Reproductive Toxicology 23 (2007) 216–225

A two-generation reproductive toxicity study of

decamethylcyclopentasiloxane (D5) in rats exposed by
whole-body vapor inhalation
Waheed H. Siddiqui a,∗ , Donald G. Stump b , Vincent L. Reynolds c ,
Kathleen P. Plotzke a , Joseph F. Holson b , Robert G. Meeks a
a Dow Corning Corporation, Midland, MI 48686, USA
b WIL Research Laboratories, Inc., Ashland, OH 44805, USA
c Eli Lilly & Company, Greenfield, IN 46140, USA

Received 13 July 2006; received in revised form 26 October 2006; accepted 8 November 2006
Available online 18 November 2006

Abstract
This two-generation reproduction study assessed the reproductive hazard potential of decamethylcyclopentasiloxane (D5 ). Sprague–Dawley rats
(30/sex/group) were exposed by whole-body vapor inhalation to a target concentration of 30, 70, or 160 ppm D5 or filtered air for 6 h/day. Exposures
for the F0 and F1 generations started at least 70 days prior to mating and lasted through weaning of the respective pups on postnatal day (PND) 21.
Female exposures were interrupted from gestation day (GD) 21 through PND 4 to allow for parturition and to permit continuous maternal care for
the early neonates. F2 pups were not directly exposed to D5 . There were no exposure-related mortalities, clinical signs of toxicity, or effects on body
weight or food consumption. There were no treatment-related gross findings or organ weight effects at the F0 and F1 necropsies. Other than minimal
alveolar histiocytosis in all exposed groups, there were no noteworthy microscopic findings. Reproductive parameters (number of days between
pairing and mating, mating and fertility indices, gestation length, and parturition), spermatogenic parameters and ovarian primordial follicle counts
and numbers of corpora lutea in the F0 and F1 parental animals were not significantly changed between treated and control groups. Mean live litter
sizes, number of pups born, sex ratios, pup body weights, postnatal pup survival and general physical condition of offspring in each generation were
not affected. The slight, but statistically significant, increase in the mean F1 male pup AGD in the 160 ppm group was not considered to be related
to treatment. Vaginal patency and balanopreputial separation were unchanged compared to controls. Thus, the No-Observed-Adverse-Effect-Level
(NOAEL) for parental and reproductive toxicity was determined to be 160 ppm D5 .
© 2006 Elsevier Inc. All rights reserved.

Keywords: Decamethylcyclopentasiloxane; D5 ; Inhalation; Reproductive toxicity; Two-generation; Siloxane; Rat

1. Introduction solubility (∼17 ppb) and unusually low surface energy of D5

Decamethylcyclopentasiloxane (D5 ; CAS # 541-02-6) is a
cyclic dimethylsiloxane with a molecular weight of 371 Da,
vapor pressure ∼0.17 mm Hg at 25 ◦ C and boiling point 211 ◦ C.
Chemically, D5 consists of alternating silicon–oxygen bonds
connected in a ring arrangement with two methyl groups cova-
lently bonded to each silicon atom (Fig. 1). The low water
∗ Corresponding author at: Dow Corning Corporation, 2200 W. Salzburg
Road, P.O. Box 994, Mail# CO3101, Midland, MI 48686-0994, USA.
Tel.: +1 989 496 4884; fax: +1 989 496 5816.
E-mail address: Waheed.Siddiqui@dowcorning.com (W.H. Siddiqui).
make this silicone material attractive for use in a broad variety
of consumer and industrial applications. D5 is used not only as
an intermediate in the manufacture of other polydimethylsilox-
anes, but also as an ingredient in consumer products including
a variety of personal care products. Consequently, persons who
may be exposed to D5 include: workers in the manufacture of D5
or personal care products containing D5 ; workers in dry cleaning
establishments that use D5 as a replacement for other cleaning
solvents; consumers who use personal care products contain-
ing D5 , including antiperspirants/deodorants (AP/Ds) and hair
care/skin care (HC/SC) products; and the general public living
in the vicinity of a plant that produces or processes these mate-
rials. Thus, there is widespread human dermal exposure to D5

