Document ID: EPA-HQ-OAR-2002-0064-0172
Agency: epa
Document Type: Supporting & Related Material
Title: 
Posted Date: 2007-03-28T04:00Z

Two Generation Toxicity Study in Sprague-Dawley Derived Rats

with n-Propyl Bromide (nPB)

Summary and Conclusions 

Two generations of Sprague-Dawley derived rats (F0 and F1) were exposed
to n-propyl bromide (nPB) for 6 hours/day, 7 days/week via whole body
inhalation for at least 70 days prior to mating, throughout mating, and
through most of gestation and lactation.

Exposure was temporarily stopped during the parturition period, from
gestation day (GD) 20 to postnatal day (PND) 5. F1 pups were culled on
PND 4 and were exposed via lactation until PND 21 and then directly via
inhalation until study termination. F2 pups were exposed via lactation
until PND 21 and sacrificed. Targeted ambient air nPB concentrations
were 0, 100, 250, 500, and 750 ppm; nominal mean exposure concentrations
were measured and shown to be close to the target concentrations: F0
generation – 0, 99, 252, 505, and 750 ppm, respectively; F1 generation
– 0, 100, 252, and 502 ppm, respectively.  No live litters were
produced in the F0 750 ppm generation; therefore, there was no F1
treatment group exposed to this dose. 

Three deaths unrelated to treatment occurred during the experiment: one
F0 500-ppm female and two F1 males (1 control and 1 at 500 ppm). At 750
ppm, F0 males displayed a decrease in mean weekly body weights and
cumulative body weight gain throughout the study; F0 females exhibited a
reduction in mean body weight during pre-mating weeks 5-10 and decreased
cumulative weight gain throughout the pre-mating period.  At 500-ppm, F0
females showed a non-significant reduction in mean body weight, weight
gain, food consumption, and food efficiency during pre-mating,
gestation, and lactation. Small, nonsignificant reductions in mean
weekly body weight gains were also observed in the F1 males and females
in this dose group. Weight loss in the F0 and F1 500-ppm females during
late gestation was associated with a reduction in the mean litter size. 

In the F0 generation, there was a statistically significant
dose-response decrease in male and female fertility indices at 500 and
750 ppm and in the mating index at 750 ppm. Infertility was 100% in the
750 ppm group and 52% in the 500 ppm group.  At 500 ppm, the mean number
of implantation sites and size of live litters was also statistically
significantly reduced.  In the F1 generation, no statistically
significant decrease in the fertility or mating index was observed at
any dose, although, there was a significant reduction in the number of
implantation sites and size of live litters at 500 ppm. 

Statistically significant changes in female reproductive parameters
included a decrease in absolute and relative ovary weights at 750 ppm in
the F0 generation and an increase in the length of the estrus cycle at
≥ 500 ppm in F0 females.  Estrus cycle length was increased at 500 ppm
in F1 females; however, the increase was not reported to be
statistically significant relative to controls.  Estrus cycling was not
observed in 2 F0 females in the 500 ppm group, 3 F0 females in the 750
ppm group, 3 F1 females in the 250 ppm group, and 4 F1 females in the
500 ppm group.  Analysis of estrus cycle length by the study authors
excluded ammenorheic females.  ICF reanalyzed these data and included
data from ammenorheic females in the analysis.  Both estrus cycle length
and mean number of estrus cycles within a 3-week period prior to mating
(following 7 weeks exposure) were evaluated.  The mean number of estrus
cycles that occurred within the 3-week period of observation was
statistically significantly decreased at ≥ 250 ppm in F0 females and
at 500 ppm in F1 females. ICF conducted a second analysis in which data
from ammenorheic females were excluded; statistically significant
decreases occurred at the same doses although the mean number of estrus
cycles was slightly increased.  ICF considered these findings to be
toxicologically significant.

The number of primordial follicles and corpora lutea were compared
between high-dose groups (750 ppm for F0 females and 500 ppm for F1
females) and controls; a statistically significant increase in
primordial follicles and a decrease in corporal lutea were observed.
However, these end points were not examined in lower dose groups and
thus, a dose-response could not be determined. 

 generation at exposures ≥ 500 ppm, and in the F1 generation at
exposures; (2) decreased number of normal sperm at exposures ≥ 250 ppm
in F0 generation and at 100 and 500 ppm in the F1 generation; (3) 
reduction in the absolute weight of the left and right cauda epididymes
at exposures ≥ 500 ppm and in the relative weight of the right cauda
epididymis at 750 ppm in the F0 generation, and in the absolute weight
of both epididymes at 500 ppm in the F1 generation; (4) decreased
absolute, but not relative, prostate weights in F0 (≥ 250 ppm) and F1
(500 ppm) males,  and (5) decreased seminal vesicle weights in F0 males
at 750 ppm and F1 males at 250 ppm males.  In general, most of the organ
weight changes were in absolute values, and were not statistically
significant when weights were expressed relative to body weight, and did
not show a clearcut dose-response.  No associated macroscopic or
microscopic tissue changes were observed except in the prostate gland
which showed acute inflammation in F1, but not F0, males exposed to 500
ppm.  The study authors concluded that the biological significance of
these findings was unclear.   Although the number of normal sperm in the
F0 generation was statistically decreased in the three highest dose
group relative to concurrent controls, these numbers were within the
normal background range and thus this finding was not considered to be
toxicologically significant. 

