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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                                                                          7

                        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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                                                                         10
                              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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                                                                         11
                            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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                                                                         12
                             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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                                                                         14



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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                                                                         21



    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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                                                                         22



 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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                                                                         23



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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                                                                         25




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 (20C)   = 254 g/100 ml;




             water (100C)  = 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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                                                                         28



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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                                                                         29



     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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                                                                         30




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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                                                                         31




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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                                                                         32



 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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                                                                         33



     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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                                                                         34

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

-------
                                                                          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 56C  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.

-------
 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

-------
                                                                         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).

-------
                                                                         51



     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).

-------
                                                                         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.

-------
                                                                         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.

-------
                                                                         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.

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                                                                          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.

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                                                                         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.

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                                                                                       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  - 24.4C.  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 - 28.3C 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 - 23.3C 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.

-------
                                                                                       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 - 25.6C 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 24.4C 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_rpsy:  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. (*) Da3 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
+ + + 8S 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 (p0.0001); 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
Pb
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
* /

uu
*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

ooo
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
          000'
                  -O-
                     oo-
                     -o
      A'
          *D
     i	1	1	1	1	1	1

     32 33 34 35 36 37 38
 OOOOOO-
                                           -OOOO
        -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.
^oo-

   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


-------