Document ID: EPA-HQ-OPP-2004-0147-0029
Agency: epa
Document Type: Supporting & Related Material
Title: 
Posted Date: 2009-05-14T04:00Z

EPA Reviewer: Robert P. Zendzian, Ph.D.			              Robert P.
Zendzian 

Toxicology Branch, HED

EPA Secondary Reviewer: Steven L. Malish, Ph.D. Toxicologist      
_____________________

Team 1, RASSB/Antimicrobial Division

		

DATA EVALUATION REPORT

STUDY TYPE: Prenatal Developmental Toxicity Study - Rat; OPPTS 870.3700
[§83-3]; OECD 414.

PC CODE:  08802	DP BARCODE: D316702, D321264, D330028         

REG:  2480-21972                                                
DECISION #: 341745

TEST MATERIAL (PURITY): Zinc pyridinethione; (98.3% a.i.)

SYNONYMS: ZPT, Zinc Pyrithione

CITATION:	Barnett, J. Jr. (2005). Percutaneous developmental toxicity
study of zinc pyridinethione (ZPT) in rats. CR-DDS Argus Division
(Horsham, Pennsylvania). Argus AEN00006, April 7, 2005. MRID 46534001.
Unpublished.

SPONSOR:	Arch Chemicals, Inc., Cheshire, Connecticut

EXECUTIVE SUMMARY:  In a developmental toxicity study (MRID 46534001),
zinc pyridinethione (98.3% a.i., batch/lot # 0108244691) was dermally
administered to 23-25 Crl:CD® (SD)IGS BR VAF/Plus® rats/dose at dose
levels of 0, 10, 15, 30, or 60 mg/kg bw/day from day 0 through 20 of
gestation. 

Dermal administration of zinc pyridinethione resulted in maternal
toxicity at 30 and 60 mg/kg/day.  Treatment-related effects at 60
mg/kg/day included clinical signs of toxicity (grade 1 erythema; grade 1
flaking; limited or no use of hindlimbs; shuffling gait; dehydration;
low carriage; chromodacyorrhea; emaciation; chromorhinorrhea; hunched
posture; decreased body weight and body weight gain, decreased uterine
weight, decreased corrected body weight and corrected body weight gain,
decreased absolute and relative feed consumption, and decreased muscle
tone and mass.  

At 30 mg/kg/day, there was an increase in the number of dams with
limited use of hindlimbs, shuffling gait, decreased body weight and body
weight gain, decreased corrected body weight and corrected body weight
gain, and decreased absolute feed consumption.  There were no
treatment-related maternal effects at 10 or 15 mg/kg/day. 

The maternal LOAEL is 30 mg/kg bw/day, based on an increase in the
number of dams with limited use of hindlimbs, shuffling gait, decreased
body weight and body weight gain, decreased corrected body weight and
corrected body weight gain, and decreased absolute feed consumption. 
The maternal NOAEL is 15 mg/kg bw/day. 

Developmental toxicity occurred at 60 mg/kg/day.  Treatment-related
findings at 60 mg/kg/day included decreased fetal weight (total, male,
and female), an increase in the percentage of litters and fetuses with
incomplete ossification of the sternal centra, an increase in the
percentage of fetuses with wavy ribs, and a decrease in the ossification
site averages for the caudal vertebrae, forelimb phalanges and
metacarpals, and hindlimb phalanges and metatarsals per fetus per
litter.  There were no treatment-related developmental effects at 10,
15, or 30 mg/kg/day. 

The developmental LOAEL is 60 mg/kg bw/day, based on decreased fetal
weight (total, male, and female), an increase in the percentage of
litters and fetuses with incomplete ossification of the sternal centra,
an increase in the percentage of fetuses with wavy ribs, and a decrease
in the ossification site averages for the caudal vertebrae, forelimb
phalanges and metacarpals, and hindlimb phalanges and metatarsals per
fetus per litter.  The developmental NOAEL is 30 mg/kg bw/day.

This developmental toxicity study in the rat is classified
ACCEPTABLE-GUIDELINE and satisfies the guideline requirement for a
developmental toxicity study (OPPTS 870.3700; OECD 414) in the rat.

COMPLIANCE:  Signed and dated GLP, Quality Assurance, Data
Confidentiality, and Flagging Statements are provided.

I. MATERIALS AND METHODS

A. MATERIALS:

1. Test Material:	

Zinc pyridinethione (ZPT)

	

Description:	

Off-white to tan powder, which was refrigerated and protected from light

	

Lot/Batch #:	

0108244691

	

Purity:	

98.3% a.i.

	

Compound Stability: 	

Not provided

	

CAS #of TGAI: 	

13463-41-7

	

Structure:	

2. Vehicle and/or positive control: Reverse osmosis membrane deionized
water (R.O. deionized water); lot number and purity not provided

3. Test animals:	

	

Species:	

Female rats

	

Strain:	

Crl:CD® (SD)IGS BR VAF/Plus®

	

Age/weight at study initiation:	

Dams were approximately 62 days old and weighed 191-236 grams.

	

Source:	

Charles River Laboratories, Inc., Raleigh, NC

	

Housing:	

Dams were individually-housed in stainless steel, wire-bottom cages
prior to mating.  During mating, each female was housed in the male
rat's cage.  Dams were then individually-housed in nesting boxes after
randomization.

	

Diet:	

Certified Rodent Diet #5002 (provided by PMI Nutrition International,
Inc., St. Louis, Missouri) was provided ad libitum; there were
reportedly no contaminants at levels exceeding the maximum concentration
limits for certified feed or deviations from expected nutritional
requirements.

