RESEARCH TRIANGLE INSTITUTE
RTI Master Protocol No. 182
RTI Study Code No. Rt85-NICL.REPRO
RTI Project No. 472U-3228-07
EPA Contract No. 68-01-7075
Date: September 23, 1988
FERTILITY AND REPRODUCTIVE PERFORMANCE OF THE Fj GENERATION
FINAL STUDY REPORT (III OF III)
TWO-GENERATION REPRODUCTION AND FERTILITY STUDY OF NICKEL CHLORIDE
ADMINISTERED TO CD RATS IN THE DRINKING WATER
Laboratory Study Dates: January 22 - July 7, 1986.
Prepared for:
Office of Solid Waste Management
U.S. Environmental Protection Agency
499 South Capitol Street, S.W.
Washington, D.C. 20003
Prepared by:
Catherine J. Price, Ph.D.
Senior Research Toxicologist 1
Center for Life Sciences and
Toxicology
Research Triangle Institute
Approved by:
_
Frederick J. de fierres. Ph.D.
Director for Life Sciences
and Toxicology
Research Triangle Institute
POST OFFICE BOX 12194 RESEARCH TRIANGLE PARK. NORTHCAROLINA 27709
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FERTILITY AND REPRODUCTIVE PERFORMANCE OF THE Fj GENERATION
FINAL STUDY REPORT (III OF III)
TWO-GENERATION REPRODUCTION AND FERTILITY STUDY OF NICKEL CHLORIDE
ADMINISTERED TO CD RATS IN THE DRINKING WATER
Catherine J. Price
Julia D. George
Melissa C. Marr
Polly E. Sanderson
Chemistry and Life Sciences
Research Triangle Institute
Post Office Box 12194
Research Triangle Park. NC 27709-2194
and
Reva Rubenstein
Gary L. Kimmel
Babasaheb R. Sonawane
Ambika Bathija
Office of Solid Waste Management
U.S. Environmental Protection Agency
499 South Capitol Street. S.W.
Washington. D.C. 20003
and
Chris de Rosa
Environmental Criteria and Assessment Office
U.S. Environmental Protection Agency
26 W. St. Clair Street
Cincinnati. OH 45268
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3
FERTILITY AND REPRODUCTIVE PERFORMANCE OF THE Fj GENERATION
FINAL STUDY REPORT (III OF III)
TWO-GENERATION REPRODUCTION AND FERTILITY STUDY OF NICKEL CHLORIDE
ADMINISTERED TO CD RATS IN THE DRINKING WATER
\<
*
/
rv
Research Triangle Institute
Catherine J. Pric>. Ph.D. Date
Study Director
Research Triangle Institute
Julia D. George. Ph.D. Date
/Co-Study Director
^Research Triangle Institute
Donald B. Feldman, D.V.M. Date
Manager. Animal Research Facility
Research Triangle Institute
Melissa c Marr / Date
Co-Laboratory Supervisor
Robert S. DeWoskin, M.S. Date
Quality Assurance Unit
Research Triangle Institute
H. Haw. M.F.A'. Date
ality Assurance Unit
Research Triangle Insliiute
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RESEARCH TRIANGLE INSTITUTE
Chemistry and Life Sciences
Quality Assurance Statement
RTI Master Protocol Number: RTI-182/472U-3228-07
TITLE: Two-Generation Reproduction and Fertility Study of Nickel Chloride
Administered to CD Rats in the Drinking Water (III of III)
Fertility and Reproductive Performance of the F Generation
SPONSOR: U.S. Environmental Protection Agency
STUDY CODE NUMBER: Rt85-NICL.REPRO (III of III); Protocol Number 182
STUDY DIRECTOR: Catherine J. Price
STUDY DATES: January 22, 1986 through July 7. 1986
Viral antibody free (VAF/Plus)-Crl:CD (SD)BR outbred albino rats were
obtained from the Charles River Breeding Laboratories, Inc., Kingston. NY.
The animals were quarantined and examined upon arrival.
Nickel chloride hexahydrate (Lot No. KM01918HM) was obtained from
Aldrich Chemical Company, Milwaukee, Wisconsin. Chemical analyses for the
identity and purity of the compound were performed by RTI.
Listed below are the dates of inspections and the dates reports were
sent to management for both the ninety day exposure study and subsequent
phases.
Critical Phases Inspection Date
Original Protocol Review
Second °rotocol Review
Test Article Receipt
Test Article Receipt Records
Animal Receipt
Quarantine
Husbandry
Dosage Formulation
07-24-85
02-17-86
05-15-85
03-25-86
06-27-85
03-11-86
03-13-86
09-18-85
03-25-86
Date Report
Signed by
Study Director
02-20-86
05-24-85
03-27-86
06-28-85
03-12-86
03-17-86
09-18-85
05-01-86
Date Report Sent
to Management
10-02-85
03-05-86
06-17-85
04-07-86
07-08-85
04-07-86
04-07-86
10-02-85
04-07-86
Post Office Box 12194 Research Triangle Park, North Carolina 27709 Telephone: 919 541-6000
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Critical Phases
Inspection Date
Date Report
Signed by
Study Director
Date Report Sent
to Management
Mating
P Cohabitation
Assign, of Pairs
F. Fetuses
Maternal Rest
Temp, and Humidity
Sacrifice
90 Day
F Pups
Pla
Sacrifice
Sacrifice
Necropsy
Records
Statistics
Analytical Data
09-18-85
09-19-85
09-27-85
11-12-85
12-04-85
10-02-85
10-31-85
12-03-85
01-03-86
04-23-86
06-11-86
06-17-86
09-18-85
04-22-86
07-10-86
03-25-86
04-22-86
09-18-85
09-19-85
09-27-85
11-12-85
12-09-85
10-02-85
11-01-85
12-04-85
01-06-86
05-15-86
09-08-86
06-18-86
09-19-85
05-01-86
07-11-86
05-15-86
05-15-86
10-02-85
10-02-85
10-02-85
12-23-85
12-23-85
10-02-85
11-08-85
12-23-85
02-07-86
05-13-86
08-18-86
08-18-86
10-02-85
05-13-86
08-18-86
04-07-86
05-13-86
The Master Protocol and Amendment No. 1 are included in Appendix I.
Two Final Report Audits and the Data Record Audit were performed by
Mr. John H. Haw.
To the best of my knowledge this Final Report accurately describes
the study methods and procedures used, and the reported results accurately
reflect the raw data.
Date
John H. Haw. QA Specialist
Quality Assurance Unit
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6
Table of Contents
Page
Title Page 2
Signature Page 3
Quality Assurance Statement 4
Table of Contents 6
List of Appendices 8
List of Text Tables 10
List of Figures 13
Abstract 14
1.0 Introduction 22
1.1 Objectives 22
1.2 Summary of the Experimental Design from Weaning of the FlD
Litter to Sacrifice of the F2D Litter on Gestational Day 20 23
2.0 Materials and Methods 26
2.1 Test Article (Nickel Chloride Hexahydrate) 26
2.1.1 Description 26
2.1.2 Safety and Handl ing 27
2.1.3 Dosage Formulation and Analysis 29
2.1.4 Treatment 32
2.2 Animals 33
2.3 Animal Husbandry 34
2.4 Toxicologic Evaluation 37
2.4.1 In-Life Evaluation 37
2.4.2 Necropsy 39
2.5 Histology and Histopathology 40
2.6 Statistical Analysis 40
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Table of Contents (continued)
Paee
2 . 7 Personnel [[[ 43
2 . 8 Storage of Records ................................................ 44
3.0 Results [[[ 45
3.1 Toxicologic Evaluation of CO Rats in the Fj Generation ............ 45
3.1.1 Clinical Status and Mortality for Fj Males and Females ...... 45
3.1.2 Body Weight. Food. Water and Nickel Consumption
for FI Females ............................................. 47
3.1.3 Body Weight. Food, Water and Nickel Consumption
for Fj Males ............................................... 48
3.2 Reproductive Performance of Fj Breeders (first mating) and
Postnatal Development of F2a Litters .............................. 50
3.2.1 General Indices of Reproductive Performance ................ 50
3.2.2 Maternal Measures During Gestation ......................... 50
3.2.3 Maternal Measures During Late Gestation and Lactation ...... 51
3.2.4 Evaluation of F2a Litters (Postnatal Days 1-21) ............ 53
3.3 Reproductive Performance of Fj Breeders (second mating)
and Embryo/Fetal Development of F2b Litters ....................... 54
3.3.1 General Indices of Reproductive Performance ................ 54
3.3.2 Maternal Measures During Gestation ......................... 54
3.3.3 Maternal Toxiclty during Gestation of the F2b Miter ....... 55
3.3.4 Evaluation of F2b Litters (Gestational Day 20) ............. 56
3.4 Necropsy and Histopathologic Evaluation of Tissues from
CD Rats in the Fj Generation ...................................... 57
3.4.1 Females [[[ 57
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List of Appendices
8
APPENDIX I:
APPENDIX II:
APPENDIX III:
APPENDIX IV:
APPENDIX V:
APPENDIX VI:
APPENDIX VII:
Protocol No. 182 and Amendment No. 1: Two-Generation
Reproduction and Fertility Study of Nickel Chloride
Administered to CD Rats In the Drinking Water
1-1
Weekly Summary of Body Weight and Food. Water
and Nickel Consumption for Fj Females in the
Two-Generation Reproduction and Fertility Study of
Nickel Chloride Administered to CD Rats in the
Drinking Water: Week 32 through Week 49
II-l
Weekly Summary of Body Weight and Food, Water and
Nickel Consumption for Fj Males in the Two-Generation
Reproduction and Fertility Study of Nickel Chloride
Arli- ' • istered to CD Rats in the Drinking Water:
Week 32 through Week 49
III-l
Clinical Signs for Fj Females in the Two-Generation
Reproduction and Fertility Study of CD Rats Exposed
to Nickel Chloride in the Drinking Water: Week 32
through Week 55
IV-1
Clinical Signs for Fj Males in the Two-Generation
Reproduction and Fertility Study of CD Rats Exposed
to Nirkel Chloride in the Drinking Water: Week 32
through Week 51
Murine Antibody Determination Reports for FH, Males
and Females on pnd 42 (2/27/86) and Flb Males and
Females at Final Necropsy (7/25/86): Microbiological
ASM-. ' tes. Inc. . Bethesda. MD
V-l
Histopathology Reports (September 4. 1986 and
December 1. 1986) from Experimental Pathology
Laboratories, Inc.. Research Triangle Park, NC.
APPENDIX VIII: Summary of Results for the Two-Generation Repro-
duction and Fertility Study of Nickel Chloride
Acln • .istered to CD Rats in the Drinking Water...
APPENDIX IX:
Graphical Representation of Pood. Water, and
Nickel (Ni++) Consumption as a Function of
Body Weight for the Fj Generation
Vl-l
VII-1
VIII-1
IX-1
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List of Appendices (continued)
9
APPENDIX X:
APPENDIX XI:
Daily Environmental Conditions in the Animal Rooms
During the Reproduction and Fertility Study of
Nir-v. > Chloride
Environmental Conditions During the Two-Generation
Reproduction and Fertility Study of Nickel Chloride
Administered to CD Rats in -the Drinking Water
X-l
XI-1
APPENDIX XII: Historical Control Data XII-1
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List of Text Tables
Page
Table 1A.
Table IB.
Table 2.
Table 3.
Table 4.
Table 5.
Table 6.
Table 7.
Table 8.
Table 9.
Table 10
Table 11
Table 12.
Table 13.
Summary of Temperature and Relative Humidity in Room 148
During the Two-Generation Reproduction and Fertility Study
of Nickel Chloride Administered to CD Rats in the Drinking
Krtler 64
Summary of Temperature and Relative Humidity in Rooms 150
and 146 During the Two-Generation Reproduction and Fertility
Study of Nickel Chloride Administered to CD Rats in the
Drinking Water 66
Fj Generation: Summary of Clinical Observations in
FJD Female CD Rats on Postnatal Days 22 through 42 68
Summary of Clinical Signs and Necropsy Findings for
FlD Females Which Died or Were Sacrificed on Postnatal
Days 22 through 42 .- 69
Fj Generation: Summary of Clinical Observations in
FlD Male CD Rats on Postnatal Days 22 through 42 74
Summary of Clinical Signs and Necropsy Findings for
FlD Males Which Died or Were Sacrificed on Postnatal
Days 22 through 42 75
PI Generation: Summary of Clinical Signs in FJD Female
CD Rats after Postnatal Day 42 80
Summary of Clinical Signs and Necropsy Findings for
FJJ, Females Which Died or Were Sacrificed after
Postnatal Day 42 81
Fj Generation: Summary of Clinical Signs in FJD Male
CD Rats after Postnatal Day 42 82
Summary of Necropsy Findings of FlD Pups on Postnatal
Day 42 for Animals not Selected as Fj Breeders 84
Summary of Reproductive Status for CD Rats in the
Fj Generation Following Continuous Exposure to Nickel
Chloride in the Drinking Water: Breeding to Produce
the F2a Litter 85
Summary of Maternal Body Weights for FlD Dams During
Gestation of the F2a Litter 87
Summary of Food Consumption for Flb Dams During Gestation
of the F2a Litter 89
Summary of Water Consumption for Flb Dams During
Gestation of the F2a Litter 90
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List of Text Tables
(continued)
Page
Table 14.
Table 15.
Table 16.
Table 17.
Table 18,
Table 19.
Table 20.
Table 21.
Table 22.
Table 23.
Table 24,
Table 25.
Table 26.
Table 27.
Table 28.
Summary of Nickel (Ni++) Consumption for Flb Dams During
Gestation of the F2a Litter 91
Summary of Maternal Body Weight for Fj Dams During Late
Gestation and Following Delivery of the F2a Litter 92
Summary of Food Consumption for Fj Dams During Late
Gestation and Following Delivery of the F2a Litter 94
Summary of Water Consumption for Fj Dams During Late
Gestation and Following Delivery of the F2a Litter 96
Summary of Nickel (Ni++) Consumption for Fj Dams During
Late Gestation and Following Delivery of the F2a Litter. 98
Summary of F2a Pup Data: Number of Live Pups per Litter
and Percent Mortality per Litter 100
Summary of F2a Pup Data: Average Pup Body Weight per
Litter 102
Summary of F2a Pup Data: Percent Male Pups per Litter.. 104
Summary of F2a Pup Data: Necropsy Findings on Postnatal
Day 21 for Male and Female Pups 105
Summary of Reproductive Status for CD Rats in the
F} Generation Following Continuous Exposure to Nickel
Chloride in the Drinking Water: Breeding to Produce
the F2b Litter 106
Summary of Fj Dam Body Weights During Gestation of the
F2b Litter 107
Summary of Food Consumption for Fj Dams During Gestation
of the F2b Litter 108
Summary of Water Consumption for Fj Dams During
Gestation of the F2b Litter 109
Summary of Nickel (Mi**) Consumption for Fj Dams During
Gestation of the F2b Litter Ill
Maternal Toxicity in CD Rats (Fj Dams) Exposed to
Nickel Chloride on Gestational Days 0 through 20 of
the F2b Litters 112
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List of Text Tables
(continued)
Paee
Table 29
Table 30.
Table 31.
Table 32.
Table 33.
Table 34.
Table 35.
Table 36.
Table 37.
Table 38.
Table 39.
Table 40.
Summary of Embryotoxicity in F2b CD Rat Fetuses
Following Maternal (Flb) Exposure to Nickel Chloride on
Gestational Days 0 through 20 115
Summary and Analysis of Teratologic Defects Observed in
F2h CD Rat Fetuses Following Exposure of Dams (FlD) to
Nickel Chloride on Gestational Days 0 through 20 122
Teratologic Defects in F2b CD Rat Fetuses Following
Exposure of Dams (FlD) to Nickel Chloride on Gestational
Days 0 through 20: Listing by Defect Type 126
Analysis of the Incidence of F2b Fetuses with Short Rib.. 129
Malformations and Variations Exhibited by F2b CD Rat
Fetuses Following Maternal (Flb Dams) Exposure to Nickel
Chloride on Gestational Days 0 Through 20: Listing by
Individual Fetus 130
Summary of Organ Weights for Pregnant Flb Female Rats
Exposed to Nickel Chloride in the Drinking Water at
Scheduled Sacrifice on gd 20 of the F2b Litter 140
Summary of Necropsy Findings for Flb Female CD. Rats
at Scheduled Sacrifice 142
Summary of Body and Organ Weights for Flb Male Rats
Exposed to Nickel Chloride in the Drinking Water 143
Summary of Necropsy Findings for Flb Male CD Rats at
Scheduled Sacrifice 146
Analysis of Nickel Chloride in Stock Solutions 147
Analysis of Nickel Chloride Dosed Drinking-Water:
Pre-Dosing Samples 148
Analysis of Nickel Chloride Dosed Drinking Water:
Postdosing Samples 156
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List of Figures
13
Page
Figure 1:
Figure 2:
Figure 3:
Figure 4:
Figure 5:
Figure 6:
Figure 7:
Figure 8:
Figure 9:
Figure 10.
Figure 11:
Average Fj Female Body Weight Expressed as a Percentage
of the Average Control Values
Average Fj Female Food Consumption (g/kg/day) Expressed
as a Percentage of the Average Control Values
Average Fj Female Water Consumption (g/kg/day) Expressed
as a Percentage of the Average Control Values
Average Fj Female Nickel Consumption (rag Ni++ /kg/day)
Average Fj Male Body Weight Expressed as a Percentage
of the Average Control Values
Average Fj Male Food Consumption (g/kg/day) Expressed
as a Percentage of the Average Control Values
Average Fj Male Water Consumption (g/kg/day) Expressed
as a Percentage of the Average Control Values
Average Fj Male Nickel Consumption (mg Ni++/kg/day)
Average Number of Live Pups or Number of Fetuses per Litter
Expressed as a Percentage of the Average Control Values
Average Percent Postnatal Mortality per Litter Expressed
as a Percentage of the Average Control Values
Average Pup Body Weight per Litter Expressed as a
162
163
164
165
166
167
168
169
170
171
Percentage of the Average Control Values.
172
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Abstract
Nickel chloride Is a potential contaminant of human drinking water. In
order to evaluate potential reproductive toxicity. a two-generation repro-
duction and fertility evaluation of nickel chloride (0, 50, 250 or 500 ppm,
Mi**) administered to CD rats in the drinking water was conducted. Data from
the FI generation animals and their offspring (F2a pups and F2b fetuses) are
reported herein (Final Report. Ill of III). Companion reports have been
issued for nonbreeder animals exposed for 90 days to 0 or 500 ppm Mi*"1" (Final
Report. I of III), and for the PQ generation and their offspring (Fla and FU,
pups) (Final Report. II of III). A summary of results for both the PQ and Fj
generations is presented in Appendix VIII (this report).
This report begins with weaning of the Flo animals at 21 days of age. At
that time. FJD males (218) and females (204) were selected as potential Fj
breeders, and all remaining pups were sacrificed. On pnd 42. Fj breeders were
selected randomly from the 0. 50, and 250 ppm groups (30 males and 30 females
per group); all surviving pups (22 males and 19 females) from the 500 ppm
group were retained as Fj breeders. All remaining FJO animals were
sacrificed.
Exposure of Fj breeders to nickel chloride was initiated indirectly via
administration to their parents (Pg generation) (Final Report II of III).
Direct exposure from the drinking water began for F^D pups before weaning, and
continued during the postweaning period until scheduled sacrifice (21 to 24
weeks of age for males and 27 to 30 weeks of age for females).
During nonreproductive phases of the study (study weeks 32-38 and 46-49).
FI females showed a decreasing pattern of fluid intake (g/kg/day) with
increasing age. Thus. Fj females in the low- through high-dose groups
consumed 9. 35 and 67 mg Ni**/kg/day during the first weekly measurement
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15
period (study week 32). as compared to 6, 23 and 44 mg/kg/day just prior to
the first cohabitation, and 5, 18 and 35 ng/kg/day just prior to the second
cohabitation. During nonreproductive phases of the study. Fj females showed
no effects on fluid intake at 50 ppm (99-107% of control consumption), but the
nid- and high-dose groups were significantly below the control group during
each week (76-84% of control at 250 ppm and 74-87% of control at 500 ppm). Fj
females showed no treatment-related reduction of food intake (g/kg/day) during
these periods. Transient increases of food intake were noted for the 500 ppm
group on week 37 (106% of control; significant trend only) and week 38 (110%
of control). For each weekly weighing, average Fj female body weight did not
differ significantly from the control group at 50 ppm (102-105% of control) or
250 ppm (95-100% of control). Body weight for females at 500 ppm (81-93% of
controls) was significantly reduced for each week during the nonreproductive
portions of the study.
During gestational days (gd) 0-6 of the F2a litter, Fj dams consumed 6. 21
and 42 rag Mi""*"/kg/day in the 50. 250 and 500 ppm groups, respectively. Mi**
intake remained stable throughout gestation. Ni++ consumption noticeably
decreased near the time of parturition, especially for 500 ppm animals (5. 16
and 15 mg/kg/day for the 50. 250 and 500 ppm groups between gd 0 and pnd 1).
Ni** consumption increased in all groups during lactation with average
consumption reaching 13. 54 and 89 mg/kg/day. respectively, for the final week
(pnd 14-21). During the F2b gestational period. Mi** consumption by Fj dams
was similar to that observed for PQ dams during gestation of the FJD litter.
During F2b gestation, ranges of 5-6. 21-22. and 35-42 mg Ni^Vkg/day were
observed for the low- through high-dose groups during measurement periods of
gd 0-6, 6-13 and 13-20. During F2a gestation, fluid intake was not affected
at 50 ppm (96-100% of control intake), but was significantly reduced at 250
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16
ppm (71-77* of control) and at 500 ppm (58-73* of control). Reduction of
fluid intake was most severe between gd 20 and pnd 1 showing decreases to 66*
and 31* of control intake at 250 ppm and 500 ppm. respectively. During F2a
lactation (pnd 1-21), fluid intake was not significantly affected at 50 ppm or
250 ppm. but was reduced at 500 ppm (73-82* of control intake). During F2b
gestation, fluid intake was reduced at all exposure levels - 88* of control
intake at 50 ppm. 65-77* of control at 250 ppm and 53-72* of control at 500
ppm. At the end of the F2a gestational period, a transient reduction of food
intake was observed at 500 ppm (93* of control intake between gd 13-20 and 45*
of control between gd 20 and pnd 1). Tl female body weight was significantly
below controls in the 500 ppm group on gd 20 (87* of control weight), and
during lactation (82-92* of control) of the F2a litters. Fj dam body weight
during the F2b gestational period was significantly below controls on gd 20
for the 500 ppm group (91* of control weights).
During the nonreproductive portions of the study (weeks 32-38 and 40-49).
Fj males showed a pattern of decreasing fluid intake (g/kg/day) with
increasing age. Thus, average Ni + * intake for the low- through high-dose
groups, was 8, 36 and 63 mg Ni**/kg/day during the first week, as compared to
4. 19 and 41 mg/kg/day just prior to the first cohabitation, and 3. 14 and 28
mg/kg/day just prior to the second cohabitation. Water intake at 50 ppm was
significantly reduced only on weeks 36 (91* of control intake) and 38 (92* of
control intake). At 250 ppm. water intake was significantly reduced on weeks
33-38 (81-87* of control), and 41-44 (87-89* of control). The 500 ppm group
was significantly below controls on weeks 33-38 (77-95* of control), weeks 41-
44 (88-92* of control), and week 48 (86* of control). Thus, for Fj males, the
predominant effects on water intake occurred at 250 and 500 ppm prior to study
week 45. Food intake (g/kg/day) for nickel-exposed males did not differ
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17
significantly from controls for weeks 33-37. For the remaining weeks (38 and
41-49), transient increases occurred at 250 ppm (105* of control on week 45).
and 500 ppm (108-121* for weeks 38 and 41-48): a significant trend on week 49
reflected a nonsignificant increase at 500 ppm (108* of control). The
absolute amount of food consumed (g/day) was significantly decreased in the
500 ppm group throughout the study due to the persistence of decreased body
weight for males in that group as described below. At all weighings. Fj male
body weight for the 500 ppm group was significantly reduced (71-86* of control
weights).
During postnatal development (pnd 22 to 42). 23 Fj males and 22 females
died with a significant increase in the Incidence of deaths at 250 and 500 ppm
for males and at 500 ppm for females. The incidence of deaths was 2/60. 4/67.
8/60. and 9/31 males and 4/58. 5/59. 4/59. and 9/28 for females in the control
through high-dose groups, respectively. In older animals (greater than 42
days of age), clinical signs observed in nickel chloride-treated animals that
survived during the study included rough hair coat, hair loss, and dental
problems such as malocclusion and chipped teeth. After 42 days of age. no
males died prior to scheduled sacrifice, but 4 females in the 250 and 500 ppm
groups died during delivery of the F2a litter. Although the incidence of
pregnancy-related deaths was not statistically significant, circumstances were
similar to maternal deaths observed in the P0 generation (Report II of III).
Based upon the absence of similar complications in pregnant historical control
animals, as well as the low incidence of mortality in nickel-exposed adult P0
and Fj females during the non-reproductive portions of this study, nickel-
exposed females in both generations showed the greatest probability for
compromised status near the time of parturition. Further evaluation is needed
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18
to determine the potential contribution of chronically reduced maternal fluid
intake to these maternal deaths.
For Fj females at scheduled necropsy (i.e.. gd 20 for F2D litters), the
only definitive effects upon organ weights occurred at 500 ppm including
decreased absolute liver weight (80* of control weight), decreased relative
liver weight (88* of control) and increased relative kidney weight (113* of
control). No treatment-related pathology was noted upon gross examination at
necropsy of Fj fn...iles. Treatment-related microscopic findings were limited
to an apparent increase in 'histlocytic cellular infiltration of the lungs at
the high dose (7*. 7*. 7* and 44* of females examined from the control through
high-dose groups, respectively).
At scheduled necropsy, Fj breeder males showed statistically significant
changes for the absolute (g) or relative weights of all weighed organs (i.e..
prostate, seminal vesicles, testicles, liver, kidneys, heart, lung and
pituitary), except for the adrenals. A clear association with nickel exposure
was noted only at 500 ppm for increased relative lung weight (114* of control)
and relative pituitary weight (123* of control). By comparison, P0-generation
males showed increased pituitary weight (both absolute and relative) at 250
and 500 ppm, but other relative organ weights were not affected. For Fj
males, no treatment-related pathology was noted upon gross examination at
necropsy. Treatment-related microscopic findings were limited to an apparent
Increase in histlocytic cellular infiltration of the lungs at the high dose
(0*. 3*. 3* and 18* of males examined from the control through high-dose
groups, respectively). Thus, the Fl generation histopathology did not differ
remarkably from the PQ data.
Thus, exposure levels of 50. 250 and 500 ppm Ni** in the drinking water
were generally well-tolerated by Fj animals with the following notable ex-
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19
ceptions: (1) water intake was reduced at 250 and 500 ppm. (2) deaths between
weaning and pnd 42 were increased at 250 and 500 ppm for Fj males and at 500
ppm for Fj females, and (3) an increased Incidence of adverse effects among
pregnant females (reduced body weight or mortality) occurred at higher dose
levels during late gestation, parturition and lactation. As also noted in the
PQ generation, susceptibility of females appeared to be greatest during the
perinatal phase of the reproductive process, in association with complications
of pregnancy.
The first mating of Fj breeding pairs resulted in no treatment-related
effects upon the % mated females. % live litters. % viable litters (pnd 1-4 or
pnd 4-21). % fertile matings, gestational length or % male pups per litter.
Live litter size on pnd 1 and 4 was significantly decreased at 500 ppm (11.40
vs. 13.63 for controls). A two-fold increase in the percent postnatal
mortality per litter (pnd 1-4 and 4-21). was observed, but statistical
significance was not attained. At 500 ppm. average pup body weight per litter
was significantly decreased on pnd 1. 4, 7. 14 and 21 (91%. 90%, 87%, 84% and
86% of control weights, respectively).