≥ 250 ppm) and F1 (≥ 100 ppm) males and in F0 females at 750 ppm and
F1 females at 500 ppm ; (2) decreased absolute pituitary weights in F0
(750 ppm) and F1 (500 ppm) males, and (3) increased absolute and
relative thymus weights in F1 males.    Most of these changes did not
show a clear dose-response and none were associated with pathologic or
histopathologic changes.  Therefore, the authors concluded that the
biological significance of these findings was unclear.  Relative kidney
weights were increased in F0 males and females at 750 ppm and absolute
kidney weight was increased in F1 males at 500 ppm. Microscopic changes
in the kidney were also observed and included significant increases in
the incidence of minimal-to-mild pelvic mineralization, and/or secondary
transitional epithelial hyperplasia following exposure to ≥ 500 ppm in
F0 females and increased mononuclear cell infiltrate at 500 ppm in F1
males; however, most of these effects did not show a clear dose-response
and were not considered to be biologically significant by the study
authors.  Hepatic effects were also observed in both F0 and F1 males and
females. An increase in mean relative liver weight occurred in F0
animals at exposures ≥ 500 ppm and in F1 animals at 500 ppm. 
Histopathology was observed in the F1 generation and consisted of an
increased incidence of minimal-to-mild centrilobular hepatocellular
degeneration at ≥ 250 ppm in males and at 500 ppm in females.  These
findings were considered to be toxicologically significant.  An increase
in hepatic glycogen was also observed at 500 ppm in F1 males and ≥ 100
ppm in F2 females; however, the biologic significance of this result is
unclear.  

Based on decreased sperm motility in F1 males, decreased mean number of
estrus cycles in F0 females, and mild liver histopathology, the study
NOAEL is 100 ppm, with a LOAEL of 250 ppm. A summary of reproductive and
developmental NOAELs for toxicologically-relevant end points for F0, F1,
and F2 generations is presented in Table S.1.   One selected
reproductive endpoint below is post-natal weight for F1 and F2
generation pups at certain exposure levels.  Pup weight was measured
every seven days and compared with controls. A statistically significant
decrease in weight was observed for both generations of the 500 ppm
females’ pups, on post-natal day (PND) 28 in the F1 generation and PND
14 and 21 in the F2 generation. 

Table S.1   Selected Reproductive and Related Endpoints

for F0, F1, and F2 Generations

F0 Generation

Reproductive Endpoints 

Female Endpoints (Measure)	NOAEL

(ppm)	LOAEL

(ppm)

Decrease in fertility Index (%)

	250	500**

Increase in mean estrus cycle length (days)b

	250	500*

Decrease in mean no. of estrus cycles within 3- week period prior to
mating – all females (N)c	100	250**

Decrease in mean no. of estrus cycles within 3- week period prior to
mating – excluding acyclic females (N)c	100	250**

Decrease in mean no. implantation sites (N/animal)	250	500**

Decrease in litter size (N/litter)	250	500**

Male Endpoints	NOAEL

(ppm)	LOAEL

(ppm) 

Decrease in fertility index (%)	250	500**

Decrease in sperm number in left. epididymis (no. sperm x 106/gram
tissue)	500	750**

Decrease in motile sperm (%)	250	500**

Selected Organ Weights (n=25)a

Male	NOAEL

(ppm)	LOAEL

(ppm)

Final mean body wt (g)  (mean % decrease)	500	750**

Decrease in relative left testis  (g/100 g)	500	750*

Decrease in absolute right cauda epididymis  (g) 	250	500**

Decrease in relative right cauda epididymis 

(g/ 100 g)	500	750**

Decrease in absolute left cauda epididymis (g)	250	500**

Decrease in absolute prostate gland (g) 	100	250**

Femalea	NOAEL

(ppm)	LOAEL

(ppm)

Decrease in relative liver (g/100 g)	500	750**

Decrease in relative kidney (g/100 g)	500	750**

Decrease in absolute ovary (g) 

	500	750**

Decrease in relative ovary  (g/100 g)  	500	750**

Other Important Endpoints	NOAEL

(ppm)	LOAEL

(ppm)

Liver histopathology	100	250*

F1 Generation

Reproductive Endpoints 

Female Endpoint (Measure)	NOAEL

(ppm)	LOAEL

(ppm)

Mean length of estrus cycles (days)	500 	N/A

Decrease in mean no. of estrus cycles within 3 weeks -all femalesc

	250	500*

Decrease in mean no. of estrus cycles within 3 weeks -excluding  acyclic
femalesd

	250	500*

Decrease in Implantation sites

	250 	500 **

Decrease in number of pups born

	250 	500 **

Male Endpoint 	NOAEL

(ppm)	LOAEL

(ppm)

Decrease in motile sperm (%)

	100 	250*

Other Important Endpoints	NOAEL

(ppm)	LOAEL

(ppm)

Liver histopathology	250	500*

Selected Organ Weights (n=25)a

Male (Measure)	NOAEL

(ppm)	LOAEL

(ppm)

Decrease in relative liver  (g/100 g)	250	500 **

Decrease in absolute right cauda epididymis (g)

	250	500**

Decrease in absolute left cauda epididymis (g)

	250	500**

Pup Weight for F1	NOAEL

(ppm)	LOAEL

(ppm)

Decreased pup weight at postnatal day 28	250	500**

F2 Generation

Pup Weight for F2	NOAEL

(ppm)	LOAEL

(ppm)

Decreased pup weight at postnatal day 14 and 21	250	500**

N/A = not available

a There were no statistically significant changes in mean final body
weights of in any of the dose groups in either generation, with the
exception of 750-ppm  F0 males. 

b Statistical analysis calculated by ICF

c Calculated by ICF Poisson regression model 

d Calculated by ICF excluding females that did not have a full estrus
cycle and analyzed using a quasi-likelihood Poisson-like regression
model

*Significantly different from control, p<0.05.	

**Significantly different from control, p<0.01.

*** Significantly different from control, p<0.001.

Materials and Methods

In a two-generation reproductive toxicity study (Stump, 2001), groups of
25 male and female rats (Crl:CD((SD) IGS BR) were exposed to 99.8% pure
n-propyl bromide (nPB) via whole-body inhalation for 6 hours/day, 7
days/week at target concentrations of 0, 100, 250, 500 or 750 ppm. 
Compound impurities were as follows: 0.015% 2-bromopropane; unknown
propyl ether 0.002%; 0.017% propyl ether; 0.004% 1,2-dibromopropane;
0.062% unknown 1,2-dibromopropanes; 0.046% unknown
1,2,3-tribromopropanes. These values are within the Office of
Prevention, Pesticides and Toxic Substances (OPPTS) health effects
guideline standards of purity and stability (OPPTS, 1998). 