0890-6238/$ – see front matter © 2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.reprotox.2006.11.006
 W.H. Siddiqui et al. / Reproductive Toxicology 23 (2007) 216–225
Fig. 1. Molecular structure of decamethylcyclopentasiloxane (D5 ).
as well as some small inhalation exposure [1]. Because of the
widespread use of D5 and the potential for human exposure, the
toxicity of D5 in laboratory animals and kinetics of D5 in both
laboratory animals and humans by relevant routes of exposure
have been assessed.
D5 has a low order of acute toxicity by oral and dermal routes.
It is not a skin or eye irritant [2,3] and is not mutagenic or a skin
sensitizer [4,5]. Dermal absorption studies demonstrated that D5
was not highly absorbed (∼0.05% absorbed) in an in vitro human
dermal absorption study [6]. Recent studies by Burns-Naas et
al. [7] demonstrated an increase in liver weight in female rats
after a 3-month nose-only exposure to D5 at concentrations of
46 and 224 ppm. There were no histopathologic changes in the
liver that could be associated with the change in liver weight.
Changes in lung pathology, including interstitial inflammation
and accumulation of alveolar macrophages were also observed.
These findings were determined to be an exacerbation of an
existing condition in control animals.
McKim et al. [8] demonstrated that after a 28-day exposure
to 160 ppm D5 by whole-body inhalation, there was a significant
increase in liver weight. Hepatic microsomal enzymes were also
induced in a pattern that resembles phenobarbital in induction
profile, although the absolute level of enzyme induction was
much lower than phenobarbital. Zhang et al. [9] showed that oral
administration of D5 in corn oil for 4 days resulted in signifi-
cant increases in relative liver weights and induction of hepatic
enzymes similar to that with phenobarbital.
Because of the potential for multi-route exposure, the phar-
macokinetics of D5 have been investigated by inhalation, oral,
and dermal routes of exposure. Dermal absorption, which is most
relevant for the use of consumer products, was small (∼0.05%
of the applied amount). In addition, the majority of the dermally
absorbed D5 (90%) was rapidly eliminated through exhala-
tion. Several studies have been performed in rats and humans
to investigate the retention, distribution, metabolism and dis-
position of D5 following inhalation exposure. The retention
of D5 from single and multiple exposures was relatively low
(∼1–2% of inhaled D5 ) and the radioactivity was widely dis-
tributed to rat tissues following 6 h/day nose-only exposures to 7
or 160 ppm 14 C-D5 [10]. The majority of systemically absorbed
D5 was eliminated in the expired volatiles. After exhalation,
the most prevalent elimination routes were through the urine
and feces.
217
In a 2-year bioassay with male and female Fischer 344
rats (10, 40 and 160 ppm D5 , 6 h/day, 5 days/week) the only
remarkable findings was an increase in endometrial epithelial
neoplasia, primarily adenocarcinomas. There was not a corre-
sponding increase in uterine endometrial hyperplasia, and no
associated dose response [11].
In a two-generation inhalation reproductive toxicity study
[12] with octamethylcyclotetrasiloxane (D4 ), a structural ana-
logue of D5 , treatment-related reduction in mean live litter
size was seen in both the first (F0 ) and second (F1 ) gener-
ations at 500 and 700 ppm. The calculated male and female
mating and fertility indices were also reduced in the 700 ppm
group following the first and second F1 mating. Subsequent
evaluation of the data indicated that the effect was a reduc-
tion in the mating indices, which was likely due to estrous
cycle disturbances (persistent diestrus). There were no effects
on male reproductive endpoints. D5 has no estrogenic activity
in the rat uterotrophic assay and it was negative in both in vitro
estrogen receptor and in vitro luciferase reporter gene assays
[13].
In order to understand and assess human health risks, if any,
associated with a number of siloxanes, Dow Corning and other
silicone manufacturers developed a program to expand the safety
database on these materials. As part of this program, a two-
generation reproductive toxicity study in rats was conducted
with D5 in accordance with the United States Environmental
Protection Agency OPPTS Health Effects Test Guideline [14].
The study was conducted in compliance with Good Laboratory
Practice (GLP) Regulations [15].
2. Materials and methods
2.1. Test material
D5 was obtained by Dow Corning. At 25 ◦ C, D5 is a clear, colorless, odorless
liquid with a vapor pressure of 0.17 mm Hg [16]. One lot (WC015338) of D5 was
used for this entire study. Purity both before and after the study was confirmed
using a GC/FID method. GC/MS also was used to confirm chemical identity.
Analytical results showed that test material purities were 99.36% and 99.37%
for the retention and residual samples, respectively.
2.2. Animals and husbandry
Virgin male and female Sprague–Dawley Crl:CD® (SD)BR rats were
obtained from Charles River Laboratories Inc., Portage, MI. The animals were
30 days old upon receipt and were allowed a 15-day acclimation interval prior to
study start. This period included a 4-day acclimation to the automatic watering
system at which time the animals were group-housed. After the 4-day acclima-
tion period, the rats were housed individually in wire-mesh cages suspended over
cage board. Food (PMI Nutrition International Inc. Certified Rodent LabDiet®
5002, in meal form) and reverse osmosis-treated municipal drinking water were
supplied ad libitum except during each daily exposure period. The supplier
performed the analyses of each feed batch for nutrient levels and possible con-
taminants. For all feed batches, nutrient levels were at or above, and contaminant
levels were below the certified levels, and therefore, judged to be suitable for
use.
The animals were maintained on a 12-h light/dark cycle (lights on at
6 am). Environmental conditions during non-exposure periods were targeted
at a temperature of 22.2 ± 2.2 ◦ C and a relative humidity of 40–70%. Following
acclimation, all animals were examined for physical abnormalities and signs
of disease. The F0 animals were 45 days old at the start of exposures. All ani-
218 W.H. Siddiqui et al. / Reproductive Toxicology 23 (2007) 216–225