The second mating of Fj breeding pairs resulted in no treatment-related
effects upon the % mated females. % fertile matings, % live litters on gd 20.
number of corpora lutea per dam. or number of implantation sites per dam. Nor
were any treatment-related effects noted for measures of prenatal growth or
viability of F2b fetuses. The percent fetuses malformed per litter was signi-
ficantly increased in the 50 ppm group (14.67% vs. 6.96% for controls), due
primarily to a higher incidence of short rib in that group. When the
incidence of fetuses with short rib was analyzed on a per litter basis, there
were no significant differences between controls and the 50 ppm group. In the
absence of similar effects at higher doses, the increased incidence of
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20
malformations at 50 ppm was viewed as a spurious result. Cumulative measures
of embryo/fetal status (* adversely affected implants and * litters with
adversely affected implants) did not differ significantly among groups.
In summary, male and female CO rats (Fj generation) were exposed continu-
ously to nickel chloride (0. 50, 250 or 500 ppm N1++) in the drinking water.
No adverse effects were noted during evaluation of the Fj generation at 50
ppm. Adverse effects during juvenile development and parturition were ob-
served in the 250 and 500 ppm groups, including 4 pregnancy-related deaths in
FI females during delivery of the Fga litter. (3 at 250 ppm and 1 at 500 ppm).
As noted for pregnancy-related deaths among P0 females, the attribution of the
pregnancy complications to nickel chloride consumption is compromised by the
reduced water intake of treated animals. Further information regarding the
effects of reduced water intake is needed in order to interpret these results.
In addition, significant reductions in live litter size and postnatal body
weight of the F2a litters were observed at 500 ppm. In the F2b litter, no
significant adverse effects on embryo/fetal development were noted. Thus, at
500 ppm. adverse effects upon the offspring from naturally-delivered F2a lit-
ters were similar to those observed for the naturally-delivered litters of the
PQ generation (Fla and FJD). However, comparable effects were not seen in
fetuses delivered by Cesarean section on gd 20 (?2b litters). Differences in
effects between the F2a and F2b litters could have been affected by differ-
ences in nickel exposure, although relative nickel intake was only slightly
lower during the F2b gestational period relative to the F2a (82-91% at 50 ppm.
90-100% at 250 and 500 ppm). Thus, adverse effects of nickel exposure upon
the offspring in the present study were expressed primarily during the peri--
natal or postnatal periods, rather than during gestation (gd 0-20).
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Evaluation of the Fj generation suggests that nickel chloride exposure
interferes primarily with the normal processes associated with late gestation.
parturition, lactation and/or postnatal development, and that the severity of
these effects shows considerable variability among Individual females and
their litters. Further studies would be needed to distinguish between the
direct effects of nickel chloride upon the physiological/endocrine processes
associated with reproduction as opposed to possible indirect effects
associated with decreased fluid intake. Also, further studies would be needed
to distinguish between direct effects of nickel chloride upon the offspring.
as opposed to effects mediated indirectly via the parental animals.
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1.0 Introduction
1.1 Objectives
The present study was designed to determine the effects of nickel chloride
on the reproduction and fertility of CD rats after continuous administration
in the drinking water to both males and females for two generations. Addi-
tional nonbreeder (NB) male and female animals were included in the control
and high-dose groups, and were sacrificed and evaluated for toxicity after 90
days of exposure. The results of the 90-day exposure have been previously
presented in part one of three (I of III) of the final study report (Price et
al.. 1986). Part II of the final study report provided a summary of the fer-
tility and reproductive performance of the P0 generation, including data col-
lected through weaning of the Fla and FlD ] it ins (Price et al. . 1988). This
report, which includes data collected after weaning of the Flb litter and
during evaluation of the F2a and F2b litters, is the third part (III of III)
of the final study report. A summary of results for the P0 and Fj generations
is presented in Appendix VIII (this report).
The presently reported portion of the study examines the effects of nickel
chloride upon general clinical condition (body weight, clinical symptoms.
morbidity, mortality or gross lesions at necropsy), food and water consump-
tion, as well as mating, fertility, and prenatal and postnatal development
through weaning of the F2a litter and examines fetal toxicity of the F2b lit-
.ter. Exposure levels of nickel chloride (0. 50. 250. and 500 ppm Ni++) in the
drinking water for the two-generation study were selected by the sponsor based
upon (1) pre-existing toxicity data obtained from the available scientific
literature. (2) the results of a dose range-finding study conducted at RTI
under the present contract (Price et al . . 1985). and (3) the outcome of expos-
ure of CD rats to 0. 50. 250. 500 or 1000 ppm N1 + + din !.•;: the first two weeks
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of the reproduction/fertility study which Indicated excessive toxlcity at the
1000 ppm level. An outline of the two-generation reproduction and fertility
study and the 90-day toxlcity study of nickel chloride administered to CD rats
in the drinking water is presented in Appendix I (Protocol Attachment I.
Figure 1 and Table 1).
1.2 Summary of the Experimental Design from Weaning of the F^h Litter
to Sacrifice of the f Litter on Gestational Day 20.
At weaning on pnd 21, 218 Fjjj males and 204 FH, f c-n . 'ns (max. 4 males and
4 females from each litter) were randomly selected as potential Fj breeders.
All remaining Fj^ pups were sacrificed and necropsied (see Study Report Part
II. Table 31). On pnd 42. 30 males and 30 females from each of the 0. 50. and
250 ppm dose groups were randomly selected as breeders; all surviving animals
(22 males and 19 females) from the 500 ppm dose group were retained as
breeders. The remaining pups were sacrificed and necropsied. The Fj breeders
were housed two per cage with same-sex litter mates until pnd 42 and singly
housed thereafter.
At approximately 42 days of age. Fj generation breeders were randomly
assigned to cohort A. B. or C for subsequent weekly evaluations on Tuesdays.
Wednesdays or Thursdays, respectively. Thus, reported weekly evaluations for
body weight and consumption of food, water and Ni++ were initiated on day 0 of
Study Week 32 which corresponded to January 28. 29 or 30, 1986. for cohorts A,
B and C respectively. By week 34. all Fj breeders had reached 42 days of age
and had been assigned to chorts A. B or C. Ten males and females were
assigned to each cohort within three of the treatment groups (0, 50. and 250
ppm Ni"). Because the number of Fj animals surviving to breeding age was
reduced at 500 ppm, only 22 treated males and 19 treated females were
available for assignment within that group, as follows: eleven treated Fj
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24
•ales each were assigned to'cohorts A and B; ten treated females Mere assigned
to cohort A and nine treated females were assigned to cohort B; no animals in
the 500 ppm group were assigned to cohort C. In order to allow evaluation of
excess male breeders, one untreated female was paired with one treate'd male in
cohort A. and two untreated females were paired with treated males in cohort
B.
All animals remained in the treatment group originally assigned to their
parents. Exposure to the test compound for Fj breeder males (22-30/treatment
group) and females (19-30/treatment group) actually began in the germ cell
stage during exposure of the PQ generation, and continued via the trans-
placental and transmammary routes for the FJD fetuses and developing juve-
niles. Direct exposure of the FJD neonates to nickel from the water bottle
began prior to weaning in the home cage of the PQ dams. Dosing in water con-
tinued for the Fj generation through the day of scheduled sacrifice (i.e.. end
of the second cohabitation period for Fj males or gd 20 of the F2b litter for
Fj females). Fj males were exposed for a total of 21-24 postnatal weeks and
females were exposed for a total of 27-30 postnatal weeks.
On pnd 74-91 (avg. = 87.3 ± 0.5 days of age) cohabitation began. During
cohabitation, one female was randomly paired with one male from the same
treatment group within the same cohort. Cohabited females were checked each
morning for evidence of mating. If sperm were observed in the vaginal lavage.
the female was segregated in a clean cage; the day of sperm detection was
designated as postcohabitatlon day (pcd) 0; for sperm-positive females with
subsequently confirmed pregnancies, the day of sperm detection was also
designated as gestational day (gd) 0. If vaginal sperm were not detected
within 14 days, cohabitation was terminated and the female was segregated in a
clean cage; the day on which breeding pairs were separated was designated as
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postcohabltation day (pcd) 0. During the postcohabitation period. F1 feaales
were evaluated for body weight, food and water consumption and clinical signs
of toxiclty until such time as a litter was delivered or until 24 days after
the breeding pair had been separated. Twenty-four days after segregation.
spern-negative females without litters were vaginally lavaged for 7 days, and
vaginal cytology was examined in order to determine whether the female
exhibited normal estrous cyclicity.
After delivery of an F2a litter, each dam was housed with her own litter
throughout the 21-day lactational period. On postnatal day (pnd) 21, all ?2a
pups were sacrificed and necropsied. Dams were allowed to rest for 14-28 days
prior to cohabitation of Fj breeding pairs to produce the F2b litters. At the
end of the second cohabitation period, all Fj m;
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26
2.0 Materials and Methods
2.1 Test Article (Nickel Chloride Hexahydrate)
2.1.1 Description
CAS Type 1 Name: Nickel chloride [NiCl2], hexahydrate (SCI)(9CI)[MF1].
CAS Number; 7791-20-0 (nickel chloride hexahydrate);
7718-54-9 (nickel chloride).
Molecular Formula: NiCl2. 6H20 (nickel chloride hexahydrate):
NiCl2 (nickel chloride).
Molecular Weight: 237.70 (nickel chloride hexahydrate);
129.61 (nickel chloride).
Nickel chloride hexahydrate contains
24.70% Ni. 29.83% Cl. and 45.47% H20.
Structure: trans-[NiC!2(H20)4]-2(H20)
Appearance: green, deliquescent crystals or crystalline powder
Solubility: water (20°C) = 254 g/100 ml;
water (100°C) = 599 g/100 ml
Chemical Reactivity: aqueous solutions are acid. pH about 4; increase in pH
above 7 results in precipitation of nickel hydroxide.
Source: Aldrich Chemical Co.
P.O. Box 355
Milwaukee. Wisconsin 53201
(800)558-9160
Lot No.: Dosed drinking water administered to F3b animals was formulated from
nickel chloride Lot Number KM01918HM (received at RTI on August 20. 1985).
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27
Purity: 99*. as reported by the vendor; >98* as documented by ICP analysis at
RTI (see Appendix I. Protocol Attachment II for method). Measured purity
of this hygroscopic compound was dependent upon the degree of dessication.
thus suggesting that the major "impurity" was water.
Identity: The identity of the test compound was verified by RTI (see Appendix
I. Protocol Attachment II for method).
Stability: Stability of aqueous solutions for 15 days under the anticipated
conditions of use had been verified prior to the initiation of this study
for concentrations of 100 ppm Mi** and greater (see Price et al., 1985).
Stability of the formulations used in the present study (i.e.. 0, 50, 250
and 500 ppm, NJ'**) here verified directly by ICP analysis of nickel in
post-dosing samples taken after the period of actual use (see Section
3.5).
Vehicle: Filtered/deionized water. Durham, NC city tap water was prefiltered
with 0.5 micron Nucleopore* membrane filters to remove fine particulate
matter. Organics were removed by charcoal adsorption. Then water was
deionized by passage through two mixed-bed ion exchangers. The filtration
and deionizlng systems were purchased from and routinely serviced by Hydro
Ultrapure Water Systems, Inc.. P. 0. Box 2855, Durham, NC 27705.
2.1.2 Safety and Handling
Nickel chloride hexahydrate is a bright green crystalline material with
no recognized fire or explosion hazard; the dusting hazard potential is
moderate. This compound does not present an extreme reactivity hazard under
routine handling conditions. The crystals are deliquescent (i.e.. have a
tendency to melt or become liquid by the absorption of moisture from the air).
and precautions were taken to avoid unnecessary opportunities for accumulation
of excess moisture. The bulk chemical was stored in sealed, light-protected
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containers at room temperature. In the presence of a solid dessicant (calciua
sulfate). Refrigeration of the bulk chemical was viewed as unnecessary due to
the expected stability of this material, and was also viewed as undesirable
since the tendency to absorb water increases when the temperature of the
crystals is below that of the ambient air. thus requiring that the bulk
chemical be warmed to room temperature prior to opening the storage container.
Persons working with the bulk chemical used procedures acceptable for
handling suspected carcinogenic agents. All operations which required
handling of the undiluted bulk chemical were conducted in a fume hood. Appro-
priate protective clothing for these operations consisted of a lab coat.
double latex gloves and eye protection. For any operations which required
handling the bulk chemical outside of the hood, respiratory protection con-
sisted of a cartridge respirator fitted with dust/particle filter.
Persons working with dilute solutions of nickel chloride (i.e.. dosage
formulations or analytical standards) outside the animal rooms wore appropri-
ate protective clothing as follows: lab coat, shoe covers, and double latex
gloves. Persons working with the dilute solutions inside the animal rooms
also wore respiratory protection consisting of a cartridge respirator fitted
with dust/particle filter, or a well-fitting dust mask. The RTI Animal
Research Facility staff wore protective clothing and respiratory protection
when working in the animal holding rooms or When handling any animal waste
materials from this study. Any environmental surfaces (e.g.. benchtops.
floor, etc.) contaminated with nickel chloride or its solutions were
decontaminated immediately. Any materials or equipment contaminated with
nickel chloride or its solutions (e.g.. glassware, gloves, etc.) were decon-
taminated or disposed of immediately after use (Appendix I).
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Recommended procedures for management of major spills and for emergency
first aid in the event of accidental exposure were provided in the study pro-
tocol (Appendix I), but no circumstances arose during the conduct of this
study which necessitated the implementation of these procedures. Waste mate-
rial contaminated with nickel chloride and excess dosage formulations were
packaged and disposed of by the RTI Safety Officer according to applicable
local, state and federal regulations.
All animal wastes, animal cages, animal feeders and any other contami-
nated materials were handled according to procedures for biohazardous materi-
als. Signs were posted on the animal holding rooms indicating that entry was
restricted solely to personnel involved in conducting the study. All animal
wastes, soiled bedding, and unused feed were double bagged outside the animal
holding room, sealed, labeled with the study code, project number, test com-
pound, date bagged and estimated maximum concentration of nickel, and removed
to an area designated for temporary storage of hazardous materials. The sealed
bags were placed inside heavy cardboard boxes with appropriate labeling and
removed for incineration or burial under the direction of the RTI Safety
Officer. Unused dosed water was either collected into one gallon glass jars.
or collected into 20 L polyethylene carboys which were emptied into a toxic
waste drum placed in the Animal Research Facility solely for that purpose.
Each glass jar or drum was labeled with the study code, project number, test
compound, date collected and estimated maximum concentration of nickel (ppm).
and was disposed of under the direction of the RTI Safety Officer.
2.1.3 Dosage Formulation and Analysis
Nickel chloride hexahydrate was dissolved in the vehicle (i.e..
filtered/deionized water) in order to formulate a concentrated stock solution
at 100.000 ppm Ni*+ (approximately 405 g Ni-Cl2-6H20/L). New batches of the
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stock solution were formulated at approximately 2-week intervals, and each
batch of the stock solution was analyzed by ICP spectrometry in order to de-
termine the actual concentration of the divalent nickel cation (per Method
detailed in Appendix I, Protocol Attachment II); the pH of each stock solu-
tion was also determined. Based upon the measured concentration of Mi** in
each batch of stock solution, the dosed drinking water was formulated by di-
luting the stock solution with filtered/deionized water such that the concen-
tration of the divalent nickel cation was 0 ppm (0 rag Ni**/L). 50 ppm (50 mg
Ni+VL). 250 ppm (250 mg Ni+VL). 500 ppm (500 mg Ni+VL) or 1000 ppm (1000 mg
Ni+VL). On each formulation date, a sufficient volume of stock solution and
dosed drinking water were formulated in order to provide a 2-week supply of
dosed drinking water at the required concentrations. Dosed water administered
to Fj males and females through scheduled sacrifice was formulated from
batches XVI through XXVI of the stock solution. All solutions of nickel
chloride hexahydrate were stored at room temperature in translucent polyethy-
lene carboys (max. capacity = 20 L).
Triplicate 20 ml aliquots were collected from each batch of the stock
solution, and from the middle of each carboy of drinking water (i.e., control
water or dilute nickel chloride solution) at the time of formulation, and
placed in polycarbonate vials with snap lids labeled with the date of
sampling, the protocol number, the color code and 5-digit random code for
concentration, and the study code. The pH of each aliquot was determined and
the pH of the control water was adjusted with hydrochloric acid to match the
range of pH observed in the nickel-dosed solutions (pH 4.0-6.0). One aliquot
each from the stock solution, from each carboy of dosed drinking water, and
from vehicle control water was analyzed by ion emission spectrometry (ICP) to
verify the concentration of nickel cation (per method described in Appendix I.
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Protocol Attachment II). Verification of stability of Ni++ concentration and
pH for all drinking water formulations was obtained by conducting ICP and pH
determinations of samples collected after the period of use (see section 3.5).
For each triplicate set of stored samples, one sample was analyzed for
nickel concentration by ionic emission spectrometry (ICP); one sample served
as a back-up analytical sample, if needed; and one sample was placed in frozen
storage as an archival reference sample (see also Section 2.8, "Storage of
Records"). Measured concentrations within a range of 90-105% of the nominal
concentration were considered acceptable for use: if the measured concentra-
tion of a pre-dosing sample was outside of this range, then the formulation
was replaced prior to administration to test animals, with minor exceptions
(see section 3.5).
The stock solutions were labeled with a batch number, a random 5-digit
code number, the date of formulation, the study code, protocol number, and the
measured concentration of divalent nickel cation. Each carboy of drinking
water was labeled with the date of preparation, protocol number, the study
code (Rt85-NICL.REPRO), a random 5-digit concentration code number, and a
color code for concentration in order to provide a partial blind for dose.
Complete anonymity of the control and of the relative concentrations of nickel
in each formulation was not possible since solutions of nickel chloride are
characterized by a green color which darkens with increasing concentration.
The carboys of vehicle control drinking water and dosed drinking water were
stored at room temperature on stainless steel work tables in the animal
holding rooms assigned to this study. Individual animal holding cages and all
relevant study records were labeled with the matching 5-digit concentration
code and color code for each dose group. Following routine sanitation, water
bottles were recycled for use in the same concentration exposure group accord-
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Ing to the color-coded tape attached to each bottle. Only the study director.
laboratory supervisor, and staff member responsible for mixing the dosed water
had access to the actual concentration represented by each concentration code.
Personnel involved in the analysis of dosage formulations for nickel concen-
tration, animal care or toxicologic evaluation were not informed of the formu-
lation concentrations represented by each code until all laboratory work had
been completed.
2.1.4 Treatment
Male and female breeder CD rats in the Fj generation remained assigned to
the dose group of their parents, i.e.. 0. 50. 250 or 500 ppm Mi** in the
drinking water. Exposure was continuous through 21 to 24 weeks of age for Fj
males (i.e.. until the end of the second cohabitation period), or 27 to 30
weeks of age for Fj females (i.e.. until sacrifice at gestational day 20 of
the F2b litter).
Treatment groups received either control water (0 ppm Ni**). or 50 ppm.
250 ppm or 500 ppm of the divalent nickel cation. Clean water bottles with
fresh solutions of control and nickel-dosed water were placed on the animal
cages at least once per week beginning on pnd 21 for Fib pups, except that
water bottles were changed on days 0. 6. 13. 20 and 24 for females during the
?2a postcohabitation periods, on days 1. 7. 14. and 21 of lactation for each
female with an F2a litter, and on days 0. 6. 13 and 20 during the F2b
postcohabitation period. Between changes of the water bottles, solutions were
added to individual water bottles as required to insure an ad libitum supply
of fluid for each animal. All unconsumed control and nickel-dosed water was
either packaged in one-gallon glass containers or transferred to a toxic waste
drum on the day of bottle-changing, and subsequently disposed of by RTI Safety
Office personnel (see also Section 2.1.2. "Safety and Handling").
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The route of exposure for animals in the present study corresponded to
the environmental route of exposure in humans which was of primary concern to
the sponsor. The range of exposure concentrations was specified by the sponsor
with the expectation that the study would provide information concerning clin-
ical symptoms of toxicity and mortality, as well as effects on reproduction
and fertility after continuous exposure of two generations to nickel chloride
in the drinking water. The high-dose exposure level (500 ppm) was expected to
produce significant toxicity in the two-generation study, but the two lower
doses (50 and 250 ppm) were selected with the goal of determining a no-
observed-effect level (NOEL) for reproductive and other toxic endpoints.
2.2 Animals
The experimental animals in the present study were viral antibody free
Crl:CD® BR VAF/Plus™ outbred albino rats supplied by the Charles River Labora-
tories. Inc. (Kingston. NY). This strain is referred to as the CD rat. Males
and females were 27-32 days old at the time of arrival at RTI for the P0 gen-
eration, and the animals for the Fl p.cneration were born at RTI. A total of
218 FI males and 204 Fj females were assigned to this investigation at weaning
(pnd 21 of the FJD litter) (sec Section 1.0 for an overview of the study
design).
The Charles River CD rat was selected as the experimental animal since
this species and strain has been a subject of choice in prior reproductive
toxicology and teratology studies at RTJ since 1976; thus a large historical
data base for reproductive performance and incidence of spontaneous malforma-
tions in control rats has been compiled at RTI. In addition, historical data
on Charles River CD controls has been summarized from teratology studies in
other laboratories, thus providing information on the frequencies and types of
spontaneous malformations in the CD rat (Banerjee and Durloo. 1973: Perraud.
1976; Woo and Hoar. 1979; Charles River. 1988).
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2.3 Animal Husbandry
At weaning (pnd 21 of the Flb litter). 218 Fj sales and 204 Fj fenales
were randomly assigned to cages, and an individually-coded tag was affixed to
one ear of each rat. All animals were housed throughout the study in solid
bottom polycarbonate or polypropylene cages (8" x 19" x 10 1/2") with
stainless steel wire lids (Laboratory Products. Rochelle Park, NJ) and molded
filter tops (Ancare Corp.. Manhasset. NY). Ab-Sorb-Dri* cage litter
(Laboratory Products. Garfield, NJ) was used in all cages. Cages were placed
on galvanized-steel racks (24" W x 60" L x 66" H) (Laboratory Products.
Rochelle Park, NJ). Animals were singly housed throughout the study, except
during a) pnd 21-42 when they were housed in same sex pairs, b) designated
periods of cohabitation (adult breeders housed 1 male and 1 female), or
c) during lactation (1 female housed with her own litter).
Purina Certified Rodent Chow® (No. 5002. pelletized) was available ad
libitum throughout the study. Rodent chow was stored at 55-60° F and the
period of use did not exceed five months after receipt at RTI. A sample from
each milling date and batch number of Purina Certified Rodent Chow8 employed
in this investigation was analyzed for nickel content with the following out-
come:
Milling Date Period of Use Nickel, ug/g
(Batch No.) (date received (mean ± S.D.; n=2)
to last issue)
12-11-85 (2A) 1-22-86 to 2-14-86 0.98 ± 0.00
121 .".6 (IB) 2-19-86 to 3-31-86 1.36 ± 0.06
2-05-86 (ID) 3-19-86 to 4-21-86 1.20 ± 0.01
2-05-86 (IE) 1.16 ± 0.09
2-19-86 (ID) 4-10-86 to 5-23-86 1.46 ± 0.00
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35
During the exposure period (see also Section 2.1.4, "Treatment"), animals
were given ad libitum access to either control water (i.e.. deionized/filtered
water with pH=4.0-6.0 adjusted with hydrochloric acid) or to an aqueous solu-
tion of nickel chloride (50. 250 or 500 ppm Ni**). Plastic water bottles with
butyl rubber stoppers and stainless steel sipper tubes were used throughout
this investigation.
The animal holding rooms assigned for this study [ARF Room Nos. 148 and
150 (16' x 12'). and Room 146 (81 x 12')] were equipped with individual
temperature controls and automatic adjustable light cycles (lights on 7:00
a.m. to 7:00 p.m.). The estimated exchange rate for air in each animal room
was 12 to 14 times per hour. Relative humidity and temperature were monitored
on Oatapod Model DP220 Electronic Hygrothermographs (Omnidata International.
Inc.. Logan. UT) located in the animal holding rooms for this study, and
summarized on a monthly basis throughout the study (Table 1A and IB). Further
details related to environmental conditions are presented in Appendices X and
XI.
On pnd 42. a total of 112 male and 109 female breeder animals (Fj genera-
tion) were randomly assigned to three cohorts, as follows. Males and females
were ordered separately within each sex on the basis of increasing ear tag
number. These animals were then randomly ordered using a randomly generated
array of integers. Within each dose group, for the 0. 50. and 250 ppm dose
groups, males and females with the highest random numbers were assigned as a
breeding pair. Breeding pairs were then sequentially assigned to cohorts A.
B. or C (10 breeding pairs per cohort per dose group). For the 500 ppm dose
group (22 males and 19 females) animals were assigned to breeding pairs as
described above (19 pairs) and assigned to cohort A or B (10 and 9 pairs.
respectively). Three untreated females were assigned as mates to the three
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36
remaining males, and these pairs were assigned to cohorts A and B (1 and 2.
respectively) such that cohorts A and B each had 11 breeding pairs. The
untreated females were included to allow the treated males (only) to be
evaluated as fully as possible in the study design.
In order to monitor the general health status of animals on study,
routine rodent health surveillance was conducted at time points designated in
the study protocol (Appendix I. Protocol Attachment I. Table 1). The
serodiagnostic test panel was conducted for FJJ, males and FlD females at 42
days of age. Serum from four male and four female rats (2 males and 2 females
each from control; 1 male and 1 female from the 50 ppm group and 1 male and 1
female from the 250 ppm group) was submitted to Microbiological Associates for
serological evaluation (Appendix I. Protocol Attachment I. Table 2). Results
of the serodiagnostic test panel were negative for 8/9 viral antibody titers.
but murine sendai virus titers were positive for 4/4 males and 4/4 females
(Appendix VI). Examination of the clinical signs recorded for this study
(Appendices IV and V) revealed no generalized symptoms which might have been
associated with sendai exposure. Discussion with Dr. Robert L. Peters. Micro-
biological Associates and Donald B. Feldman. D.V.M.. RTI. indicated that CD
rats exposed to murine sendai may develop antibodies but remain essentially
symptom free, as was apparently the case in this study; thus, no threat to the
integrity of the present study is implied by positive antibody determinations
in these cases. At final sacrifice of Fj males (following the end of the
second cohabitation period) and Fj females (on gd 20 of the Fgfc litter), serum
was collected from sentinel animals for evaluation in the serodiagnostic test
panel (2 males each from the control and 250 ppm groups: 2 females each from
the control and 500 ppm groups). Results of the serodiagnostic test panel
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37
were negative for 9/9 viral antibody titers. including aurine sendai. for all
animals evaluated.
2.4 Toxicologic Evaluation
2.4.1 In-Life Evaluation
Live body weight (g). was recorded for individual Fj mnlrx and females
between 8:30 a.m. and 10:30 a.m. at least weekly between pnd 21 and pnd 42.
Beginning on study week 32. Fj animals which were at least 42 days of age were
assigned to cohorts in order to establish a weekly weighing schedule (see
Section 1.2); all Fj animals reached 42 days of age and were assigned to
cohorts by week 34 (Appendices II and III). Thus, beginning on study week 32.
body weight for Fj males and females was recorded once per week for the
remainder of the study except during cohabitation or during gestation and
lactation when female body weight was recorded on postcohabitation days (pcd)
0. 6. 13. and 20 (if a litter had not been delivered by pcd 24. body weight
was also recorded on that day), as well as on postnatal days (pnd) 1. 4, 7.
14. and 21 (dams were weighed on the designated postnatal days only if a
litter was present on the day of measurement). Food consumption (g) and water
consumption (g) were recorded at each occasion when body weights were recorded
(except on pud 4. per the study protocol) or when food or water was added to
the cages. Average daily consumption of food and water (g/animal/day and
g/kg/day) was calculated for each dose group. During cohabitation, collective
food and water consumption was recorded for each breeding pair. During
lactation, food and water consumption were expressed as the total intake per
dam recognizing that intake by the offspring contributed to this measure
during the later stages of lactation.
Throughout the study, each male and female was observed dally between
8:30 a.m. and 10:30 a.m. for clinical signs of toxicity. morbidity, or mor-
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38
tality. beginning on day 0, and continuing until scheduled sacrifice.
Clinical signs observed during handling of the animal were recorded at the
tine of weighing; on all other days, recorded observations were based upon
cage-side observations. For any P1 animal found dead or sacrificed In
extremis, the date and time of the observation was recorded, the animal was
weighed and necropsied as soon as possible for external and internal signs of
toxicity. and a final measurement of food and water Intake was taken. Necrop-
sies for pups which died or were sacrificed in extremis before pnd 21 were
specified in the study protocol, but were not performed due to a misunder-
standing by the technical staff.