	

Water: 	

Local water that was processed by passage through a reverse osmosis
membrane was provided ad libitum.  Water was analyzed twice annually for
possible chemical contamination and monthly for possible bacterial
contamination.  There were reportedly no contaminants found that
interfered with the study results.

	

Environmental conditions:	

Temperature:

Humidity:

Air changes:

Photoperiod:	

Temperature was targeted at 18-26(C; the actual range was 20.8-22.1(C.

Humidity was targeted at 30-70%; the actual range was 38.5-64.6%.

10/hr

12-hours light/12-hours dark

	

Acclimation period:	

Females were acclimatized for 5 days.  Dams also were acclimatized to
Elizabethan collars for 3 days (for at least 30 minutes, two hours, and
four hours, respectively) during the week prior to the start of
treatment.

B. PROCEDURES AND STUDY DESIGN

1. In life dates:

Start:	December 14, 2004		End:	January 10-15, 2005

2. Mating:  After acclimation, female rats were placed with male rats at
a 1:1 ratio.  The mating period lasted 6 days.  The day on which
spermatozoa were present in the vaginal smear and/or a copulatory plug
was observed in situ was considered to be gestation day (gd) 0.

3. Animal Assignment:  Animals were placed into dose groups using a
computer-generated (weight-ordered) randomization procedure based on
body weights recorded on gd 0 as indicated in Table 1.

TABLE 1.  Animal Assignment

Dose (mg/kg bw/day)	

0	

10	

15	

30	

60

# Females	

23	

24	

24	

24	

25

4. Dose Selection Rationale:  The dose levels were selected based on the
results from a range-finding study (Argus AEN00005) where percutaneous
administration of up to 400 mg/kg/day resulted in the following clinical
signs at the top doses (400 and 200 mg/kg/day): flaking (grades 1 and
2), erythema (grade 1), scabbing at the administration site,
dehydration, shuffling gait, limited use of hindlimbs, low carriage,
emaciation, chromodacyorrhea, urine-stained abdominal fur,
chormorhinorrhea, no use of hindlimbs, red perivaginal substance,
lacrimation, and splayed limbs.  Additional observations that were noted
only at the 200-mg/kg/day dose level were  hunched posture, mydriasis
and scabbing on the right flank; at the 400-mg/kg/day dose level only,
localized alopecia (limbs), ungroomed coat, red fur on the lower midline
and hindlimbs, ptosis and scabbing on the right hindlimb were noted. 
Animals at the top doses (400 and 200 mg/kg/day) also exhibited
decreased body weight, reduced feed consumption, reduction in muscle
tone, reduction in muscle mass, and increased resorptions.  Due to the
toxic effects observed, dams administered the top doses were sacrificed
after 11 to 14 daily dosages.  

Clinical observations considered treatment-related in the remaining
dose groups (100, 30, and 15 mg/kg/day) were flaking (grade 1),
dehydration, shuffling gain and limited use of the hindlimbs.  Dams
administered 30 or 100 mg/kg/day also exhibited erythema (grade 1), low
carriage, emaciation, and hunched posture, while dams administered 100
mg/kg/day also exhibited chromodacyorrhea, urine stained abdominal fur,
chromorhinorrhea, no use of the hindlimbs, red perivaginal substance,
lacrimation, splayed hindlimbs, and mydriasis.  Additionally,
dose-dependent reductions in body weight gain occurred at 100, 30, and
15 mg/kg/day for the entire dose period.  Decreased average gravid
uterine weights (100 mg/kg/day), decreased absolute and relative feed
consumption (30 and 100 mg/kg/day), decreased relative feed consumption
values over the entire treatment period (100 mg/kg/day), reduced muscle
tone (30 and 100 mg/kg/day), increased postimplantation loss (100
mg/kg/day), increased percentage of dams with resorptions and increased
percentage of resorbed conceptuses per litter (100 mg/kg/day), decreased
overall litter size and live fetuses (100 mg/kg/day), and decreased
fetal weights (30 and 100 mg/kg/day) also were noted.

5. Dosage Preparation and Analysis:  Test formulations were prepared
once and stored under refrigeration, protected from light.  Samples of
the test formulations were analyzed for stability, homogeneity, and
achieved concentration. 

Stability was evaluated using samples prepared for 10- and 60-mg/mL dose
formulation concentrations and stored under refrigeration protected from
light.  The concentration at 7, 14, 21, and 28 days was compared to the
concentration immediately after preparation, and stability was expressed
as percent error.

All dose formulation concentration levels were evaluated for
homogeneity.  Samples taken from the top, middle, and bottom were
measured for mean concentration.  Relative standard deviation (RSD) was
calculated based on the three mean values.

Samples from all dose concentration levels were obtained at the start
and end of the study.  The actual and nominal concentrations were
compared to determine the percent error.

Results	Homogeneity Analysis:  Initial analyses showed samples were not
within the acceptable limits of (5% RSD.  The values were 7.5%, 7.1%,
6.1%, and 6.1% for the 10, 15, 30, and 60 mg/mL formulations,
respectively.  The results from the backup homogeneity samples from the
start of the study, however, were within the acceptable range, except
for the 10 mg/mL concentration (14.5% RSD).

Stability Analysis:  Formulations were stable for up to 28 days when
stored refrigerated and protected from light.