The animals were acclimatized for 15 days after receipt; all animals
were housed singly except at mating and weaning.  Animal husbandry and
care were in accordance with appropriate OPPTS health effects harmonized
guidelines (OPPTS, 1998). Exposure chambers were constructed of wire
mesh and the animals were not given either water or food during
exposures periods. The concentrations were monitored during exposure and
the measured mean exposure concentrations were as follows: F0 generation
– 0, 99, 252, 505, and 750 ppm; F1 generation – 0, 100, 252, and 502
ppm.  There was no 750 ppm group for the F1 generation because no live
litters were produced at this exposure level in the F0 generation. 
Homogeneity of the test article’s distribution within 4 different
areas in the exposure chamber was measured during method development
prior to exposures. The test article was distributed evenly in the
exposure chamber, with a mean percent deviation from the reference
concentration ranging from – 4.6% to + 4.3%. 

The F0  generation was ~ 6 weeks old at the start of exposure and
exposed for 70 days prior to mating (1:1).  Exposure to male animals of
both generations continued throughout mating to the day prior to study
termination.  Exposure to female animals in both generations continued
throughout mating, from gestation day (GD) 1- 21, and from postnatal
(PND) 4 through the day prior to study termination.  Thus, exposure was
temporarily stopped around the time of parturition (GD 21-PND4).   In
the F1 generation, pups were directly exposed via whole body inhalation
to nPB on PND 22, following weaning on PND 21.  Selected F1 generation
animals (4 pups/sex/litter, culled on PND 4) were exposed for 70 days
prior to mating; females were not exposed during the period GD 21-PND 4.
 F2 pups were exposed via lactation until PND 21 and sacrificed at that
time.  

All animals were observed twice daily for clinical signs, appearance,
behavior, and mortality. Body weights, food consumption values were
recorded daily. Developmental landmarks (balanopreputial separation and
vaginal patency) were also evaluated.  Culled F1 pups were necropsied on
PND 21 or 28 and culled F2 pups were necropsied on PND 21.  Parental
animals were necropsied after weaning and selected organs were weighed
and/or examined macroscopically and microscopically. Measured
reproductive endpoints included sperm motility, morphology and numbers
for males in all dose groups and ovarian primordial follicle counts and
corpora lutea counts in the control and high-dose F0 and F1 females. 
Estrus cycle stage and length were monitored during the 3-week period
prior to mating (following 7 weeks exposure) in both F0 and F1 groups

Statistical Analyses

Most analyses by Stump were conducted using two-tailed tests for a
minimum significance level of 5% comparing each treated group to the
control group.  Each mean was presented with the standard deviation and
the number of animals used to calculate the mean.  Data obtained from
nongravid animals were excluded from statistical analyses following the
mating period.  Statistical analyses were not performed when weekly food
or body weight data for one or more animals were not available because
the animals remained in the lactation phase.  Statistical tests were
performed using appropriate computing devises or programs and are
referenced in the report tables. The following analyses used different
statistical tests: 

Table 1.  Statistical Tests Used in Analyses

Statistical Test

	Parameter

Chi-square test with Yates correction factor	Parental Mating and
Fertility Indices

One-way ANOVA with Dunnet’s Test	Parental Weekly Body Weights and
Weight Changes, Gestation and Lactation Body Weights, Parental Food
Consumption, Food Efficiency, Gestation length, Pre-coital Interval,
Implantation Sites, Unaccounted Sites, Offspring Weights and Weight
Changes, Absolute and Relative Organ Weights, Live Litter Size, Sperm
Production Rate, Epididymal and Testicular Sperm Numbers, Ovarian
Primordial Follicle Counts, Number of Pups Born, Balanopreputial
Separation, Vaginal Patency

Kruskal-Wallis Test with Mann-Whitney U-Test	Sperm Motility, Percent
Morphologically Normal Sperm, Pup Sexes at birth (% Males per litter),
Proportional Postnatal Survival

Fisher’s Exact Test	Histopathological Findings

Study Results

F0 Generation

One 500-ppm F0 female was euthanized in extremis due to an abnormality
of the eye that was not considered to be treatment-related. No clinical
signs of toxicity were noted that were considered to be
treatment-related. In the F0 animals, mean body weights in the 750-ppm
males were reduced by 5.4-12.7% from week 3 through study termination
(at week 19), which correlated with sporadic decreases in weekly weight
gains and food efficiency.  The 750-ppm males displayed reduced
cumulative weight gains throughout the study. Generally, the 750-ppm F0
males and females displayed comparable food consumption as compared to
controls, showing only sporadic reductions in food efficiency. The mean
body weight of 750-ppm females was reduced by 5.0-5.8% during weeks
5-10, as compared to controls, with associated reductions in cumulative
weight gains during the pre-mating period.  The 750-ppm females were
never pregnant; therefore no data on weight and weight gain during
parturition and/or lactation were available. The 500-ppm males displayed
a general decrease in weight (4.7-6.3% during study weeks 12-19) and
weight gain, but the data were only sporadically statistically different
from controls. The 500-, 250-, and 100-ppm females and 250- and 100-ppm
males did not display a treatment-related reduction in weight, weight
gain, food consumption, or food efficiency.  During gestation, a
reduction in weight gain was observed on GD 11-14, 14-20, and 0-20 for
the 500- and 250-ppm females.  However, the only statistically
significant finding was a decrease at 500 ppm on GD 14-20 and GD 0-20;
these results correlated with a statistically significant reduction in
overall food consumption during these periods. Reduced gestation weights
were statistically significant in the 500-ppm females on GD 14 and 20,
and were attributed to a reduction in litter size. During lactation,
slight nonsignificant reductions in body weight were observed in the
500-ppm females (Lactation Day (LD) 1-4 and LD 7-14). 