During evaluation of Fj dams and F2a litters, the following measures were
recorded: length of gestation, number of pups per litter, number of live pups
per litter, number of dead pups per litter, percentage of male pups per lit-
ter, individual pup body weight (pnd 1. 4. 7, 14. and 21). external (gross)
anatomical malformations, and any other clinically abnormal condition ob-
servable at cage-side or at weighing. On pnd 4, litters were culled to a
maximum size of 10 giving consideration to equal representation of males and
females when possible. At normal weaning age (pnd 21) all F2a pups were sac-
rificed and a gross necropsy performed.
Fetuses from the F2b litters were collected at sacrifice of the dam on gd
20. The parameters measured were number of corpora lutea per dam, implanta-
tion sites per litter, resorptions per litter, dead fetuses per litter, non-
live Implants per litter (dead fetuses plus resorptions). adversely affected
implants per litter (nonlive implants or malformed live fetuses), live fetuses
per litter, sex of fetus and individual fetal body weight. Fetuses were
examined for external, visceral, and skeletal abnormalities, as well as other
anatomical variations.
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39
2.4.2 Necropsy
At scheduled sacrifice, animals selected for the animal health surveil-
lance serodiagnostic panel (see Section 2.3. Animal Husbandry), were
anesthetized with carbon dioxide, blood samples were collected by cardiac
puncture, and each animal was sacrificed by exsanguination. Serum was diluted
1:5 in phosphate buffered saline or equivalent, and heated at 56°C for 30
minutes. Minimal samples of 1.5 ml diluted serum were stored in tightly-
sealed screw-cap vials, and shipped for evaluation in a serodiagnostic test
panel (Appendix I. Attachment I. Table 2) performed by Microbiological Associ-
ates. Bethesda. MD. All other animals were anesthetized with carbon dioxide
and sacrificed by cervical dislocation.
Post-mortem body weight was taken immediately after sacrifice. A gross
necropsy was performed, including external and internal examination. At
scheduled sacrifice, specifJed tissues (Appendix I. Protocol Attachment I,
Table 3) were excised and weighed, the weights were recorded, and these organs
fixed in neutral buffered 10% formalin, except for testes which were fixed in
Bouin's solution. Any other organs exhibiting gross lesions and/or abnormal
masses were also dissected and saved in neutral buffered 10% formalin.
Organs were stored in 8 oz. Nalgene* bottles, except for ovaries and
testes which were stored in 20 ml glass scintillation vials. Each storage
container was identified by study code, concentration code, color code, animal
number, sex. and date of sacrifice. Following examination, all other organs
and carcasses were destroyed by incineration.
For gravid females (gd 20). the gravid uterine weight was recorded. The
fetuses were counted, weighed and the implantation sites identified according
to the protocol (Salewski. 1964).
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40
All scheduled necropsies were supervised by a Board Certified Veterinary
Pathologist (Experimental Pathology Laboratories. Inc.. Research Triangle
Park. NC).
2.5 Histology and Histopathology
Histology and hlstopathology were conducted at Experimental Pathology
Laboratories. Inc., Research Triangle Park, NC. All procedures were in com-
pliance with the Good Laboratory Practice Regulations and were performed as
outlined in the Experimental Pathology Laboratories. Inc., Standard Operating
Procedures.
Routine hematoxylin and eosin-stained tissue sections (6 micron thick-
ness) were prepared from the following organs: the testes with attached
epididymides, seminal vesicles, and prostate from male animals; uterus.
vagina, and ovaries from female animals; and liver, kidneys, pituitary, lungs.
adrenals, and heart from both male and female animals (Appendix I. Protocol
Attachment I. Table 3). Tissue sections from any unusual masses or gross
lesions found in necropsied animals were also prepared and examined micro-
scopically.
Tissues were examined histopathologically by a Board Certified Veterinary
Pathologist. The pathology reports of these findings include an interpretive
narrative summary, summary incidence tables, individual animal incidence
tables, and tables correlating gross observations with microscopic findings.
The pathology reports have been appended to and incorporated into the present
study report (Appendix VII).
2.6 Statistical Analysis
For data collected during the second generation (e.g.. Fj adults and F2a
and F2D litters), a standard statistical paradigm was followed that examined
the experiment-wise effect of dose and the dose response trend. If the
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41
experiment-wise test revealed a significant difference among the doses then a
test of pair-wise differences between the control and each dosed group was
performed. For parameters that were expected to conform to the parametric
assumptions of normality and homogeneity of variance, the following tests were
used to examine the experiment-wise effect of dose, dose response trend, and
pair-wise effect of dose, respectively: one-way analysis of variance (Snedecor
and Cochran, 1967), simple linear regression (Snedecor and Cochran, 1967),
Williams' Test (Williams. 1971 and 1972) and Dunnett's Tests (Dunnett, 1955
and 1964). The outcome of both Williams' and Dunnett's tests have been re-
ported for each parameter, and in most cases the results of these tests were
in agreement. For those cases in which the significance of these tests dif-
fered. Williams' test is considered to give the best evaluation of the data
when the means are ordered across groups with respect to dose. If the re-
sponse was not ordered across groups, then Dunnett's Test is considered to
give the best evaluation of the data, with regard to differences between in-
dividual Ni""•'-exposed groups and the control group. When appropriate, a co-
variate (e.g.. litter size) was used to adjust the dependent variable (e.g..
pup body weight) and allow the effect of dosn to be determined more precisely
(Snedecor and Cochran. 1967). Bartlett's Test for Homogeneity of Variance
(p<0.001) was used to ensure that the variable being analyzed was
homoscedastic (Snedecor and Cochran, 1967). If the variances in the groups to
be tested were not equal then equivalent nonparametric tests were considered.
In each case the use of parametric statistical evaluation was viewed as
reasonable since the ANOVA procedure is generally considered to be robust even
though some biological endpoints deviate from the assumptions of homogeneity
of variance and normality (Winer, 1962; Hays. 1963).
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42
When the data were expected to violate the assumptions of the parametric
tests, then similar nonparametrie tests were used. When data were continuous
or approximately continuous but not normally distributed, the Kruskal-
Wallis (Siegel. 1956). Mann-Whitney U (Siegel. 1956). and Jonckheere's
(Jonckheere. 1954) tests were used to examine the experiment-wise effect of
dose, the pair-wise effect of dose, and the dose response trend, respec-
tively. For nonparametric pair-wise comparisons of Individual groups against
the controls, the following tests were used: (1) for data with a significant
Kruskal-Wallis Test (p<0.05) and evidence of a trend (p<0.10. Jonckheere's
Test). Shirley's multiple,.comparison procedure (Shirley, 1977) was used to
determine the smallest dose at which there was a statistically significant
effect, or (2) for data with a significant Kruskal-Wallis Test (p<0.05) and no
evidence of a trend (p>0.10. Jonckheere's Test), Dunn's Test, a nonparametric
analogue to Dunnett's Test was applied (Dunn. 1964). Index (categorical) data
were analyzed using the Chi-Square Test for Independence (Snedecor and
Cochran. 1967), Fisher's Exact Probability Test (Snedecor and Cochran. 1967).
and the Test for Linear Trend on Proportions (Snedecor and Cochran, 1967) for
the detection of the experiment-wise, pair-wise, and trend effects of dose.
respectively.
The particular variables which were analyzed for Fj adults and their
litters (F2a pups through weaning and F2D fetuses at pnd 20), and the tests
used to analyze each variable are presented in Appendix I, Protocol Attachment
I, Table 4. The analysis of the data was conducted using the SAS software
(SAS Institute, Inc., 1982a, 1982b) available on an IBM 3081 at the Triangle
Universities Computation Center (TUCC). Research Triangle Park. NC and on a
VAX 11/785 at RTI.
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2.7 Personnel
Technical Monitors:
Study Director:
Co-Study Director:
Senior Chemist:
St;<> istical Advisor:
Statistician/Programmer:
Co-Laboratory Supervisor:
Co-Laboratory Supervisor:
Biologist II:
Biologist I:
Assistant Biologists:
Biological Laboratory Assistants:
Draft Report Preparation:
Appendix XI Author
43
Dr. Reva Rubenstein. USEPA
Dr. Gary L. Kiiaael. USEPA
Dr. Babasaheb R. Sonawane, USEPA
Dr. Anbika Bathija. USEPA
Dr. Chris De Rosa. USEPA
Dr. Catherine J. Price
Dr. Julia D. George
Dr. David A. Binstock
Mr. Steven K. Seilkop,
Analytical Sciences. Inc.
Kr-i-.-irch Triangle Park. NC
Dr. Brian M. Sadler
Ms. Melissa C. Marr
Ms. Polly E. Sanderson
Ms. Christina B. Myers
Ms. Mary M. Parker
Ms. Ellen B. Hahn
Ms. Mertie V. Snead
Ms. Margaret R. Shilling
Mr. Steven C. Morgart
Ms. Vickie I. Wilson
Mr. Fred D. Cole
Ms. Melody F. Cower
Ns r-illie M. Sumrell
Ms Elizabeth M. Gulll
Ms. Pamela L. Moody
Dr. Patricia A. Fail
Dr. Elizabeth A. Field
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44
2.8 Storage of Records
All original study records, including all original data sheets, have been
bound and stored in the Research Triangle Institute Archives under the control
of the RTI Quality Assurance Officer. For biological samples which were col-
lected during the course of the study and examined microscopically by EPL. the
corresponding tissue blocks and slides have been placed in secure storage at
the Research Services Building. Research Triangle Institute. Work sheets and
computer printouts from the Research Triangle Institute Computer Applications
Center generated in the statistical analysis of data have also been stored at
the Research Services Building. Research Triangle Institute. Copies of the
final study report will be filed with the Chemistry and Life Sciences Unit,
Research Triangle Institute as well as with the contracting agency. U. S.
Environmental Protection Agency. In accordance with the EPA Pesticide Pro-
grams. Good Laboratory Practice Standards. Final Rule. (Federal Register Part
IV. pp. 53962-53969. November 29. 1983) all study records, data and reports
can be maintained in secure storage for a minimum of five years following
signed approval of the final study report; however, the sponsor anticipates
that a minimum storage period of two years will be adequate for the purposes
of the present investigation. Thus, chemical and biological samples will be
m;i i nt nined for a minimum of two years or for as long as the quality of the
preparation affords evaluation, whichever is less. No archival materials will
be destroyed without 90 day prior notification of the sponsor. Archival
materials may be shipped to an alternate storage site at the written request
of the sponsor at any time after the completion of this study.
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45
3.0 Results
3.1 Toxicologic Evaluation of CD Rats in the fl Generation
3.1.1 Clinical Status and Mortality for Ft Males and Females
Male and female CD rats In the Fj gi-m i.it ion were derived from the Flo
litters (Final Report. II of III) and remained assigned to the dose group of
their parents. Thus, the Fj animals had been exposed to nickel chloride as
germ cells in the PQ generation, via the transplacental route as fetuses, via
the transmammary route during lactation, and via the drinking water during the
later stages of lactation. Fl exposure to nickel chloride (0. 50. 250 or 500
ppm Ni*+) in the drinking water was continuous from weaning (pnd 21) until
scheduled sacrifice. As previously noted in the P0 generation report (Final
Report. II of III), average pup body weight for FH, litters (both sexes com-
bined) was significantly below controls on pnd 21 at 250 ppm (87% of control
weight: p<0.05) and at 500 ppm (75% of control weights; p<0.01). At weaning
on day 21. 218 males and 204 females were selected as potential breeders, and
allowed to mature. On pnd 42. animals were selected for breeding, singly
housed, and food and water consumption and body weight data were collected for
7 weeks prior to cohabitation. At the first weekly weighing after final
selection and individual housing of Fj brooders (week 32), body weight for the
500 ppm animals was still significantly decreased (81% of control weight for
females and 72% for males; both p<0.01). but minor reductions in body weight
at 250 ppm (95% of control weights for both sexes) were no longer statisti-
cally significant (Appendices II and III; Figures 1 and 5). All Fj animals
not assigned as breeders were necropsied on pnd 21 or 42.
Monthly averages of temperature and relative humidity in treatment rooms
throughout the study are presented in Tables 1A and IB. Dally averages of
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46
temperature and relative humidity in the treatment rooms throughout the study
are presented in Appendix X.
During the period of Juvenile development (days 22-42) for selected Fj
pups. 23 males and 22 females died or were sacrificed j_n extremis (11% of the
total population of 218 males and 204 females). The incidence of deaths was
•\
significantly increased at 250 ppm (p<0.05) and 500 ppm (p<0.01) for males
(2/60. 4/67. 8/60 and 9/31 animals, in the control through high-dose groups)
and at 500 ppm (p<0.01) for females (4/58, 5/59, 4/59 and 9/28) (Tables 2, 3,
4 and 5). Necropsy at pnd 42 of animals not assigned as breeders revealed no
apparent association between treatment and the incidence of gross lesions
(Table 9). As adults (after 42 days of age), all Fj males survived to
scheduled sacrifice; among females, four deaths occurred at parturition of the
F2a litter (3/30 females at 250 ppm and 1/19 at 500 ppm) (Tables 6 and 7).
Although the incidence of maternal deaths was not strictly dose-related.
deaths had been observed under similar circumstances for nickel-exposed
females in the PQ generation, but not in the PQ or Fj control groups. In
addition, death during delivery is an unexpected finding based upon historical
control data from this laboratory (0 maternal deaths/147 control-group
pregnancies with natural delivery of single litters) or based upon data
supplied by the vendor (0 maternal deaths/258 naturally-delivered litters from
reproduction studies of varying designs) (Charles River, 1988). Collectively.
the Incidence of these deaths suggest that nickel exposure was associated with
compromised status of pregnant females around the time of parturition, and
further evaluation of this effect appears to be warranted in order to
determine how this effect was mediated. Clinical signs observed for both
males (Table 8) and females (Table 6) included rough hair coat, piloerertion.
and dental problems. Clinical signs observed were not notably different than
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47
for the P0 gunf-ration (see p. 43, Final study report Part II. Tables 2 and 3).
In general, necropsy of dead or sacrificed Moribund animals did not reveal the
cause of death (Tables 3 and 5). In summary, deaths and moribund sacrifices
among Fj adults (after pnd 42) can be categorized as follows: (1) no deaths
or moribund sacrifices in Fj males. (2) for'Fj females, deaths associated with
pregnancy occurred with an incidence of 0/30. 0/30. 3/30. and 1/19 in the
control through high-dose groups, respectively); (3) no injuries or tumors
were noted. As also noted in the P0 gn.r-ration, susceptibility of adult F!
females appeared to be greatest during the perinatal phase of the reproductive
process, in association with complications of pregnancy.
3.1.2 Body Weight. Food. Water and Nickel Consumption for fl
Females (see also Appendices II and IX)
fen Tj fn. 'ns. body weight and consumption of food, water and nickel
were analyzed weekly during the nonreproductive portions of the study (Figures
1-4 and Appendix II). These weekly evaluations excluded periods of
cohabitation, gestation and lactation (e.g.. weeks 39-45 and 50-55). These
data for females during gestation and lactation are described in sections 3.2
and 3.3 below.
During the nonreproductive portions of this study, average daily intake
of Mi*"*" (mg/kg/day) tended to decrease across weeks (Figure 4) due to
decreased relative fluid intake (g/kg/day) in all groups (Appendix II). This
effect was also noted for male rats (see section 3.1.3). and appeared to be
age-related. Average Ni++ intake (Figure 4 and Appendix II) for Fj females in
the low- through high-concentration groups, respectively, was 8.65. 34.84.
66.51 mg/kg/day during week 33. 5.82, 22.56, and 44.41 mg/kg/day during week
38 (prior to first cohabitation) and 4.61. 17.89 and 35.31 mg/kg/day during
week 49 (prior to second cohabitation).
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48
Water intake (g/kg/day) for females during the nonreproductive portions
of the study was not affected at 50 ppm (99-107% of control Intake), but was
significantly reduced (p<0.01) at 250 ppm (76-84% of control) and 500 ppn (74-
87% of control) during each week of the exposure period (Figure 3 and Appendix
II).
Food consumption (g/kg/day) for Fj females exhibited a significant
(p<0.05) increasing trend on week 37, due solely to a nonsignificant increase
at 500 ppm (106% of control). On week 38 the 500 ppm was significantly
increased above controls (110% of control; p<0.05 or 0.01) (Figure 2 and
Appendix II). No other significant effects were noted for food intake of
females during the nonreproductive portions of the study.
Average body weight for Fj females in the 50 ppm group (102-105% of
control weight) and the 250 ppm group (95-100% of control) were not
significantly different from the control group (Figure 1 and Appendix II).
The 500 ppm group exhibited average weights which were significantly below the
control group (81-93% of control weights; p<0.05 or 0.01) for each week during
the nonreproductive portions of the study (Appendix II). Graphical
representation of Fj female food, water, and nickel consumption as a function
of body weight during the nonreproductive portions of the study is presented
in Appendix IX.
3.1.3 Body Weight. Food. Water and Nickel Consumption for F1
Males (see also Appendix III and IX)
For Fj males, body weight and consumption of food, water and nickel were
analyzed weekly during the nonreproductive portions of the study (Figures 5-8
and Appendix III). These weekly evaluations excluded periods of cohabitation.
so that no data are presented for weeks 39. 50 and 51. and only body weights
are presented for week 40. Average daily Intake of water for each group, and
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49
therefore of Ni** in each exposed group, tended to decrease across weeks for
male rats (Appendix III). Thus, average Ni** intake for the low through high
concentrations, respectively, was 7.63. 35.83 and 63.05 rag/kg/day during week
33. and 2.95. 14.45 and 28.49 mg/kg/day during week 49 (Appendix III).
Water intake (g/kg/day) for males showed a significant decreasing trend
(p<0.05. 0.01 or 0.001) across groups within each week of the study, except
for weeks 37. 45-47 and 49 (Appendix III). Average water intake at 50 ppm was
slightly below controls for each measurement period (91-97% of control
intake), but only the pairwise comparisons on week 36 (91%) and 38 (92%) were
significant (p<0.05 or 0.01). At 250 ppm. water intake was significantly
reduced (82-89% of control intake; p<0.05 or 0.01) on weeks 33-38. and 41-44.
but not on weeks 45-49 (90-95% of control intake). The 500 ppm group was
significantly below controls (77-95% of control intake; p<0.01) on weeks 33-
38. 41-44 and 48. No statistically significant effects upon water intake were
observed for weeks 45-47 or 49. Thus, the predominant effects on water intake
occurred at 250 and 500 ppm prior to study week 45. In older animals (weeks
45-49), water intake was decreased only sporadically in the 500 ppm group.
Food intake (g/kg/day) for nickel-exposed males did not differ signifi-
cantly from the controls for weeks 33-37. For the remaining weeks (38 and 41-
49). no effects were observed on food Intake at 50 or 250 ppm, except for a
transient increase (105% of control intake; p<0.05) at 250 ppm on week 45. At
500 ppm, relative food intake (g/kg/day) was significantly increased (108-121%
of control intake; p<0.01) on weeks 38 and 41-48; a significant trend (p<0.05)
on week 49 reflected a nonsignificant increase (108% of control) at 500 ppm
(Figure 6 and Appendix III). The absolute amount of food consumed (g/day) was
significantly decreased (p<0.05 or 0.01) in the 500 ppm group throughout the
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50
study, except for weeks 47 and 49. due to the persistence of decreased body
weight for males in that group as described below (Appendix III).
Throughout the period of measurement (weeks 32-49). average male body
weight was 106-109% of controls at 50 ppm and 95-99* of controls at 250 ppm,
but these minor differences did not reach statistical significance. Decreased
male body weight at 500 ppm (71-86% of control weight) attained statistical
significance (p<0.01) for each observation from week 32-49 (Figure 5 and
Appendix III). Graphical representation of Pl male food, water, and nickel
consumption as a function of body weight during the nonreproductive portions
of the study is presented in Appendix IX.
3.2 Reproductive Performance of Fj Breeders (first mating) and
Postnatal Development of Fga Litters
3. 'f i General Indices of Reproductive Performance
Following 7 weeks of exposure, randomly selected Fj breeding pairs (1
male: 1 female) within each treatment group were cohabited for a maximum of
two weeks or until sperm were detected in the daily vaginal lavage. Offspring
of this mating were designated as the ?2a * '''Prs• No adverse effects of
treatment were observed for the mating index (* mated females), fertility
index (% fertile matings), gestational index (% live litters), perinatal
viability index (* viable litters on pnd 4). or the lactational index (*
viable litters on pnd 21) (Table 10).
3.2.2 Maternal Measures During Gestation
1h< i.i- tational period was not significantly lengthened by nickel treat-
ment in this study (Table 11) as it was in the P0 generation. The proportion
of litters delivered on or before gd 21 vs. on or after gd 22 did not differ
statistically across groups (Table 11).
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On gd 0, 6 and 13, gestational body weight for timed-orated females with
litters showed significant trends (p<0.05) due to nonsignificant decreases at
500 ppm (96% of control weight). On gd 20. the 500 ppm group was
significantly below controls (87* of control weight; p<0.01) (Table 11 and
Figure 1).
Relative food consumption (g/kg/day) for Fj dams during gestation showed
no adverse treatment-related effects on gd 0-6 and gd 6-13. However, food
consumption was significantly decreased (93% of control intake; p<0.01) at 500
ppm on gd 13-20 (Table 12 and Figure 2).
Water consumption (g/kg/day) during gestation was not affected by nickel
exposure at 50 ppm (Table 13 and Figure 3). However, water consumption in the
250 and 500 ppm groups was significantly below controls (p<0.01) during gd 0-
6. gd 6-13 and gd 13-20 (71-77% of control intake at 250 ppm and 58-73% of
control at 500 ppm) (Table 13 and Figure 3). Within individual groups,
average daily fluid intake did not vary greatly across different periods of
gestation (gd 0-6, gd 6-13. or gd 13-20) (Table 13). Thus, average daily
nickel consumption was relatively stable throughout these periods, e.g., 5.79
-6.42 mg/kg/day at 50 ppm, 20.97 - 24.69 at 250 ppm, and 38.69 - 45.42 at 500
ppm (Table 14 and Figure 4). Graphical representation of Fj female food.
water, and nickel consumption as a function of body weight during gestation of
the F2a litter is presented in Appendix IX.
3.2.3 Maternal Measures During Late Gestation and Lactation
Dm 'me. late gestation and lactation. Fj female body weight was 102-103%
of controls at 50 ppm (nonsignificant), and 94-96% of controls at 250 ppm
(nonsignificant). The 500 ppm group was significantly below controls (82-92%
of controls; p<0.05 or 0.01) for maternal body weight at each of these time
points (Table 15 and Figure 1).
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52
Maternal food consumption (g/kg/day) was significantly decreased only at
500 ppm (p<0.01) to 45% of the control Intake during late gestation (gd 20 -
pnd 1). No effects related to nickel exposure were observed for maternal food
intake for the periods pnd 1-7. pnd 7-14 or pnd 14-21 (Table 16 and Figure 2).
Maternal water intake (g/kg/day) at 50 ppm was not affected during late
gestation (gd 20-pnd 1), and throughout lactation (pnd 1-7, pnd 7-14 and pnd
14-21) (Table 17 and Figure 3). At 250 ppm. water intake was significantly
reduced to 66% of control intake (p<0.01) on gd 20 - pnd 1. On pnd 1-7. 7-14
and 14-21. the 250 ppm group consumed 89-93% as much as controls which
appeared to contribute to the downward trend, but pair-wise comparisons to the
control group were not significant. The 500 ppm group showed significantly
reduced water consumption (p<0.05 or 0.01) for all periods of measurement from
gd 20 - pnd 21 (Table 17 and Figure 3). The most severe reduction (31% of
control intake) occurred between gd 20 and pnd 1; reductions during pnd 1-21
ranged from 73-82% of control intake. As normally occurs in mammalian species
(Appendix XII). fluid intake showed a slight reduction during late gestation.
and a large, systematic increase during lactation, recognizing that intake by
the offspring contributes to this measure during the later stages of lacta-
tion. This characteristic pattern of fluid intake was observed despite the
treatment-related decreases noted above, and resulted in a comparable systema-
tic increase in nickel average consumption per dam (i.e.. dam-litter unit), as
follows: the 50 ppm group increased from 4.97 to 13.08 mg/kg/day; the 250 ppm
group increased from 16.28 to 54.69 mg/kg/day; and the 500 ppm group increased
from 15.17 to 89.54 mg/kg/day (Table 18 and Figure 4). Notably, the
volumetric increase in fluid intake was similar in all groups (approximately
150 g/kg/day) during lactation. A graphical representation of food, water.
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53
and nickel consumption as a function of Maternal body weight during lactation
of the ?2a Jitirr is presented in Appendix IX.
During evaluation of the P0 generation (Section 3.2.3, Study Report Part
II of III), the need was recognized to monitor food and water intake at more
frequent intervals during lactation than proposed in the study protocol. This
deviation from the protocol was required to assure an adequate supply of food
and water during this period of increased consumption for Fj females and their
F2a litter.
3.2.4 Evaluation of Fga Litters (Postnatal Days 1-21) .
On pnd 1 and 4 (before culling), as well as on pnd 14 and 21 (e.g.,
after culling to a maximum litter size of 10 live pups), significant decreas-
ing trends (p<0.01 or 0.001) for live litter size (live pups/litter) were
observed. The low dose was a no-observed-adverse-effect level (104-110% of
control litter size), the mid-dose litter size was not significantly reduced
(91-97% of control), and the high-dose group was significantly below the con-
trols (67-84% of control) (Table 19 and Figure 9). A similar relation among
doses was observed on pnd 7 (p<0.05, trend), but the decrease at the high dose
to 81% of control litter size was not significant. No statistically signifi-
cant effects were noted for neonatal mortality (pnd 1-4) or mortality of pups
during the remainder of lactation (pnd 4-21) (Table 19 and Figure 10).
Average pup body weight per litter was not affected at 50 or 250 ppra
(Table 20 and Figure 11). At 500 ppm. average pup body weight per litter was
reduced to 91%. 90%. 87%. 84% and 86% of the average control weight on pnd 1.
4. 7, 14. and 21. respectively (Table 20 and Figure 11). The proportion of
male pups per litter on pnd 1, 4, 7. 14 or 21 (Table 21) was not significantly
affected by treatment.
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54
At scheduled necropsy, no treatment-related findings were observed
(Table 22). The observed abnormalities (dilatation of the renal pelvis) did
not occur with a dose-related incidence, and were considered to be spontaneous
lesions.
3.3 Reproductive Performance of Pi Breeders (second Bating) and
Embrvo/fetal Development of Fgh Litters
3.3.1 General Indices of Reproductive Performance
Following 18 weeks of exposure, randomly selected Fj breeding pairs (1
male: 1-female) within each treatment group were cohabited for a maximum of
two weeks or until sperm were detected in the daily vaginal lavage. The day
of sperm detection was designated as gestational day (gd) 0. The offspring of
timed-mated females (the F2o littirs) were collected by Cesarean section on gd
20. No differences among groups were observed for the mating index (% mated
females), fertility index (* fertile matings). or gestational index (% live
litters) (Table 23).
3.3.2. Maternal Measures During Gestation
Because the study was designed to assess continuous exposure to nickel
for 2 generations, timed-mated females remained in their assigned dose groups.
Thus, body weights of timed-mated females exhibited differences among nickel-
exposed groups on gd 0 (p<0.05). showing 108%. 101* and 98% of the control
weight at 50. 250 and 500 ppm, respectively (Table 24 and Figure 1). Gesta-
tional body weight for nickel-exposed females (timed-mated with litters) did
not differ from controls on gd 0. 6 or 13. On gd 20. the 500 ppm group was
significantly below controls (91% of control weight; p<0.05) (Table 24 and
Figure 1).
Relative food consumption (g/kg/day) showed no significanttreatment-
related effects during gestation of" the F2b litters (Table 25 and Figure 2).
-------
55
although the decreases observed during gd 13-20 of the F2a and F2b litter were
comparable when expressed as a percentage (93%) of the control Intake (Tables
12 and 25 and Figure 2).