Concentration Analysis:  All concentration analyses from the start of
the study were within the acceptable range (±10%), except for the
10-mg/mL concentration that was -15%; all concentration analyses from
the end of the study were within the acceptable ranges, except for the
60-mg/mL concentration that was 10.9%. 

The analytical data indicated that the mixing procedure was adequate and
that the variance between nominal and actual dosage to the study animals
was acceptable.

6. Dosage Administration:  All doses were administered dermally once
daily on gd 0-20.  The duration of each exposure was at least 6 hours,
and the same dosage site was used each day.  Dosing was based on the
most recent body weight determination prior to administration.

The following method was used when dermally applying the test
formulations.  A 3 cm x 4 cm site was demarcated with a permanent marker
on the clipped area for the back.  The interior edges of a 3M Reston(
Self-Adhering Foam Pad were then aligned to the markings on the back and
secured.  The appropriate test formulation was applied to the pad and
evenly distributed within the dosage site at the center of the
rectangular area using a syringe with attached gavage needle.  

Once the test formulation was applied, the 3M Reston( Self-Adhering Foam
Pad was covered with Johnson & Johnson Nu Gauze( general-use gauze.  The
general-use gauze did not touch the site of dosage administration.  On
either side of the 3M Reston( Self-Adhering Foam Pad, 3M Micropore( tape
and/or 3M Vetrap( bandaging tape was placed (without impairing the
mobility of the rat) in order to secure the foam padding and gauze on
the rat's back for the exposure period.  An Elizabethan collar was
placed on the rat's neck, preventing access to the dosage site.

At the exposure period completion, the Elizabethan collars, 3M Reston(
Self-Adhering Foam Pad, and Johnson & Johnson Nu Gauze( general-use
gauze were removed from the back.  The back of each rat was then washed
with soapy water and rinsed with warm water three times to ensure the
removal of the test substance.  Bedding was changed, and the cage was
wiped down with Zephiran® chloride solution (1:750) to remove any test
formulation that may have been present in the cage.

C. OBSERVATIONS

1. Maternal Observations and Evaluations:  The animals were checked for
morbidity at least twice a day.  General appearance was recorded daily
before and after treatment.  Clinical observations, abortions, premature
deliveries, and deaths were recorded 60±10 minutes after rinsing on gd
0-7, 9-11, 13-15, and 17-19.  A detailed post-treatment observation was
performed on all animals on gd 8, 12, 16, and 20.

Prior to the first daily application, and at 24-hour intervals each day
thereafter, each skin site was observed for signs of skin irritation and
graded using the endpoints described by Draize and the National Research
Council.  A hindlimb neurological evaluation was performed just prior to
dosing on gd 8, 12, 16, and 20, in which dams were assessed for muscle
tone and mass of the calf muscles.  Body weight data were recorded on gd
0 and daily during the treatment/post-treatment periods.  Feed
consumption was recorded on gd 0, 3, 6, 9, 12, 15, 18, and 20, and feed
left was recorded on the day of sacrifice.  

All surviving dams were sacrificed on gd 21 via CO2 asphyxiation.  A
caesarean section was performed, and a gross necropsy examination was
conducted of the thoracic, abdominal, and pelvic viscera.  Gross lesions
were retained in neutral buffered 10% formalin for possible future
evaluation.  

The number and distribution of the corpora lutea were recorded.  The
uterus of each dam was removed, weighed, and examined for pregnancy,
number and distribution of implantation sites, live and dead fetuses,
and early (defined as a conceptus in which organogenesis was not grossly
evident) and late (defined as one in which the occurrence of
organogensis was grossly evident) resorptions.  Uteri of nonpregnant
rats were stained with 10% ammonium sulfide to confirm the absence of
implantation sites.  Nonresponding term fetuses were considered dead. 
Dead fetuses and late resorptions were differentiated by the degree of
autolysis present; marked to extreme autolysis indicated that the fetus
was a late resorption.  Placentae were examined for size, color, and
shape.  

Dams that died prior to the schedule necropsy were examined for the
cause of death on the day that mortality was observed.  These animals
were examined for gross lesions, and the pregnancy status and uterine
contents were recorded.  Concepti in utero were examined to the extent
possible, using the same methods described for term fetuses.  Uteri of
nonpregnant rats were stained with 10% ammonium sulfide to confirm the
absence of implantation sites.

2. Fetal Evaluations:  After removal from the uterus, fetuses were
placed in individual containers and individually identified with a tag
noting the study number, litter number, uterine distribution, and
fixative.  Fetuses were then weighed, examined for gross lesions, and
sexed.  Live fetuses were sacrificed via an intraperitoneal injection of
sodium pentobarbital.  Approximately half of the fetuses per litter were
fixed in Bouin's solution and examined for visceral abnormalities by
using a variation of the microdissection technique of Staples.  The
heads subsequently were examined by free-hand sectioning, and the
sections were stored in alcohol.  Decapitated carcasses were not
retained.  A skeletal examination was performed on the remaining
fetuses, which had been fixed in alcohol and stained with alizarin red
S.  Skeletal preparations were retained in glycerin with thymol added as
a preservative.  Representative photographs of alterations (gross,
visceral, and skeletal) were taken. 

Fetal abnormalities were classified as malformations (irreversible
changes that occur at low incidences in this species or strain) or
variations (common findings in this species and strain and reversible
delays or accelerations in developments).