Table 2. F0 Females Reproductive Endpointsa

Endpoint	0 ppm	100 ppm	250 ppm 	500 ppm	750 ppm

Fertility index (%)

N	92.0

25	100

25	88.0

25	52.0**

25	0.0**

25

Mating index (%)

N	96.0

25	100

25	100

25	84.0

25	68.0*

25

Evidence of mating w/out delivery (no.)	1	0	3	10	17

Estrus cycle length (days)b

N	4.2(0.49

25	4.5(1.05

25	4.7(0.90

25	5.5(2.17*

23	5.6(1.79*

22

Mean no. of estrus cycles within 3 weeksc

N	3.96(0.54

25	3.84(0.62

25	3.52(0.65**

25	2.88(1.17***

25	2.56(1.26***

25

Mean no. of estrus cycles within 3 weeks excluding  ammenorheic femalesd

N	3.96(0.54

25	3.84(0.62

25	3.52(0.65**

25	3.00(1.02***

24	2.78(1.04***

23

Implantation sites

N	15.3(2.53

23	14.3(3.09

25	13.8(4.23

22	9.0(4.54**

11	NA

Number of born

N	15.0(2.42

23	13.6(3.09

25	12.5(4.27

22	8.5(4.41**

11	NA

Unaccounted Sites

N	0.3(0.57

23	0.7(0.95

25	1.3(1.36**

22	0.5(0.69

11	NA

a Data were provided on pp. 123-124, 207

b Statistical analysis calculated by ICF

c Calculated by ICF and analyzed using a quasi-likelihood Poisson-like
regression model

d Calculated by ICF excluding females that did not have a full estrus
cycle and analyzed using a quasi-likelihood Poisson-like regression
model

*Significantly different from control, p<0.05.	

**Significantly different from control, p<0.01.

*** Significantly different from control, p<0.001.

Table 3. Primordial Follicles and Corpora Luteaa for the F0 Animals

N=25	0 ppm	500 ppm	750 ppm

Primordial follicles	107.8(55.80	NA	134.2(58.29

Corpora lutea	178.9(51.61	156.8(56.03	126.9(67.49**

a Data were derived from pp. 245 of the study report

**Significantly different from control, p<0.01.

A decrease in reproductive performance was statistically significant in
the 500- and 750-ppm animals with fertility indices (both male and
female) of 52.0 and 0.0%, respectively. No live litters were produced at
750 ppm.  Historical control fertility indices for males and females
were 89.3% and 90.4%, respectively. Mating indices (both male and
female) for the 500- and 750-ppm animals were 84.0% and 68.0%,
respectively; however, only the 750-ppm value reached statistical
significance as compared to controls (96.0%) and was lower than
historical control data (96.0 and 97.0% for males  and females,
respectively). An increase in the number of females that displayed
evidence of mating without delivery was observed and 2 females from the
500-ppm group were nongravid (i.e., showed no evidence of implantation).
The mean number of days between pairing and coitus were extended in the
500- and 750-ppm groups, to 4.3 and 4.8 days, respectively, as compared
to concurrent control (mean of 3.4 days) and historical control mean of
2.0-3.5 days) data.  The mating interval could not be determined for 1
female in the control group, 4 females in the 500 ppm group, and 8
females in the 750-ppm group, because there was no evidence of mating.
Implantation sites were reduced in the 500-ppm females and the mean
number of unaccounted-for sites was comparable to controls. An increase
in the number of unaccounted- for sites in the 250-ppm females was
statistically significant with a slight decrease in the number of
implantation sites; however, a clear dose-response was not apparent.  

An increase in estrus cycle length was statistically significant in the
500- and 750-ppm females; the data are summarized in Table 2. However,
the estrus cycle data reported by the study authors excluded females who
showed no evidence of cycling (2 and 3 for the 500- and 750-ppm groups,
respectively). Therefore, ICF evaluated the individual data by (1)
counting the number of estrus cycles within the three-week period prior
to mating; (2) including females who did not complete an estrus cycle;
and (3) conducting statistical analysis of dose-response using a Poisson
regression model.  ICF also analyzed the F1 generation estrus cycle data
in a similar manner (See F1 generation section for these results).  A
dose-dependent decrease in the number of estrus cycles was observed in
females exposed to  ( 250-ppm.  For comparison purposes, ICF conducted a
second analysis of the estrus cycle data, excluding acyclic females.
Although exclusion of these individuals skewed the data, the statistical
significance of the findings stayed at the same concentration levels.  A
similar dose response was observed that was significantly higher than
controls at ( 250 ppm.

Table 4. F0 Males Reproductive Endpointsa

Endpoint 	0 ppm	100 ppm	250 ppm 	500 ppm	750 ppm

Fertility index (%)

N	92.0

25	100

25	88.0

25	52.0**

25	0.0**

25

Mating index (%)

N	96.0

25	100

25	100

25	84.0

25	68.0*

25

Evidence of mating w/out delivery (no.)	1	0	3	8	17

L. Epididymis (no. sperm in million/gram tissue)

N	471.9(81.11

25	459.4(101.94

25	480.1(80.21

25	429.3(101.46

25	369.6(90.66**

25

Motile sperm (%)

N	86.8(11.90

25	88.8(7.22

25	83.4(10.41

25	71.9(9.27**

23	53.2(19.59**

15

Normal sperm

N	99.7(0.6

25	99.7(0.52

25	99.3(0.83*

25	98.2(2.59**

24	90.6(8.74**

24

Normal shaped head separated from flagellum

N	0.2(0.46

25	0.2(0.36

25	0.4(0.54

25	1.4(2.15

24	4.8(4.28

24

Head absent from flagellum

N	0.1(0.22

25	0.1(0.24

25	0.3(0.46

25	0.5(0.77

24	4.6(5.91

24

a Data were provided on pp. 123, 195, 197-198

*Significantly different from control, p<0.05.	