During gd 0-6. gd 6-13 and gd 13-20. water consumption (g/kg/day) was
significantly decreased (p<0.05 or 0.01) at 50 ppn (88% of control), 250 ppm
(65-77% of control) and 500 ppm (53-72% of control) (Table 26 and Figure 3).
Within individual groups, average daily fluid intake did not vary greatly
across different periods of gestation (gd 0-6, gd 6-13, or gd 13-20) (Table
26), and average daily nickel consumption was relatively stable throughout
these periods, e.g., 4.74 - 5.79 mg/kg/day at 50 ppm, 20.89 - 22.24 at 250
ppm. and 34.64 - 41.82 at 500 ppm (Table 27 and Figure 4). Thus, gestational
exposure to nickel (mg/kg/day) was comparable for the F2a and F2b litters
(Tables 18 and 27 and Figure 4). Graphical representation of food, water, and
nickel consumption as a function of Fj maternal body weight during gestation
of the F2b litter is presented in Appendix IX.
3.3.3 Maternal Toxicity during Gestation of the Fg^ Litter
During gestation, maternal body weight was not affected except for a
significant (p<0.05) reduction to 91% of the average control weight on gd 20
at 500 ppm (Tables 24 and 28 and Figure 1). Maternal weight gain during
gestation was 103% of control gain at 50 ppm (nonsignificant). 92% at 250 ppm
(nonsignificant) and 74% at 500 ppm (p<0.01) (Table 28). Corrected maternal
gain (maternal weight gain during gestation minus gravid uterine weight)
exhibited a dose-related decreasing trend (p<0.001) across all groups with
reduction to 94% of control weight gain at 50 ppm (nonsignificant). 73% at 250
ppm (p<0.05) and 34% at 500 ppm (p<0.01) (Table 28).
-------
56
3.3.4 Evaluation of F?h Litters (Gestational Day 20)
A comparison of ?2b litters across groups (n=14-20 litters/group) on
gestational day (gd) 20 revealed no significant dose-related effects on Meas-
ures of maternal reproductive status (number of corpora lutea per dan) or
embryo/fetal toxlcity (number of implantation sites per litter, % preimplanta-
tlon loss. % resorptions per litter. * litters with resorptions. % dead
fetuses per litter. % litters with dead fetuses. % nonlive implants (resorp-
tions plus dead fetuses). % litters with nonlive implants, % adversely
affected implants (nonlive plus malformed), % litters with adversely affected
implants, number of live fetuses per litter, % male fetuses per litter, or
average fetal body weight) (Table 29). The incidence of malformed fetuses was
significantly elevated (p<0.05) in the 50 ppm dose group which contained an
average of 14.67% fetuses malformed per litter as compared to an average of
6.96% in the control group (Table 30). In contrast, the 250 and 500 ppm
groups contained averages of 2.34% and 3.75% malformed fetuses per litter
(Table 30). There was no significant effect of treatment on the incidence of
litters with externally, viscerally or skeletally malformed fetuses relative
to the control group (Table 31). The characteristic malformation at 50 ppm
was short rib. When the incidence of fetuses per litter with short rib was
analyzed separately, there was no significant difference between controls and
any Ni++-treated group (Table 32). These results suggest that the increased
incidence of short rib at 50 ppm was a spurious result, since an increased
incidence of malformations was not observed at 250 or 500 ppm. A listing by
individual fetus of malformations and variations is presented in Table 33.
-------
57
3.4 Necropsy and Histopathologic Evaluation of Tissues from CD Rats in
the FI Generation
3.4.1 Females
At scheduled sacrifice on gd 20 of the F2b litters, timed-mated females
exhibited reduced body weight at 500 ppm, as reported above (Tables 24 and 28;
Sections 3.3.2 and 3.3.3). However, no statistically significant differences
among groups were noted for the following organ weights: gravid uterine weight
(absolute), ovarian weight (absolute or relative), kidney weight (absolute).
adrenal weight (absolute), heart weight (absolute or relative), lung weight
(absolute) or pituitary weight (absolute or relative) (Table 34). Absolute
liver weight (g) exhibited a significant decreasing trend (p<0.001). within
which 50 ppm was 103% of control weight, 250 ppm was 94% of control weight
(nonsignificant) and 500 ppm was significantly below controls (80% of control
weight: p<0.01). Relative liver weight (% body weight) also exhibited a
decreasing trend across all groups (p<0.001). with reductions of 98%
(nonsignificant). 96% (nonsignificant) and 88% (p<0.01) of control weights at
50. 250 and 500 ppm. respectively (Table 34). Relative kidney weight (% body
weight) exhibited a significant increasing trend across all groups (p<0.001).
showing 101% (nonsignificant). 105% (nonsignificant) and 113% (p<0.01) of
control values for the low. mid- and high-dose groups, respectively (Table 34).
For relative adrenal weight (% body weight), the only evidence of a dose effect
was a marginal increase at 500 ppm (117% of control). Relative lung weight (%
body weight) exhibited a significant increasing trend (p<0.01). within which 50
ppm was a no-observed-effect level (91% of control) and increases at 250 and
500 ppm to 103% or 115% of control weight were not statistically different from
controls (Table 34). Thus, the only definitive effects upon organ weights
occurred at 500 ppm (decreased absolute and relative liver weight and increased
-------
58
relative kidney weight), with suggestive evidence of increased relative adrenal
and relative lung weights also at 500 ppm.
No treatment-related pathology was noted upon gross examination at
necropsy (Table 35). Treament-related microscopic findings were limited to an
apparent increase in hlstiocytic cellular infiltration of the lungs at the high
dose (7%, 7%, 7% and 44% of females examined from the control through high-dose
groups, respectively) (Appendix VII).
3.4.2 Males
As scheduled, Fj breeder males were sacrificed following the second co-
habitation period. Body weight for males was 106% and 98% of control weight.
for the 50 and 250 ppm groups respectively (both nonsignificant). The 500 ppm
group was significantly reduced to 86% of the average control weight (p<0.01)
(Table 36). No differences among groups were observed for adrenal weights
(absolute or relative) (Table 36). For all other weighed organs, statistically
significant tests were obtained for either the absolute (g) or relative (% body
weight) weights as described below (Table 36). For prostate and heart weights
(g). group averages generally followed the pattern for body weight, and no
differences among groups were observed for relative organ weights. For
testicle weight (g). group averages generally followed the pattern of body
weight; relative weight at the high dose (103% of control) contributed to an
increasing trend (p<0.05). but this group did not differ from the control group
by pair-wise comparison. Liver weight (g) generally followed the pattern for
body weight; relative weights at the low- and mid-dose levels were 106% of the
controls (p<0.05). but the absence of a clear dose-related pattern suggests
that this may have been a spurious result. Kidney weight (g) was significantly
higher at 50 ppm (112% of control; p<0.01). but did not differ from controls at
-------
59
250 or 500 ppn; relative kidney weight was significantly higher than controls
in aJJ groups (p<0.05 or 0.01), but a clear association with dose was not
observed (106%. Ill* and 108* of control values in the low through aid-dose
groups, respectively). Lung weight (g) did not differ among groups, but
relative lung weight was increased (114* of control; p<0.05) at 500 ppm.
Pituitary weight (g) did not differ among groups; relative pituitary weight
showed an increasing trend (p<0.001) for which 50 ppm was a no-observed-effect
level (95* of control), 250 ppm was not significantly increased (109* of
control) and 500 ppm was above the control group (123* of control; p<0.01).
Thus, differences in organ weights among groups failed to show a clear
association with nickel exposure, with the exception of increased relative lung
and pituitary weights at 500 ppm (Table 36). By comparison. P0-generation
males showed increased pituitary weight (both absolute and relative) at 250 and
500 ppm. but other relative organ weights were not affected (Final Report. II
of III).
No treatment-related pathology was noted upon gross examination at
necropsy (Table 37). Treatment-related microscopic findings were limited to an
apparent increase in histiocytic cellular infiltration of the lungs at the high
dose (0*. 3*. 3* and 18* of males examined from the control through high-dose
groups, respectively) (Appendix VII).
3.5 Chemical Analysis of Dosage Formulations.
Aliquots of nickel chloride solutions in water were analyzed by ICP
spectrometry as described in the study protocol (Appendix I). Samples were
analyzed from (a) batches XVI-XXVI of the concentrated stock solution (Table
38). (b) from each carboy in each batch dilution (XVI-XXVI) of dosed-drinking
water before administration to animals on study (Table 39). and (c) from
representative carboys (batches XVI-XXVI) after the period of use (Table 40).
-------
60
Measured concentrations for dosage formulations were within the acceptable
range of 90-105* of the theoretical concentration with only Minor exceptions
(Table 38. 39, and 40).
-------
61
4.0 References
Banerjee. B. N. and R. S. Durloo. Incidence of Teratological Anomalies in
Control Charles River CD Strain Rats. Toxicology 1. 151-154. 1973.
Charles River, Embryo and Fetal Developmental Toxicity (Teratology) Control
Data in the Charles River Crl:CD« BR Rat. Charles River Laboratories.
Inc.. Wilmington. MA. 1988.
Dunn. 0. J.. Multiple Comparisons Using Rank Sums. Technometrics 6, 241-252.
1964.
Dunnett. C. W.. A Multiple Comparison Procedure for Comparing Several Treat-
ments with a Control. J. Am. Stat. Assoc. 50. 1096-1121. 1955.
Dunnett. C. W.. New Table for Multiple Comparisons with a Control. Biometrics
20. 482-491, 1964.
Hays. W. L.. Statistics. Holt. Rinehart and Winston. Inc.. New York. NY. 1963.
Jonckheere, A. R.. A Distribution-Free k-Sample Test Against Ordered Alterna-
tives. Biometrika 41.. 133-145. 1954.
Perraud, J., Levels of Spontaneous Malformations in the CD Rat and the
CD-I Mouse, Lab. An. Sci. 26. 293-300. 1976.
Price. C. J.. J. D. George. M. C. Marr, G. L. Kimmel. B. R. Sonawane, and R.
Rubenstein. Dose-Range Finding Study of Nickel Chloride Administered to
CD Rats in the Drinking Water - Final Study Report. EPA Contract No. 68-
01-7075. Work Assignment No. 28. November 15. 1985.
Price. C. J.. J. D. George. M. C. Marr. P. E. Sanderson. G. L. Kimmel. B. R.
Sonawane. and R. Rubenstein. Ninety-Day Exposure of CD Rats to Nickel
Chloride Administered in the Drinking Water - Final Study Report (I of
III) for the Two-Generation Reproduction and Fertility Study of Nickel
Chloride Administered to CD Rats in the Drinking Water. EPA Contract No.
68-01-7075. Work Assignment 28. September 24. 1986.
-------
62
Price. C. J.. J. D. George. M. C. Marr. P. E. Sanderson. R. Rubenstein, G. L.
Kitnmel. B. R. Sonawane. and A. Bathija. Fertility and Reproductive Per-
formance of the P0 - Final Study Report (II of III) for the Two-Genera-
tion Reproduction and Fertility Study of Nickel Chloride Administered to
CD Rats in the Drinking Water. EPA Contract No. 68-01-7075. Work Assign-
ment 28, September 23. 1988.
Salewski, E. . Farbemethode zum Nakroskopischen Nachweis von Implantations-
Stellen am Uterus Der Ratte. Naunyn-Schmiedebergs. Arch. Exp. Pathol.
Pharmakol. 247. 367. 1964.
SAS Institute Inc.. SAS User's Guide: Basics. 1982 edition. SAS Institute
Inc.. Box 8000. Gary. NC. 1982a.
SAS Institute Inc.. SAS User's Guide: Statistics. 1982 edition. SAS Institute
Inc.. Box 8000. Gary. NC. 1982b.
Shirley. E.. A Non-Parametric Equivalent of William's Test for Contrasting
Increasing Dose Levels of a Treatment. Biometrics 33. 386-389. 1977.
Siegel. S. . Nonparametric Statistics for the Behavioral Sciences. McGraw-Hill
Book Co.. NY. 1956.
Snedecor. G. W. and W. G. Cochran, Statistical Methods. 6th edition. Iowa
State University Press. Ames. Iowa. 1967.
Williams. D. A.. A Test for Differences Between Treatment Means When Several
Dose Levels are Compared with a Zero Dose Control. Biometrics 27. 103-
117, 1971.
Williams, D. A., The Comparison of Several Dose Levels with a Zero Dose Con-
trol. Biometrics 28. 519-531, 1972
Winer. B. J.. Statistical Principles in Experimental Design. McGraw-Hill Book
Co.. Inc.. New York. NY. 1962.
-------
63
Woo. D. C. and R. M. Hoar, Reproductive Performance and Spontaneous
Malformations in Control Charles River CD Rats. A Joint Study by
MARTA. Teratology 19. 54A. 1979.
-------
64
Table 1A.
Summary of Temperature and Relative Humidity in Room 148 During
the Two-Generation Reproduction and Fertility Study of Nickel
Chloride Administered to CD Rats in the Drinking Water.8
Recording Period
June 18-30. 1985?
July. 1985
August, 1985
September. 1985
October. 1985
November. 1985
December 1-5. 1985k
December 11-31. 1985k
January. 1986
February. 1986
March. 1986
April. 1986
May. 1986
June. 1986
July 1-7. 198CU
Temperature
(°C)b Range0
20,0
20.1
20.6
20.5
20.6
21.2
24.2
21.7
20.6
20.4
20.4
19.4
19.5
19.8
19.8
+
*
+
*
*
*
*
i
+
+
+
+•
*
t
+
0.8
0.8
0.5
0.6
0.4
0.6
1.4
0.3
0.6
0.2
0.7
0.8
0.8
0.4
0.7
17
15
16
16
19
20
22
21
19
18
17
15
15
16
' 17
.0-22
.5-22
.0-21
.0-21
.0-22
.0-24
.5-26
.0-22
.5-22
.5-21
.5-21
.5-20
.0-21
.5-21
.0-21
.0
.5
.5
.5
.0
.0
.0
.0
.5
.0
.5
.5
.5
.0
.0
No.
Readingsd
75/75
185/185
185/185
179/179
181/1856
128/1801
0/30*
80/126"
185/185
168/168
184/184
179/180<1
182/1858
180/180
39/39
Humidity
(* RH)b-e
59
65
64
60
55
49
38
35
39
51
48
53
58
63
68
± 4
1 4
± 2
1 6
i 7
1 8
* 18
* 9
t 5
i 5
: 6
* 4
i 4
i 4
i 6
Range0
50-68
52-74
56-76
42-71
24-69.
29-70
22-55
23-53
23-49
41-58
27-60
44-70
42-72
56-74
57-76
No.
Readingsd
75/75
185/185
185/185
179/179
173/185h
152/180-"
0/30m
80/126°
177/185P
168/168
184/18'
179/18v
182/185t
180/180
39/39
a See Appendix X in Part III of this report for more detailed information.
b Mean ± SD for daily average temperature and relative humidity. Unless otherwise
specified, data were obtained from a Datapod Model DP220 Electronic Hygrothermograph
(Omnidata International. Inc., Logan. UT). which recorded average conditions at 4-hour
intervals. "Short dump" readings from the datapod were manually recorded once a day
and reflect the conditions in effect at. the time the recording was made (these values
were used on a limited basis as the best available estimate of average daily conditions
as specified in the footnotes below).
c Range of datapod readings for temperature or relative humidity for the entire recording
period.
Number of usable datapod readings/maximum number of readings for the recording period
(i.e.. one reading every 4 hours).
e Relative humidity values have been corrected according to the calibration curve reported
in Appendix X in Part III of this report. According to the calibration curve presen'
in Appendix X. a reading of 0% RH corresponds to an actua^ value of 22* RH or below.
These readings were excluded from calculations of the mean and range since they could
not be specified (the incidence of these findings is noted below).
-------
65
Table 1A. (concluded)
f Dosing was initiated on June 18. 1985.
% Four datapod readings were discarded as unrealistic, probably due to a naif unction of
the datapod storage unit.
n A reading of 0* RH was obtained in 12/185 cases.
A Fifty-two of the datapod readings were discarded as unrealistic, probably due to a
malfunction of the datapod storage unit. Thus, "short dump" readings were used for
days on which the datapod readings were discarded. The range of temperatures according
to an Ertco maximum/minimum thermometer in the room was 21.1 - 24.4°C. See also Table
1. Appendix X. Part III of this report.
-' Twenty-four of the datapod readings were discarded as unrealistic, probably due to
malfunction of the datapod storage unit. Thus, "short dump" readings were used for four
days. A reading of 0% RH was obtained in 4/156 of the remaining readings.
k Females were temporarily relocated to Room 150 on December 5. 1985 and were returned to
Room 148 on December 11. 1985.
£ Thirty of the datapod readings were discarded as unrealistic, probably due to a mal-
function of the datapod storage unit. Thus, "short dump" readings were used for this
recording period. The range of temperatures according to an Ertco maximum/minimum
hermometer in the room was 22.2 - 28.3°C for the recording period. See also Table 1.
X. Part III of this report.
m Thirty of the dataood readings were discarded as unrealistic, probably due to malfunc-
tion of the datapod storage unit. Thus, "short dump" readings were used for five days.
n Due to an soparent malfunction of the datapod storage unit, no readings were stored for
December 11-17. 1985. and four readings were not stored on December 18. 1985: "short
dump" readings were used for December 11-17, 1985. The range of temperatures according
to an Ertco maximum/minimum thermometer in the room was 17.2 - 23.3°C for this
recording period. See also Table 1. Appendix X. Part III of this report.
0 Due to an apparent malfunction of the datapod storage unit, readings were not stored for
December 11-17. 1985. and four readings were not stored on December 18. 1985. Thus.
"short dump" readings were used as the best available estimate of average
humidity on December 11-17. 1985.
P A reading of 0% RH was obtained in 8/185 readings.
c- One datapod reading was discarded as unrealistic, probably due to a malfunction of the
datapod storage unit.
r One datapod reading was discarded as unrealistic, probably due to a malfunction of the
datapod storage unit.
s Three datapod readings were discarded as unrealistic, probably due to a malfunction of
the datapod storage unit.
1 Three datapod readings were discarded as unrealistic, probably due to a malfunction of
le datapod storage unit.
* inal sacrifice for F^ female breeders was completed on July 7. 1986.
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66
Table IB. Summary of Temperature and Relative Humidity in Rooms 150 and 146 During
the Two-Generation Reproduction and Fertility Study of Nickel Chloride
Administered to CD Rats in the Drinking Water.3
Recording Period
June 18-30. 1985?
July. 1985
August. 1985
September , 1985
October. 1985
November. 1985
December 1-10. 1985?
January 1-21. 1986 J
January 22-31, 1986™
February, 1986
March 1-5. 1986
March 6-31. 1986m
April. 1986
May. 1986
June 1-11. 1986n
Temperature
(°C)b
21.5
21.5
22.1
22.1
22.6
22.9
23.6
20.1
21.8
22.0
22.1
20.6
20.4
20.4
21.0
± 1.4
t 1.3
± 0.8
± 1.0
± 0.4
t 0.2
± 0.5
i 0.3
± 0.3
± 0.4
± 0.8
r 0.4
l 0.8
* 0.9
± 0.6
Range0
16.0-23.0
16.0-23.0
15.5-23.0
16.0-23.5
19.5-23.5
21.0-23.5
23.0-27.0
19.5-21.5
21.0-22.0
20.0-22.5
20.5-23.0
17.5-21 .5
16.0-21.5
15.5-21.5
17.0-22.0
No.
Readings'1
74/74
186/186
185/185
179/179
186/186
180/180
28/60h
67/126k
60/60
168/168
30/30
155/155
180/180
186/186
66/66
Humidity
(* RH)b-e
54
59
57
54
50
48
31
46
44
48
43
48
52
57
61
* 5
* 7
± 2
t 5
± 6
± 5
± 9
* 7
± 5
± 4
t 2
: 7
* 4
± 5
± 3
Range0
46-70
48-75
50-76
39-66
33-68
31-57
23-52
33-60
33-52
38-55
40-46
25-60
42-70
40-72
56-69
No.
Readings'1
74/74
186/186
185/185
179/179
186/186
180/180
25/60 *
67/126*
60/60
168/168
30/30
155/15,,
180/180
186/186
66/66
a See Appendix X in Hart III of this report for more detailed information. Data apply
to Room 150, unless otherwise noted.
b Mean j_ SO for daily average temperature (°C) and relative humidity (* RH). Unless
otherwise specified, data were obtained from a Datapod Model DP220 Electronic
Hygrothermograph (Omnidata International, Inc.. Logan. UT). which recorded average
conditions at 4 hour intervals. "Short dump" readings from the datapod were manually
recorded once a day and reflect the conditions in effect at the time the recording was
made ("Short Dump" values were used or a limited basis as the best available estimate
of average daily conditions as specified in the footnotes below).
c Range of datapod readings for temperature or relative humidity for the entire recording
period.
° Number of usable datapod readings/maximum number of readings for the recording period
(i.e.. one reading every 4 hours).
e Relative humidity values have been corrected according to the calibration curve
reported in Appendix X in Part III of this report. According to the calibration cur
presented in Appendix X. a reading of 0% RH corresponds to an actual value of 22% RH or
below. These readings were excluded from calculations of the mean and range since they
could not be specifled (the incidence of these findings is noted below).
-------
67
Table IB (concluded)
f Dosing was initiated on June 18. 1985.
£ Final sacrifice for PQ males was held on December 5. 1985. PQ females were relocated
from Room 148 to Room 150 from December 5-10. 1985 and returned to Room 148 on
December 11. 1985.
n Due to an apparent malfunction of the datapod storage unit, 32 readings were not stored:
"short dump" readings were used for 5 days. The range of temperatures according to an
Ertco maximum/minimum thermometer in the room was 21.7 - 25.6°C for this recording
period. See also Table 2. Appendix X. Part III of this report.
* Due to an apparent malfunction of the datapod storage unit. 32 readings were not
stored: "short dump" readings were used for five days. A reading of 0% RH was obtained
in three cases.
J Due to space limitations in Room 148. some FJD litters were moved from Room 148 to Room
150 on the day of weaning, beginning January 1, 1985.
k A datapod unit was not activated until January 9. 1986; thus, 48 readings were missing.
The range of temperatures according to the Ertco maximum/minimum thermometer in the
room was 20.0 to 24.4°C from January 1-9. 1986. Due to an apparent malfunction of the
datapod storage unit during January 9-21, 1986. 11 readings were not stored; "short
dump" readings were used for one day.
aatapod unit was not activated until January 9. 1986. Thus. 48 readings were
.issing. Due to an apparent malfunction of the datapod-storage unit during January
9-2.'.. 11 readings were not stored; "short dump" readings were used for one day.
m Fjo weanlings which had been housed in Room 150 were transferred to Room 146 or Room
148 on January 22. 1986. Fjo breeders were returned to Room 150 on March 6. 1987. five
days prior to the cohabitation period for the F2a litter.
n Final sacrifice for- Fj male breeders was completed on June 11. 1986.
-------
Table 2. F Generation: Summary of Clinical Observations In
F Female CD Rats on Postnatal Days 22 through 42.
Group Postnatal
(Ni^. ppm) Day
0 25
27
28
29
30
31
50 23
24
25
26
250 22
23
26
28
500 24
25
26
Animal
Number
646
482
480
732
732
732
732
826
828
650
474
478
806
604
668
622
472
572
776
780
692
766
768
778
774
Clinical Observation
Death 1-8-86; no prior clinical signs noted.
Death 1-8-86; no prior clinical signs noted.
Dehydration, lethargy, moribund, sacrificed on 1-8-86.
Dehydration, lethargy, emaciation 1-12-86.
Dehydration, lethargy, emaciation 1-13-86.
Dehydration, lethargy, emaciation 1-14-86.
Death 1-15-86.
Death 1-10-86; no prior clinical signs noted.
Death 1-11-86; no prior clinical signs noted.
Death 1-6-86; no prior clinical signs noted.
Death 1-6-86; no prior clinical signs noted.
Death 1-6-86; no prior clinical signs noted.
Moribund, sacrificed on 1-7-86.
Death 1-5-86; no prior clinical signs noted.
Death 1-9-86; no prior clinical signs noted.
Death 1-10-86; no prior clinical signs noted.
Death 1-4-86; no prior clinical signs noted.
Death 1-6-86; no prior clinical signs noted.
Moribund, sacrificed on 1-8-86.
Moribund, sacrificed on 1-8-86.
Death -8-86; no prior clinical signs noted.
Death -9-86; no prior clinical signs noted.
Death -9-86; no prior clinical signs noted.
Death -9-86; no prior clinical signs noted.
Death -10-86; no prior clinical signs noted.
O)
CO
-------
Table 3. Summary of Clinical Signs and Necropsy Findings for F . Females
Which Died or Were Sacrificed on Postnatal Days 22 through 42.
Group
*+, ppm)
Animal
Number
Reason for
Necropsy
Summary
0
482
Death
480
Moribund
646
Death
732
Death
Cljnlcnl: Found dead on 1-8-86 (pnd 27); no prior
clinical signs noted.
Necropsy: No food In stomach, a little food In
Intestine, and full bladder. No gross abnormalities
noted.
Cllnjhcal: Dehydration, lethargy 1-8-86. Sacrificed on
1-8-86 (pnd 27).
Necropsy: Food In stomach and Intestine and bladder
full. No gross abnormalities noted.
cnn.lca.1: Found dead on 1-8-86 (pnd 25); no prior
clinical signs noted.
Necrpjjsy: No food stomach, no gross abnormalities.
C_l_lnica_l: Dehydration, lethargy, moribund, emaciation
on 1-12-86, 1-13-86 and 1-14-86. Found dead on 1-15-86
(pnd 31).
Necropsy: Little food In stomach and empty bladder.
No gross abnormalities.
(continued)
o>
-------
TabJo 3. (continued)
Group
++. ppm)
Animal
Number
Reason for
Necropsy
Summary
50
478
474
650
826
828
250
604
Death
Death
Death
Death
Death
Death
Cljjiical: Found dead on 1-6-86 (pnd 26); no prior
clinical signs noted.
Necnnpjsy: Very little food in stomach, feces in large
intestine very hard. No gross abnormal 1 ties.
Clinical: Found dead on 1-6-86 (pnd 26); no prior
clinical signs noted.
Necropjy: No food in stomach; no gross abnormalities.
.ClJulPAl: Found dead on 1-8-86 (pnd 25); no prior
clinical signs noted.
Nec_rp£sy: No food in stomach; no gross abnormalities.
Clinical : Found dead on 1-10-86 (pnd 23); no prior
clinical signs noted.
Necropsy.: Little food in stomach, full bladder. No
gross abnormalities.
Clinical; Found dead on 1-11-86 (pnd 24); no prior
clinical signs noted.
Necropsy: No food in stomach or small intestine.
Bilateral pelvic dilatation of the kidneys.
C_y_n_j_cal: Found dead on 1-5-86 (pnd 23); no prior
clinical signs noted.
Necropsy: No food in stomach; no gross abnormalities.
(cont«-
-------
Table 3. (continued)
Group
*\ ppm)
Animal
Number
Reason for
Necropsy
Summary
500
806
668
622
472
572
Moribund
Death
Death
Death
Death
Clinical: Moribund, sacrificed on 1-7-86 (pnd 22): no
prior clinical signs noted.
Necropsy: No food In stomach; no gross abnormalities
noted.
Cljnical: Found dead on 1-9-86 (pnd 26); no prior
clinical signs noted.
Necrojjsy: Food In stomach; no gross abnormalities.
Clinical: Found dead on 1-10-86 (pnd 28); no prior
clinical signs noted.
Necropsy: Little food in stomach; no gross
abnormalities.
Clinical; Found dead on 1-4-86 (pnd 24); no prior
clinical signs noted.
Necropsy: No food in stomach; no gross abnormalities.
Clinical: Found dead on 1-6-86 (pnd 24); no prior
clinical signs noted.
Necropsy: No food In stomach, hard dry boll in large
Intestine. No gross abnormalities.
(continued)
-------
Table 3. (continued)
Group
f+, ppm)
Animal
Number
Reason for
Necropsy
Summary
776
780
692
766
768
Moribund
Moribund
Death
Death
Death
Clinical: Moribund 1-8-86; sacrifice on 1-8-86 (pnd
24).
Necropsy: Empty stomach, food in intestines, full
bladder. Pelvic dilatation of the right kidney.
Clinical: Moribund 1-8-86; sacrificed on 1-8-86 (pnd
24)7"
Necrojjsjf: No food in stomach, full intestine and
bladder. No gross abnormalities noted.