D. DATA ANALYSIS

1. Statistical Analyses: Clinical observations and other proportional
data were analyzed using the Variance Test for Homogeneity of the
Binomial Distribution.  The Bartlett's Test of Homogeneity of Variances
was used to analyze continuous data (e.g., body weights, body weight
changes, feed consumption values, litter averages for percent male
fetuses, percent resorbed conceptuses, fetal body weights and fetal
anomaly data).  If the Bartlett's Test of Homogeneity of Variances was
not significant (p>0.001), Analysis of Variance (ANOVA) was used.  If
the ANOVA was significant (p(0.05), a Dunnett's Test was used to
identify the statistical significance of the individual group compared
to the control.  If the Bartlett's Test was significant  (p(0.001), the
Kruskal-Wallis Test was used when (75% of ties were present.  The Dunn's
Method of Multiple Comparisons was used to identify the statistical
significance of each individual group compared to the control where the
Kruskal-Wallis Test was statistically significant (p(0.05).  If there
were >75% ties, the Fisher's Exact Test was used.  Count data obtained
at the caesarean-sectioning were evaluated using the procedures
described above for the Kruskal-Wallis Test.

2. Indices: Index formulas were not reported in this study; pre- and
post-implantation loss were calculated in this study by the reviewer.

3. Historical control data:  Historical control data are provided in
Appendix H of the study to allow comparison with concurrent controls. 
Summaries of reproductive indices, maternal necropsy observations, fetal
gross external alterations, fetal soft tissue alterations, fetal
skeletal alterations, and fetal ossification sites are provided.

II.  RESULTS

A. MATERNAL TOXICITY

1. Mortality and Clinical Observations:  There were no treatment-related
mortalities.  One 15-mg/kg/day dam and one 60-mg/kg/day dam were found
dead on gd 5.  There were no observed adverse clinical signs for either
of these dams, and weight gain and feed consumption values were
comparable to other rats in the respective dosage groups.  Additionally,
all tissues for both dams appeared normal at necropsy.

Significant clinical observations are presented in Table 2.  There was a
significant (p(0.01) increase in the number of 60-mg/kg/day dams
exhibiting the following clinical signs: flaking (grade 1); limited use
of hindlimbs; shuffling gait; dehydration; ungroomed coat; urine stained
abdominal fur; low carriage; chromodacyorrhea; emaciation;
chromorhinorrhea; and hunched posture.  Dams administered 60 mg/kg/day
also exhibited erythema (grade 1) and no use of hindlimbs, which were
considered treatment-related, although not statistically significant. 
These clinical signs resulted in a significant (p(0.01) increase in the
number of 60-mg/kg/day dams with abnormal detailed post-treatment
clinical signs.  Dams administered 30 mg/kg/day also exhibited a slight
but statistically nonsignificant increase in the incidence of limited
use of hind limbs and shuffling gait.  There were no treatment-related
clinical observations reported at 10 or 15 mg/kg/day.

TABLE 2.  Significant Clinical Observationsa

Observations	

Dose (mg/kg bw/day)

	

0	

10	

15	

30	

60

Maximum possible incidence	

506/23b	

528/24	

512/24	

528/24	

534/25

Mortality	

0	

0	

1c	

0	

1d

Skin Reaction

Erythema (grade 1)	

5/1e	

0/0	

0/0	

0/0	

2/2

Flaking (grade 1)	

2/1	

0/0	

0/0	

2/1	

56/14**

Clinical Observations

Limited use of hindlimbs	

0/0	

0/0	

0/0	

3/2	

133/24**

Shuffling gait	

0/0	

0/0	

0/0	

5/1	

136/22**

Dehydration	

0/0	

0/0	

0/0	

2/1	

198/21**

Ungroomed coat	

0/0	

0/0	

0/0	

0/0	

161/19**

Urine-stained abdominal fur	

0/0	

0/0	

0/0	

0/0	

83/12**

Low carriage	

0/0	

0/0	

0/0	

0/0	

47/11**

Chormodacryorrhea	

0/0	

0/0	

1/1	

0/0	

20/9**

Emaciation	

0/0	

0/0	

0/0	

0/0	

37/7**

Chromorhinorrhea	

0/0	

0/0	

2/1	

0/0	

19/8**

Hunched posture	

0/0	

0/0	

0/0	

0/0	

6/4**

No use of hindlimbs	

0/0	

0/0	

0/0	

0/0	

4/1

Detailed Post-treatment Observations

Appeared normal	

89/23	

94/24	

91/23	

92/24	

31/21**

Were not normal	

3/2	

2/2	

1/1	

3/3	

65/24**

a   Data obtained from page 38-39 in the study report.

b   Maximum possible incidence = (days x rats)/number of rats examined
per group on gd 0-21.

c   Dam 2337 was found dead on gd 5.

d   Dam 2332 was found dead on day 5 of presumed gestation.

e   N/N = total number of observations/number of rats with observations

** Statistically different (p <0.01) from the control.

2. Body Weight: Body weights were significantly decreased in 30- and
60-mg/kg/day dams on gd 13-21 and gd 7-21, respectively.

Body weight gain data are summarized in Table 3.  Treatment-related,
significant decreases in body weight gains occurred in both 30- and
60-mg/kg/day dams throughout the entire treatment period and the
post-treatment period.  For the 30-mg/kg/day dams, significant (p(0.05
or p(0.01) decreases in body weight gains occurred during gd 12-15,
15-18,18-20, and 20-21.  For 60-mg/kg/day dams, significant (p(0.01)
decreases in body weight gains occurred at all measured intervals except
gd 3-6.  Body weight gains for 10- and 15-mg/kg/day dams were comparable
to the control group throughout the treatment period.