mal handling was performed in accordance with the guidelines of the National exposure cages that were placed into the inhalation chambers. Cage positions in
Research Council [17]. the exposure chambers were rotated daily to prevent bias due to location within
the chamber. Following each daily exposure period, the rats were returned to
2.3. Inhalation exposure systems, test atmosphere generation and their home cages.
monitoring
2.6. Estrous cyclicity determinations
The highest exposure concentration was 160 ppm, which was the highest
D5 exposure concentration that could be reliably generated and maintained as Vaginal smears were prepared daily from all F0 and F1 females selected for
a vapor without interference by aerosol formation. Exposure concentrations of breeding, beginning 21 days prior to pairing and continuing until either evidence
30 and 70 ppm were selected as the two lower concentrations. Control ani- of mating was detected or the mating interval elapsed. The smears were examined
mals were exposed to filtered air. Test atmospheres were generated in 2 m3 under a light microscope to determine the stage of estrus.
(2000 l) stainless steel and glass whole-body inhalation chambers. Chambers
were operated with 12–15 air changes per hour (400–500 l/min) using air sup- 2.7. Pairing, mating, and parturition
plied by a HEPA- and charcoal-filtered source. Environmental conditions within
the chambers were targeted at a temperature of 18–26 ◦ C and a relative humidity
The F0 rats were approximately 16 weeks old when paired 1:1 in the home
of 30–60%. Chamber ventilation rate, temperature, and relative humidity were
cage of the male. Mating pairs were examined daily for evidence of mating
monitored continuously and recorded at approximately 30-min intervals with
determined by the presence of a copulatory plug or sperm in a vaginal smear.
LabVIEW® for Windows® Data Acquisition software (National Instruments,
Positive evidence of mating was designated as GD 0. The mating pairs were then
Austin, TX).
separated, and the females were housed individually in plastic maternity caging
Vapor atmospheres of D5 were generated using metering pumps (Fluid
with nesting material (Bed-O’Cobs® , The Andersons Inc., Maumee, OH) until
Metering Inc., Oyster Bay, NY) to transfer test material at a known, con-
weaning on PND 21. If no evidence of mating was detected during the allotted
stant rate to glass vaporization columns, 20 cm long × 24 mm i.d. (Ace Glass
14-day mating interval, the pair was separated and the female was housed in
Inc., Vineland, NJ), filled with a mixture of 8 and 12 mm glass beads. The
maternity caging as noted above. Dams were closely observed during the time
columns were maintained at 87–90 ◦ C with 206-W electric heating tape (Glass-
of expected parturition for signs of dystocia and to determine when parturition
Col Inc., Terre Haute, IN) attached to a digital temperature controller (Omega
was complete. Individual gestation durations were calculated using the date
Engineering, Stanford, CA). Chamber inlet pipes were also wrapped with heat-
that delivery initiated. The day that parturition was judged to be complete was
ing tape and heated to 40 ◦ C. D5 concentrations were measured automatically
designated PND 0.
at 35-min intervals using a sample loop and computer-controlled multiposi-
Procedures for pairing and mating the F1 rats were analogous to those used
tion gas sampling valve attached to a GC/FID (Hewlett Packard 5890 Series
for the F0 animals. The F1 rats were 13–15 weeks old at the initiation of pairing.
II) equipped with a J&W DB-5 Megabore® column (15 m × 0.530 mm i.d.
Sibling pairings were avoided.
with 1.5 ␮m film thickness) and a 3396 Series II Integrator. Prior to initia-
tion of animal exposures, an industrial dust sensor (Model #IDS-10) was used
to demonstrate that aerosol formation did not occur under exposure chamber 2.8. Litter data collection and lactation
conditions.
Litters were sexed, pups were examined for gross malformations, and the
numbers of live and stillborn pups were recorded upon completion of parturi-
2.4. Group assignments and exposure regimen
tion. All pups were identified using tattoo markings (AIMS Inc., Piscataway,
NJ) on the digits on PND 0. Litters were culled to eight pups (4 per sex where
A computerized randomization based on body weight stratification in a block
possible) on PND 4 using a computerized randomization program. Pups were
design was used to assign the F0 rats to either the control or exposure groups
examined twice daily for survival. Each pup was weighed and received a detailed
(n = 30/sex/group). Exposures were 6 h/day for at least 70 consecutive days prior
physical examination on PND 1, 4, 7, 14 and 21 and at weekly intervals there-
to pairing and continued throughout mating, gestation and lactation until the
after until euthanasia. Intact pups dying from PND 0–4 were necropsied using
day prior to euthanasia following offspring weaning on PND 21. Female expo-
a modification of the Stuckhardt and Poppe [18] fresh dissection technique.
sure was suspended from gestation day (GD) 21 through PND 4 to prevent
Pups with external abnormalities that warranted further evaluation were cleared
parturition from occurring within the inhalation chamber and to avoid separat-
and stained for skeletal exams as described by Dawson [19]. A detailed gross
ing the dams from their offspring during early neonatal life. Direct inhalation
necropsy was done on all pups dying between PND 4 and weaning. Gross lesions
exposures commenced for randomly selected F1 animals on PND 22 and con-
from these animals were preserved in 10% neutral-buffered formalin for possible
tinued for at least 70 consecutive days prior to pairing and mating to produce
histopathologic evaluation.
the F2 generation. F1 exposures continued through the mating interval (up to
14 days) and gestation through GD 20 at which time exposures for the dams
were suspended until resuming on lactation day 5, as for the F0 generation. 2.9. Weaning and selection
The F1 maternal and paternal exposures then continued throughout lactation
until weaning on PND 21. The F2 animals did not receive any direct inhalation A computerized randomization program was used to select 60 F1
exposure. pups/sex/group at weaning on PND 21. These animals were directly exposed to
D5 or filtered air for 6 h/day, starting on PND 22. From those pups selected at
2.5. In-life methods and observations weaning, 30 rats/sex/group were selected on PND 28 using the same randomiza-
tion procedure. When available, a minimum of one male and one female from
each litter were selected. The rats selected on PND 28 were used to produce the
Individual F0 and F1 body weights were recorded weekly, beginning with
F2 generation. The F2 pups were culled on PND 4 and reared to weaning on
the initiation of exposure and continuing until euthanization. All animals were
PND 21 using procedures analogous to those used for the F1 .
checked twice daily for general appearance, behavior, moribundity and mortal-
ity. Detailed physical exams were performed weekly. The rats were observed
for clinical signs of toxicity during and approximately 1 h after cessation of 2.10. Sexual maturation
each daily exposure session. Following detection of evidence of mating, female
body weights were recorded on GD 0, 7, 10, 14 and 20, as well as on lactation On PND 1, anogenital distance (AGD) was measured for all F1 pups in the
days 1, 4, 7, 14 and 21. Food consumption was measured weekly until pairing. control and 160 ppm groups and for all F2 pups in all exposure groups. Each
Following mating, female food consumption was measured on GD 0, 7, 10, 14 selected F1 and F2 female was observed for vaginal patency starting on PND 30
and 20, as well as on lactation days 1, 4, 7, 14 and 21. For the exposures, the [20]. Each selected F1 and F2 male was observed for balanopreputial separation
rats were transferred from their home cages to racks of individual wire-mesh starting on PND 40 [21].
 W.H. Siddiqui et al. / Reproductive Toxicology 23 (2007) 216–225
2.11. Spermatogenic evaluations
Immediately upon euthanasia, the reproductive tract of the F0 and F1 males
used for breeding was exposed by a ventral mid-line incision. The right epi-
didymis was excised and weighed, and an incision was made in the distal right
cauda epididymis. The right cauda epididymis was then placed in 40 ml Dul-
becco’s phosphate-buffered saline (pH 7.0–7.6; 37 ◦ C) with 10 mg/ml bovine
serum albumin for a 10 min period. At least 200 spermatozoa, motile and
non-motile, from each male (if possible) were analyzed in all control and expo-
sure groups. Motility was evaluated at constant temperature (∼37 ◦ C) using
a Hamilton-Thorne HTM-IVOS version 10 computer-assisted sperm-analysis
(CASA) system.
Sperm morphology for the F0 males was performed using a dry-mount pro-
cedure. Duplicate smears of epididymal sperm from the cauda were prepared on
microscope slides and dried. The slides were then stained with Weigert’s-iron
hematoxylin, counter-stained with eosin-phloxin, cover-slipped, and evaluated.
F1 sperm morphology was evaluated by light microscopy using a modification
of the wet-mount procedure described by Linder et al. [22]. Abnormal sperm
(double heads, double tails, microcephalic or megacephalic sperm, etc.) were
recorded from a differential count of 200 spermatozoa per animal where possible.
Sperm parameters (including testicular and epididymal sperm numbers and
sperm production rate) were assessed in all F0 and F1 males used for breeding.
The left testis and epididymis were weighed, frozen, homogenized, and then
evaluated for homogenization-resistant spermatid counts and production rates
[23].
2.12. Euthanasia, necropsy and histopathology
All surviving F0 and F1 adults were euthanized by intravenous injection
of sodium pentobarbital after weaning of their offspring. A complete necropsy
was done on all parental animals (F0 and F1 ) dying spontaneously, euthanized
in extremis, or surviving to their scheduled euthanasia. The necropsy included
macroscopic examination of external surfaces, all orifices, the cranial cavity,
external surfaces of the brain and spinal cord, the thoracic, abdominal and
pelvic cavities and organ weights. Tissues from all groups were preserved in
formalin. A complete set of organs was weighed from all F0 and F1 animals.
A microscopic examination was performed on hematoxylin and eosin stained
tissue sections from the control and 160 ppm groups, and for all unscheduled
necropsies [24,25]. The following tissues were examined: adrenal gland, brain,
cervix, coagulating gland, epididymis (right caput, corpus, and cauda), kidneys,
liver, lungs, ovaries, penis, pituitary gland, prepuce, preputial gland, prostate,
seminal vesicles, testis (right), thyroid, uterus, vagina, vas deferens and all gross
internal lesions. Initial analysis indicated that the lung was a target organ of
toxicity, so lung sections from all groups were examined. The right testis and
epididymis (caput, corpus, and cauda) were fixed, embedded, and stained with
PAS and hematoxylin. Ovaries from F0 and F1 control and high exposure rats
were fixed, trimmed, and embedded in paraffin as described above. Five sections
at least 100 ␮m apart were taken from the inner third of each ovary. The sections
were mounted on slides, stained with hematoxylin and eosin, and evaluated to
determine the numbers of primordial follicles and presence/absence of grow-
ing follicles and corpora lutea [26,27]. All F2 weanlings were euthanized and
necropsied on PND 21.
2.13. Statistical analyses
Unless otherwise noted, all analyses were two-tailed using a minimum signif-
icance level of 5% (p < 0.05). Means and standard deviations (S.D.) are presented
with the number of animals (N) used to calculate the mean. Statistical analyses
were not performed if the number of animals was 2 or less. The following tests
were performed: χ2 test with Yates’ correction factor for parental mating and fer-
tility indices [28]; one-way analysis of variance (ANOVA) [29] with Dunnett’s
test [30] for precoital intervals, parental weekly, gestational, and lactational body
weights and food consumption data, gestation lengths, sperm numbers, sperm
production rates, organ weights, ovarian primordial follicle counts, numbers of
pups born (experimental unit = litter), live litter sizes (experimental unit = litter),
AGDs (experimental unit = litter), pup body weights (experimental unit = litter),
time to vaginal patency, and time to balanopreputial separation; Kruskal–Wallis
219
test with Mann–Whitney U test [31] for sperm motility, sperm morphology,
pup sexes at birth (% males per litter; experimental unit = litter), and postnatal
survival (% per litter; experimental unit = litter); and Kolmogorov–Smirnov test
[32] (one-tailed) for histopathological findings. The AGD data were normalized
using the ratio of AGD/cube root of body weight as discussed by Gallavan et al.
[33].
3. Results
3.1. Exposure concentrations
The overall mean measured D5 concentrations (±S.D.)
for the F0 generation were 31 (±0.7), 71 (±1.4), and 162
(±2.3) ppm for the 30, 70 and 160 ppm groups, respectively.
The corresponding values for the F1 generation were 31 (±0.8),
71 (±1.6), and 162 (±3.2) ppm.
3.2. General health parameters
3.2.1. Clinical signs and mortalities
No exposure-related clinical signs were detected in either
the F0 or F1 generation. Six mortalities occurred among the F0
animals. In the 30 and 160 ppm groups, respectively, one and
two F0 males and one and one females were found dead. In
addition, one F0 female in the 70 ppm group was euthanized in
extremis. However, no clear exposure–response relationship was
evident and no consistent target organ could be identified for the
F0 mortalities, and therefore, the deaths were not attributed to
D5 exposure. This conclusion is supported by the fact that all
selected F1 rats survived to their scheduled euthanasia.
3.2.2. Body weight and food consumption
There were no exposure-related effects on body weight or
body weight gain during the premating, gestation, or lactation
intervals for either the F0 or F1 generations. Small, statistically
significant increases and decreases in weekly body weight gains
were seen sporadically among the 30, 70, and 160 ppm groups
in the F0 and F1 generations. They were not considered to be
exposure-related because these differences were small, transient,
and unaccompanied by a consistent exposure–response relation-
ship. Weekly food consumption during the premating interval,
gestation, and lactation was not affected by D5 exposure in either
generation (data not shown).
3.2.3. Necropsy, organ weights, and histopathology
No exposure-related gross findings were noted at the sched-
uled necropsies of the F0 and F1 rats of either sex. There were no
differences in mean organ weight or organ:body weight ratio data
that could be attributed to D5 exposure. A significant increase in
mean brain weight relative to body weight was seen in 160 ppm
F1 females. However, this was judged not to be related to D5
exposure since the mean absolute brain weight at 160 ppm for the
F1 (1.96 ± 0.088 g) was not significantly different from the con-
trol F1 (1.91 ± 0.112 g). Moreover, the brain:body weight ratios
were not decreased significantly among the F0 females. Statis-
tically significant increase in mean liver weight was observed
in the 160 ppm F0 females. However, no remarkable differences
220 W.H. Siddiqui et al. / Reproductive Toxicology 23 (2007) 216–225