Clinjcal: Found dead on 1-8-86 (pnd 25); no prior
clinical signs noted.
Necropsy: No food in stomach, full bladder, and some
food In intestine. No gross abnormalities noted.
Clinical: Found dead on 1-9-86 (pnd 25); no prior
clinical signs noted.
Necropsy: Little food in stomach, no gross
abnormalities.
Found dead on 1-9-86 (pnd 25); no prior
clinical signs noted.
Necropsy: No food in stomach; no gross abnormalities.
(continued)
to
-------
Table 3. (continued)
Group Animal Reason for
(Nl++. ppm) Number Necropsy Summary
778 Death Clinical: Found dead on 1-9-86 (pnd 25); no prior
clinical signs noted.
Necropsy: Some food In stomach; no gross
abnormalItles.
774 Death Clinical: Found dead on 1-10-86 (pnd 26); no prior
clinical signs noted.
Necropsy: Little food in stomach; no gross
abnormalItles.
CO
-------
Table 4. F Generation: Summary of Clinical Observations In
F Male CD Rats on Postnatal Days 22 through 42.
Group Postnatal
(Nl , ppm) Day
0 25
35
50 23
26
35
36
250 22
24
26
27
28
34
500 24
25
26
27
35
36
37
38
39
42
Animal
Number
627
717
719
835
605
607
757
757
819
817
735
465
463
633
635
667
475
477
787
697
695
699
789
571
857
857
857
857
857
703
Clinical Observation
Death 1-8-86; no prior clinical signs noted.
Dehydration
Death 1-19-86; no prior clinical signs noted.
Death 1-10-86; no prior clinical signs noted.
Death 1-8-86; no prior clinical signs noted.
Death 1-8-86; no prior clinical signs noted.
Paralysis In lefthind limb
Sacrificed on 1-20-86.
Death 1-7-86; no prior clinical signs noted.
Moribund, sacrificed on 1-7-86.
Death 1-8-86; no prior clinical signs noted.
Death 1-6-86: no prior clinical signs noted.
Death 1-6-86; no prior clinical signs noted.
Death 1-10-86; no prior clinical signs noted.
Doath 1-11-86; no prior clinical signs noted.
Death 1-17-86; no prior clinical signs noted.
Death 1-4-86; no prior clinical signs noted.
Death 1-4-86; no prior clinical signs noted.
Moribund, sacrificed on 1-8-86.
Death -8-86; no prior clinical signs noted.
Death -8-86; no prior clinical signs noted.
Death -8-86; no prior clinical signs noted.
Death -10-86; no prior clinical signs noted.
Death -9-86; no prior clinical signs noted.
Rough coat 1-30-86.
Rough coat 1-31-86.
Rough coat 2-1-86.
Rough coat 2-2-86.
Rniifrh coat 2-3-86.
Death 1-25-86; no prior clinical signs noted.
-a
-------
Table 5. Summary of Clinical Signs and Necropsy Findings for F Males
Which Died or Were Sacrificed on Postnatal Days 22 through 42.
Group
(Nl . ppm)
Animal
Number
Reason for
Necropsy
Summary
627
719
50
605
Death
Death
Death
.c.LAnJc_al: Found dead on 1-8-86 (pnd 25); no prior
clinical signs noted.
Necropsy: Stomach and bladder empty, food In
intestine. No gross abnormalities noted.
Clinical: Found dead on 1-19-86 (pnd 35); no prior
clinical signs noted.
Necropsy: Animal very necrotic. food in stomach, and
dehydration (water bottle slpper tube blocked). No
gross abnormalities noted.
Clinical: Found dead on 1-8-86 (pnd 26); no prior
clinical signs noted.
Necropsy: Little food in stomach; no gross
abnormalities.
607
835
Death
Death
Clinical: Found dead on 1-8-86 (pnd 26); no prior
clinical signs noted.
Necropsy: Food present in stomach and Intestines,
bladder empty. No gross abnormalities noted.
Clinical: Found dead on 1-10-86 (pnd 23); no prior
clinical signs noted.
Little food In stomach and full bladder. No
gross abnormalities.
(continued)
cn
-------
Table 5. (continued)
Group
f*. ppm)
Animal
Number
Reason for
Necropsy
Summary
250
757
465
463
Sacrificed
Death
Death
Clln.l.ca.1.: Paralyzed In left hindlimb 1-19-86 (pnd 35),
Sacrificed on 1-20-86 (pnd 36).
Necropsy; Pood present In stomach and Intestine, left
hindlimb not broken, no gross abnormalities.
CH.nlcal: Found dead on 1-6-86 (pnd 26); no prior
clinical signs noted.
Necropsy: Little food In stomach, no gross
abnormalities.
Clinical; Found dead on 1-16-86 (pnd 26); no prior
819
817
clinical signs noted.
Necropsy: Little food
abnormalities.
Death Clinical : Found dead
clinical signs noted.
Necropsy; No food in
noted.
Moribund Clinical: Moribund on
(pnd 22).
Necropsy: No food in
noted.
In stomach, no gross
on 1-7-86 (pnd 22): no prior
stomach, no gross abnormalities
1-7-86; sacrificed on 1-7-86
stomach, no gross abnormalities
(continued)
en
-------
Table 5. (continued)
Group
*+. ppm)
Animal
Number
Reason for
Necropsy
Summary
735
633
635
667
500
475
Death
Death
Death
Death
Death
Clinical: Found dead on 1-8-86 (pnd 24); no prior
clinical signs noted.
Nejsrpjjsy: Gastrointestinal tract and organs necrotlc.
bladder empty, no gross abnormalities noted.
Clinical: Found dead on 1-10-86 (pnd 27); no prior
clinical signs noted.
Necropsy; Little food In stomach, no gross
abnormal Itles.
Clinical; Found dead on 1-11-86 (pnd 28); no prior
clinical signs noted.
Necropsy: No food in stomach, empty bladder, gaseous
gastrointestinal tract. No gross abnormalities noted.
Clinical: Found dead on 1-17-86 (pnd 34); no prior
clinical signs noted.
Necropsy: Little food In stomach, bladder not
completely empty, no gross abnormalities.
Clinical : Found dead1 on 1-4-86 (pnd 24); no prior
clinical signs noted.
Necropsy: Found dead on 1-4-86 (pnd 24); no prior
clinical signs noted.
(continued)
-------
Table 5. (continued)
Group
+*. ppm)
Animal
Number
Reason for
Necropsy
Summary
477
787
697
695
699
Death
Moribund
Death
Death
Death
Cl.inlcaJ: Found dead on 1-4-86 (pnd) 24; no prior
clinical signs noted.
Necropsy: No food In stomach; no gross abnormalities
noted.
Clinical: Moribund on 1-8-86; sacrificed on 1-8-86
(pnd 24J.
Necropsy: No food in stomach, gas filled intestines.
full blndder. No gross abnormalities noted.
Clinical; Found dead on 1-8-86 (pnd 25); no prior
clinical signs noted.
Necropsy; No food in stomach, little food in
Intestine, bladder empty, gas in intestine. No gross
abnormalities noted.
Clinical; Found dead'on 1-8-86 (pnd 25); no prior
clinical signs noted.
Necropsy: No food in stomach, food in intestine,
bladder empty. No gross abnormalities noted.
Clinical: Found dead on 1-8-86 (pnd 25); no prior
clinical signs noted.
Necropsy: No food in stomach or Intestine, empty
bladder, slightly necrotic gastrointestinal tract and
organs. No gross abnormalities noted.
(continued)
-3
oo
-------
Table 5. (continued)
(Ni . ppra)
Animal
Number
Rnason Tor
Necropsy
Summary
571
789
703
Death
Death
Death
Clinical: Found dead on 1-9-86 (pnd 27); no prior
clinical signs noted.
Necropsy: No food In stomach, no gross abnormalities.
cn.n_lcal: Found dead on 1-10-86 (pnd 26); no prior
clinical signs noted.
Necropsy: Little food In stomach, no gross
abnormalities.
Cjin_ical: Found dead on 1-25-86 (pnd 42); no prior
clinical signs.
Necropsy; Food In stomach, full bladder, no gross
abnormalities.
(0
-------
Table 6.
F- Generation:
Summary of Clinical In Signs in
F~b Female CD Rats after Postnatal Day 42.
80
Group Study
(Ni**. ppm) Week
0 36
37
39
40
41
42
43
50 41
49
50
51
52
250 42
500 34
35
Animal
No.
728
728
488
488
488
488
486
830
818
818
818
818
468
802
810
576
376
576
Clinical Observations
chromodacryorrhea
chromodacryorrhea
Wounded left shoulder 3-14-86.
sore on left shoulder 3-16-86
sore on left shoulder
sore on left shoulder
sore on left shoulder 4-4-86
healing 4-6-86. healed 4-8-86
diarrhea
weight loss (-12.30 g. gd 0-6)
chromodacryorrhea
chromodacryorrhea
chromodacryorrhea
chromodacryorrhea
delivered two dead pups and died
during delivery on 4-6-86.
delivered three pups and died
during delivery on 4-7-86.
delivered two dead pups and died
during delivery on 4-9-86.
rough coat
piloerection
piloerection. rough coat.
36
37
38
39
42
43
45
48
51
576
576
576
578
580
568
688
764
772
690
690
576
growth under chin, hunched
piloerection. hunched.
growth under chin, rough coat
rough coat, sore under chin, maloc-
clussion (upper incisors clipped).
alopecia under chin
rough coat, alopecia under chin.
lost 1 lower incisor
right upper incisor chipped and both
lower incisors chipped
delivered one dead pup and died
during delivery on 4-4-86.
all pups dead postnatal day 1.
emaciated, rough coat, bloody
vaginal discharge
rough coat
rough coat
rough coat, emaciated, weight loss
(-51.07. pnd 1-7)
diarrhea
diarrhea
teeth clipped
-------
Table 7. Summary of Clinical Signs and Necropsy Findings for F .
Females Which Died or Were Sacrificed after Postnatal Bay 42.
Group
(Nl . ppm)
Animal
Number
Reason for
Nrcropsy
Summary
250
468
Death
Clinical: Delivered 2 dead pups and died during
delivery on gestatlonal day 22 (4-6-86; week 42). No
prior clinical signs noted.
Necropsy: Ten fetuses In utero. No gross
abnormal it ins noted.
802
Death
CJlnJcajh Delivered 3 dead pups and died during
delivery on gestatlonal day 23 (4-7-86; week 42). No
prior clinical signs noted.
Necropsy: Hemorrhage at cervix, ten fetuses In utero
(7 left, 3 right).
810
Death
Clinical: Delivered 2 dead pups and died during
delivery on gestatlonal day 24 (4-9-86; week 42). No
prior clinical signs noted.
Necropsy: Ten fetuses In utero. 1 breach In vaginal
canal, mesentary around intestine yellowed, contents
were dark green liquid.
500
580
Death
Clinical; One dead pup delivered, on partially
delivered, died during delivery on gestatlonal day 23
(4-4-86; week 42). No prior clinical signs noted.
Necropsy: Thirteen fetuses In utero. No gross
abnormalities noted.
00
-------
82
Table 8. F Generation: Summary of Clinical Signs
in Flb Male CD Rats after Postnatal Day 42.
+Group Study
(Ni . ppm) Week
0 33
50 34
47
48
250 33
46
47
48
51
500 33
34
35
36
37
38
Animal
No.
457
491
631
749
461
721
651
721
651
581
551
551
467
551
561
469
777
777
777
575
777
575
855
853
855
777
575
853
777
855
853
785
Clinical Observations
diarrhea
diarrhea
diarrhea
diarrhea
diarrhea
diarrhea
diarrhea
diarrhea
diarrhea
diarrhea
diarrhea
diarrhea
diarrhea
diarrhea
excessive salivation
rough coat
rough coat, chromodacryorrhea
rough coat
piloerection. rough coat.
dehydrated, weight loss (-20.73 g)
piloerection. dehydrated.
rough coat
piloerection. emaciated.
rough coat, dehydrated
rough coat
rough coat, weight loss (-8.71 g)
rough coat
rough coat, raalocclusion (upper
incisors clipped)
rough coat, sores on forepaws.
swollen penis, dehydrated, ma 1 oc-
clusion (upper incisors clipped)
rough coat
rough coat, swollen penis.
malocclusion (upper incisor
clipped)
rough coat, swollen penis
rough coat
rough coat (one lower incisor broken
off at gum line) swollen penis
malocclusion (right upper
incisor broken off at gum line)
(continued)
-------
Table 8. (continued)
83
Group
(Ni**. ppm)
Study
Week
Animal
No.
Clinical Observations
39
40
45
47
50
51
785
855
785
773
773
777
853
naloccluslon (both upper Incisors
broken off at gum line)
rough coat
rough coat
diarrhea
diarrhea
diarrhea
rough coat
-------
84
Table 9. Summary of Necropsy Findings of FID Pups on Postnatal
Day 42 for Animals not Selected as Fj Breeders.3
Nickel Chloride (pom)
50 250 500
Number of Males Necropsied 26 29 21 0°
•\
Number (*) of males with
no gross abnormalities 26(100) 28(97) 20(95)
Kidney:
Pelvic dilatation: right Oil
Number of Females Necropsied 22 23 24 Ob
Number (*) of females with
no gross abnormalities 21(95) 21(91) 24(100)
Kidney:
Pelvic dilatation: right 020
bilateral 1 0 0
aTherr were no records of necropsy maintained for 7 male and 4 female
pups (4. 2 and 1 males and 2, 1 and 1 females in the control through
mid-dose groups, respectively): however, records do indicate that these
pups were culled on postnatal day 42.
1 pups from the 500 ppm group were saved for breeding.
-------
Table 10. Summary of Reproductive Status for CD Rats in the F Generation
Following Continuous Exposure to Nickel Chloride in the Drinking
Water: Breeding to Produce the F0 Litter.
23
85
Nickel Chloride (ppm)
50
250
500
(No. F-. Breeding Pairs to
Produce F2g Litters)
MATING INDEX
(30)
(30)
(30)
(19)
No. Mated (sperm-positive and/or
confirmed pregnant)
No. Not Mated (sperm-negative
28
28
29
19
and not pregnant )
Total Breeding Pairs
% Mated Females
FERTILITY INDEX
No. Confirmed Pregnancies
No. Females Not Pregnant
Total Mated Females
* Fertile Matings
GESTATIONAL INDEX
No. Live Litters
No. Dead Litters
Total Litters
% Live Litters
PERINATAL VIABILITY INDEX
No. Live Litters on pnd 1
No. Live Litters on pnd 4
* Viable Litters on pnd 4
LACTATIONAL INDEX
No. Live Litters pnd 1
No. Live Litters pnd 21
% Viable Litters pnd 21
2
30
NS 93
24
4
28
* 86
24
0
24
NS 100
24
23
NS 96
24
23
NS 96
2
30
93
28
0
28
100
28
0
28
100
28
28
100
28
28
100
1
30
97
28c.d.e
1
29
97
26*
2d.e
28
93
26
23
88
26
23
88
0
19
100
17f
2
19
89
•15-
2f.g
17
88
15
14
93
15
13
87
(continued)
-------
86
Table 10. (continued)
Pregnancy was confirmed by delivery of a litter, or by uterine Inspection at
necropsy. The circumstances under which pregnancy was confirmed have been
explained in the table footnotes below, if other than delivery of a litter.
Viexposed females were mated to 3 males from the 500 ppm group, bringing the
total no. breeding pairs to 22. Each unexposed female was found to be sperm
positive. Two of these produced a live litter which was viable through pnd
21. The third female failed to deliver and uterine examination revealed no
implant sites. These litters are excluded from further consideration in the
present table since only one member of the breeding pair had been exposed to
Q
One female died during delivery, but delivered 2 live pups and had 10 pups in
utero. —
d
One female died during delivery, but delivered 2 dead pups and had 10 pups in
utero. —
One female died during delivery, but delivered 3 dead pups and had 10 pups in
utero. —
One female died during delivery, but delivered 1 dead pup. had 1 pup partially
delivered, and had 13 pups In utero.
80ne live female delivered all dead pups.
NS. nonsignificant. p>0.05.
p<0.05; Chi-Square Test for Independence.
-------
Table 11. Summary of Maternal Body Weights for Fjb Dams During Gestation of the F2a Litter.
ANOVA TRENDS
or Chi -Square
SUBJECTS (N)
GESTATIONAL BODY HEIGHT (g)
gd Oa 4 + §
gd 6 + §
gd 13 * 5
gd 20 +++ §5§
GESTATIONAL LENGTH (days) NA NA
Nickel
0
21
260.1
i 6.2
285.4
1 6.6
317.5
* 7.5
397.8
1 10.0
21.57
i 0.15
Chloride (ppm)
50
28
277.9
i 6.0
304.0
t 6.4
336.5
i 6.3
415.3
i 7.9
21.54
i 0.10
250
27
259.5
i 3.9
283.3
t 4.5
316.2
i 4.9
379.0
f 6.1
21.74b
» 0.15
500
16
248.5
i 8.5
273.1
* 9.1
303.2
t 10.0
345.3***«
i 10.0
21.69C
1 0.18
(continued)
00
-j
-------
Table 11. (concluded)
Nickel Chloride (pom)
DAY OP DELIVERY
[No. (*) Da«3 per
Rd 20
Rd 21
Bd 22
?d 23
(,'d 24
gd 22
ANOVA
or Chi -Square
Group]
NA
NA
NA
NA
NA
NS
TRENDS
NA
NA
NA
NA
NA
NA
0
1 (5)
8(38)
11(52)
1 (5)
0 (0)
9/12
50
0 (0)
13(46)
15(54)
0 (0)
0 (0)
13/15
250
0 (0)
11(41)
13(48)
2 (7)
1 (4)
11/16
500
0 (0)
7(44)
7(44)
2(13)
0 (0)
7/9
a(?(l-£RStalional day.
''Three fomaJes died during delivery on gestationaj days 22, 23 and 24.
C0ne female delivered a litter of all dead pups on gestatlonal day 23.
NS. nonsignificant. p>0.05.
NA. not applicable; no statistical tests reported.
**D<0.01; Dunnett's Test.
ftD<0.01: Williams' Test.
*D<0.05; ANOVA.
Mo<0.01; ANOVA.
*f*D<0.001; ANOVA.
sa<0.05: Test for Linear Trend.
<0.001; Test for Linear Trend.
CO
00
-------
Table 12. Summary of Pood Consumption for F . Dams During Gestation of the F Litter.
1D £&
Nickel Chloride (ppm)
SUBJECTS
ANOVA
(N)
TRENDS 0
21
50
28
250
27
500
16
POOD CONSUMPTION (g/day)
gd 0 -
gd 6 -
gd 13
gd 6a NS
gd 13 NS
- gd 20 •+++
NS 22.76
1 0.72
NS 25.34
i 0.69
§§§ 26.88
t 0.73
24.76
i 0.50
26.42
1 0.54
27.75
t 0.52
23.51
i 0.57
25.87
i 0.54
26.15
i 0.47
23.41
i 1.14
25.18
» 0.99
22.69**f*
i 0.74
FOOD CONSUMPTION (g/kg/day)
gd 0 -
Fd 6 -
gd 13
gd 6 NS
gd 13 NS
- gd 20 +
S 83.37
i 1.48
§ 84.07
i 1.08
§ 75.18
i 0.97
85.42
t 1.28
82.72
t 1.23
73.98
i 0.92
86.68
* 1.73
86.32
* 1.16
75.31
i 0.96
90.15
t 3.88
87.34
i 2.41
70.23**f
i 1.57
a
gd = gestatlonal day.
NS, nonsignificant, p>0.05.
*
p<0.0f»; Dunnett's Test.
**p<0.01; Dunnett's Test.
ftp<0.01; Williams' Test.
§
§§§
p<0.05; ANOVA.
p<0.001; ANOVA.
p<0.05; Test for Linear Trend.
p<0.001; Test for Linear Trend,
00
-------
Table 13. Summary of Water Consumption for F Dams During Gestation of the F Litter.
•1 D bd
Nickel Chloride (oom)
ANOVA
SUBJECTS (N)
WATER
gd
gd
gd
WATER
gd
gd
gd
CONSUMPTION (g/day)
0 - gd 6a +++
i
6 - gd 13 + + •*
13 - gd 20 +++
CONSUMPTION (g/kg/day)
0 - gd 6 +++
6 - gd 13 +++
13 - gd 20 +++
TRENDS 0
21
§§§ 31.54
i 1.23
§§§ 38.78
i 1.73
§§§ 47.70
i 2.32
§§§ 116.1
i 4.4
§§§ 128.8
1 5.1
§§§ 133.3
i 5.4
50
28
33.39
i 1.09
39.67
i 1.09
47.81
i 1.25
115.7
t 3.9
125.1
i 4.1
128.4
t 4.2
250
27
22.81****
i 1.07
29.63****
i 1.01
32.72****
i 0.93
83.9****
i 3.7
98.8****
* 2.8
94.2****
i 2.3
500
16
21.78b****
i 0.85
25.92°****
i 1.44
24.73****
* 0.72
84.5b***»
* 4.0
90.8C***»
i 5.0
77.4****
i 3.4
gd = gestatlonal day.
n=15; Water consumption could not be determined for one animal because the water bottle spilled prior
to weighing on day 6.
£
n=15; Water consumption could not be determined for one animal because the water bottle leaked.
**
**
p<0.01; Dunnett's Test.
p<0.01; Williams' Test.
§66
p<0.001; ANOVA.
p<0.001; Test for.'Linear Trend.
CO
o
-------
table 14. Summary of Nickel (Ni ) Consumption for F . Dams During Gestation of the F Litter.
Nickel Chloride (pom)
SUBJECTS (N)
NICKEL CONSUMPTION fag/day)
gd 0 - gd 6
gd 6 - gd 13
gd 13 - gd 20
NICKEL CONSUMPTION (ag/kg/day)
gd 0 - gd 6
gd 6 - gd 13
gd 13 - gd 20
50
28
1.67
i 0.05
1.98
i 0.06
2.39
* 0.06
5.79
i 0.19
6.26
t 0.21
6.42
i 0.21
250
27
5.70
t 0.27
7.41
i 0.25
8.18
t 0.23
20.97
t 0.92
24.69
i 0.69
23.54
i 0.56
500
16
10.89b
t 0.42
12.96°
i 0.72
12.36
i 0.36
42.23b
i 2.00
45.42°
i 2.51
38.69
i 1.68
gd = gestational day.
n=15; Water consumption could not be determined for one animal because the water bottle spilled
prior to weighing on day 6.
°n=15; water consumption could not be doterminrd for one animal because the water bottle leaked.
-------
Table 15. Summary of Maternal Body Weight for Fj Dams During Late
Gestation and Following Delivery of the F2a Litter.
1
Nickel Chloride (ppm)
ANOVA TRENDS
No. Live Litters on pnd 1
No. of Timed-Pregnant Females
with a Live Litter on pnd 1
GESTATIONAL BODY WEIGHT (g)
pd 20r| +4+ §§fi
POSTNATAL BODY WEIGHT (g)
pnd 1R +++ §§§
nrul 4 + -n S§§
pnd 7 +n §§§
pnd 14 *++ §§§
pnd 21 -M §§§
0
24a
21
397.8
i 10.0
312.5
* 7.3
324.3
t 7.0
335.8
i 7.0
348.4
± 7.1
339.9
i 6.6
50
28
28
415.3
* 7.9
321.3
i 6.0
334.5
* 6.2
341.0
i 5.8
357.5
i 5.7
349.6
i 5.4
250
25b
24
379.6
i 6.1
296.7
i 5.7
305.7
i 6.6
315.9
1 5.7
332.6
t 5.6
325.0
t 6.6
500
15C
15
346.0***f
* 10.7
257.2**ff
i 10.6
269.4**»#
t 11.3
281.7**f»
t 11.6
312.0**t»
t 10.0
312. 7*#
t 7.8
(continued)
CO
-------
Table 15. (concluded)
aall pups in one litter were missing (i.e.. presumed c.innJhali/ed) on postnatal day 4; n=23 on pnd 4, 7,
14 and 21.
"All pups in two litters were missing on postnatal day 4: n=23 on pnd 4. 7, 14 and 21.
CA11 pups in one litter died before postnatal day 4 (n=14 on pnd 4 and 7). All pups in one additional
litter died before postnatal day 14 (n=13 on pnd 14 and. 21)).
('gd - gestational day.
epnd - postnatal day.
*p<0.05: Uunnett's Test.
**p<0.01: Dunnett'a Test.
**p<0.01; Williams' Test.
'*p<0.01; ANOVA.
***p<0.001: ANOVA.
sssp<0.001: Test for Linear Trend.
-------
Table 16. Summary of Food Consumption for F Dams During Late
Gestation and Following Delivery of the F Litter.
Nickel Chloride (nnm)
ANOVA
No. Live Litters on pnd 1
No. Timed-Pregnant Females
with a Live Litter on
pnd la
No. of Females with Live
Litters on pnd 7
No. of Females with Live
Litters on pnd 14
FOOD CONSUMPTION (g/day)
gd 20 - pnd 1 +++
pnd 1 - pnd 7 ++
pnd 7 - pnd 14 +
pnd 14 - pnd 21 ++
POOD CONSUMPTION (g/kg/day)
gd 20 - pnd 1 +++
TRENDS 0
24
21
23
23
§SS 22.51
t 0.93
§§§ 36.85
i 1.44
§§ 53.83
± 2.20
§§ 63. 0G
i 2.4
§§§ 63.87
i 2.34
50
28
28
28
28
22.44
i 0.53
36.97
i 1.09
54.90
i 1.77
68.5
f 1.9
61.21
i 1.48
250
25
24
23
23
21.48
i 1.80
32.55*
i 1.53
52.71
» 1.91
62.3
i 2.8
62.72
t 4.22
500
15
15
14
13
8.90****
* 1.45
29.99***
* 2.03
44.89***
i 2.31
54.7
i 3.4
29.04****
t 4.30
(continued)
-------
Table 16. (continued)
Nickel Chloride (ppm)
ANOVA TRENDS 0
pnd 1 - pnd 7 NS NS 114.1
1 4.4
pnd 7 - pnd 14 NS NS 158.1
± 6.4
pnd 14 - pnd 21 NS NS 183. 6e
! 6.9
50
111.9
i 3.1
157.7
1 5.0
193.4
i 4.7
250
110.1
i 4.4
163.2
* 6.0
189.7
i 8.4
500
111.4
1 6.6
150.4
i 7.4
175.2
* 10.0
Only timed-pregnant females are represented for gd 20 - pnd 1.
Only females with a live litter on pnd 7. are represented for pnd 1-7.
C0nly females with a live litter on pnd 14 are represented for pnd 7-14 and pnd 14-21. All of these
litters still contained live pups on pnd 21.
gd = gestatlonal day; pnd = postnatal day.
en-22: One animal's food was inadvertently weighed on the wrong day.
NS, nonsignificant, p>0.05.
*
p<0.05; Dunnett's Test.
**
p<0.01; Dunnett's Test.
tp<0.05; Williams' Test.
**p<0.01; Williams' Test.
*p<0.05; ANOVA.
**p<0.01; ANOVA.
f+ f
§§
p<0.001; ANOVA.
p<0.01; Test for Linear Trend.
§§§
p<0.001; Test for Linear Trend.
-------
Table 17. Summary of Water Consumption for f. Dams During Late
Gestation and Following Delivery or the F Litter.