Uterine weights were significantly (p(0.01) decreased in 60-mg/kg/day
dams.  Corrected body weights were significantly decreased (p(0.01) at
30 and 60 mg/kg/day.  Corrected body weight gains were significantly
reduced (p(0.01) at 30 mg/kg/day for gd 0-21 and at 60 mg/kg/day for gd
0-21 and 20-21.  Uterine weight, corrected body weight, and corrected
body weight gains for 10- and 15-mg/kg/day dams were comparable to the
control group.

TABLE 3.  Mean (±SD) Maternal Body Weight Gain (g)a

Interval	

Dose in mg/kg bw/day (# of Dams)

	

0 (23)	

10 (23)	

15 (24b)	

30 (24)	

60 (23)

Treatment:

GD 0-3	

18.5±5.7	

17.7±5.3	

14.7±6.5	

14.8±6.1	

12.3±7.0**

GD 3-6	

7.7±9.9	

6.2±8.4	

9.2±3.4	

6.9±7.2	

14.1±7.3

GD 6-9	

14.6±10.2	

15.0±6.7	

14.0±3.9	

12.6±8.4	

5.2±13.4**

GD 9-12	

19.2±8.1	

19.0±3.6	

17.2±6.0	

15.3±6.3	

-4.1±19.0**

GD 12-15	

20.6±5.1	

19.4±7.9	

19.7±5.2	

12.4±6.8**	

-8.0±11.7**

GD 15-18	

35.8±8.4	

35.3±6.4	

32.3±7.4	

29.2±7.8*	

11.0±11.9**

GD 18-20	

29.4±7.2	

29.4±8.6	

27.6±6.2	

22.2±9.5**	

21.2±9.0**

Post-treatment:

GD 20-21	

16.8±6.0	

15.3±6.6	

17.4±5.2	

12.3±5.5*	

7.1±8.5**

Overall Body Weight Gain

GD 0-21	

162.6±22.2	

157.3±21.5	

151.8±20.6	

125.8±24.2**	

48.8±28.5**

Uterine Weight	

99.10±15.66	

100.93±12.27	

96.54±16.73	

95.65±12.82	

76.09±14.38**

Corrected BW Gain

GD  0-21	

63.6±14.4	

56.4±14.8	

55.3±13.6	

30.1±19.3**	

-27.3±17.3**

GD 20-21	

-82.3±16.6	

-85.6±10.7	

-79.1±15.5	

-83.3±14.3	

-69.0±12.9**

a   Data obtained from page 42-43 in the study report.

b   N=23 beginning on gd 3-6.  Calculations exclude values for dam 2337
because the dam was found dead on gd 5.

*   Statistically different (p <0.05) from the control.

** Statistically different (p <0.01) from the control.

3. Food Consumption:  Absolute and relative feed consumption data are
summarized in Tables 4a and 4b.  Treatment-related, significant
decreases in feed consumption (gd 0-21) occurred in both 30- and
60-mg/kg/day dams.  For the 30-mg/kg/day dams, significant (p(0.01)
decreases in feed consumption values occurred during gd 18-20 and 20-21.
 For 60-mg/kg/day dams, significant (p(0.01) decreases in absolute feed
consumption values occurred during 6-9, 9-12, 12-15, 15-18, 18-20, and
20-21.  Absolute feed consumption values for 10- and 15-mg/kg/day dams
were comparable to the control group throughout the treatment period.

Significant decreases (p(0.01) in relative feed consumption values
occurred at 60 mg/kg/day for the overall treatment period (gd 0-21) and
during gd 9-12, 12-15, and 15-18.  Relative feed consumption values for
10-, 15-, and 30-mg/kg/day dams were comparable to the control group
throughout the treatment period.

TABLE 4a.  Mean (±SD) Absolute Maternal Feed Consumption (g/day)a

Interval	

Dose in mg/kg bw/day (# of Dams)

	

0 (23)	

10 (23)	

15 (24b)	

30 (24)	

60 (23)

Treatment:

Days 0-3	

18.8±2.1	

18.4±1.7	

18.2±2.1	

18.2±2.0	

17.9±2.3

Days 3-6	

20.4±2.5	

20.0±1.9	

19.7±2.1	

19.2±2.0	

19.6±2.0

Days 6-9	

22.6±2.7	

22.0±1.9	

21.9±2.0	

21.2±1.9d	

20.5±3.2**

Days 9-12		

24.3±2.7	

24.2±2.0	

23.7±2.6	

23.6±2.5	

17.7±6.2**

Days 12-15	

26.0±3.3	

25.7±2.6	

24.3±3.3c	

23.7±2.9	

16.6±5.8**

Days 15-18	

28.4±3.6c	

27.4±2.9	

26.4±3.4c	

26.1±3.3	

17.2±5.0**

Days 18-20	

29.0±4.0	

27.6±3.3	

27.5±2.5	

25.4±3.3**	

21.4±5.0**

Days 20-21	

25.8±3.4	

24.7±4.7c	

25.6±3.8	

20.8±3.9**	

17.2±5.8**

Overall Feed Consumption

Days 0-21	

24.0±2.4	

23.5±1.7c	

23.0±1.9	

22.3±1.6**	

18.5±2.5**

a   Data obtained from page 44 in the study report.

b   N=23 beginning on gd 3-6.  Calculations exclude values for dam 2337
because the dam was found dead on gd 5.

c   N=22 because values that appeared incorrectly recorded, as well as
those that were associated with spillage or set/soiled feed, were
excluded.

d   N=23 because values that appeared incorrectly recorded, as well as
those that were associated with spillage or set/soiled feed, were
excluded.