Table 1
Summary of selected F0 and F1 parental absolute (g) and relative (g/100 g) organ weights
Males Females

0 30 70 160 0 30 70 160

Final body weight F0 536 ± 54 539 ± 41 543 ± 45 559 ± 52 311 ± 26 320 ± 34 320 ± 38 320 ± 30
F1 530 ± 50 528 ± 57 530 ± 59 520 ± 60 302 ± 27 292 ± 20 295 ± 27 292 ± 20

Liver
Absolute F0 17.90 ± 2.17 18.34 ± 1.89 18.37 ± 2.33 19.25 ± 2.39 11.14 ± 1.48 11.83 ± 1.12 12.09 ± 1.82 12.44 ± 1.72**
Relative 3.35 ± 0.30 3.41 ± 0.32 3.38 ± 0.29 3.44 ± 0.21 3.59 ± 0.39 3.71 ± 0.32 3.78 ± 0.34 3.76 ± 0.33
Absolute F1 18.12 ± 2.36 18.09 ± 2.78 17.81 ± 2.31 17.71 ± 2.61 11.51 ± 1.57 10.41 ± 1.41* 11.13 ± 1.53 11.13 ± 1.27
Relative 3.42 ± 0.28 3.42 ± 0.28 3.37 ± 0.28 3.40 ± 0.23 3.81 ± 0.42 3.56 ± 0.37* 3.78 ± 0.41 3.82 ± 0.36