2a
Nickel Chloride (DDID)
ANOVA
No. of Live Litters on pnd 1
No. Timed-Pregnant Females
with a Live Litter on
pnd 1
No. of Females with Live
Litters on pnd 7
No. of Females with Live
Litters on pnd 14
WATER CONSUMPTION (g/day)
gd 20 - pnd 1 +++
pnd 1 - pnd 7 +++
pnd 7 - pnd 14 +++
pnd 14 - pnd 21 + «•*
WATER CONSUMPTION (g/kg/day)
gd 20 - pnd 1 +++
TRENDS 0
24
21
23
23
§§8 34.82
1 1.91
SS§ 45.88
i 1.86
§§§ 65.94
1 3.05
§§§ 84. 8C
i 4.2
§SS 98. 4
» 4.5
50
28
28
28
28
36.08
t 2.19
45.51
i 1.66
68.59
i 1.98
92.5
1 3.1
99.4
* 6.8
250
25
24
23
23
22.11**ff
i 1.17
37.86***
i 2.21
58.33*
t 2.73
72.1**
i 4.1
65.1****
i 3.0
500
15
15
14
13
9.19****
t 1.34
31.48****
i 2.59
45.76****
t 2.34
55.7****
1 3.5
30.3****
i 4.1
(continued)
a>
-------
Table 17. (continued)
Nickel Chloride (ppm)
pnd 1 - pnd 7
pnd 7 - pnd 14
pnd 14 - pnd 21
ANOVA TRENDS 0
+ §<5 142.6
i 6.3
++ S§§ 194.0
i 9.4
+ «•+ §S§ 247. 0R
i 11.7
50
137.8
i 5.1
196.9
* 5.5
261.7
i 8.3
250
128.2
± 6.1
180.3
t 8.1
218.8
i 11.8
500
116.4**
* 8.7
154.6***
* 9.8
179.1****
t 11.5
Only timed-pregnant females are represented for gd 20 - pnd 1.
Only females with a live litter on pnd 7 are represented for pnd 1-7.
C0nly females with a live litter on pnd 14 are represented for pnd 7-14 and pnd 14-21. All of these
litters still contained live pups on pnd 21.
gd = gestatlonal day; pnd - postnatal day.
en=22; One animal's water was Inadvertently weighed on the wrong day.
NS, nonsignificant, p>0.05.
**
**
p<0.05; Dunnett's Test.
«
p<0.01; Dunnett's Test.
rp<0.05: Williams' Test.
p<0.01; Williams' Test.
p<0.05; ANOVA.
**p<0.01; ANOVA.
p<0.001, ANOVA.
§§
§§§
p
-------
+ 4 ,
Table 18. Summary of Nickel (Ni ) Consumption for F Dams During
Late Gestation and Following Delivery of tne F. Litter.
50
Nickel Chloride (ppm)
250 " 500
No. Females with Live
Litters on pnd 1
28
25
15
No. Timed-Pregnant Females
with a Live Litter on pnd 1*
28
24
15
No. of Females witji Live
Litters on pnd 7
28
23
14
No. of Females with Live
Litters on pnd 14°
NICKEL CONSUMPTION (ng/day)
28
23
13
gd 20
pnd 1
pnd 7
pnd 14
- pnd 1
- pnd 7
- pnd 14
- pnd 21
1.80
i 0.11
2.28
i 0.08
3.43
i 0.10
4.62
i 0.15
5.53
i 0.29
9.47
i 0.55
14.58
i 0.68
18.02
i 1.03
4.60
t 0.67
15.74
t 1.30
22.88
t 1.17
27.83
i 1.75
NICKEL CONSUMPTION (ng/kg/day)
Ed 20
- pnd 1
4.97
1 0.33
16.28
t 0.75
15.17
i 2.05
(continued)
CD
00
-------
Table 18. (continued)
Nickel Chloride loon)
pnd 1 - pnd 7
pnd 7 - pnd 14
pnd 14 - pnd 21
50
6.89
t 0.26
9.85
i 0.2R
13.08
* 0.41
250
32.04
1 1.51
45.07
± 2.04
54.69
* 2.96
500
58.22
1 4.36
77.32
t 4.91
89.54
* 5.76
Q
Only timed-pregnant females are represented for gd 20 - pnd 1.
Only females with a live litter on pnd 7 are represented for pnd 1-7.
°0nly females with a live Utters on pnd 14 are represented for pnd 7-14 and
pnd 14-21. All of these litters still contained live pups on pnd 21.
Ed = gestational day; pnd - postnatal day.
-------
Table 19. Summary of F
Data: Number of Live Pups per Litter and Percent Mortality per Litter.
NO. OP LITTERS
NO. OP LIVE PUPS PER LITTER
PRIOR TO CULLING ON PND 4d
pnd le
pnd 4
NO. OP LIVE PUPS PER LITTER
AFTER CULLING ON PND 4d
pnd 7
pnd 14
pnd 21
PERCENT MORTALITY PER
LITTER (*)f
pml 1 pnd 4
pnd 4 - pnd 21
Nickel Chloride
ANOVA Linear Trend
or or
Kruskal-Wal 1 Is Jonckheere 0
24a
+ §§ 13.63
i 0.64
++ §§S 12.29
* 0.85
NS § 8 . 79
t 0.49
* §§ 8.33
i 0.52
f §§ 8.33
* 0.52
NS& NS 12.9
i 5.3
NS NS 6.68
i 2.25
(ppm)
Nickel Chloride (ppm)
50
28
14.18
i 0.58
13.50
i 0.61
9.43
i 0.29
9.14
± 0.33
9.11
± 0.33
4.4
t 2.0
4.28
+ 1.78
250
25b
12.40
t 0.84
11.72
±0.98
i 8.28
1 * 0.67
8.08
1 0.67
8.08
i 0.67
9.8
i 5.5
6.53
i 3.37
500
15C
11.40*
± 0.72
8.27***
±1.05
7.13
±0.83
6.13***
± 0.91
6.13***
±0.91
28.6
i 8.5
14.63
±6.06
ontlnued)
o
o
-------
Tablo 19. (concluded)
aAll pups in one litter were missing (i.e.. presumed cannibalized) on postnatal day 4; n=23 on pnd 4, 7, 14
and 21.
bAll pups in two litters were missing on postnatal day 4; n=23 on pnd 4. 7. 14, and 21.
°A]J pups in one Utter were dead or missing on postnatal day 4 (n=14 on pnd 4 an 7); all pups in one
additional litter were dead or missing on postnatal day 14 (n=13 on pnd 14 and 21).
''statistical analyses included ANOVA, Test for Linear Trend. Dunnett's Test, and Williams' Test.
epnd - postnatal day.
Statistical analyses Included Kruskal-Wallis Test. Jonckheere's Test, and Shirley's or Dunn's Test.
Pp=^0.0500 for Kruskal-Wallis; pairwise comparisons of nickel-treated groups against the control group
were all nonsignificant (p>0.05).
NS, nonsignificant, p>0.05.
*p<0.05; Uunnett's Test.
*p<0.05: Williams' Test.
**p<0.01: Williams' Test.
*p<0.05: ANOVA.
ffp<0.01; ANOVA.
Sp<0.05; Test for Linear Trend.
sso<0.01; Test for Linear Trend.
sssp<0.001; Test for Linear Trend.
-------
Table 20. Summary of F Pup Data: Average Pup Body Weight per Litter.
ANCOVA
TRF.NDS
N J eke 1_ Chi pri de__( ppm)_
0 50~
250
500
NO. OF LITTERS
24'
28
25
15'
AVG. PUP BODY NT.
PER LITTER (g)C
pnd 1
pnd 4
pnd 7
pnd 14
pnd 21
+ + * §SS 6.03
• 0.14
+ «•+ SSS 7.80a
i 0.24
+ + 5SS 11.52
i 0.40
+ + + 8§S 24.93
i 0.65
+++ §§§ 37.76
t 0.77
6.18
1 0.13
8.02
i 0.25
11.82
1 0.44
25.19
i 0.73
38.77
i 0.91
6.10
! 0.12
7.84
t 0.25
11.66
1 0.38
24.03
i 0.57
36.04
i 0.87
5.47**
t 0.20
7.01**
t 0.33
10.03**
t 0.60
21.04**
t 1.20
32.30**
1 1.44
All pups in one litter were missing (i.e.. presumed cannibalized) on postnatal day 4; n=23 on pnd 4. 7',
14 and 21.
All pups in two litters were missing on postnatal day 4; n=23 on pnd 4, 7, 14, and 21.
All pups in one litter were dead or missing on postnatal day 4 (n=14 on pnd 4 and 7). All pups in one
additional litter were dead or missing on postnatal day 14 (n=13 on pnd 14 and 21).
Means adjusted for live? litter size using analysis of covariance. Sidak t-test used for pairwise
comparisons.
'pnd=postnatal day.
NS. nonsignificant. p>0.05.
itinued)
o
ro
-------
Tabli; 20. (continued)
**
p<0.01; SJdak t-test.
p<0.01: Analysis of Covariance (ANCOVA).
p<0.001; Analysis of Covariance (ANCOVA).
SRK
p<0.001: Multiple Linear Regression (MLR).
o
CO
-------
Table 21. Summary of P Pup Data: Percent Male Pups per Litter.
Nickel Chloride (ppm)
ANOVA TRENDS
NO. OP LITTERS
PERCENT MALE PUPS PER
LITTER (*)
pnd ld NS NS
pnd 4 NS NS
pnd 7 NS NS
pnd 14 NS NS
pnd 21 NS NS
0
243
52.1
* 2.5
53.1
i 2.5
51.0
t 1.6
50.9
i 1.7
50.9
1 1.7
50
28
49.5
i 2.5
49.9
* 2.5
49.5
i 1.7
49.6
i 2.1
49.4
i 2.1
250
25b
45.4
t 4.2
44.4
t 3.8
44.4
t 3.7
45.4
i 3.7
45.4
t 3.7
500
15°
51.2
i 3.7
53.4
i 4.5
53.8
i 5.1
58.9
t 6.3
58.9
i 6.3
3A11 pups In one litter were missing (i.e.. presumed cannibalized) on postnatal day 4; n=23 on
pnd 4, 7. 14 and 21.
bAll pups In two litters were missing on postnatal day 4; n=23 on pnd 4, 7, 14 and 21.
°A11 pups In one litter were dead or missing on postnatal day 4 (n=14 on pnd 4 and 7); all pups in one
additional litter were dead or missing on postnatal day 4 (n=13 on pnd 14 and 21).
pnd = postnatal day.
NS, nonsignificant. p>0.05.
-------
Table 22. Summary of F Pup Data: Necropsy Findings on
Postnatal Day 21 for Male and Female Pups.
105
Number of Males
Number (*) of males with
no gross abnormalities
Kidney:
Pelvic dilatation
right
left
bilateral
Number of Females
Number (%) of females with
no gross abnormalities
Kidney:
Pelvic dilatation
right
left
bilateral
Nickel
0
102
101(99)
1
0
0
99
94(95)
3
0
2
Chlori
50
127
116(91)
7
1
3
135
119(88)
7
1
7
de ( com )
250
93
90(97)
2
0
1
109
104(95)
1
0
4
500
50
49(98)
1
0
0
35
33(94)
1
0
1
2 mm tan subcutaneous focus
Eye: right corneal opacity
0
0
0
0
0
0
-------
1 Ofi
Table 23. Summary of Reproductive Status for CD Rats in the F Generation
Following Continuous Exposure to Nickel Chloride in the Drinking
Water: Breeding to Produce the F Litter.
(No. FJb Breeding Pairs to
Produce F2b Litters)
MATING INDEX
No. Mated (sperm-positive and/or
confirmed pregnant)9
No. Not Mated (sperm-negative
and not pregnant)
Total Breeding Pairs
% Mated Females NS
FERTILITY INDEX
No. Confirmed Pregnancies3
No. Females Not Pregnant
Total Mated Females
% Fertile Matings NS
GESTATIONAL I HDP*
No. Live Litters
No. Dead Litters
Total Litters
% Live Litters NS
Nickel
0
(30)
22
8
30
73.3
21
1
22
95.5
21
0
21
100
Chloride
50
(29)
21
8
29
72.4
20
1
21
95.2
20
0
20
100
(DDm)
250
(27)
23
4
27
85.2
20
3
23
87.0
20
0
20
100
500
(18)
17
1
18
94.4
15
2
17
88.2
15
0
15
100
a
Pregnancy was confirmed by a litter on gd 20 sacrifice or delivery of a litter
NS. nonsignificant. p>0.05.
-------
Table 24. Summary of F Dam Body Weights During Gestation of the F2b Litter.
Nickel Chloride (ppm)
ANOVA
SUBJECTS (N)
GESTATIONAL BODY HEIGHT (g)
gd oa +
gd 6 NS
gd 13 NS
gd 20 +++
TRENDS 0
17
NS 322.4
1 7.3
NS 339.8
1 7.9
NS 367.1
* 8.7
§S§ 450.4
i 11.6
50
20
346.7
» 8.4
366. 9b
t 9.3
393.9
* 9.0
478.3
± 10.8
250
19
325.1
i 6.8
346.1
* 7.4
370.8
* 8.4
443.0
i 9.7
500
14
316.2
i 6.0
340.4
± 6.3
367.7
i 6.9
410. 5*«
* 7.0
§§§
gd=gestational day.
n=19; One animal was weighed on day 7, not day 6.
*
p<0.05; Dunnett's Test.
•*p<0.05; Williams' Test.
*p<0.05; ANOVA.
p<0.001; ANOVA.
p<0.001; Test for Linear Trend.
-------
Table 25. Summary of Pood Consumption for F Dams During Gestation of the P . Litter.
Nickel Chloride (ppm)
SUBJECTS
ANOVA
(N)
TRENDS 0
17
50
20
250
19
500
14
POOD CONSUMPTION (g/day)
gd 0 -
gd 8 -
gd 13
gd 63 NS
gd 13 NS
- gd 20 +
NS 20.28
1 0.83
NS 25.20
i 0.81
SS 26.50
i 1.09
22.55b
1 0.72
26.21b
i 0.69
26.99
t 0.73
22.20
i 0.55
25.53
i 0.69
25.33
» 0.57
21.90
t 1.36
26.17
i 0.76
23.43**
i 0.83
POOD CONSUMPTION (g/kg/day)
gd 0 -
gd 6 -
gd 13
gd 6 NS
gd 13 NS
- gd 20 NS
NS 61.18
i 1.96
§ 71.22
* 1.30
NS 64.62
t 1.53
63.38
i 1.73
69.08
1 1.12
61.89
t 0.93
66.31
i 1.42
71.19b
i 1.01
62.37
i 0.95
66.92
i 4.18
73.97
* 1.81
60.21
i 1.79
gd = gestatlonal day.
n=19; food consumption could not be determined for one animal because the food was weighed on day 7,
not day 6.
NS. nonsignificant p>0.05.
*
p<0.05; Dunnett's Test.
*p<0.05; Williams' Test.
§§
p<0.05; ANOVA.
5
p<0.05; Test for Linear Trend.
p<0.01; Test for Linear Trend.
o
oo
-------
Table 26. Summary of Water Consumption for F Dams During Gestation of the F Litter.
J 2b
Nickel Chloride (ppm)
ANOVA
SUBJECTS (N)
NATER
gd
gd
gd
WATER
gd
gd
gd
CONSUMPTION (g/day)
0 - gd 6 +++
6 - gd 13 +++
13 - gd 20 +++
CONSUMPTION (g/kg/day)
0 - gd 6 ++ +
6 - gd 13 +++
13 - gd 20 +++
TRENDS 0
17
SS§ 35.80
i 1.75
SSS 45.40
t 2.54
§SS 53.65
t 2.96
§§§ 108.2
i 5.1
§§S 128.2
i 6.7
§§§ 130.9
± 6.3
50
20
33.58b
i 1.90
42.83b
1 1.55
50.14
! 1.49
•
94. 9b*
i 5.7
113.3b*f
i 3.9
115.8**
t 3.8
250
19
27.87°****
i 0.98 i
31.99***#
i 1.22 i
34.79****
1 1.23 i
83.5°****
i 2.2 t
89.0****
i 2.2 i
85.5****
t 2.4 i
500
14
25.59****
1.16
29.60****
1.46
26.98****
1.36
78.2****
3.6
83.6****
3.8
69.3****
3.2
a
gd = gestatlonal day.
n=19; Water consumption could not be determined for one animal because the water bottle was weighed on
on day 7, not day 6.
^
'n=18; Water consumption could not be determined for one animal because the water bottled leaked.
(continued)
o
CO
-------
Table 26. (continued)
*
p<0.05; Dunnett's Test.
**
p<0.01; Dunnett's Test.
*p<0.05; Williams1 Test.
**p<0.01; Williams' Test.
***p<0.001; ANOVA.
§§§
p<0.001; Test for Linear Trend.
-------
Table 27. Summary of Nickel (Nl++) Consumption for F, Dams During Gestation of the P ^ Litter
I 2b
Nickel Chloride (ppm)
SUBJECTS (N)
NICKEL CONSUMPTION (mg/day)
a
gd 0 - gd 6
gd 6 - gd 13
gd 13 - gd 20
NICKEL CONSUMPTION (mg/kg/day)
gd 0 - gd 6
gd 6 - gd 13
gd 13 - gd 20
50
20
1.68b
i 0.10
2.14b
£ 0.08
2.51
£ 0.08
4.74b
£ 0.28
5.67b
i 0.19
5.79
£ 0.19
250
19
6.97C
i 0.25
8.00
i 0.31
8.70
i 0.31
20.89°
£ 0.55
22.24
i 0.56
21.37
£ 0.60
500
14
12.80
£ 0.58
14.80
£ 0.73
13.49
i 0.68
39.09
£ 1.78
41.82
£ 1.90
34.64
£ 1.59
gd = gestatlonal day.
n=19; Hater consumption could not be determined for one animal because the water bottle was
weighed on day 7, not day 6.
"n=18; Water consumption could not be determined for one animal because the water bottle leaked.
-------
, Maternal ToxjcUy in CD Rats (P Dams) E*ppsed to Nickel
Chloride qn OflstaUonal Days 0 thrpMgh 20 of the pgb Utters
Nickel Chloride (ppm)
0
SUBJECTS (Dams)
Total Treated 18
No. Removed8 0
No. Dead 0
50 250
22 22
1 0
0 0
500
16
0
0
No. Nonpregnant at Sacrifice
1132
No. (*) Pregnant at Sacrifice
17( 94.4) 20( 95.2) 19( 86.4) 14( 87.5)
Maternal Body Weight (gd 0) (g)
322.40 346.65 325.04 316.23
i 7.29 i 8.42 * 6.83 i 6.02
ANOVA STATS: Bartlett's (p=0.2850); DOSE (p=0.0257).
Maternal Body Weight (gd 20) (g)
450.42S§§ 478.25 443.03 410.47*f
* 11.62 i 10.77 i 9.66 * 7.05
ANOVA STATS: Bartlett's (p=0.1442); DOSE (p=0.0004).
(cont d)
ro
-------
Table 28. (continued)
Nickel Chloride (ppm)
50
250
500
Maternal Body Weight (gd 0 and 20)
See Above for Means and Standard Errors.
ANOVA STATS: Bartlett's (p=0.0270); DOSExOAY (C).
Maternal Weight Gain (gestation) (g)
128.02§§§ 131.60
t 6.71 * 4.84
117.99
i 4.04
ANOVA STATS: Bartlett's (p"0.0784); DOSE (p<0.0001).
94.24**tf
i 3.83
Maternal Weight Gain (corrected) (R
51.34§§§
i 2.82
b.d
48.21
i 3.25
37.37*f
i 3.66
17.71***f
i 6.39
ANOVA STATS: Bartlett's (p=0.0435); DOSE (p<0.0001).
aOne dam was removed for the 50 ppm dose group after she received drinking water from the
0 ppm dose group on gestatlonal day 6 through 13.
Includes all dams pregnant at sacrifice; mean t S.E.M.
°DOSExDAY interaction was significant (p<0.0001); See above for results of two-way ANOVA.
weight gain during gestation minus gravid uterine weight.
(continued)
-------
Table 28. (continued)
§§§
p<0.001; Test for Linear Trend.
*
p<0.05; Dunnett's Test.
**
p<0.01; Dunnett's Test.
*p<0.05; Williams' Test.
**p<0.01; Williams' Test.
-------
Table 29. Summary of Embryotoxlclty in F . CD Rats Fetuses Following Maternal
(F .) Exposure to Nickel ChJonHe on Gestatlonal Days 0 through 20.
Nickel Chloride (ppm)
0 50 250 500
ALL LITTERS3 (17) (20) (19) (14)
No. Corpora Lutea per Dam
16.88 17.20 17.53 17.71
0.61 t 0.67 t 1.11 i 0.76
ANOVA STATS: Bartlett's (p-0.0264); DOSE (p-0.9081).
No. Implantation Sites per Litter
15.12 16.40 16.00 15.14
1.01 ! 0.44 * 0.99 i 0.98
ANOVA STATS: Bartlett's (p=0.0111); DOSE (p-0.6512).
Percent Prelmplantatlon Loss
11.88 6.60 12.70 15.11
4.75 i 2.29 * 5.08 i 5.78
ANOVA STATS: Bartlett's (p=0.2907); DOSE (p=0.5536).
No. Resorptlons per Litter
0.82 1.15 1.37 1.36
0.25 i 0.29 * 0.31 * 0.32
(continued)
>->
i-»
en
-------
Table 29. (continued)
Nickel Chloride (ppm)
0 50 250
500
Percent Resorptions per Litter
ANOVA STATS
4.86 7.00 7.83
* 1.35 * 1.80 i 1.74
: Bartlett's (p=0.7174); DOSE (p-0.3998).
10.50
t 2.74
No. Litters with Resorptions
% Litters wl
10 12 14
th Resorptions
58.8 60.0 73.7
10
71.4
Dead per Litter
Percent Dead
ANOVA STATS
0.06 0.00 0.00
i 0.06 i 0.00 * 0.00
per Litter
0.37 0.00 0.00
i 0.37 1 0.00 i 0.00
: Bartlett's (f); DOSE (p=0.3812).
0.00
i 0.00
0.00
i 0.00
(continued)
CD
-------
Tablr 29. (continued)
Nickel Chloride (DDRI)
0 50 250
No. Litters with Dead
1 0 0
* Litters with Dead
5.9 0.0 0.0
b c
No. Nonllve Implants per Litter '
0.88 1.15 1.37
i 0.26 * 0.29 t 0.31
b c
Percent Nonllve Implants per Litter '
5.23 7.00 7.83
i 1.42 i 1.80 i 1.74
ANOVA STATS: Bartlett's (p-0.7805); DOSE (p=0.4578).
£
No. Litters with Nonllve Implants
10 12 14
% Litters with Nonllve Implants
58.8 60.0 73.7
500
0
0.0
1.36
t 0.32
10.50
i 2.74
10
71.4
(continued)
-------
Table 29. (continued)
Nickel Chloride (ppm)
50 250 500
No. Adversely Affected Implants per Litter '
1.94 3.30 1.79 1.93
i 0.60 i 0.54 ±0.36 0.44
Percent Adversely Affected Implants per Litter '
11.44 20.58 10.11 14.05
i 3.17 i 3.56 i 1.96 * 3.11
ANOVA STATS: Bartlett's (p=0.4977); DOSE (p-0.1019).
No. Litters with Affected Implants
12 18 16 12
% Litters with Affected Implants
70.6 90.0 84.2 85.7
LIVE LITTERS
(No. Litters with Live Fetuses)
(17) (20) (19) (14)
(continued)
-------
TabIP 29. (continued)
Nickrl Chloride (ppm)
0 50 250
500
No. Live Fetuses per Litter
ANOVA STATS:
14.24 15.25 14.63
i 0.91 * 0.51 t 0.88
Bartlett's (p-0.1109); DOSE (p-0.6493).
13.79
i 1.02
No. Male Fetuses per Litter
7.00 8.10 6.84
t 0.58 t 0.49 1 0.84
7.71
i 0.77
No. Female Fetuses per Litter
Percent Male
ANOVA STATS:
7.24 7.05 7.79
i 0.76 i 0.51 t 0.63
Fetuses per Litter
50.14 53.56e 43.82
i 3.41 t 3.15 i 4.62
Bartlett's (p-0.0629); DOSE (p=»0.1045).
6.07
i 0.95
56.97
! 4.84
(continued)
-------
Table 29. (continued)
Nickel Chloride (ppm)
0 50 250 500
Average Petal Body Weight (g) per Litter
3.345 3.321 3.316 3.482
1 0.063 «. 0.042 ± 0.055 * 0.068
ANOVA STATS: Bartlett's (p=0.5607); DOSE (p--0.1792).
Average* Male Fetal Body Weight (g) per Litter
3.449 3.397 3.335 3.575
1 0.069 * 0.042 i 0.065 * 0.069
ANOVA STATS: Bartlett's (p=0.3006); DOSE (p=0.0611).
Average Female Petal Body Weight (g) per Litter
3.243 3.233 3.256 3.372
1 0.058 1 0.045 * 0.054 1 0.075
ANOVA STATS: Bartlett's (p=0.6250); DOSE (p=0.3397).
ANOVA STATS: Bartlett's (p=0.5758); DOSE (p-0.1837); SEX (p£0.0001); DOSExSEX (p=0.3108)
(continued)
to
o
-------
Table 29. (continued)
alncludes all dams pregnant at sacrifice; litter size = no. implantation sites per dam.
Reported as mean i S.E.M.
Q
Nonllve = dead plus resorbed.
Affected = nonllve plus malformed.
elncludes only dams with live fetuses; litter si/p - no. live fetuses per dam.
Zero variance In one or more groups - test not done.
eOne fetus not sexed In two litters.
CO
-------
Table 30. Summary and Analysis of Teratologlc Defects Observed In
F . CD Rat Fetuses Following Exposure of Dams (F ) to Nickel
Chloride on Gestatlonal Days 0 through 20.
Nickel Chloride (ppm)
50
250
500
No. Fetuses Examined
a
242
305
278
193
No. Litters Examined
17
20
19
14
No. Gross Malformations per Litter
0.00 0.00
t 0.00 t 0.00
0.05 0.00
i 0.05 t 0.00
No. Visceral Malformations per Litter
0.00
0 . 00
0.05
i 0.05
0 . 05
i 0.05
0 . 00
t 0.00
No. Skeletal Malformations per Litter
c.d
1.06
J 0.39
2.10
± 0.49
0.32
i 0.27
0.57
t 0.36
No. Fetuses Malformed per Litter
1.06
i 0.39
2.15
t 0.48
0.42
* 0.27
0.57
i 0.36
(conti-
to
-------
Table 30. (continued)
Nickel Chloride (ppm)
0 50 250
Q
Percent Fetuses Malformed per Litter
6.96SS 14.67* 2.34
* 2.52 i 3.33 t 1.49
ANOVA STATS: Bartlett's (p=0.1869); DOSE (p=0.0010).
No. Fetuses Malformed
18 43 8
* Fetuses Malformed
7.44 14.10 2.88
No. Litters with Malformations6
8 15 4
% Litters with Malformations
47.06++ 75.00 21.05
o
No. Males Malformed per Litter
0.53 1.10 0.17
t 0.24 1 0.27 * 0.09
500
3.75
i 2.39
8
4.15
4
28.57
0.29
i 0.22
(continued)
w
-------
Table 30. (continued)
Nickel Chloride (ppm)
50 " 250 500
No. Females Malformed per Litter
0.53 1.05
i 0.21 t 0.28
0.26
i 0.21
0.29
i 0.16
Percent Males Malformed per Litter
7.31§
i 3.75
16.35*
i 4.19
1.79
i 1.06
ANOVA STATS: Bartlett's (p=0.0029); DOSE (p=0.0027).
2.74
t 2.18
Percent Females Malformed per Litter
5.81 16.16*
i 2.18 i 5.07
2.51
* 1.87
4.54
* 2.96
ANOVA STATS: Bartlett's (p-0.0126); DOSK (p=0.0130).
ANOVA STATS: Bartlett's (p=0.0007); DOSE (p=0.0010); SEX (p=0.8710); DOSExSEX (p=0.9396)
Only live fetuses were examined for malformations.
Includes only litters with live fetuses.
^Reported as mean * S.E.M. for all live fetuses
Fetuses with one or more malformations.
g
Litter with one or more malformed fetuses.
-------
Table 30. (continued)
p<0.05; Test for Linear Trend.
§§
p<0.01; Test for Linear Trend.
«
p<0.05; Dunnett's Test.
p<0.01; Chi-Square Test.
IV)
en
-------
Table 31. Teratologlc Defects in
Nickel Chloride on Gesta
F . CD Rat Fntusos Following Exposure of Dams (F..) to
ntional Days 0 through 20: Listing by Defect Type .