*   Statistically different (p <0.05) from the control.

** Statistically different (p <0.01) from the control.

TABLE 4b.  Mean (±SD) Relative Maternal Feed Consumption (g/kg/day)a

Interval	

Dose in mg/kg bw/day (# of Dams)

	

0 (23)	

10 (23)	

15 (24b)	

30 (24)	

60 (23)

Treatment:

Days 0-3	

78.7±6.6	

77.1±5.7	

76.6±7.2	

77.0±6.8	

75.4±8.4

Days 3-6	

80.8±7.7	

80.0±6.1	

79.3±6.1	

77.7±6.6	

80.0±6.4

Days 6-9	

86.0±7.3	

84.4±6.0	

84.5±5.7	

82.7±5.5d	

82.0±11.8

Days 9-12	

86.8±7.1	

87.0±4.8	

86.0±7.1	

87.2±7.6	

69.1±21.5**

Days 12-15	

86.9±7.8	

86.5±6.7	

83.0±9.9c	

83.4±7.8	

67.4±19.8**

Days 15-18	

87.2±7.5c	

85.0±7.6	

83.4±9.4c	

86.0±7.9	

70.9±19.8**

Days 18-20	

80.5±8.0	

77.4±6.5	

78.5±5.3	

77.1±8.2	

82.5±19.0

Days 20-21	

67.5±8.1	

65.0±10.0c	

68.8±9.4	

59.8±11.0	

62.2±18.4

Overall Feed Consumption

Days 0-21	

82.7±4.9	

81.5±3.4c	

80.7±4.4	

80.4±3.0	

74.3±5.3**

a   Data obtained from page 45 in the study report.

b   N=23 beginning on gd 3-6.  Calculations exclude values for dam 2337
because the dam was found dead on gd 5.

c   N=22 because values that appeared incorrectly recorded, as well as
those that were associated with spillage or set/soiled feed, were
excluded.

d   N=23 because values that appeared incorrectly recorded, as well as
those that were associated with spillage or set/soiled feed, were
excluded.

*   Statistically different (p <0.05) from the control.

** Statistically different (p <0.01) from the control.

4. Hindlimb Evaluation: Results from the hindlimb evaluation are
presented in Tables 5a and 5b.  At 60 mg/kg/day, there was a significant
(p(0.01) increase in the number of dams with low muscle tone, and a
corresponding significant (p(0.01) decrease in the number of dams with
moderate muscle tone on gd 12, 16, and 20.

Muscle mass was slightly decreased in one dam administered 60 mg/kg/day
on gd 12.  There was a significant (p(0.01) increase in the number of
60-mg/kg/day dams that exhibited slightly reduced muscle mass, and a
corresponding significant decrease (p(0.01) in the number of
60-mg/kg/day dams that exhibited normal muscle mass on gd 16 and 20.  

At 30 mg/kg/day, low muscle tone was observed in 2 out of the 24 dams on
gd 20.  This observation was not considered treatment-related because:
1) there was a low frequency of occurrence; 2) there was no
corresponding loss of muscle mass; and 3) the measurement was not
sufficient alone to demonstrate an adverse effect.  Muscle tone and
muscle mass for 10- and  15-mg/kg/day dams were comparable to the
control group throughout the treatment period.

TABLE 5a.  Muscle Tone (N)a

	

Dose in mg/kg bw/day (# of Dams)

	

0 (23)	

10 (24)	

15 (23b)	

30 (24)	

60 (24b)

GD 8 - Muscle Tone

None	

0	

0	

0	

0	

0

Low	

0	

0	

0	

0	

1

Moderate	

23	

24	

23	

24	

23

High	

0	

0	

0	

0	

0

GD 12 - Muscle Tone

None	

0	

0	

0	

0	

1d

Low	

0	

0	

0	

0	

15d**

Moderate	

21c	

22c	

22d	

23d	

7d**

High	

0	

0	

0	

0	

0

GD 16 - Muscle Tone

None	

0	

0	

0	

0	

2

Low	

0	

0	

0	

0	

21**

Moderate	

23	

24	

23	

24	

1**

High	

0	

0	

0	

0	

0

GD 20 - Muscle Tone

None	

0	

0	

0	

0	

0

Low	

0	

0	

0	

2	

21c**

Moderate	

23	

24	

23	

22	

2c**

High	

0	

0	

0	

0	

0

a   Data obtained from page 46-47 in the study report.

b  Excludes dams that were found dead on gd 5.

c  Excludes two rats in which evaluation was not performed.

d  Excludes one rat in which evaluation was not performed.

** Statistically different (p <0.01) from the control.

TABLE 5b.  Muscle Mass (N) a

Interval	

Dose in mg/kg bw/day (# of Dams)

	

0 (23)	

10 (24)	

15 (23b)	

30 (24)	

60 (23)

GD 8 - Muscle Tone

Greatly reduced	

0	

0	

0	

0	

0

Slightly reduced	

0	

0	

0	

0	

0

Normal	

23	

24	

23	

24	

24

GD 12 - Muscle Tone

Greatly reduced	

0	

0	

0	

0	

0

Slightly reduced	

0	

0	

0	

0	

1d

Normal	

21c	

22c	

22d	

23d	

22d

GD 16 - Muscle Tone

Greatly reduced	

0	

0	

0	

0	

0

Slightly reduced	

0	

0	

0	

0	

6**

Normal	

23	

24	

23	

24	

18**

GD 20 - Muscle Tone

Greatly reduced	

0	

0	

0	

0	

0

Slightly reduced	

0	

0	

0	

0	

12c**

Normal	

23	

24	

23 	

24	

11c**

a   Data obtained from page 46-47 in the study report.

b  Excludes dams that were found dead on gd 5.

c  Excludes two rats in which evaluation was not performed.

d  Excludes one rat in which evaluation was not performed.