Lungs
Absolute F0 2.22 ± 0.60 2.20 ± 0.63 2.21 ± 0.67 2.27 ± 0.61 1.96 ± 0.52 1.78 ± 0.49 1.83 ± 0.50 1.72 ± 0.51
Relative 0.42 ± 0.13 0.41 ± 0.12 0.41 ± 0.12 0.41 ± 0.12 0.64 ± 0.20 0.56 ± 0.16 0.58 ± 0.17 0.52 ± 0.15
Absolute F1 2.12 ± 0.45 2.30 ± 0.71 2.24 ± 0.69 2.12 ± 0.58 1.77 ± 0.45 1.69 ± 0.46 1.65 ± 0.39 1.70 ± 0.41
Relative 0.40 ± 0.07 0.44 ± 0.15 0.43 ± 0.15 0.43 ± 0.15 0.59 ± 0.15 0.58 ± 0.16 0.56 ± 0.14 0.58 ± 0.15

Right testis
Absolute F0 1.76 ± 0.18 1.72 ± 0.20 1.77 ± 0.11 1.74 ± 0.19
Relative 0.33 ± 0.04 0.32 ± 0.04 0.33 ± 0.04 0.31 ± 0.04
Absolute F1 1.78 ± 0.17 1.68 ± 0.14 1.75 ± 0.14 1.70 ± 0.22
Relative 0.34 ± 0.04 0.32 ± 0.03 0.33 ± 0.04 0.33 ± 0.05

Left testis
Absolute F0 1.78 ± 0.19 1.74 ± 0.19 1.80 ± 0.10 1.75 ± 0.21
Relative 0.33 ± 0.04 0.32 ± 0.04 0.33 ± 0.04 0.32 ± 0.05
Absolute F1 1.78 ± 0.17 1.76 ± 0.28 1.78 ± 0.13 1.72 ± 0.28
Relative 0.34 ± 0.04 0.33 ± 0.05 0.34 ± 0.04 0.33 ± 0.06

Ovaries
Absolute F0 0.149 ± 0.020 0.141 ± 0.028 0.147 ± 0.023 0.141 ± 0.018
Relative 0.048 ± 0.008 0.044 ± 0.009 0.047 ± 0.010 0.043 ± 0.007
Absolute F1 0.161 ± 0.017 0.147 ± 0.024** 0.153 ± 0.016 0.152 ± 0.018
Relative 0.054 ± 0.006 0.050 ± 0.009 0.053 ± 0.007 0.052 ± 0.005

0, 30, 70 and 160 denote the exposure level in ppm.

* Significantly different from control group at p ≤ 0.05 using Dunnett’s test.
** Significantly different from control group at p ≤ 0.01 using Dunnett’s test.

in mean liver weight were noted at this concentration in the

F1 females. Additionally, the mean liver weight relative to final
Table 2
body weight in the 160 ppm F0 females was similar to that in the Histopathological findings in the F0 and F1 generationsa
control group (Table 1). Other significant differences in mean
Finding Exposure group
organ:body weight ratios relative to controls animals were seen
in the 30 ppm F1 females for the liver and spleen. These were Control 30 ppm 70 ppm 160 ppm
not accompanied by an exposure–response relationship, and no Alveolar histiocytosis (minimal)
such changes were noted in the F0 generation. F0 males 5/30 5/29 7/30 6/28
Noteworthy findings from the histopathological examina- F0 females 0/30 5/29 4/29 10/29*
tions following scheduled necropsies of the F0 and F1 rats are F1 males 2/30 4/30 6/30 7/30
F1 females 3/30 10/30 8/30 13/30*
listed in Table 2. Significantly increased incidences of minimal
alveolar histiocytosis were seen in the high-concentration F0 and Pulmonary vascular mineralization (minimal)
F1 females; a non-significant increased incidence also was noted F0 males 13/30 17/29 23/30* 4/28
F0 females 7/30 20/29* 22/29b,* 10/29
in the F1 males. This lesion was evident as small sporadic groups F1 males 6/30 28/30* 27/30* 16/30*
of foamy macrophages in the alveolar spaces, primarily in the F1 females 13/30 22/30 24/30* 4/30
periphery of the lobe. A significant minimal increase in the inci- a Data are expressed as no. of animals with finding/no. of animals examined.
dence of pulmonary vascular mineralization was seen in all F0 b One of these cases was graded as mild.
and F1 animals. However, no exposure–response relationship in * p < 0.05, as compared to control values using the Kolmogorov–Smirnov test

either incidence or severity among the exposed groups was evi- (one-tailed).
 W.H. Siddiqui et al. / Reproductive Toxicology 23 (2007) 216–225 221

Table 3
Reproductive parameters and litter data in rats exposed to D5
Parametera F0 generation F1 generation