Nickel Chloride (ppm)
50 "~250 500
Total Fetuses Examined
^
Total Litters Examined
I. EXTERNAL MALFORMATIONS
242
17
305
20
278
19
193
14
No. Fetuses with Malformations
No. Litters with Malformations
Edema
II. VISCERAL MALFORMATIONS
(0)
(0)
(0)
(0)
(1)
(1)
(0)
(0)
No. Fetuses with Malformations
No. Litters with Malformations
Missing Ductus Arteriosus
Pulmonary Artery Malformation
Renal Agenesis (left)
e
(0)
(0)
(1)
(1)
(1)
(1)
(0)
(0)
(continued)
o>
-------
Table 31. (continued)
Nickel Chloride (pom)
0
50
250
500
III. SKELETAL MALFORMATIONS
No. Fetuses with Malformations
No. Litters with Malformations ++
(18)
(8)
(42)
(14)
(7)
(3)
(8)
(4)
Short Rib
Missing Rib
Ribs Fused to Each Other: Cartilage to Cartilage
Bone to Done
Missing Vertebrae: Lumbar
Thoracic
Missing Lumbar Centra
Missing Thoracic Arch
Unilateral Ossification of Thoracic Centra
Lumbar Arch Fused to Sacral Arch
Lumbar Arch Six Cartilage Fused to Illlum
Split Sternum Cartilage
IV. VARIATIONS
No. Fetuses with Variations
No. Litters with Variations6
13
1
1
5
1
1
1
1
(30)
(12)
34
3
1
4
1
1
1
2
1
(30)
(15)
5
2
1
1
2
1
(31)
(13)
8
1
(12)
(9)
1
Incomplete Ossification. Cartilage Present
Misaligned Sternebrae
Bipartite Thoracic Centra
20
8
3
16
6
3
18
5
11
4
1
6
(continued)
-------
Table 31. (continued)
Nickel Chloride (ppm)
50
250
500
Centra and Cartilage Split
Split Centrum Cartilage
Unossified Cervical Centra,
Distended Ureter(s)
Missing Innominate Artery
Hematoma (head)
Very Soft Tissue - Kidney
Cartilage Normal
1
2
1
1
1
8A single fetus may be represented more than once in listing Individual defects.
Defects are listed in order of frequency (high to low) for all dose groups combined.
Only live fetuses were examined for malformations.
CIncludes only litters with live fetuses.
Fetuses with one or more malformations.
eLitters with one or more malformed fetuses.
Fetuses with one or more variations.
^Litters with one or more fetuses with variations.
Pulmonary artery one-half normal width with no apparent connection to vena cava.
One dam in the 0 ppm dose group had an incompletely ossified interparletal bone. In
the 50 ppm dose group two dams had unosslfled publs bone and one had an unossifled
thoracic centra. Two dams in the 250 ppm dose group had unossifled publs bones. All
other Incomplete ossifications involved frontal and parietal bones.
p<0.01; Chi-Square Test
10
00
-------
129
Table 32. Analysis of the Incidence of F . Fetuses with Short Rib.
No. of Litters Examined3
Percent Fetuses per Litter
with Short Rib 'c
Nickel
ANOVA TRENDS 0
17
+ NS 4.
1 2.
Chloride
50
20
95 12.
44 ± 3.
(ppm)
250
19
03 1.46
14 ±1.46
500
14
3
* 2
.75
.39
aOnly litters with live fetuses were examined for malformations.
Only live fetuses were examined for malformations.
Q
Reported as mean ± SEN for all live fetuses.
NS. nonsignificant. p>0.05.
*p<0.05; ANOVA.
-------
Table 33. Malformations and Variations Exhibited by F CD Rat Fetuses Following Maternal (F . Dams)
Exposure to Nickel Chloride on Gestational Days 0 Through 20: Listing by Individual Fetus.
Average Fetal Fetus
Dose Rep Damid Live/Implants Body Weight Number
0 1 452 12/12 3.61 5
8
456 14/16 3.37 2
484 13/14 3.33 3
11
13
486 11/12 3.09 3
628 15/19 3.23 1
5
7
9
12
14
15
676 16/16 3.55 11
678 17/18 3.83 2
Fetus
Weip.ht
3.32
3.85
3.12
3.31
3.14
3.06
2.72
2.69
3.21
3.29
3.62
3.57
2.82
3.45
3.40
3.54
Defects
Type Description
Skeletal
Variation
Variation
Variation
Variation
Skeletal
Variation
Variation
Skeletal
Skeletal
Skeletal
Skeletal
Variation
Skeletal
Skeletal
Skeletal
Skeletal
Skeletal
Variation
Variation
Skeletal
Skeletal
Skeletal
Short Rib
Misaligned Sternebrae
Misaligned Sternebrae
Incomplete Ossification, Cartilage Present -
Frontals and Parietals
Incomplete Ossification, Cartilage Present -
Frontals and Parietals
Lumbar Arch Fused to Sacral Arch
Incomplete Ossification, Cartilage Present -
Frontals and Parletals
Misaligned Sternebrae
Split Sternum Cartilage
Short Rib
Short Rib
Short Rib
Misaligned Sternebrae
Short Rib
Missing Lumbar Centra
Missing Lumbar Arch
Short Rib
Short Rib
Incomplete Ossification, Cartilage Present -
Frontals and Parletals
Incomplete Ossification, Cartilage Present -
Frontals and Parietals
Short Rib
Missing Lumbar Arch
Missing Lumbar Centra
(continued)
CO
0
-------
Table 33. (continued)
Average Fetal Fetus
Dose Rep Dam id Live/Implants Body Weight Number
678 17/8 3.83 13
17
704 16/16 3.38 2
12
13
730 15/15 3.45 1
3
4
5
7
9
11
13
15
734 19/20 2.91 1
7
Fetus
Weight
3.99
3.77
3.19
3.45
3.34
3 . 56
3.26
3.33
3.48
3.23
3.53
3.38
3.56
3.40
2.41
3.25
Defects
Type Description
Skeletal
Skeletal
Skeletal
Skeletal
Skeletal
Variation
Skeletal
Skeletal
Variation
Variation
Variation
Variation
Variation
Variation
Variation
Variation
Variation
Variation
Variation
Missing Lumbar Arch
Missing Lumbar Centra
Short Rib
Missing Lumbar Arch
Missing Lumbar Centra
Misaligned Sternebrae
Short Kib
Short Rib
Incomplete Ossification,
Frontals and Parietals
Incomplete Ossification,
Frontals and Parietals
Bipartite Thoracic Centra
Incomplete Ossification,
Frontals and Parietals
Incomplete Ossification,
Frontals and Parietals
Incomplete Ossification,
Frontals and Parietals
Incomplete Ossification.
Frontals and Parietals
Incomplete Ossification,
Frontals and Parietals
Incomplete Ossification,
Frontals and Parietals
Incomplete Ossification,
Frontals and Parietals
Incomplete Ossification,
Frontals and Parietals
Cartilage Present -
Cartilage Present -
Cartilage Present -
Cartilage Present -
Cartilage Present -
Cartilage Present -
Cartilage Present -
Cartilage Present -
Cartilage Present -
Cartilage Present -
(continued)
-------
Table 33. (continued)
Average Fetal Fetus
Dose Rep Damld Live/Implants Body Weight Number
734 19/20 2.91 11
12
17
19
738 16/18 2.99 8
11
13
14
852 22/23 3.13 12
13
854 6/6 3.32 1
50 1 514 19/20 3.29 6
13
19
518 16/16 3.48 8
Fetus
Weight
2.73
2.07
2.91
3.38
3.15
3.04
3.01
2.75
2.49
3.32
3.27
3.29
3.03
3.44
3.43
Defects
Type Description
Variation
Variation
Skeletal
Skeletal
Skeletal
Variation
Variation
Variation
Skeletal
Skeletal
Skeletal
Skeletal
Variation
Skeletal
Variation
Variation
Variation
Variation
Variation
Variation
Variation
Variation
Incomplete Ossification, Cartilage Present -
Frontals and Parietals
Bipartite Thoracic Centra
Missing Thoracic Arch
Missing Rib
Fused Rib Cartilage
Incomplete Ossification, Cartilage Present -
Frontals and Parietals
Bipartite Thoracic Centra
Incomplete Ossification, Cartilage Present -
Frontals and Parietals
Short Rib
Short Rib
Missing Lumbar Centra
Missing Lumbar Arch
Misaligned Sternebrae
Lumbar Arch Six Cartilage Fused to Ilium
Misaligned Sternebrae
Misaligned Sternebrae
Incomplete Ossification. Cartilage Present -
Interparietal
Incomplete Ossification, Cartilage Present -
Frontals and Parietals
Very Soft Tissue - Kidney
Incomplete Ossification. Cartilage Present -
Frontals and Parietals
Incomplete Ossification, Cartilage Present -
Pubis
Misaligned Sternebrae
(continued)
ro
-------
Table 33. (continued)
Average Frtal I'otus
Dose Rep Dam id Live/Implants Body Weight Number
520 12/16 3.58 1
3
5
7
9
522 14/14 3.46 2
3
8
9
10
12
14
532 16/16 3.22 6
536 15/15 3.38 5
11
540 20/20 3.48 1
9
12
16
Fetus
Weight
3.40
3.71
3.50
3.88
3.77
3.32
3.45
3 . 35
3.18
3.34
3 . 30
3.80
3 . 35
3. 17
3.68
3.64
3.49
3.60
3.74
Defects
Type Description
Skeletal
Skeletal
Variation
Vnriat ion
Skeletal
Vnriat Jon
Varint ion
Skeletal
Skeletal
Skeletal
Skeletal
Skeletal
Skeletal
Skeletal
Skeletal
Skeletal
Variation
Skeletal
Variation
Skeletal
Skeletal
Variation
Skeletal
Skeletal
Skeletal
Skeletal
Short Rib
Short Rib
Incomplete Ossification. Cartilage Present -
Frontals and Parietals
Incomplete Ossification. Cartilage Present -
Frontals and Parietals
Short Rib
Incomplete Ossification. Cartilage Present -
Frontals and Parietals
Incomplete Ossification. Cartilage Present -
Frontals and Parietals
Short Rib
Short Rib
Missing Rib
Missing Thoracic Arch
Missing Thoracic Centra
Short Rib
Short Rib
Short Rib
Short Rib
Bipartite Thoracic Centra
Short Rib
Incomplete Ossification. Cartilage Present -
Frontals and Parietals
Lumbar Arch Fused to Sacral Arch
Lumbar Arch Fused to Sacral Arch
Misaligned Sternebrae
Missing Lumbar Arch
Missing Lumbar Centra
Missing Lumbar Arch
Missing Lumbar Centra
(continued)
w
w
-------
Table 33. (continued)
Average Petal Fetus
Dose Rep Dam id Live/ Implants Body Weight Number
612 13/13 3.21 8
9
10
12
656 13/15 2.93 2
3
9
11
13
658 16/17 3.01 1
3
5
7
9
13
694 12/16 3.22 12
712 15/16 3.49 9
11
Fetus
WeiRht
3 . 55
3.00
3.34
3.38
2.71
2.79
2.92
3.38
3.01
2.00
1
2.03
3.60
3.26
2.55
3.24
3.49
3.58
3.38
Defects
Type Description
Variation
Skeletal
Skeletal
Skeletal
Skeletal
Skeletal
Skeletal
Skeletal
Skeletal
Variation
Variation
Variation
Variation
Variation
Variation
Variation
Skeletal
Skeletal
Skeletal
Skeletal
Skeletal
Variation
Variation
Distended Ureter(s)
Missing Rib
Short Rib
Short Rib
Short Rib
Short Rib
Missing Rib
Missing Lumbar Arch
Missing Lumbar Centra
Incomplete Ossification, Cartilage Present -
Prontals and Parietals
ft
Incomplete Ossification, Cartilage Present -
Frontals and Parietals
Incomplete Ossification, Cartilage Present -
Frontals and Parietals
Incomplete Ossification. Cartilage Present -
Frontals and Parietals
Bipartite Thoracic Centra
Incomplete Ossification, Cartilage Present -
Thoracic Centra
Incomplete Ossification, Cartilage Present -
Pub is
Short Rib
Short Rib
Short Rib
Short Rib
Lumbar Arch Six Cartilage Fused to Ilium
Incomplete Ossification, Cartilage Present -
Frontals and Parietals
Incomplete Ossification. Cartilage Present -
Frontals and Parietals
(continued)
-------
Table 33. (continued)
Average FetaJ Fetus
Dose Rep Dam Id Live/Implants Body Weight Number
716 16/17 3.19 6
8
9
14
744 12/14 3.43 1
4
6
8
10
748 17/18 3.33 1
4
9
11
12
13
15
784 15/16 3.41 1
3
4
11
12
824 15/15 3.60 1
6
13
Fetus
Weight
3.65
2.84
2.92
3.29
3.74
3.55
3.06
3.28
3.40
3 . 25
2.91
3.61
3.24
3.32
3.53
3.33
3.19
3.27
3.47
3.44
3.57
3.35
3 . 52
3.72
Defects
Type Description
Skeletal
Skeletal
Variation
Variation
Skeletal
Skeletal
Skeletal
Skeletal
Skeletal
Variation
Skeletal
Skeletal
Variation
Skeletal
Skeletal
Variation
Skeletal
Variation
Skeletal
Skeletal
Skeletal
Skeletal
Skeletal
Skeletal
Skeletal
Variation
Variation
Variation
Skeletal
Variation
Short Rib
Short Rib
Hematoma (head)
Misaligned Sternebrae
Short Rib
Short Rib
Short Rib
Short Rib
Short Rib
Misaligned Sternebrae
Short Rib
Short Rib
Misaligned Sternebrae
Ribs Fused to Each Other
Unilateral Ossification of Thoracic Centra
Thoracic Centra and Cartilage Split
Missing Lumbar Centra
Misaligned Sternebrae
Short Rib
Missing Lumbar Arch
Missing Lumbar Centra
Short Rib
Short Rib
Short Rib
Short Rib
Missing Innominate Artery
Distended Ureter(s)
Bipartite Thoracic Centra
Short Rib
Incomplete Ossification, Cartilage Present -
Frontals and Parietals
(continued)
CO
en
-------
Table 33. (continued)
Average Fetal Fetus Fetus
Dose Rep Damld Live/Implants Body Weight Number Weight Type
Defects
Description
846 18/18 3.01 18
850 17/20 3.27 3
250 1 462 15/16 3.47 14
560 20/23 3.43 1
562 20/22 3.26 8
14
19
584 12/15 3.65 1
11
592 3/ 3 3.91 I
598 14/15 3.10 3
6
7
9
620 16/17 2.96 5
6
2.86
3.48
3.45
3.49
2.48
3.37
3.30
3.50
3 . 59
3.59
3.04
3.32
3.15
3.07
2.85
2.71
Visceral
Visceral
Variation
Skeletal
Variation
Variation
Variation
Variation
Skeletal
Variation
Variation
Variation
Variation
Variation
Variation
Variation
Variation
Variation
Variation
Variation
Pulmonary Artery One-half Normal Width with no
Apparent Connection to Vena Cava
Missing Ductus Arteriosus
Unosslfied Cervical Centra, Cartilage Normal
Short Rib
Misaligned Sternebrae
Incomplete Ossification, Cartilage Present -
Frontals and Parietals
Misaligned Sternebrae
Bipartite Thoracic Centra
Fused Rib Cartilage
Incomplete Ossification, Cartilage Present -
Frontals and Parietals
Incomplete Ossification, Cartilage Present -
Frontals and Parietals
Incomplete Ossification, Cartilage Present -
Frontals and Parietals
Incomplete Ossification, Cartilage Present -
Frontals and Parietals
Bipartite Thoracic Centra
Bipartite Thoracic Centra
Incomplete Ossification, Cartilage Present -
Frontals and Parietals
Incomplete Ossification. Cartilage Present -
Frontals and Parietals
Bipartite Thoracic Centra
Misaligned Sternebrae
Incomplete Ossification, Cartilage Present -
Pub is
(continued)
u
at
-------
Table 33. (continued)
Average Fetal Pot us
Dose Rep Dam id Live/Implants Body Weight Number
636 16/19 3.04 3
6
11
662 17/17 3.33 14
722 18/18 3.07 3
5
9
13
15
16
18
.752 12/17 3.55 4
6
792 15/16 3.24 1
5
7
Fetus
Weight
3.28
2.99
2.97
2.42
2.71
3.16
2.98
3.16
2.97
3.15
3.04
3.38
3.80
2.94
3.46
3.83
Defects
Type Description
Variation
Variation
Variation
Gross
Variation
Skeletal
Skeletal
Skeletal
Variation
Skeletal
Skeletal
Skeletal
Skeletal
Skeletal
Variation
Skeletal
Skeletal
Skeletal
Variation
Skeletal
Variation
Variation
Variation
Variation
Variation
Bipartite Thoracic Centra
Bipartite Thoracic Centra
Bipartite Thoracic Centra
Edema
Bipartite Thoracic Centra
Missing Rib
Missing Lumbar Centra
Missing Lumbar Arch
Incomplete Ossification, Cartilage Present -
Publs
Short Rib
Missing Lumbar Centra
Short Rib
Short Rib
Short Rib
Misaligned Sternebrae
Short Rib
Missing Rib
Unilateral Ossification of Thoracic Centra
Bipartite Thoracic Centra
Missing Lumbar Centra
Bipartite Thoracic Centra
Misaligned Sternebrae
Incomplete Ossification, Cartilage Present -
Frontals and Parletals
Incomplete Ossification, Cartilage Present -
Frontals and Parietals
Incomplete Ossification, Cartilage Present -
Frontals and Parletals
(continued)
CO
-------
Table 33. (continued)
Average Fetal Fetus Fetus
Dose Rep Damid Live/Implants Body Weight Number Weight Type
Defects
Description
792 15/16
796 17/19
808 13/13
500 1 564 18/18
566 15/15
570 15/17
582 15/16
688 15/1C
690 11/15
758 17/17
3.24 9
11
13
ir>
3.53 3
3.36 11
13
3.24 8
10
3.41 15
3.67 2
10
3.19 2
3
5
7
8
15
3.58 3
3.71 1
3.32 3
9
3.16 Variation
Variation
3.05 Variation
3.23 Variation
2.99 Variation
3.26 Visceral
3.59 Variation
3.38 Variation
3.68 Variation
3.17 Variation
3.53 Skeletal
Skeletal
3.31 Variation
3.21 Skeletal
2.95 Skeletal
3.39 Skeletal
3.53 Variation
2.92 Skeletal
3.00 Skeletal
3.34 Skeletal
4.08 Variation
3.72 Variation
3.17 Variation
3.01 Skeletal
Incomplete Ossification, Cartilage Present -
Frontals and Parletals
Bipartite Thoracic Centra
Incomplete Ossification, Cartilage Present -
Prontals and Parietals
Incomplete Ossification, Cartilage Present -
Frontals and Parletals
Incomplete Ossification. Cartilage Present -
Frontals and Parletals
Renal Agenesis (left)
Incomplete Ossification, Cartilage Present, -
Frontals and Parietals
Incomplete Ossification, Cartilage Present -
Frontals and Parietals
Bipartite Thoracic Centra
Bipartite Thoracic Centra
Short Rib
Lumbar Arch Six Cartilage Fused to Ilium
Bipartite Thoracic Centra
Short Rib
Short Rib
Short Rib
Bipartite Thoracic Centra
Short Rib
Short Rib
Short Rib
Bipartite Thoracic Centra
Bipartite Thoracic Centra
Incomplete Ossification, Cartilage Present -
Frontals and Parietals
Short Rib
(continued)
oo
-------
Table 33. (continued)
Average Fetal Fetus Fetus Defects
Dose Rep Damid Live/Implants Body Weight Number Weight Type Description
762 16/18 3.44 13 3.23 Variation Split Cervical Centrum Cartilage
770 13/15 3.66 2 3.70 Variation Misaligned Sternebrae
772 13/15 3.32 1 3.05 Variation Incomplete Ossification, Cartilage Present -
Frontals and Parietals
11 2.91 Variation Incomplete Ossification. Cartilage Present -
Frontals and Parietals
13 3.34 Variation Incomplete Ossification, Cartilage Present -
Frontals and Parietals
W
(0
-------
Table 34.
Summary of Organ Weights for Pregnant F . Female Rats Exposed to Nickel Chloride
in the Drinking Water at Scheduled Sacrifice on gd 20 of the F_. Litter.
9h
ANOVA TRENDS
Subjects (N)
Gravid Uterine Weight NS NS
Ovarian Weight (g) NS NS
Relative Ovarian Weight NS NS
(% body weight)
Liver Weight (g) +++ §§§
Relative Liver Weight +t §§§
(* body weight)
Kidney Weight (g) NS NS
Relative Kidney Weight +++ §§§
(* body weight)
Adrenal Weight (g) NS NS
Relative Adrenal Weight ++ S§
(% body weight)
Nickel
0
17
76.7
i 5.4
0 . 234
i 0.013
0.052
i 0.002
16.18
t 0.53
3.59
t 0.06
2.24
i ' 0.07
0.498
! 0.011
0.082
i 0 . 003
0.018
t 0.001
Chloride (cone.
50
20
83.4
* 2.8
0.236
i 0.013
0.049
i 0.002
16.72
t 0.37
3.51
i 0.06
2.40
i 0.06
0.502
i 0.006
0.081
± 0.004
0.017
i 0.001
in drinking
250
19
80.6
i 4.3
0.210
i 0.008
0.048
i 0.002
15.17
i 0.38
3.45
i 0.10
2.30
i 0.05
0.521
i 0.009
0.080
i 0.003
0.018
i 0.001
water , ppm)
500
14
76.5
t 5.3
0.219
i 0.011
0.053
* 0.002
12.99*»f»
i 0.44
3.16**t#
i 0.08
2.31
i 0.08
0.562***»
t 0.014
0.084
t 0.003
0.021
i 0.001
-------
Table 34. (continued)
ANOVA TRENDS
Heart Weight (g) NS NS
Relative Heart Weight NS NS
(% body weight)
Lung Weight (g) NS NS
Relative Lung Weight + §§
(* body weight)
Pituitary Weight (g) NS NS
Relative Pituitary Weight NS NS
(% body weight)
Nickel
0
1.22
* 0.04
0.272
i 0.009 ,
1.56
+ 0.06
0.350
* 6.018
0.0155
i 0.0007
0.0035
t 0 . 0002
Chloride (cone, in drinking
50
1.28
+ 0.03
0.268
i 0 . 006
1.52
i 0.06
0.319
1 0.012
0.0155
i 0.0007
0.0032
i 0.0001
250
1.22
i 0.04
0.275
+ 0.007
1.59
i 0.08
0.362
i 0.019
0.0155
i 0.0006
0.0036
i 0.0002
water, ppm)
500
1.19
1 0.07
0.289
i 0.016
1.65
i 0.10
0.402
i 0.025
0.0148°
i 0 . 0006
0.0036°
t 0.0002
n=13; one pituitary lost prior to weighing.
NS. nonsignificant.
**
**
4- +
§§
§§§
p<0,OJ; Dunnett's Test.
p<0.01; Williams' Test.
*p<0.05; ANOVA.
p<0.01; ANOVA.
p<0.001; ANOVA.
p<0.01; Test for Linear Trend.
p<0.001; Test for Linear Trend.
-------
Table 35.. Summary-of Necropsy Findings for F Female
CD Rats at Scheduled Sacrifice.
142
Nickel Chloride (ppm)
NUMBER OF FEMALES
Number (*) of Females with
no gross abnormalities
OBSERVATIONS:
Lung
mottling, red. diffuse
multiple 1-3 mm red foci
Liver
accentuated lobu-lar pattern
pale
cyst
2 mm white foci
Kidney
pelvic dilitation: bilateral
right
left
renal cortex left, brown
pinpoint focus
renal cortex left. 0.5 mm
clear cyst
renal cortex right. 2 mm
clear cyst
Uterus, serosa multiple red foci
0
17
9(53)
4
0
0
0
Ia
0
1
1
1
0
0
0
0
50
20
14(20)
2
2
0
0.
lb
0
1
0
0
1
0
0
0
250
19
10(53)
3
5
0
0
0
1
0
0
0
0
1
1
0
500
14
7(50)
2
1
3
1
0
0
0
0
0
0
0
0
1
a
Animal 220 had a 4 mm clear cyst.
Animal 216 had a 4 mm light tan cyst.
-------
Table 36. Summary of Body and Organ Weights for Flb Male Rats Exposed to Nickel Chloride In the Drinking Water.
ANOVA TRENDS
Subjects (N)
Body Weight (g) +<+ SSS
Prostate Weight (g) NS §
Relative Prostrate Weight NS NS
(* body weight)
Seminal Vesicle Weight (g) NS NS
Relative Seminal Vesicle Weight NS §
(\ body weight)
Testicle Weight (g) +++• SSS
Relative Testicle Weight + §
(* body weight)
Livnr Weight (g) +«+ §S§
Relative Liver Weight + NS
(* body weight)
Kidney Weight (g) + + + S
Nickel
0
30
625 . 9
13.0
1.36
0.06
0.220
0.010
1.97
0.07
0.318
0.013
5.33
0.09
0.86
0.01
21.75
0.60
3.47
0.05
3.80
0.09
Chloride (cone.
50
30
665.3
11.6
1.43
0.06
0.215
0.009
2.06
0.07
0.312
0.012
5.38
0.10
0.81
0.01
24.49**
0.72
3.67*
0.07
4.24**
0.08
In drinking
250
30
613.9
12.2
1.33
0.06
0.219
0.011
2.06
0.10
0.336
0.016
5.18
0.11
0.85
0.02
22.56
0.65
3.67*
0.06
4.11
0.11
water, ppm)
500
22
539.9**
15.9
1.22
0.07
0.225
0.012
1.92
0.09
0.362
0.018
4.76*
0.10
0.89
0.02
18.65**
0.53
3.47
0.05
3.54
0.13
w
-------
Table 36. (continued)
ANOVA TRENDS
Relative Kidney Weight *+ §
(\ body weight)
Adrenal Weight (g) NS NS
Relative Adrenal Weight NS NS
(% body weight)
Heart Weight (g) + + + §§§
Relative Heart Weight NS NS
(* body weight)
Lung Weight (g) NS NS
Relative Lung Weight + §§
(% body weight)
Pituitary Weight (g) NS NS
Relative Pituitary Weight +++ §§§
(* body weight)
Nickel
0
0.607
0.008
0.080
0.004
0.0129
0.0007
1.72
0.04
0.277
0.006
2.09a
0.04
0 . 338a
0.010
0.014b
0.00046
0.0022b
0.0001
Chloride (cone, in drinking
50
0.64U
0.013
0.087
0.006
0.0132
0.0009
1.81
0.04
0.273
0.004
2.19
0.07
0.330
0.010
0.014
0.00047
0.0021
0.0001
250
0.671***
0.014
0.094
0.008
0.0155
0.0014
1.78
0.04
0.293
0.008
2.07
0.06
0.339C
0.008
0.014°
0.00034
0.0024°
0.0001
water. ppm)
500
0.654*t
0.013
0.083
0.012
0.0149
0.0017
1.52**
0.04
0.283
0.006
2.09
0.14
0.386*
0.023
0.015
0.00059
0.0027**
0.0001
-------
36. (continued)
a
n=29; 1 lung not weighed.
n=29; 1 pituitary gland lost prior to weighing.
°n=29; 1 pituitary gland lost prior to writhing.
NS. nonsignificant. p>0.05.
*
p<0.05; Dunnett's Test.
**
p<0.01; Dunnett's Test.
*p<0.05: ANOVA.
**p<0.01; ANOVA.
***p<0.001; ANOVA.
§
p<0.05; Test for Linear Trend.
§§
p<0.01; Test for Linear Trend.
KRK
p<0.001; Test for Linear Trend.
*p<0.05: Williams' Test.
Ul
-------
Table 37. Summary of Necropsy Findings for F . Male
CD Rats at Scheduled Sacrifice.
146
Nickel Chloride (pom)
NUMBER OF MALES
Number (%) of Males with
no gross abnormalities
OBSERVATIONS:
Lung
mottling, red. diffuse
multiple 1-3 mm red foci
left diaphramatic lobe, focus
Liver
accentuated lobular pattern
pale
focus
Thymus
multiple 1-2 mm red foci
mottling, red. marked, diffuse
Kidney
pelvic dilitation: right
left
left renal lymph node, redness
left cortex. 1 mm red foci
right cortex. 1 mm red-black foci
Testicle: left soft and watery
right enlarged
Mesentary. mass
Omen turn, mass
0
30
20(67)
1
2
0
3
0.
lb
0
0
3
0
3
0
0
0
0
0
0
50
30
12(40)
5
6
I3
2
0
0
4
1
2
1
2
0
0
1
0
0
0
250
30
13(43)
1
4
0
0
2
1C
1
3
3
1
0
1
0
1
1
0
0
500
30
14(64)
2
1
0
2
0
0
0
0
0
0
0
2
1
0
0
1*
26
Animal 356 had a 10 x 5 mm white irregular focus.