**Significantly different from control, p<0.01.

Spermatogenic analysis showed a reduction in sperm motility and in the
mean number of  morphologically normal sperm from the 750-ppm males. 
There were two types of sperm abnormalities; one in which abnormal heads
were separated from the flagellum and another in which normal flagella
were without the head. The 500-ppm males displayed a reduction in sperm
motility and number of morphologically normal sperm cells,  similarly
abnormal sperm cell types. The number of normal sperm was statistically
decreased in the 250-ppm males; however, they were very close to control
values and not considered to be toxicologically significant.  Relative
to controls, the right epididymes were small on two 750- and one 500-ppm
males. A 750-ppm male had a small left epididymis. The testes were small
in one 500- and 750-ppm males and one 500-ppm male had soft testes.

Table 5a. Selected F0 Male Organ Weights (n=25)a

Organ	0 ppm	100 ppm	250 ppm	500 ppm	750 ppm

Final Body Wt (g)	548(56.0	526(52.3	530(63.3	515(61.3	483(44.6**

Brain (g)	2.19(0.091	2.15(0.114	2.08(0.087**	2.10(0.177*	2.05(0.091**

Relative brain (g/100 g)	0.403(0.0401	0.411(0.0384	0.398(0.0554
0.410(0.0446	0.428(0.0453

Liver (g)	20.09(3.206	18.63(3.008	19.42(2.934	20.85(4.147	21.25(2.953

Relative liver (g/100 g)	3.662(0.4292	3.538(0.4238	3.675(0.4573
4.034(0.4949*	4.401(0.4206**

Kidney (g)	3.77(0.418	3.58(0.358	3.59(0.274	3.62(0.334	3.62(0.329

Relative kidney 

(g/100 g)	0.690(0.0649	0.684(0.0654	0.684(0.0629	0.708(0.0862
0.7454(0.0706**

Spleen (g)	0.91(0.167	0.86(0.144	0.85(0.120	0.87(0.110	0.92(0.157

Relative spleen 

(g/100 g)	0.167(0.0291	0.164(0.0231	0.162(0.0212	0.170(0.0231
0.192(0.0353**

L. testis (g)	1.79(0.186	1.79(0.217	1.69(0.146	1.77(0.227	1.75(0.188

Relative L. testis (g/100 g)	0.329(0.0413	0.343(0.0474	0.323(0.0422
0.348(0.0666	0.364(0.0426*

R. Cauda Epididymis (g)	0.3327(0.03631	0.3311(0.4453	0.3053(0.04188
0.2912(0.05206**	0.2405(0.04804**

R. Cauda Epididymis 

(g/ 100 g)	0.61(0.0096	0.064(0.0121	0.059(0.0098	0.057(0.0120
0.050(0.0097**

L Cauda Epididymis (g)	0.3252(0.03673	0.3242(0.03149	0.3050(0.03556
0.2877(0.03170**	0.2401(0.03529**

L. Cauda Epididymis 

(g/ 100 g)	0.060(0.0100	0.062(0.0087	0.058(0.0096	0.056(0.0084
0.050(0.0068

Prostate (g)	1.33(0.247	1.27(0.241	1.14(0.169**	1.14(0.232*	1.14(0.178**

Prostate 

(g/ 100 g)	0.244(0.0525	0.245(0.0597	0.218(0.0442	0.224(0.0503
0.237(0.0405

a Data were provided on pp.208-211 and 214-216 of the study report

*Significantly different from control, p<0.05.	

**Significantly different from control, p<0.01.

A decrease in the absolute brain weight was statistically significant in
males at  (250 ppm as compared to controls; the study authors attributed
this finding to a decrease in body weight and did not consider it to be
toxicologically significant. However, the extent of the decrease in
brain weight prompted further histopathological examinations of the
brain to determine if any lesions could explain the weight loss.  No
corresponding lesions were found. A decrease in the absolute adrenal and
relative spleen weights of the 750-ppm males was statistically
significant; these decreases were also attributed to general weight loss
relative to controls as no macroscopic or microscopic lesions were noted
at necropsy.  

Decreases in the absolute prostate ((250 ppm), right epididymis ((500
ppm), pituitary (750 ppm), and seminal vesicles/coagulating gland (750
ppm) weights were statistically significant although relative weights of
these organs/tissues were not.  Further, none of these decreases were
dose related. A decrease in the left and right cauda epididymes absolute
weights was statistically significant as compared to the controls. These
decreases were considered to be treatment-related based on that
spermatogenic effects observed in the 500- and 750-ppm males. Other
organ weight changes included an increase in the relative left testis
weight (750 ppm) and in relative liver ((500 ppm), kidney (750 ppm), and
spleen (750 ppm) weights as compared to concurrent controls. The liver
and kidneys displayed microscopic lesions; these findings are summarized
in Table 5a and 5b and discussed further below. 

Table 5b. Selected F0 Female Organ Weights (n=25)a

Organ	0 ppm	100 ppm	250 ppm	500 ppmb	750 ppm

Final Body Wt (g)	331(20.7	330(22.3	327(24.8	332(38.3	319(25.5

Brain (g)	1.96(0.078	1.92(0.094	1.94(0.084	1.89(0.105*	1.86(0.072**

Relative brain (g/100 g)	0.595(0.0446

	0.585(0.0458	0.595(0.0454	0.577(0.0674

	0.586(0.0431

Liver (g)	12.55(1.292	13.08(1.443	12.91(1.199	12.86(1.595	13.70(1.735

Relative liver 

(g/100 g)	3.800(0.3414	3.958(0.3716	3.948(0.2402	3.888(0.3862
4.302(0.4402**

Kidney (g)	2.22(0.211	2.27(0.244	2.25(0.177	2.27(0.217	2.37(0.253

Relative kidney 

(g/100 g)	0.672(0.0548	0.686(0.0660	0.692(0.0734	0.688(0.0646
0.746(0.0861**

Ovaries (g)	0.1227(0.02592	0.1265(0.02404	0.1152(0.02360	0.1119(0.01514
0.09575(0.0.02798**

Relative ovaries

 (g/100 g)	0.037(0.0078	0.038(0.0068	0.035(0.0072	0.034(0.0056
0.031(0.0079**

a Data were provided on pp.212-213 and 218-219 of the study report

b N=24

*Significantly different from control, p<0.05.	