** Statistically different (p <0.01) from the control.

5. Gross Pathology: There were no treatment-related gross pathology
changes observed at any dose level.

6. Cesarean Section Data: Data are as summarized in Table 6.  There were
no treatment-related changes in any of the following reproductive
parameters at any dose level: corpora lutea, implantations, litter
sizes, live fetuses, early and late resorptions, percentage of dead or
resorbed conceptuses, and the percentage of live male fetuses.  At 60
mg/kg/day, there was a significant (p(0.01) decrease in fetal weight
(total, male, and female).  Fetal weights recorded for 10-, 15-, and
30-mg/kg/day pups were comparable to the control group.  Pre- and post
implantation losses (as calculated by our reviewers) appear to be
comparable among groups, mainly due to the large standard deviations.

TABLE 6.  Cesarean Section Observations a

Observation	

Dose (mg/kg bw/day)

	

0	

10	

15	

30	

60

# Animals Assigned (Mated)	

23	

24	

24	

24	

25

# Animals Pregnant	

23	

23	

24	

24	

23

  	Pregnancy Rate (%)	

100.0	

95.8	

100.0	

100.0	

92.0

# Nonpregnant	

0	

1	

0	

0	

2

Maternal Wastage	

	

	

	

	

  	# Died	

0	

0	

1	

0	

1

# Died Pregnant	

0	

0	

1	

0	

0

# Died Nonpregnant	

0	

0	

0	

0	

1

# Aborted	

0	

0	

0	

0	

0

# Premature Delivery	

0	

0	

0	

0	

0

Total # Corpora Lutea

Corpora Lutea/Dam	

357

15.5±1.9	

370

16.1± 2.0	

348

15.1± 2.6	

355

14.8± 2.3	

361

15.7± 2.4

Total # Implantations

Implantations/Dam	

334

14.5± 1.9	

339

14.7±1.7	

322

14.0±2.3	

337

14.0±1.8	

324

14.1±2.4

Total # Litters	

23	

23	

24	

24	

23

Total # Live Fetuses

Live Fetuses/Dam	

315

13.7±2.4	

328

14.3±1.7	

310

13.5±2.4	

323

13.4±2.1	

308

13.4±2.4

Total # Dead Fetuses

Dead Fetuses/Dam	

0

0.0±0.0	

0

0.0±0.0	

0

0.0±0.0	

0

0.0±0.0	

0

0.0±0.0

Total # Resorptions	

Total	

19	

11	

12	

14	

16

Early	

19	

10	

12	

12	

15

Late	

0	

1	

0	

2	

1

Resorptions/Dam

Total	

0.8±1.4	

1.5±0.9	

0.5±0.8	

0.6±0.7	

0.7±0.9

Early	

0.8±1.4	

0.4±0.8	

0.5±0.8	

0.5±0.7	

0.6±0.9

Late	

0.0±0.0	

0.0±0.2	

0.0±0.0	

0.1±0.3	

0.0±0.2

Litters with Total Resorptions	

0	

0	

0	

0	

0

Mean Fetal Weight (g)	

	

	

	

	

Total	

5.35±0.26	

5.25±0.45	

5.28±0.29	

5.31±0.44	

4.31±0.47**

Males	

5.52±0.29	

5.41±0.46	

5.44±0.27	

5.44±0.45	

4.39±0.49**

Females	

5.15±0.30	

5.07±0.44	

5.10±0.31	

5.17±0.44	

4.23±0.48**

Sex Ratio (% Male)	

51.9±16.7	

52.2±13.8	

52.1±12.7	

47.6±15.3 	

47.5±14.9

Preimplantation Loss (%)	

6.38±6.0	

8.11±5.97	

7.03±8.20	

4.59±6.06	

10.21±10.11

Postimplantation Loss (%)	

5.79± 9.33	

3.10±5.54	

3.76±5.57	

4.32±5.53	

4.86±6.29

a   Data obtained from pages 48-49 in the study report.

b   Preimplantation loss ([(number of corpora lutea - number of
implantations)]/number of corpora lutea) X 100) and postimplantation
loss ([(number of implantations - number of live young)]/number of
implantations) X 100) were calculated by our reviewers; no statistical
analyses was conducted.

*   Statistically different (p <0.05) from the control.

** Statistically different (p <0.01) from the control.

B. DEVELOPMENTAL TOXICITY 

1. External Examination:  There were no treatment-related gross external
alterations (malformations or variations) at any dose level.

2. Visceral Examination:  There were no treatment-related visceral
alterations (malformations or variations) at any dose level.

3. Skeletal Examination:  Significant skeletal findings are presented in
Tables 7a and 7b.  There were no treatment-related skeletal
malformations identified.  At 60 mg/kg/day, there was a significant
(p(0.01) increase in the percentage of litters and fetuses with
incomplete ossification of the sternal central.  There also was a
significant increase in the percentage of fetuses (but not litters) with
wavy ribs.  These increases were correlated to significant reductions in
ossification site averages for the caudal vertebrae, forelimb phalanges
and metacarpals, and hindlimb phalanges and metatarsals per fetus per
litter at 60 mg/kg/day.  Skeletal alterations recorded for the 10-, 15-,
and 30-mg/kg/day fetuses were comparable to the control group.

