-propylbromide (10/group) in whole-body inhalation chambers for 8
hours/day, 7 days/week for 12 weeks. For three weeks prior to exposure,
the estrous cycles of the rats were monitored.  Measured concentrations
of nPB in the dose groups were the following:  0, 200±8, 415±19, and
813±28 ppm (mean ± SD).  After 7 weeks of exposure, all the rats at
800 ppm became seriously ill and were sacrificed.  Rats from the
remaining dose groups were decapitated on the day of diestrus I in the
13th week. 

The estrus cycle was monitored during the study period via the analysis
of vaginal smears taken at the same time each day.  The estrus cycle was
divided into four periods: proestrus, estrus, diestrus I, and diestrus
II.  Normal estrus cycles were those containing each defined period,
which typically lasted 4-6 days.  The 15-week study period was divided
into the three week pre-exposure period, and 4 exposure periods of 3
weeks in length.  If a cycle overlapped two consecutive study periods,
then 0.5 cycle was counted in each period.  Cycles were defined as
regular (3-5 cycles/period), irregular (0.5-2.5 cycles/period), or
absent (no cycle; 0 cycles/period).

μm serial sections).  Differential follicle counts were determined as
primordial (types 1-3b), growing, (types 4-5b), and antral (types 6-8). 
The follicles were differentiated by the following pathologic
characteristics: primordial, oocytes either with or without a complete
one-layer ring of granulosa cells; growing, oocytes with several layers
of surrounding granulosa cells without antrum (cavity within the
follicle) formation; antral, large oocytes with multiple layers of
surrounding granulosa cells and fluid-filled antrum.

Study Results

Body weights were significantly decreased in all females at 800 ppm
starting at week 2 and continuing to week 7 (the rats lost 17% of their
pre-exposure body weight, rather than gaining weight during the exposure
period); the animals were sacrificed during week 8 because of
moribundity.  The body weights of the other exposure groups did not
differ significantly from the controls during any exposure week.  Forty
percent of the high-dose rats (4/10) had irregular estrous cycles during
weeks 1-3 of exposure, with the remaining females (6/10) having regular
cycles.  This trend toward cycle irregularity continued in the high-dose
females, with 50% having irregular cycles at 4-6 weeks of exposure and
50% having no cycle during this period.  After 1-3 weeks exposure, 1/10
females at 200 ppm nPB had an irregular cycle, with 9/10 at this
concentration having a regular cycle.  All of the females at 400 ppm had
regular cycles after 1-3 weeks exposure.  At every subsequent exposure
period, the females at 200 ppm exhibited regular cycles.  After 4-6
weeks exposure to nPB, one female at 400 ppm which had no cycle, while
the remaining 9/10 had regular cycles.  At 7-9 weeks, 3/10 females at
400 ppm had irregular cycles, 7/10 had regular cycles.  At 10-12 weeks
exposure, 4/10 had regular cycles, 5/10 had irregular cycles, and 1/10
had no cycle.  The data are presented in Table 1 below.

Table 1.  Changes in Estrus Cycle in Female Rats Exposed to
n-Propylbromide

Dose

(ppm)	Before Exp.	1-3 Weeks	4-6 Weeks	7-9 Weeks	10-12 Weeks

	Reg	Irreg	None	Reg	Irreg	None	Reg	Irreg	None	Reg	Irreg	None	Reg	Irreg
None

0a	9	0	0	9	0	0	9	0	0	9	0	0	9	0	0

200	10	0	0	9	1	0	10	0	0	10	0	0	10	0	0

400	10	0	0	10	0	0	9	0	1	7	3	0**	4	5	1**

800	10	0	0	6	4	0*	0	5	5**







Numbers per dose group were as follows: Control, 9; 200 ppm, 10; 400
ppm, 10; 800 ppm, 10.

*p<0.05; **p<0.01

  

Reproductive organ weights in the 200- and 400-ppm groups did not differ
from the controls.  Because the 800-ppm rats were sacrificed early,
their body and organ weights were not included in the statistical
analysis.  Absolute and relative liver and kidney weights were increased
in the 200- and 400-ppm females compared to the controls.  No abnormal
histopathological findings in these organs were reported in these dose
groups, however.  In the 800-ppm females, mild dilatation of the
proximal tubules was noted in the kidneys as well as scattered
cytoplasmic degeneration in hepatocytes located around the central veins
in the liver, which was accompanied by nuclear pyknosis.  No serious
changes (e.g., diffuse or focal necrosis) were reported. 

Changes in the number of the different types of ovarian follicles were
noted among the exposure groups (data in Table 2 below).  The 200-ppm
group did not exhibit any significant histopathology in the ovaries, but
the number of antral follicales was significantly decreased (59%)
compared to controls.  The number of growing and primordial follicles
was not significantly different at this dose.  The number of growing and
primordial follicles at 400 ppm was significantly decreased compared to
controls (76% and 33%, respectively).  The numbers of developed
follicles was further decreased in the high-dose females sacrificed
after 7 weeks, but it is unclear whether this is because of the younger
developmental age of the rats or because of the exposure, or both.  No
changes in LH or FSH were noted in the 200- and 400-ppm dose groups.

Table 2.  Changes in Ovarian Follicle Development in nPB-Exposed Female
Rats

	Control (n=8)	200 ppm (n=9)	400 ppm (n=9)	800 ppm (n=9)

Exposure Duration	12 weeks	12 weeks	12 weeks	7 weeks

Primordial	176.8±48.8	157.8±49.4	206.0±66.6	423.1±140

Growing	70.0±20.3	53.4±17.9	47.2±17.3*	30.1±15.1

Antral	30.1±22.4	12.6±4.82*	7.44±6.52**	3.78±3.87



These data suggest that nPB is affecting the maturation of ovarian
follicles; the mechanism is unclear, based on the data.  Although there
was a significant decrease in antral follicles at 200 ppm, there were no
changes in estrus cycling at this exposure concentration.  A decrease in
the number of female rats exhibiting regular estrus cycles was observed
in 400-ppm females during 7-9 weeks of exposure and later.  Therefore, a
NOAEL of 200 ppm is identified with a LOAEL of 400 ppm for the changes
in estrus cycles.  The data from this study can be used to determine an
alternate benchmark dose for female reproductive effects.  Without
performing the benchmark dose analysis, it is not clear whether these
data provide a more sensitive endpoint of nPB-induced reproductive
toxicity than sperm motility in exposed males.

References

Yamada T, Ichihara G, Wang H, et al. 2003.  Exposure to 1-bromopropane
causes ovarian dysfunction in rats.  Toxicol Sci 71:96-103.