0 30 70 160 0 30 70 160

No. mating pairs 30 30 30 29 30 30 30 30

Precoital intervalb 3.0 ± 1.44 3.7 ± 2.70 3.6 ± 2.39 2.4 ± 1.19 4.0 ± 2.91 3.1 ± 2.71 3.4 ± 2.68 3.7 ± 2.08
Mating index (%)c 100 83.3 93.3 93.1 96.7 90.0 100.0 100.0
Fertility index (%)c 93.3 76.7 86.7 79.3 96.7 80.0 100.0 96.7
Gestation lengthd 21.8 ± 0.39 21.6 ± 0.49 21.6 ± 0.50 21.9 ± 0.81 21.5 ± 0.51 21.4 ± 0.49 21.4 ± 0.50 21.5 ± 0.50
Total no. of pups/litter 13.7 ± 2.52 13.8 ± 2.19 14.2 ± 2.07 12.3 ± 3.93 13.6 ± 2.19 13.1 ± 1.80 13.3 ± 1.68 14.0 ± 1.78
No. of live pups/litter 13.4 ± 2.25 13.4 ± 2.73 13.9 ± 2.11 12.1 ± 3.93 13.6 ± 2.18 13.0 ± 1.90 13.1 ± 1.82 14.0 ± 1.80
Sex ratioe 46.5 ± 13.05 52.9 ± 13.86 51.1 ± 13.83 49.6 ± 15.82 51.8 ± 14.59 50.3 ± 11.83 48.6 ± 12.18 46.6 ± 12.88
Pup survivalf
PND 0 98.0 (±4.86) 96.5 (±7.77) 97.5 (±4.13) 94.2 (±20.9) 99.8 ± 1.24 99.3 ± 3.40 98.2 ± 4.57 99.5 ± 1.90
PND 0–1 99.6 (±1.89) 97.3 (±7.24) 98.7 (±3.13) 98.8 (±2.68) 99.8 ± 1.33 95.5 ± 8.75** 98.6 ± 3.38 99.5 ± 1.86
PND 1–4 (precull) 99.4 (±2.04) 98.7 (±2.98) 99.5 (±1.74) 99.3 (±2.17) 99.8 ± 1.33 99.4 ± 1.95 99.7 ± 1.40 99.3 ± 2.13
PND 0–4 (precull) 97.1 (±5.51) 93.0 (±12.00) 95.7 (±5.40) 92.5 (±20.6) 99.3 ± 2.77 94.3 ± 9.53** 96.5 ± 5.23** 98.3 ± 3.50
PND 4 (postcull)–21 99.1 (±3.28) 99.4 (±2.67) 99.5 (±2.50) 100 (±0.00) 99.1 ± 3.22 100.0 ± 0.00 100.0 ± 0.00 100.0 ± 0.00
No. of total litter losses 0 0 0 1g 0 0 0 0
a Unless otherwise noted, data are expressed as the mean ± S.D.
b Precoital interval is expressed in days.
c Mating index = [number of males (or females) with evidence of mating/total number of males (or females) paired] × 100. Male fertility index = [number of

males impregnating a female/total number of males paired] × 100. Female fertility index = [number of females with a confirmed pregnancy/total number of females
paired] × 100. Because each male was paired with only one female, the male and female mating and fertility indices are identical.
d Gestation length is expressed in days.
e Sex ratio is expressed as % males per litter.
f Pup survival is expressed as % per litter for the indicated time interval.
g One female had a litter consisting of only one pup that was found dead on PND 0 and these data were not included in the analyses.
** Significantly different from control values at p ≤ 0.01.

dent. Significant increases in non-suppurative inflammation was
found in the 30 and 70 ppm F1 males, but there was no associated
dose response. No other associated pulmonary histopathology or
changes in lung weights were seen in either generation. The inci-
dence of lesions in other organs was not significantly different
from the incidence in the controls.
3.3. Reproductive parameters, litter data, and sexual
development
3.3.1. Reproductive parameters
Reproductive performance was not adversely affected by
exposure to D5 in either the F0 or F1 generations (Table 3). There
were no exposure-related effects on the regularity or duration
of estrous cycles, number of days between pairing and mating,
mating or fertility indices, duration of gestation, or parturition.
3.3.2. Sperm assessments and ovarian histopathology
D5 exposure did not adversely affect any spermatogenic
endpoint (viz., sperm number, production rate, motility, mor-
phology) evaluated in either the F0 or F1 (Table 4). A significant
decrease in epididymal sperm number was seen in the F0 males
at 70 ppm. However, this was not attributed to D5 exposure
since it was not accompanied by an exposure–response rela-
tionship. No effects on epididymal sperm number were seen in
any of the F1 groups. Histopathological evaluation of the ovaries
revealed no statistically significant exposure-related differences
in mean ovarian primordial follicle counts. Ovarian sections
with growing follicles were noted with similar frequency in the
control and 160 ppm groups of both the F0 and F1 generations
(Table 4).
3.3.3. Litter data
The total number of F1 and F2 pups born per litter, mean live
litter size, and sex ratio of litters (percentage of males per litter)
were unaffected by parental exposure to D5 (Table 3). No mean-
ingful exposure-related differences between treated and control
groups were seen in pup survival and general health in any post-
natal time interval examined. One F0 female at 160 ppm had only
one pup which was partially cannibalized. However, because no
exposure-related decreases in live litter size or in postnatal sur-
vival were noted in any treatment group, when compared with
the controls, this finding was not attributed to D5 exposure.
3.3.4. Developmental endpoints
On PND 1, there was a slight, but statistically significant,
increase in the mean F1 male pup AGD (absolute and relative to
cube root of body weight) in the 160 ppm group, however, there
was no change in the mean F1 female pup AGD or across all
exposure groups in the F2 generation (Table 5).
No effects were seen on the time for offspring to reach sexual
maturity in either the F1 or the F2 generation (Table 6). A statis-
tically significant (p < 0.05) increase in the mean time to achieve
vaginal patency was noted in the F1 females at 30 ppm. How-
ever, no differences from control were evident at 70 or 160 ppm,
and the increase at 30 ppm was not judged to be related to D5
222 W.H. Siddiqui et al. / Reproductive Toxicology 23 (2007) 216–225

Table 4
Spermatogenic assessments and ovarian histopathology
Parametera F0 generation F1 generation

0 30 70 160 0 30 70 160

Sperm parameters
No. of rats examined 30 29 30 28 30 30 30 30
Sperm numberb
Left testis 93.3 ± 17.73 87.5 ± 14.97 91.1 ± 23.84 100.1 ± 26.60 85.7 ± 14.29 78.6 ± 15.73 82.4 ± 13.33 79.5 ± 22.84
Left epididymis 529.9 ± 113.36 508.2 ± 133.81 424.1 ± 93.20* 564.0 ± 194.48 478.8 ± 65.70 511.2 ± 92.48 476.9 ± 85.44 444.5 ± 130.10
Production ratec 15.3 ± 2.91 14.3 ± 2.46 14.9 ± 3.90 16.4 ± 4.36 14.0 ± 2.33 12.9 ± 2.58 13.5 ± 2.18 13.0 ± 3.74
Motility (%)d 88.3 ± 4.91 87.3 ± 5.45 84.5 ± 12.64 84.5 ± 8.10 77.7 ± 11.60 77.5 ± 14.02 77.1 ± 11.05 76.6 ± 12.66
Morphologye 99.3 ± 0.56 98.7 ± 1.12 98.8 ± 1.30 99.1 ± 0.91 98.9 ± 1.19 98.8 ± 1.33 98.0 ± 2.37 99.1 ± 0.93