TABLE 7a.  Skeletal Examinationsa

Observationsb	

Dose (mg/kg bw/day)

	

0	

10	

15	

30	

60

#Fetuses (litters) examined	

164 (23)	

171 (23)	

161 (23)	

168 (24)	

160 (23)

#Fetuses (litters) affected	

5 (4)	

2 (1)	

2 (2)	

1 (1)	

10 (6)

Wavy Ribs

Litter Percentage

Fetal Percentage	

0 (0)c

0.0

0.0	

0 (0)

0.0

0.0	

0 (0)

0.0

0.0	

0 (0

0.0

0.0	

3 (2)

8.7

1.9**d,e

Incompletely Ossified Sternal Centra

Litter Percentage

Fetal Percentage	

0 (0)

0.0

0.0

	

0 (0)

0.0

0.0

	

0 (0)

0.0

0.0

	

0 (0)

0.0

0.0

	

5 (3)

13.0**

3.1**e,f

a   Data obtained from pages 53-54 and 127-137 in the study report.

b   Some observations may be grouped together.

c   Fetal (litter) incidence

d   Fetus 2371-1 had other skeletal alterations.

e   Fetus 2399-13 had other skeletal alterations.

f    Fetus 2385-1 had other skeletal alteraions.

** Statistically different (p <0.01) from the control.

TABLE 7b.  Fetal Ossification Sites/Littera

Observation

	

Fetal Ossification Sites/Litter

------------------------------------------------------------------------
---------

Dose in mg/kg bw/day

	

0	

10	

15	

30	

60

Vertebrae

Caudal	

7.40±0.63	

7.48±0.70	

7.25±0.69	

7.58±0.92	

6.34±0.73**

Forelimbb

Metacarpals	

4.00±0.00	

4.00±0.00	

4.00±0.00	

4.00±0.00	

3.97±0.08*

Phalanges	

7.80±0.73	

7.88±0.73	

7.54±1.08	

8.01±0.78	

7.24±0.84*

Hindlimbsb

Metatarsals	

4.72±0.26	

4.78±0.25	

4.72±0.25	

4.77±0.29	

4.22±0.26**

Phalanges	

5.60±0.82	

5.84±0.99	

5.75±0.90	

6.04±1.22	

5.18±0.43*

a   Data obtained from pages 55 in the study report.

b  Calculated as average per limb.

*   Statistically different (p <0.05) from the control.

** Statistically different (p <0.01) from the control.

III. DISCUSSION and CONCLUSIONS

A. Maternal toxicity: Treatment related effects were seen at 30 and 60
mg/kg/day. Treatment-related effects at 60 mg/kg/day included clinical
signs of toxicity (grade 1 erythema; grade 1 flaking, limited or no use
of hindlimbs; shuffling gait; dehydration; ungroomed coat; urine stained
abdominal fur; low carriage; chromodacryorrhea; emaciation;
chromorhinorrhea; hunched posture; and no use of hindlimbs), decreased
body weight and body weight gain, decreased uterine weight, decreased
corrected body weight and corrected body weight gain, decreased absolute
and relative feed consumption, and decreased muscle tone and mass.  

At 30 mg/kg/day, there was an increase in the number of dams with
limited use of hindlimbs, shuffling gait; decreased body weight and body
weight gain, decreased corrected body weight and corrected body weight
gain, and decreased absolute feed consumption. 

The maternal LOAEL is 30 mg/kg bw/day, based on an increase in the
number of dams with limited use of hindlimbs, shuffling gait, decreased
body weight and body weight gain, decreased corrected body weight and
corrected body weight gain, and decreased absolute feed consumption. 
The maternal NOAEL is 15 mg/kg bw/day. 

B. Developmental toxicity:

a. Deaths/Resorptions: There were no treatment-related effects on fetal
deaths.

b. Altered Growth: At 60 mg/kg/day, there was a significant decrease in
fetal weight (total, male, and female).

c. Developmental Variations: There were no treatment-related visceral
variations.  At 60 mg/kg/day, there was an increase in the percentage of
litters and fetuses with incomplete ossification of the sternal centra,
an increase in the percentage of fetuses with wavy ribs, and a decrease
in the ossification site averages for the caudal vertebrae, forelimb
phalanges and metacarpals, and hindlimb phalanges and metatarsals per
fetus per litter. 

d. Malformations: There were no treatment-related external, visceral, or
skeletal malformations.

The developmental LOAEL is 60 mg/kg bw/day, based on decreased fetal
weight (total, male, and female), an increase in the percentage of
litters and fetuses with incomplete ossification of the sternal centra,
an increase in the percentage of fetuses with wavy ribs, and a decrease
in the ossification site averages for the caudal vertebrae, forelimb
phalanges and metacarpals, and hindlimb phalanges and metatarsals per
fetus per litter.  The developmental NOAEL is 30 mg/kg bw/day.

IV. STUDY DEFICIENCIES:  Pre- and postimplantation losses were
calculated; by the reviewer. There are no significant differences
between the treatment and control groups, mainly due to the large
standard deviations observed.  

V. STUDY CLASSIFICATION: This study is classified as
ACCEPTABLE-GUIDELINE and satisfies the guideline requirements for a
developmental toxicity study (OPPTS 870.3700; OECD 414) in rats. 

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Zinc Pyrithione	870.3700 Prenatal Developmental Toxicity Study/Rat 

	 

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