**Significantly different from control, p<0.01.

An increase in the relative liver and kidney weights was statistically
significant in the 750-ppm females. The 750-ppm females also displayed a
significant decrease in relative ovary weights that correlated with the
infertility of these animals. The 750-ppm females displayed a decrease
in absolute brain weight that was attributed to the decreased weight of
these animals and was not considered by the study authors to be
toxicologically significant. A dose-dependent decrease in absolute (22%)
and relative ovary weights (16%) was observed in the 750-ppm females,
relative to controls.

Table 6. Selected Histopathology Data for the F0 Animals (n=25)a

	Male	Female

	0 ppm	100 ppm	250 ppm	500 ppm	750 ppm	0 ppm	100 ppm	250 ppm	500 ppmb
750 ppm

Kidneys

Pelvic Mineralization	1	0	1	2	6	2	3	5	12*	14*

Transitional Cell Hyperplasia	0	0	1	3	4	1	0	2	6*	5

Testes

Seminiferous Tubules Degeneration 	1	2	0	3	6	N/A

Ovaries

Decreased Corpora Lutea	N/A	3	0	3	6	11*

Luteinized Follicular Cyst	N/A	2	4	3	5	9*

Follicular Cyst	N/A	7	1*	3	3	6

Interstitial Hyperplasia	N/A	3	0	0	3	7

Liver

Centrilobular Hepatocellular Vacuolation	0	0	7*	22*	24*	0	0	0	6*	16*

Increased Glycogen	14	14	20	21	24*	15	18	22	23*	23*

a Data were derived from pp. 227-244 of the study report

*Significantly different from control, p<0.05.

The males manifested a slight nonsignificant increase in minimal
mineralization of the pelvic kidney and the females displayed a
minimal-to-mild increase that was statistically significant at (500 ppm.
The kidneys of treated males had a slight nonsignificant increase in the
incidence of minimal transitional cell hyperplasia. The females
displayed a more pronounced effect and the incidence for the 500-ppm
females was statistically significant from controls.  However,
statistical significance for this end point was not achieved at 750 ppm,
indicating a lack of dose-response.  Statistically significant increases
in the incidence of mild-to-minimal centrilobular hepatocellular
vacuolation was noted in males ((250 ppm) and females ((500 ppm). An
increase in liver glycogen was statistically significant for males at
750 ppm and for females at (500 ppm. 

Increases in incidence and severity of seminiferous tubules degeneration
were noted in the treated males at (500 ppm; although these incidences
were not statistically significantly different from controls, the study
authors considered these changes to be treatment-related because of
spermatogenic effects observed at these doses. A statistically
significant decrease in corpora lutea and an increase in luteinized
follicular cysts were observed in the ovaries of 750 ppm females.
Microscopic determination of the number of primordial follicles and
corpora lutea in the ovaries are presented in Table 6; a significant
increase in primordial follicles and decrease in corpora lutea was
observed at 750 ppm, relative to controls. However, lower doses were not
analyzed for these end points; therefore, no dose-response conclusions
can be made.  

F1 Generation 

One control male (at week 22) and one 50-ppm male (at week 18) were
euthanized in extremis and the deaths were not attributed to treatment.
In the F1 animals, the 500-ppm group displayed a significant reduction
in live litter size, attributed to reductions in body weight.  Postnatal
survival was unaffected by treatment. Body weights were increased in the
500-ppm pups during PND 1 (both sexes) and 4 (females), and decreased in
the 250- and 500-ppm males on PND 28. Body weight gains were decreased
in 500-ppm males on PND 4-7, 7-14, and 21-28; decreases in body weight
gains were also observed at PND 21-28 in (100-ppm males and 750-ppm
females, although mean body weights did not differ from controls. The
absolute brain weights of the ( 100-ppm F1 males and 100-ppm females
sacrificed at PND 21 displayed a statistically significant reduction;
however, relative weights were unaffected. An increase in the average
day at balanopreputial separation of the 500-ppm males was statistically
significant as compared with concurrent controls (9% increase); however,
there was no dose-response. Vaginal patency was increased in the 500-ppm
females by 5% compared with concurrent controls; however, this delay was
not statistically significant. 

Mean body weight gains were reduced in the 500-ppm F1 males (weeks 19-20
and 20-21) as compared to controls; percent decrease in body weights
gains ranged from 9.3-18.5% during weeks 19-37; 500-ppm females
displayed reduced body weight gains (weeks 19-28) with a 6.7-11.8%
decrease in body weight being observed, as compared to controls, during
weeks 19-28. The 100-ppm males displayed a reduction in body weight
during weeks 26-37; however, the decrease was not dose-related and not
attributed to treatment. F1 500-ppm females displayed reduced weight
gain (during GD 7-11, 11-14, 14-20, and 0-20) and body weight (at GD 14
and 20) that coincided with a decrease in litter size (42%), number of
implantations (37%), and food efficiency.  Further, mean body weights
and food consumption were reduced in the F1 500-ppm females through LD
4, 7, and 14. The 250-ppm females displayed a reduction in mean body
weight gains during GD 14-20 with a slight reduction in food efficiency
(GD 7-11, 11-14, and 14-20) and a significant overall reduction in food
efficiency (GD 0-20).  The 100-ppm females displayed a reduction in mean
body weight gains during GD 4-7 with a slight reduction in food
efficiency (GD 4-7 and 11-14). However, weight gain and food efficiency
data of the 250- and 100-ppm females were comparable to controls. 