Ovarian primordial follicle counts

No. of rats examined 30 ND ND 30 ND ND 30
No. of folliclesf 56.7 ± 29.52 ND ND 48.7 ± 18.55 56.6 ± 31.46 ND ND 51.3 ± 23.75
a Data are expressed as mean ± S.D.
b Sperm number is given in millions of sperm per gram of tissue.
c Sperm production rate is calculated as number of sperm (in millions) per gram of tissue divided by 6.1 days (the rate of turnover of the germinal epithelium).
d Sperm motility data are expressed as % motile sperm. A minimum of 200 spermatozoa (motile and non-motile) per animal were evaluated. Because males with

insufficient numbers of sperm were excluded from the analysis, the number of animals examined was 29, 28, 30, and 27 for the control, 30, 70, and 160 ppm groups
in the F1 , and 30, 30, 30, and 29 for these groups in the F1 .
e Sperm morphology data are expressed as the % of sperm with no visible morphological abnormalities.
f Ovarian follicle counts are presented as the mean number obtained by evaluating 10 sections per animal as described in Section 2.
* Statistically different from control at p < 0.05 using one-way ANOVA and Dunnett’s test.

Table 5
Anogenital distance (AGD) on PND 1 in neonatal rats from dams exposed to D5
AGD F1 pups F2 pups

0 30 70 160 0 30 70 160

Absolute AGD (mm)a

Males 5.6 ± 0.50 NDb ND 6.1 ± 0.77* 5.0 ± 0.31 5.1 ± 0.31 5.0 ± 0.36 5.0 ± 0.33
Females 3.7 ± 0.34 ND ND 3.9 ± 0.71 3.4 ± 0.37 3.3 ± 0.37 3.4 ± 0.44 3.4 ± 0.33
AGD normalized to the cube root of body weighta
Males 2.96 ± 0.24 ND ND 3.16 ± 0.34* 2.63 ± 0.14 2.71 ± 0.15 2.68 ± 0.16 2.64 ± 0.16
Females 1.98 ± 0.20 ND ND 2.08 ± 0.35 1.85 ± 0.19 1.80 ± 0.18 1.83 ± 0.26 1.83 ± 0.17
a Data are expressed in mean ± S.D.
b Not done.
* These values differed significantly (p < 0.05) from control group values when evaluated using one-way ANOVA (BMDP, 1979) with Dunnett’s test.

Table 6
Effect of D5 exposure on the time to developmental markers of sexual maturity
Generation Exposure concentration (ppm)

0 30 70 160

Females: mean days of age at vaginal patencya

F1 34.1 ± 2.02 35.8 ± 2.89* 34.8 ± 2.24 35.1 ± 2.29
F2 36.9 ± 4.36 37.4 ± 3.64 35.8 ± 3.28 36.4 ± 5.06
Males: mean days of age at balanopreputial separationa
F1 44.9 ± 2.11 46.7 ± 3.50 45.9 ± 2.83 46.1 ± 2.50
F2 46.4 ± 4.78 46.9 ± 4.92 47.6 ± 3.61 47.0 ± 3.56
a Data are expressed as the mean age at the time the milestone was first evident.
* Significantly different from control group at p < 0.05 using one-way ANOVA and Dunnett’s test.