Animal 278 had a 4 mm irregular red-black focus.
Animal 374 had a 5 mm red-black focus.
Animal 212 had a 4 x 1.5 x 1.5 cm firm. tan. multilobulated mass.
Animal 272 had a 1.5 x 1 x 0.5 cm firm red irregular mass and animal
212 had a 5 x 3 x 2 mm firm yellow mass.
-------
Table 38. Analysis of Nickel Chloride in Stock Solutions'
147
Batch
Number
XVI
XVII
XVIII
XIX
XX
XXI
XXII
XXIII
XXIV
XXV
VI
Date of
Formulation
1-8-86
1-22-86
2-4-86
2-18-86
3-4-86
3-19-86
4-3-86
4-16-86
5-1-86
5-16-86
6-2-86
Date of
Analysis
1-8-86
1-22-86
2-4-86
2-19-86
3-4-86
3-19-86
4-4-86
4-16-86
5-1-86
5-16-86
6-2-86
Theoretical
Concentration
(ppm. Ni~)
100
100
100
100
100
100
100
100
100
100
100
.000
.000
.000
.000
.000
.000
.000
.000
.000
.000
.000
Measured
Concentration
(ppm. Ni*")°
88
93
92
90
91
93
91
92
90
92
91
.400
.050
.600
.700
,150
.100
.950
.400
.750
.800
.000
% Theoretical
Concentration pH
88
93
92
90
91
93
92
92
90
92
91
.4
.1
.6
.7
.2
.1
.0
.4
.8
.8
.0
2.
2.
2.
2.
2.
2.
2.
2.
2.
2.
2.
c
25
99
16
20
10
22
47
53
43
23
40
Batch Nos. XVI - XXVI were formulated from nickel chloride hexahydrate.
Lot No. KM01918HM (received at RTI on August 20. 1985) (see also section 2.0).
3
Mean value of two analytical samples.
Mean value of three samples.
-------
Table 39. Analysis of Nickel Chloride Dosed Drinking Water: Pre-Dosing Samples.
Dose Level ppm Batch Dosing Date of Date of
(Formulation Code) Number8 Period Formulation Analysis
0 (73952) XVI 1-14-86 1-13-86 1-13-86
to
1-30-86
50 (93352) XVI 1-14-86 1-13-86 1-13-86
to 1-13-86 1-27-86
1-30-86 1-13-86 1-13-86
1-13-86 1-13-86
250 (08252) XVI 1-14-86 1-13-86 1-13-86
to
1-30-86
500 (44880) XVI 1-14-86 1-13-86 1-13-86
to
1-30-86
0 (73952) XVII 1-28-86 1-22-86 1-27-86
to
2-13-86
50 (93352) XVII 1-28-86 1-22-86 1-27-86
to
2-13-86
Theoretical
Concentration Carboy
(ppm) Number
0 1
2
3
4
50 1
2
3
4
250 1
2
3
4
500 1
2
3
4
0 1
2
3
4
50 1
2
3
4
Measured
Concentration
(ppm)
0
0
0
0
51.5
50.0
51.5
50.3
252
254
238.
252
503
511
516
511
0
0
0
0
51.1
51.0
49.9
50.3
% Theoretical
Concentration
_
-
-
—
103.0
100.0
103.0
100.6
100.8
101.6
95.2
100.8
100.6
102.2
103.2
102.2
-
—
-
—
102.2
102.0
99.8
100.6
PH"
4.63
4.38
4.39
4.36
4.25
3.32
4.19
4.17
.28
.3*0
.34
.30
.11
.09
.11
.12
3.58
3.64
3.57
3.38
3.04
3.08
3.05
3.01
(continued)
00
-------
Table 39. (continued)
Dose Level ppm Batch
(Formulation Code) Number9
250 (08252) XVII
500 (44880) XVII
0 (78952) XVIII
50 (93352) XVIII
250 (08252) XVIII
500 (44880) XVIII
0 (78952) XIX
Dosing Date of Date of
Period Formulation Analysis
1-28-86 1-22-86 1-27-86
to
2-13-86
1-28-86 1-22-86 1-27-86
to
2-13-86 2-6-86° 2-10-86
2-11-86 2-7-86 2-10-86
to
2-27-86
2-11-86 2-7-86 2-10-86
to
2-27-86
2-11-86 2-7-86 2-10-86
to
2-27-86
2-11-86 2-7-86 2-10-86
to
2-27-86
2-25-86 2-24-86 2-24-86
to
3-13-86
Theoretical
Concentration Carboy
(ppm) Number
250 1
2
3
4
500 1
2
3
0 1
2
3
4
50 1
2
3
4
250 1
2
3
4
500 1
2
3
0 1
2
3
4
Measured
Concentration
(ppm)
242
244
244
244
483
485
484
0
0
0
0
51.6
52.0
50.1
50.4
242
240
238
237
480
484
480
0
0
0
0
% Theoretical
Concentration
96.8
97.6
97.6
97.6
96.6
97.0
96.8
-
-
-
-
103.2
104.0
100.2
100.8
96.8
96.0
95.2
94.8
96.0
96.8
96.0
—
-
-
PH"
3.24
3.12
3.09
3.01
3.19
3.19
5.43
4.76
4.83
4.99
5.39
4.72
4.68
4.68
4.68
4.55
4.37
4.08
4.47
4.44
4.50
4.36
d
-
-
(continued)
to
-------
Table 39. (continued)
Dose Level ppm Batch
(Formulation Code) Number
50 (93352) XIX
250 (08252) XIX
500 (44880) XIX
0 (78952) XX
50 (93352) XX
250 (08252) XX
500 (44880) XX
Dosing Date of Date of
Period Formulation Analysis
2-25-86 2-24-86 2-24-86
to
3-13-86
2-25-86 2-24-86 2-24-86
to
3-13-86
2-25-86 2-24-6 2-24-86
to
3-13-86
3-11-86 3-6-86 3-7-86
to
3-27-86
3-11-86 3-6-86 3-7-86
to 3-14 86C 3-17-86
3-27-86
3-11-86 3-6-86 3-7-86
to
3-27-86
3-11-86 3--G-86 3-7-86
to
3-27-86
Theoretical
Concentration Carboy
(ppm) Number
50 ]
2
3
4
250 1
2
3
4
500 1
2
3
0 1
2
3
4
50 1
2
3
4
250 1
2
3
4
500 1
2
3
Measured
Concentration
(ppm)
49.6
49.8
49.2
51.0
239
244
242
246
500
497
496
0
0
0
0
51.0
51.0
50.2
51.0
246
240
244
243
486
520
495
% Theoretical
Concentration
99.2
99.6
98.4
102.0
95.6
97.6
96.8
98.4
100.0
99.4
99.2
_
-
-
-
102.0
102.0
100.4
102.0
98.4
96.0
97.6
97.2
97.2
104.0
99.0
pHb
-
-
-
_
-
-
-
_
-
-
5.22
5.02
4.72
4.70
4.19
4.64
4.45
4.53
4.56
4.25
4.23
4.26
Inued)
-------
Table 39. (continued)
Dose Level ppm Batch Dosing
."oriiiu lilt ion Code) Number Period
0 (78952) XXI 3-25-86
to
4-10-86
50 (93352) XXI 3-25-86
to
4-10-86
250 (08252) XXI 3-25-86
to
4-10-86
500 (44880) XXI 3-25-86
to
4-10-86
0 (78952) XXII 4-8-86
to
4-24-86
50 (93352) XXII 4-8-86
to
4-24-86
Theoretical
Date of Date of Concentration Carboy
Formul at Inn Analysis (ppm) Number
3-20-86 3-24-86 0 1
2
3
4
3-20-86 3-24 86 50 1
2
3
4
3-20-86 3-24-86 250 1
2
3
4
3-20-86 3-24-86 500 1
2
3
4-8-86 4-9-86 0 1
2
3
4
4-8-86 4-9-86 50 1
2
3
4
Measured
Concentration
(ppm)
0
0
0
0
50.0
51.8
50.6
50.3
244
243
242
242
490
486
487
0
0
0
0
51.1
50.2
49.1
50.2
% Theoretical
Concentration
-
-
-
100.0
103.6
101.2
100.6
97.6
97.2
96.8
96.8
98.0
97.2
97.4
_
-
-
-
102.2
100.4
98.2
100.4
PH"
5.10
4.97
4.91
4.85
3.70
3.75
3.88
3.97
4.06
4.27
.35
.12
.18
.20
.22
6.00
5.67
5.77
5.90
5.17
5.33
5.43
5.27
(continued)
en
-------
Tab Jo 39. (nont Jriued)
)ose Level ppm Batch
(Formulation Code) Number
250 (08252) XXII
500 (44880) XXII
0 (78952) XXIII
50 (93352) XXIII
•
250 (08252) XXIII
500 (44880) XXIII
Dosing Date of Date of
Period Formulation Analysis
4-8-86 4-8-86 4-9-86
to
4-24-86
4-8-86 4-8-86 4 9-86
to
4-24-86
4-22-86B 4-20-86 4-22-86
to
5-5-86
f
4-29-86 5-1-86
4-22-86 4-20-86 4-22-86
to
5-5-86
f
4-29-86 5-1-86
4-22-86 4-20-86 4-22-86
to
5-5-86
4-22-86 4-20-86 4-22-86
to
5-5-86
Theoretical
Concentration Carboy
(ppm) Number
250 1
2
3
4
500 1
2
3
0 1
2
3
4
5
50 1
2
3
4
5
250 1
2
3
4
500 1
2
3
4
Measured
Concentration
(ppm)
240
239
244
240
490
490
492
0
0
0
0
0
52.2
52.2
50.4
50.7
48.0
232
233
237
237
485
487
490
486
% Theoretical
Concentration
96.0
95.6
97.6
96.0
98.0
98.0
98.4
_
-
-
-
—
104.4
104.4
100.8
101.4
96.0
92.8
93.2
94.8
94.8
97.0
97.4
98.0
97.2
PH"
5.23
5.13
5.17
5.17
4.97
5.07
5.03
4.63
4.87
5.13
4.97
4.93
5.03
5.03
5.27
5.50
5.23
5.17
.87
.70
.67
.73
.77
4.77
(continued)
en
ro
-------
Table 39. (continued)
Dose Level ppm Batch
(Formulation Code) Number3
0 '(78952) XXIV
50 (93352) XXIV
250 (082*52) XXIV
500 (44880) XXIV
0 (78952) XXV
50 (93352) XXV
Dosing
Period
5-6-86
to
5-15-86
5-6-86
to
5-15-86
5-6-86
to
5-15-86
5-6-86
to
5-15-86
5-13-86
to
6-3-86
5-13-86
to
6-3-86
Theori'lical
Date of Date of Concentration Carboy
Formulation Analysis (ppm) Number
5-6-86 5-8-86 0 1
2
3
4
5-6-86 5-8-86 50 1
2
3
4
5-6-86 5-8-86 250 1
2
3
4
5-6-86 5-8-86 500 1
2
3
i
5-21-86 5-23-86 0 1
2
3
4
5-21-86 5-23-86 50 1
2
3
4
Measured
Concentration
(ppm)
0
0
0
0
51.0
50.9
49.9
51.0
243
240
236
245
490
488
500
0
0
0
0
51.6
51.9
50.3
50.2
% Theoretical
Concentration
_
-
-
102.0
101.8
99.8
102.0
97.2
96.0
94.4
98.0
98.0
97.6
100.0
-
-
-
-
103.2
103.8
100.6
100.4
PH"
_d
_
-
-
—
_
-
-
_
_
-
-
_
_
-
6.30
5.83
5.73
5.53
5.63
5.27
5.40
5.33
(continued)
Oi
CO
-------
Table 39. (continued)
Dose Level ppm Batch
(Formulation Code) Number
250 (08252) XXV
vV (44880) XXV
.1 (78952) XXVI
50 (93352) XXVI
250 (08252) XXVI
500 (44880) XXVI
Dosing Date of Date of
Period Formulation Analysis
5-13-86 5-21-86 5-23 86
to
6-3-86
5-13-86 5-21-86 5-23-86
to
6-3-86
6-4-86 6 4 -86 6-4-86
to
7-7-86
6-4-86 6-4-86 6-4-86
to
7-7-86
6-4-86 6-4-86 6-4-86
to
7 7-86
6-4-86 6-4-86 6-4-86
to
7-7-86
Theoretical
Concentration Carboy
(ppm) Number
250 1
2
3
4
500 1
2
3
0 1
2
3
4
50 1
2
3
4
250 1
2
3
4
500 1
2
3
Measured
Concentration
(ppm)
241
238
245
257
508
506
488
0
0
0
0
52.9
52.1
47.7
50.3
252
248
246
244
503
483
494
* Theoretical
Concentration
96.4
95.2
98.0
102.8
101.6
101.2
97.6
-
-
-
-
106.0
104.0
95.0
101.0
101.0
99.2
98.4
97.6
101.0
96.6
98.8
P«b
5.00
4.97
5.10
5.03
4.87
4.87
4.90
5.03
4.. 70
4.53
4.50
4.87
4.87
4.47
4.53
4.47
4.47
4.40
4.35
4.10
4.47
4.37
(continued)
01
-------
Table 39. (continued)
The batch number of drinking water formulations is the same as the stock solution batch number.
pH of 0 ppm dosing solutions adjusted with HC1 Is necessary to achieve pH of less than 7.0.
Q
Carboy 3 for dose level was reformulated due to low measured concentration.
pH not taken.
g
Carboys 2. 3. and 4 were reformulated due to high measured concentration.
Due to an insufficient amount of dose formulations, additional carboys were formulated for dose level 78952
(yellow) and 93352 (purple).
el)osing period for Batch XIII was extended to 5-5-86.
01
en
-------
40. Analysis pf NJpKpJ chlnrjdp Posprt Drinking Water; PostdosJng Samples,
Theoretical Measured
Dose Level ppm Batch Dosing Date of Date of . Concentration Carboy Concentration
(Formulation Code) Number3 Period Formulation Analysis (ppm) Number (ppm)
0 (78952)
50 (93352)
250 (08252)
500 (44880)
0 (78952)
XVI 1-14-86 1-13-86 2-4-86 040
to
1-30-86
XVI 1-14-86 1-13-86 2-4-86 50 4 50.5
to
1-30-86
XVI 1-14-86 1-13-86 2-4-86 250 4 250
to
1-30-86
XVI 1-14-86 1-13-86 2-4-86 500 4 512
to
1-30-86
XVII 1-28-86 1-22-86 2-13-86 040
% Theoretical
Concentration pH
4.29
101.0 3.04
100.0 4.09
102.4 4.18
3.27
to
2-13-86
f>0 (93352) XVII 1-28-86 1-22-86 2-13-86 50 4 50.5 101.0 2.89
to
2-13-86
2.0 (08252) XVII 1-28-86 1-22--86 2--13-86 250 4 246 98.4 2.55
to
2-13-86
5f)0 (448RO) XVII 1-28-86 2-6-86 2-13-86 500 3 491 98.2 3.79
to
2-13-86
(continued)
i->
en
o>
-------
Table 40. (continued)
Theoretical Measured
Dose Level ppm Batch Dosing Date of Date of Concentration Carboy Concentration * Theoretical
(Formulation Code) Number Period Formulation Analysis (ppm) Number (ppm) Concentration pH
0 (78952) XVIII 2-11-86 2-7-86 2-27-86 040 - 4.12
to
2-27-86
50 (93352) XVIII 2-11-86 2-7-86 2-27-86 50 4 50.9 101.8 3.76
to
2-27-86
250 (08252) XVIII 2-11-86 2-7-86 2-27-86 250 4 239 95.6 3.55
to
2-27-86
500 (44880) XVIII 2-11-86 2-7-86 2-27-86 500 3 482 96.4 3.97
to
2-27-86
0 (78952) XIX 2-25-86 2~24-8fi 3-14-86 040 - 4.22
to
3-13--R6
•>C (93352) XIX 2-25-86 2-24-86 3 14-86 50 4 52.1 104.2 4.02
to
3-13-86
?'>0 (08252) XIX 2-25-86 2-24-86 3-14-86 250 4 246 98.4 4.11
to
3-13-86
500 (44880) XIX 2-25-86 2-24-86 3-14-86 500 3 505 101.0 4.09
to
3-13-86
(continued)
M
01
-a
-------
Tnblo 40. (continued)
Theoretical Measured
Oose Level ppm Batch Dosing Date of Date of Concentration Carboy Concentration * Theoretical
(Formulation Code) Number Period Formulation Analysis (ppm) Number (ppm) Concentration pH
0 (78952) XX 3-11-86 3-6-86 C 0 4 -
to
3-27-86
50 (93352) XX 3-11-86 3-6-86 ° 50 4 - -
to
3-27-86
250 (08252) XX 3-11-86 3-6-86 C 250 4 - - -
to
3-27-86
500 (44880) XX 3-11-86 3-6-86 ° 500 3 - -
to
3-27-86
0 (78952) XXI 3-25-86 3-20-86 4-16-86 040 - 5.47
to
4-10-86
50 (93352) XXI 3-25-86 3-20-86 4-16-86 50 4 51.5 103.0 5.47
to
4-10-86
250 (08252) XXI 3-25-86 3-20-86 4-16-86 250 4 243 97.2 5.37
to
4-10-86
500 (44880) XXI 3-25-86 3-20-86 4-16-86 500 3 496 ' 99.2 6.00
to
4-10-86
(continued)
^^l
in
oo
-------
Table 40. (continued)
Dose Level ppm Batch
(Formula! Jon Code) Number
0 (78952) XXII
50 (93352) XXII
250 (08252) XXII
500 (44880) XXII
0 (78952) XXIII
Dosing
Period
4-8-86
to
4-24-86
4-8-86
to
4-24-86
4-8-86
to
4-24-86
4-8-86
to
4-24-86
4-22-86
Theoretical
Date of Date of . Concentration
Formulation Analysis (ppm)
4-8-86 4-22-H6 0
4-8-86 4-22 «6 50
4-8-86 4-22-86 250
4-8-86 4-22-86 500
4-20-86 5-8-86 0
Measured
Carboy Concentration % Theoretical
Number (ppm) Concentration
40
4 50.2 100.4
4 234 93.6
3 484 96.8
40
pH
5.40
4.30
4.40
4.60
7.27
to
5-1-86
50 (93352) XXIII 4-22-86 4-20-86 5-8-86 50 4 51.2 102.4 6.53
to
5-1-86
250 (08252) XXIII 4-22-86 4-20-86 5-8-86 250 4 236 94.4 5.90
to
5-1-86
500 (44880) XXIII 4-22-86 4-20-86 5-8-86 500 3 484 96.8 5.70
to
5-1-86
(continued)
t-»
01
-------
Table 40. (continued)
Dose Level ppm Batch
(Formulation Code) Number
0 (78952) XXIV
50 (93352) XXIV
250 (08252) XXIV
500 (44880) XXIV
0 (78952) XXV
50 (93352) XXV
250 (08252) XXV
500 (44880) XXV
Dosing
Period
4-29-86
to
5-15-86
4-29-86
to
5-15-86
4-29-86
to
5-15-86
4-29-86
to
5-15-86
5-13-86
to
6-3-86
5-13-86
to
6-3-86
5-13-86
to
6-3-86
5-13-86
to
6-3-86
Theoretical Measured
Date of Date of Concentration Carboy Concentration % Theoretical
Formulation Analysis (ppm) Number (ppm) Concentration pH
4-20-86 C 0 4 - -
4-20-86 ° 50 4 - -
4-20-86 ° 250 4 - -
4-20-86 ° 500 3 -
5-6-86 7-14-86 040 - 6.58
5-6-86 7-14-86 50 4 49.9 99.8 4.47
5-6-86 7-14-86 250 4 240 96 4.35
5-6-86 7-14-86 500 3 480 96 4.08
(continued)
o>
o
-------
Table 40. (continued)
Theoretical Measured
Dose Level ppm Batch Dosing Date of Date of Concentration Carboy Concentration
(Formulation Code) Number8 Period Formulation Analysis (ppm) Number (ppm)
0 (78952)
50 (93352)
250 (08252)
500 (44880)
XXVI 6-4-86 6-4-86 7-14-86 040
to
7-7-86
XXVI 6-4-86 6-4-86 7-14-86 50 3 47.4
to
7-7-86
XXVI 6-4-86 6-4-86 7-14-86 250 3 245
to
7-7-86
XXVI 6-4-86 6-4-86 7-14-86 500 3 492
to
7-7-86
* Theoretical
Concentration pH
4.76
94.8 4.34
98.0 4.34
»
98.4 3.94
3The batch number of drinking water formulations is the same as the stock solution batch number.
Samples analyzed after all dosing was completed.
°Postdosing samples for this Batch were not collected prior to disposal of the formulations.
o>
-------
o
o
a>
o
JH
(D
PH
Average F, Female Body Weight Expressed as a Percentage
of the Average Control Values
Premating F2a F2a
Exposure for F2a Gestation Lactation
Rest F2b
Period Gestation
11U -
105 -
100 -
95 -
90 -
B5 -
80-
75 -
7f» -
O — O O
o o~— o o
A^
A "S*A"' ""A
A
Vi *n
* /
u—u
*D
*D
o — o 50 ppm
A — -A 250 ppm
D D 500 ppm
"^o
\
o
A^A-"AV
\
D-D-D \
\ A
\
\
\
\
\
\
*D
O — O^ ^.O— O
^0"^
^A
A%_^ ^A
A — A
^
D
*D
/
/
J
. D^
o-o-o-o
A-A-A^^
*
D
\* * *
D-D-D
O-O^
Ov"o
A
A ^A
D-CK
D \ A
\
\
\
\
\
n
* •
32 33 34 35 36 37 38 0 6 13 20 147 14 21 46 47 48 49 0 6 13 20
(study week) (day) (day) (study week) (day)
FIGl'"" 1
-------
o
o
14-1
o
-4-i
Q)
O
fc
120
Average F^^ Female Food Consumption (g/kg/day) Expressed
as a Percentage of the Average Control Values
Premating F2a F2a Rest F2b
Exposure for F2a Gestation Lactation Period Gestation
no -
100 -
eo -
80 -
70 -
60 -
50 -
40
D
/
D
D
o — o 50 ppm
A — A 250 ppm
n —D 500 ppm
33 34 35 36 37 38
(study week)
-------
Average F, Female Water Consumption (g/kg/day) Expressed
as a Percentage of the Average Control Values
no -
100 -
i-H
O
£ 90 -
O
O 80 -
«4-t
P
+-» 7°-
4^HV,
Lj
Q>
O 80 -
CD
^H 50 -
40 -
30 -
Premating F2a F2a Rest F2b
Exposure for F2a Gestation Lactation Period Gestation
Q
o-" \0^ ~~~°
*
*a~7D-D^A
o — o 50 ppm
A A OC f\ _ — —
A «sou ppm
D — a 500 ppm
— i — i — i — i — i — i —
o
^o — o
*A
* \
\
LJ
1 1 1
O
0 0
0
<^x
K
' 1
^
o\Q
\
A\*
* "7D~i
*o-b-fe
*^
\
V
D
33 34 35 38 37 38
» «
o
«
47 48 48
3
o
CM
(study week)
(day) (day)
Fir 3
(study week) (day)
OJ
-------
cti
4-
Average FI Female Nickel Consumption (mg Ni4"+/kg/day)
Premating F2a F2a Rest
Exposure for F2a Gestation Lactation . Period
9 " -
ttO
F2b
Gestation
LUU ~
90 -
80 -
70 -
60 -
50 -
40 -
30 -
20 -
10 -
0 -
O^^ C ^\ _. _
o uU ppm
A A **"> C rt
A «sou ppm
Df— i c r\f\ .
u ouu ppm
D
\
\
' • i
—0
A-A^
O— O— O
— 1 1 1
47 48 49
D \
D
A— A— A
o—o—o
1 1 1 — 1
«D n o
1 •? w
I
n
(study week)
(day)
(day)
(study week) (day)
FIGURE 4
en
-------
o
J-.
-4->
ti
o
o
d
0)
o
PL,
Average
Male Body Weight Expressed as a Percentage
of the Average Control Values
110
Pre-Mating Exposure for F2a Pre-Mating Exposure for F2b
100-
90-
80-
70-
60
0—0—0'
-O-
•o—o-
-o
A'
*D—
—i 1 1 1 1 1 1—
32 33 34 35 36 37 38
O—O—O—O—O—O-
-O—O—O—O
-D-"-1
o o 50 ppm
A A 250 ppm
n D500 ppm
—I 1 T 1 1 1 1 1 1 1—
40 41 42 43 44 45 46 47 48 49
(study week)
FIG1' 5
01
O)
-------
O
J-H
•*->
a
o
o
0)
o
SH
(D
ou
Average FI Male Food Consumption (g/kg/day) Expressed
as a Percentage of the Average Control Values
Pre-Mating Exposure for F2a Pre-Mating Exposure for F2b
1 fcjtj
120-
115-
110-
105-
100-
95-
90-
85-
RO-
*D
D
^K/
S^ \ \ A
VA A
A
*m * * *
LJ l_J \4c
*AX
/^v A . A
A*^"
6f~) (^ .f~}~ ,/~*\ /~\
^-U D 0 0^Q 0
^v^ /
o o 50 ppm
A A O Pv f*\ •*-*. t-*. »-»->
A <:ou ppm
n D500 ppm
1 1 1 r 1 1 1 1 — i —
-D
^A
33 34 35 36 37 38 41 42 43 44 45 46 47 48 49
(study week)
FIGURE 6
o>
-i
-------
o
SH
O
o
0)
o
!H
CD
PH
100
Average F< Male Water Consumption (g/kg/day) Expressed
as a Percentage of the Average Control Values
Pre-Mating Exposure for F2a Pre-Mating Exposure for F2b
95-
90 i
85-
80-
75 H
70
o
I I I
O.
^o—o-
u.D
0
-O-
D
D
D
o — o 50 ppm
A A 250 ppm
D Q500 ppm
33 34 35 36 37 38 41 42 43 44 45 46 47 48 49
(study week)
o>
-------
cd
T>
bfl
Average Fj_ Male Nickel Consumption (mg Ni++/kg/day)
Pre-Mating Exposure for F2a Pre-Mating Exposure for F2b
1 \J -
60-
50-
40-
30-
20-
10-
0-
D
\
n
x
\
i i — i
i i i i
\
D
A
\
\
A\
XA^A-A
o
~~ — 'O— — r*» r*\
(J O -Q
o — o 50 ppm
A A 250 ppm
Dm C" f\ i~\ __ _
LJ ouu ppm
D
"D~D^D^D~D^D_ _D
A A — —A A A
A — A — A A — A — A— .A
A
f~) (~)
1 ^T 1 1 1— 1 1 1 — — w—
33 34 35 36 37 38 41 42 43 44 45 46 47 48 49
(study week)
FIGURE 8
o>
co
-------
o
o
0)
o
*-.
CD
PU
Average Number of Live Pups or Number of Fetuses per Litter
Expressed as a Percentage of the Average Control Values
120
no -
100 -
90 -
80 -
70 -
60 -
50
F2a
D-
*~ *
o o 50 ppm
A—-A 250 ppm
n — D500 ppm
4 7 14
(postnatal day)
21
F2b Teratology
Evaluation
O
A
D
20
(gestational day)
.-^F g
V-*
-3
O
-------
o
CJ
0)
o
u
0)
PH
Average Percent Postnatal Mortality per Litter Expressed
as a Percentage of the Average Control Values
250
200 -
150 -
100 -
50 -
D-
o — o 50 ppm
A A 250 ppm
D D500 ppm
1-4
F2a
(postnatal day)
-D
O
4-21
FIGURE 10
-------
Average Pup Body Weight per Litter Expressed
as a Percentage of the Average Control Values
F2a
F2b Teratology
Evaluation
no -•
,__i
O 100 -
"S
o
o
O 90 "
"3
OJ
o
x
O CO
11,5
5' 3 «» I
(0^2 = CD
|l8R
3 8J Q.^3
n -• ® c
So oj»
^Sro
§|2§
§® i
1 S
(postnatal day)
(gestational day)
11
to
------- |