Table 7. F1 Females Reproductive Endpointsa

Endpoint	0 ppm	100 ppm	250 ppm 	500 ppm

Fertility index (%)

N	100.0

25	84.0

25	80.0

25	100.0

25

Mating index (%)

N	88.0

25	68.0

25	64.0

25	72.0

25

Evidence of mating w/out delivery (no.)	3	4	4	8

Estrus cycle length (days)b

N	4.5(1.25

24	4.5(0.91

24	4.9(1.43

22	5.1(1.68

21

Mean no. of estrus cycles within 3 weeksc

N	3.64(1.15

25	3.68(1.11

25	2.88(1.36

25	2.68(1.35*

25

Mean no. of estrus cycles within 3 weeks excluding  ammenorheic femalesd

N	3.64(1.15

25	3.68(1.11

25	3.13(1.10

23	2.91(1.12*

23

Implantation sites

N	15.5(2.11

22	15.8(3.29

17	13.5(4.12

16	9.8(4.93**

17

Number of born

N	14.9(1.97

22	15.1(3.35

17	13.1(4.12

16	8.6(4.51**

17

Unaccounted Sites

N	0.5(0.86

22	0.6(1.22

17	0.4(0.63

16	1.2(1.09

17

a Data were provided on pp. 272-275 and 356 of the study report

b Statistical analysis calculated by ICF

c Calculated by ICF Poisson regression model 

d Calculated by ICF excluding females that did not have a full estrus
cycle and analyzed using a quasi-likelihood Poisson-like regression
model

*Significantly different from control, p<0.05.	

**Significantly different from control, p<0.01.

Mating, fertility, and number of unaccounted-for implantation sites were
unaffected by treatment. Estrus cycle length was extended by 13% in the
500-ppm females (historical controls range is 4.1-5.1 days); however,
the data were not significantly different from concurrent controls.
Three 250-ppm females and four 500-ppm females did not complete an
estrus cycle; the study authors excluded the data from these individuals
from their statistical analysis. ICF re-evaluated the estrus cycle data
by determining the number of estrus cycles within the 3 weeks prior to
mating, including data from all females, and performing a statistical
analysis of dose-response using a Poisson regression model. A
significant reduction in estrus cycle number was observed at 500 ppm. 
ICF performed further analysis of the estrus cycle data; results from
acyclic females were excluded from this analysis.  Although exclusion of
the results from these individuals skewed the distribution of the data,
the statistical significance remained at the same concentration levels.
Gestation length was not affected by treatment; only one 250-ppm female
experienced dystocia, which was not considered by the study authors to
be treatment-related. 

Table 8. F1 Males Reproductive Endpointsa

Endpoint 	0 ppm	100 ppm	250 ppm 	500 ppm

Fertility index (%)

N	87.5

24	68.0

25	64.0

25	70.8

24

Mating index (%)

N	100.0

24	84.0

25	80.0

25	100.0

24

Evidence of mating w/out delivery (no.)	3	8	9	7

L. Epididymis (no. sperm in million/gram tissue)

N	640.1(141.82

23	609.1(115.22

23	573.7(97.17

24	635.3(160.50

24

Motile sperm (%)

N	88.9(4.52

24	86.4(4.96

25	84.8(6.02*

25	74.4(14.06**

24

Normal sperm (%)

N	99.5(0.79

24	98.9(0.95**

25	99.1(1.13

25	95.3(6.51**

24

Normal shaped head separated from flagellum (%)

N	0.2(0.47

24	0.5(0.68

25	0.4(0.73

25	1.6(4.12

24

Head absent from flagellum (%)

N	0.2(0.57

24	0.6(0.75

25	0.4(0.61

25	2.0(2.99

24

a Data were provided on pp. 274, 345-348 of the study report

*Significantly different from control, p<0.05.	

**Significantly different from control, p<0.01.

Spermatogenic analysis indicated a reduction in sperm motility and the
number of morphologically normal sperm in 500-ppm males. There were two
types of sperm abnormalities; one in which sperm had a normal head
separated from the flagellum and another in which normal flagella were
without the head. The mean testicular and epididymal sperm counts were
comparable among all treatment and control groups. The 250-ppm males
displayed a significant reduction in sperm motility. At 100-ppm, males
also showed a significant decrease in number of morphologically normal
sperm cells; however, this value was within the range of historical
control values, and there was no clear dose-response. Therefore, this
finding was not considered to be toxicologically significant.

Table 9a. Selected F1 Male Organ Weightsa

Organ	0 ppm	100 ppm	250 ppm	500 ppm

Final Body Wt (g)

N	583(65.7

24	543(63.9

25	562(55.3

25	526(43.3

24

Brain (g)

N	2.21(0.092

24	2.11(0.111**

25	2.12(0.109**

25	2.01(0.079**

24

Relative brain (g/100 g)

N	0.382(0.0379

24	0.392(0.0401

25	0.379(0.0365	0.384(0.0313

24

Liver (g)

N	20.18(3.353

24	18.54(2.304

25	19.94(2.994

25	21.25(4.126

24

Relative liver (g/100 g)

N	3.455(0.3775

24	3.424(0.3348

25	3.538(0.3198	4.017(0.5570**

24

Kidney (g)

N	3.73(0.455

24	3.37(0.354**

25	3.52(0.349

25	3.40(0.450*

24

Relative kidney (g/100 g)

N	0.640(0.0548

24	0.624(0.0672

25	0.627(0.0523

25	0.645(0.0587

24

R. Cauda Epididymis (g)

N	0.3178(0.03778

24	0.3129(0.0.3862

25	0.3029(0.03885

25	0.2720(0.03787**

24

Relative r. Cauda Epididymis (g/ 100 g)	0.055(0.0075

24	0.058(0.0104

25	0.054(0.0083

25	0.052(0.0073

24

L Cauda Epididymis (g)

N	0.3304(0.03279

24	0.3325(0.03912

25	0.3221(0.03981

25	0.2967(0.04462**

24

Relative l. Cauda Epididymis (g/ 100 g)

N	0.057(0.0061

24	0.062(0.0111

25	0.058(0.0089

25	0.056(0.0075

24

Thymus (g)

N	0.2319(0.05952

24	0.2463(0.06420

25	0.2702(0.05165

25	0.2762(0.05771*

24

Relative thymus (g/100 g)

N	0.040(0.0114

24	0.045(0.0110

25	0.048(0.0091*

25	0.052(0.0102**

24

Prostate (g)

N	1.16(0.258

24	1.00(0.285

25	1.06(0.243

25	1.05(0.205

24

 Relative prostate (g/100 g)

N	0.200(0.0405

24	0.187(0.0595

25	0.190(0.0502

25	0.201(0.0371

24

a Data were provided on pp. 357-360, 363-366 of the study report

*Significantly different from control, p<0.05.	