exposure. The times for F2 females to exhibit vaginal patency 4. Discussion

and for F1 or F2 males to demonstrate balanopreputial sepa-
ration did not differ significantly between exposed and control D5 is used in a broad range of industrial and consumer
rats. applications. Because of the potential for widespread human
 W.H. Siddiqui et al. / Reproductive Toxicology 23 (2007) 216–225
exposure, D5 was selected for extensive safety testing. This
study was conducted to identify potential reproductive hazards
associated with exposure to D5 .
An extensive exposure assessment established that the two
main routes of human exposure to D5 are (1) inhalation of D5
vapor in workplace settings, and (2) dermal contact with D5
present in personal care products [1]. The major route of expo-
sure was via inhalation for workers and the general public. Both
the dermal and inhalation routes of exposure were considered for
personal care users. There is essentially no oral exposure to D5 .
Due to its physical properties, D5 has been widely used in con-
sumer products as a carrier, emollient and lubricant. Consumer
products evaluated included AP/Ds, HC products (shampoo,
rinse-out conditioner, leave-in conditioner and hair spray) and
SC products (mascara, moisturizer, nail care and foundation).
While most AP/Ds and some SC products may be formulated
with moderate levels (0–50%) of D5 , this is not the case for the
majority of HC/SC products. Often D5 is only present in small
amounts in materials that are used to make HC/SC products so
the remaining level of D5 in the final product is <10%.
Thus, either dermal applications or inhalation exposures
would be the most directly relevant to humans. The choice
of inhalation rather than dermal for the pivotal safety studies
was justified on practical as well as metabolic, kinetic, and sys-
temic exposure grounds. A repeated-dose study via the dermal
route could not be seriously considered because of the surface-
spreading characteristics of D5 , the volatility, and the dosing
requirements in such a study. In addition, extensive pharmacoki-
netic studies have been completed with D5 as well as another
cyclic siloxane, octamethylcyclotetrasiloxane (D4 ) following
several routes of administration in rats and limited studies on
inhalation and dermal exposures in human subjects. For absorp-
tion and retention of D4 and D5 in the body, there are three
important attributes that control the disposition of these com-
pounds. The pharmacokinetics of these compounds are heavily
influenced by an unusual set of properties, high lipid partition-
ing coupled with very high blood clearance due to exhalation
and metabolism. Inhalation and dermal absorption lead to simi-
lar pharmacokinetics, presumably since they provide uptake of
molecular forms of the siloxanes. Oral dosing leads to more com-
plex pharmacokinetic that appears to be associated with uptake
of siloxanes as microemulsions. During dermal exposures to D4
and D5 , these chemicals are rapidly absorbed into the outer layers
of skin, but they evaporate back out of the skin before signifi-
cant systemic absorption can occur. In vivo rat dermal absorption
studies showed that ∼90% of either D4 or D5 volatilize from
the skin surface and that the remaining D4 or D5 in the skin
at the end of the 24 h dosing period actually migrated to the
skin surface and continued to evaporate, significantly decreas-
ing the amount remaining in the skin to ∼0.09% D4 and 0.03%
D5 [34,35]. Physiologically based pharmacokinetic modeling of
the percutaneous absorption data from an in vivo human dermal
absorption study also predicts dermal absorption of D4 and D5
to be about 0.5% and 0.05%, respectively [36]. Of the amount
systemically absorbed, ∼80% D4 and 90% of D5 is exhaled
unchanged. Thus, systemic concentrations for these materials
following dermal exposure would be minimal.
223
The exposure concentrations used in this study were cho-
sen based on physical properties of D5 and potential exposure
to humans. Based on the vapor pressure of D5 , the 160 ppm
group is the highest concentration of D5 that could be reliably
generated and controlled without interference by aerosol forma-
tion. Aerosols are not generated in any of the applications and
uses identified in the exposure study. The low exposure con-
centration in this study (30 ppm) is significantly higher than the
average measured concentrations of around 1 ppm of D5 in the
manufacturing workplace [1].
No significant toxicological effects on either general (i.e.,
non-reproductive) or reproductive endpoints were identified in
this study. Clinical signs of toxicity, animal appearance and
behavior, body weight and body weight gain, food consump-
tion, gross necropsy findings, and organ and tissue weights were
unaffected by D5 exposure. The only remarkable histopatholog-
ical finding was an increased incidence of pulmonary alveolar
histiocytosis (graded minimal in all cases; see Table 2). This
finding, which is indicative of an inflammatory response by pul-
monary macrophages, was characterized by occasional small
groups of foamy macrophages within the alveolar spaces, pri-
marily at the periphery of the lobe. However, this finding was
not accompanied by any significant changes in absolute or rela-
tive lung weights. The alveolar histiocytosis was considered to
be an exposure-related exacerbation of the spontaneous lesion
described by Boorman et al. [37] and Mohr [38]. Rather than
being viewed as a frankly adverse effect, this finding was most
probably a compensatory response to the presence of material
delivered to the lungs by inhalation. Although the focal alveo-
lar macrophage accumulations were more prevalent in females
than in males in this study, this is in contrast to other studies
in Fischer 344 rats that found males to be more responsive than
females in this regard [39]. Similar lesions were reported in a 90-
day study with D5 in male and female rats exposed to 224 ppm
via inhalation [7]. However, these authors noted that an aerosol
formed at 224 ppm and that the macrophage influx may have
been a clearance mechanism for removal of aerosol particles.
D5 did not elicit any adverse effects on the reproductive
endpoints examined. Estrous cyclicity, sperm parameters, repro-
ductive performance, and litter parameters were not affected
by D5 exposure (Tables 3 and 4). The slight, but statistically
significant, increase in the mean F1 male pup AGD (absolute
and adjusted) in the 160 ppm group was not considered to be
treatment-related (Table 5). If prenatal D5 exposure causes an
increase in PND 1 male pup AGD, this alteration should have
been evident in both the F1 and F2 generations of prenatally
exposed pups. An increase in perinatal male AGD would typ-
ically be considered an androgenic response (since the growth
of this peritoneum is androgen-regulated) and a concomitant
increase in perinatal female AGD should be apparent [40,41]. If
there was a true increase in male AGD and not female AGD, the
toxicological significance of this observation in only one of the
two generations would be difficult to explain and would unlikely
be considered adverse effect in this animal model without other
signals of developmental, reproductive or neurobehavioral tox-
icity. In addition, D5 whole-body vapor exposure to castrated
male F-344 rats at a concentration of 160 ppm did not demon-
224 W.H. Siddiqui et al. / Reproductive Toxicology 23 (2007) 216–225
strate androgenic activity when evaluated in a Hershberger
assay [13]. Furthermore, there is no supporting data in the cur-
rent study to suggest that D5 affects other endpoints of male
reproductive development (e.g., organ weights, histopathology,
sperm assessment). Based on the above rationale and since the
more conclusive F2 generation AGD data was virtually identical
across all exposure groups, the alteration in the F1 male AGD
was not considered related to D5 exposure.
A statistically significant increase in the mean time to achieve
vaginal patency was noted in the F1 females at 30 ppm. However,
no differences from control were evident at 70 or 160 ppm, and
the increase at 30 ppm was not considered to be related to D5
exposure. The times for F2 females to exhibit vaginal patency
and for F1 or F2 males to demonstrate balanopreputial separa-
tion did not differ significantly between exposed and control rats.
The historical control mean for vaginal patency for 17 studies
conducted in the same strain and in the same time frame of the
D5 study was 33.2 ± 1.63 (31.0–38.2) days of age. The histori-
cal control mean for balanopreputial separation was 43.5 ± 1.44
(40.3–46.5) days of age. The values in this study for both param-
eters were slightly higher than the historical control values. We
have seen this consistently in inhalation studies relative to oral
studies and have attributed this to the fact that the inhalation
animals tend to have lower weights than animals in the oral
studies. The mean values in the D5 study are consistent with
the WIL historical control data for inhalation studies. There are
eight inhalation studies in the historical control data for IGS rats.
The mean for vaginal patency is 35.2 ± 2.08 (32.5–38.8) days
of age. The mean for balanopreputial separation is 46.3 ± 1.55
(44.2–49.0) days of age.
The data presented here demonstrate an absence of adverse
reproductive effects in rats exposed to D5 . These findings stand
in contrast to results obtained in reproductive toxicity assess-
ments of octamethylcyclotetrasiloxane (D4 ) [12]. Although D4
is a structural analogue of D5 , and the overall kinetic behavior
is similar to D4 , it differs substantially from D5 in many phys-
ical properties. For example, D5 has a larger molecular weight
and size, a lower vapor pressure (limiting the highest concentra-
tion that you can reliably generate to 160 ppm D5 compared to
700 ppm for D4 ), and higher viscosity which all limit systemic
bioavailability compared to D4 . Whether these differences in
physical and chemical properties play a role in the observed
reproductive toxicity with D4 is unknown at this time.
In summary, no adverse effects on reproductive function in
Sprague–Dawley rats were associated with prolonged expo-
sure to 160 ppm D5 . The NOAEL (No-Observed-Adverse-Effect
Level) for both parental and reproductive toxicity was deter-
mined to be 160 ppm D5 in this study.
Acknowledgements
The excellent performance by the technical staff at WIL
Research Laboratories is gratefully acknowledged. We thank
Rachel Dankert for assistance in manuscript preparation. This
work was supported in part by the Silicones Environmental,
Health and Safety Council of North America.
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