**Significantly different from control, p<0.01.

The kidneys (100- and 500-ppm males) displayed a decrease in absolute
weight that was not considered to be treatment-related or
toxicologically significant, due lack of a dose-response and absence of
macroscopic or microscopic kidney lesions. Mean absolute brain weights
were reduced in a non-dose related fashion for the (100-ppm males and
500-ppm females; therefore, these change were not considered to be
toxicologically significant. A decrease in absolute pituitary weight was
statistically significant in the 500-ppm males; however, no lesions were
found that suggested an association with treatment. No microscopic or
macroscopic lesions were observed in the epididymides but the effect was
similar to those observed in the F0 males. Absolute and relative thymus
weights were increased in the 500-ppm F1 males; however, no macroscopic
or microscopic lesions were noted in this gland. 

 

Table 9b. Selected F1 Female Organ Weights (N=25)a

Organ	0 ppm	100 ppm	250 ppm	500 ppm

Final Body Wt (g)	321(27.3	325(28.1	318(26.7	309(29.5

Brain (g)	1.97(0.076	1.96(0.073	1.92(0.067	1.89(0.102**

Relative brain (g/100 g)	0.615(0.0.520	0.606(0.0549	0.608(0.0470
0.617(0.0588

Liver (g)	12.27(1.571	12.57(1.559	12.44(1.641	12.53(1.894

Relative liver (g/ 100 g)	3.811(0.2847	3.873(0.3744	3.914(0.4246
4.059(0.4546

Ovaries (g)	0.1131(0.01554	0.1077(0.03170	0.1056(0.02791	0.1062(0.02302

Relative ovaries (g/100 g)	0.035(0.0027	0.022(0.0032	0.022(0.0042
0.021(0.0045

a Data were provided on pp. 361-362, 367-369 of the study report

**Significantly different from control, p<0.01.

The F1 males and females exposed to 500 ppm displayed an increase in
relative liver weight as compared to controls; however, the values for
females were not statistically significant.  These increases were
considered treatment-related, because associated histopathology was
observed, consisting of an increase in hepatic centrilobular
vacuolation. An statistically significant increase in the number of
primordial follicles (19%) relative to controls was observed in the 500
ppm group. However, this end point was not measured in lower dose
groups, and thus a dose-response could not be determined. 

Table 10. Primordial Follicles and Corpora Luteaa for F1 Animals

N=25	0 ppm	500 ppm

Primordial follicles	105.6(44.96	141.8(50.26*

Corpora lutea	171.7(40.89	171.7(49.18

a Data were derived from p. 390 of the study report

**Significantly different from control, p<0.01.

The liver ((250-ppm males and (100-ppm females) and kidneys (500-ppm
males and (250-ppm females) displayed treatment-related lesions that
correlated with lesions found in the F0 generations. 

Table 11. Selected Histopathology Data for the F1 Animalsa

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㓿ۖĀ̊l倃曾ĴᄀPelvic Mineralization	0	1	0	3	4	5	7	8

Mononuclear Infiltrate	11	9	1*	20*	5	5	5	7

Transitional Cell Hyperplasia	1	1	2	1	2	3	2	2

Prostate

Acute Inflammation	3	-	-	13*

	Ovaries

Decreased Corpora Lutea

3	3	7	4

Luteinized Follicular Cyst

2	3	2	3

Follicular Cyst

5	5	7	10

Interstitial Hyperplasia

12	10	13	18

Liver

Total Centrilobular Hepatocellular Vacuolation	0	0	15*	23*	0	0	2	6*

Increased glycogen	19	18	17	24*	16	24*	23*	23*

a Data were provided on pp. 375- of the study report. N=24, 25, 25, 24
males treated with 0, 100, 250, 500 ppm of nPB and 25 all treated
females

*Significantly different from control, p<0.05.

**Significantly different from control, p<0.01.

In the F2 animals, a 41% decrease in live animals born per dam (PND 0)
was observed in the 500-ppm pups. The 500-ppm pup weights were reduced
at PND 14 (16% males and 14% females) and PND 21 (18% males and 15%
females); weight gain was reduced in the 500-ppm pups at PND 4-7 (33%
males and 31%), 7-14 (26% males and 24% females), and 14-21 (20% males
and 16% females). The absolute brain, thymus, and spleen weights were
reduced in the 500-ppm F2 males and the absolute thymus and splean
weights were decreased in 500-ppm females. A decrease in the relative
brain and spleen weights in 500 ppm males and in relative spleen weights
in 500-ppm females was also statistically significant. 

						

References

National Research Council (1996) Guide to the Care and USE of Laboratory
Animals, Institute of Laboratory Animal Resources, Commission of Life
Sciences. National Academy Press, Washington, D.C. 

The Office of Prevention, Pesticides and Toxic Substances (OPPTS)
Harmonized Test Guidelines, Series 870 Health Effects Test Guidelines --
Final Guidelines EPA 712–C–98–189 August 1998

 

Stump D.G.  2001.  An Inhalation Two-Generation Reproductive Toxicity
Study of 1-Bromopropane in Rats.  Conducted by WIL Research
Laboratories, Inc., Sponsored by Brominated Solvents Consortium.  May
24, 2001.

Appendix 1:  List of Acronyms

ANOVA	 Analysis of Variance

Crl:CD((SD) IGS BR	 Type of Sprague Dawley Rat Species

FSH	 Follicle Stimulating Hormones

GD	 Gestation Day

LD	 Lactation Day

LOAEL	 Low Observable Adverse Effect Level

nPB	 normal-Propyl Bromide

PND	 Postnatal Day

PPM	 Parts per Million

NOAEL	 No Observable Adverse Effect Level

OPPTS	The Office of Prevention, Pesticides and Toxic Substances

September 24, 2004 

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