EPA-600/1-83-007
June 1983
FET0T0XIC EFFECTS OF NICKEL IN DRINKING WATER IN MICE
Ezra Berman and Blair Rehnberg
Experimental Biology Division
Health Effects Research Laboratory
U.S. Environmental Protection Agency
Research Triangle Park, North Carolina 27711
Project Officer
Ezra Berman
Experimental Biology Division
Health Effects Research Laboratory
Research Triangle Park, N.C. 27711
Health Effects Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Research Triangle Park, North Carolina 27711

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NOTICE
This document has been reviewed in accordance with
U.S. Environmental Protection Agency policy and
approved for publication. Mention of trade names
or commercial products does not constitute endorse-
ment or recommendation for use.

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FOREWORD
The many benefits of our modern, developing, industrial society are
accompanied by certain hazards. Careful assessment of the relative risk
of existing and new man-made environmental hazards is necessary for the
establishment of sound regulatory policy. These regulations serve to
enhance the quality of our environment in order to promote the public
health and welfare and the productive capacity of our nation's population.
The Health Effects Research Laboratory, Research Triangle Park,
conducts a coordinated envi ronmnental health research program in toxicology,
epidemiology, and clinical studies using human volunteer subjects. These
studies address problems in air pollution, non-ionizing radiation, environ-
mental carcinogenesis and the toxicology of pesticides as well as other
chemical pokllutants. The Laboratory participates in the development
and revision of air quality criteria documents on pollutants for which
national air quality standards exist or are proposed, provides the data
for registration of new pesticides or proposed suspension of those already
in use, conducts research on hazardous and toxic materials, and is primarily
responsible for providing the health basis for nonionizing radiation
standards. Direct support to the regulatory function of the Agency is
provided in the form of expert testimony and preparation of affidavits as
well as expert advice to the Administrator to assure the adequacy of health .
care and surveillance of persons having suffered imminent and substantial
endangerment of their health.
The nickel-plating industry is the largest consumer of nickel. Nickel
is also converted to an environmental pollutant when organic fuels are
consumed. In these processes, there are always opportunities for the loss
of nickel into the environment. It is, therefore, pertinent to determine
the safety of these compounds for human health.
F.G. Hueter, Ph.D.
Di rector
Health Effects Research Laboratory
i i i

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ABSTRACT
Nickel chloride was administered in drinking water to pregnant mice
from the 2nd through the 17th day of gestation at nickel doses of 0, 500,
or 1000 ppm. Fetal or maternal toxicity was not seen after administration
of 500 ppm of nickel. However, the higher dose caused spontaneous abortions,
loss of fetal mass in survivors, and loss of maternal mass. The oral route
of administration via drinking water was at least 2.7 times less effective
than parenteral routes in producing fetal effects.
~tv-.

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SECTION 1
CONCLUSIONS
The oral dosage of nickel (Ni) required to produce fetal toxicity or
teratology may have been overestimated when based on studies using parenteral
administration. A factor as large as 2.7 may be needed to correlate the
effects of parenterally administered Ni with those of orally administered Ni.
1

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SECTION 2
EXPERIMENTAL PROCEDURES
MATERIALS AND METHODS
Time-bred female mice (CD-I, Charles River Breeding Labs, Inc.,
Wilmington, Mass.) were used in these experiments. Mice were bred and shipped
on the 1st day of gestation. (Presence of a copulatory plug is considered the
1st day of pregnancy.) Upon arrival, the mice were randomly assigned to sham
or treatment groups in equal numbers, weighed, and housed in groups of four in
shoe-box type cages. On the 18th day of pregnancy, each dam was killed by
suffocation with CO? gas and weighed. The uterus was removed and counts were
made of dead (resorbed and newly dead) and live fetuses. The live fetuses were
removed, examined for gross abnormalities, blotted dry, and weighed. Two of
every three fetuses were fixed in Bouin's solution, and the third fetus was
preserved in alcohol.
In initial dose-finding experiments, nickel chloride (Mi CI 2) water
solutions were offered as drinking water ad lib to stock adult male CD-I mice.
The means of body weights over days were compared to those of similar sham mice
on ordinary tap water. A 10,000-ppm Mi solution adjusted to a pH of 6 with
phosphate buffer could not be used due to observable precipitation; however,
acetate buffer sustained a 10,000-ppm solution at pH 6.2-6.5. When compared to
tap-water controls, mice maintained with this solution as drinking water lost
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26% of their body weight within 3 days (4 nice/group). Consumption of this Ni
solution was insignificant {2% of body weight); shams consumed tap water equal
to 16% of their mass.in this same period. After a switch between the tap water
and the Ni solution in these animals, the mice that were previously on tap
water but were subsequently changed over to Ni solution lost 14? of their body
weight in 1 day, while the mice that were switched to tap water almost regained
their initial weights. Clearly, the 10,000-ppm Ni solution was not acceptable
to mice as drinking water.
An additional attempt was made to induce consumption of the 10,000-ppm Ni
solution by adding an artificial sweetener to water at concentrations of 1 and
10%, but water consumption was not increased, and'severe loss of body weight
ensued. However, mice did tolerate well a lOCO-ppm Ni solution (pH 6-6.5)
without sweetener, and, after an initial small but noticeable loss, the masses
were approximately equal to initial size in 7 days. The 1000-ppm solution was
then considered as the maximum tolerated dose in drinking water for this
experiment.
Pilot groups of time-bred CD-I mice were used to test the effects of
gestational exposure to Ni solutions (pH 6-6.5) as drinking water. Tap or
drinking water with Ni concentrations of 1000, 1500, 2000, 3000, or 5000 ppm
were given to groups of 8 bred mice. The effects of dose on survival of the
dam and incidence of pregnancy in survivors are shown in Table 1. The dose
threshold for lethality of dams in this regimen was between 2000 and 3000 ppm.
The threshold for total early lethality of the conceptuses resulting in a
non-pregnant female was 1000 to 1500 ppm of Ni. Because a dose of 1000 ppm did
3

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TABLE 1. SURVIVAL AND PREGNANCY RATES OF TIME-BRED CD-I MICE
GIVEN VARYING CONCENTRATIONS OF NI IN DRINKING WATER
ON DAYS 2-17 OF GESTATION


Pregnant
Pregnant
Ni
Survived
Survivors
Survivors
(ppm)
(No.)
(No.)
m
1000
8/8
7/8
88
1500
8/8
4/8
50
2000
8/8
1/8
12
3000
4/8
0/4
0
5000
1/8
' 0/1
0
not appear to be lethal to either the pregnancy or the dam, our choice of this
as the maximum tolerable dose appeared to be reasonable.
Since the mice were avoiding water with concentrations of Ni above
1000 ppm, to achieve voluntary consumption at this level we added Ni CI 2 to
the mouse feed (Bioserv, Frenchtown, NJ) and fed it to time-bred mice. All
subjects avoided the feed containing 10,000-ppm Ni and showed severe loss of
weight. The idea of attempting to force the consumption of Ni in the diet did
not appear to be a successful strategy. The highest level of Ni we could
attain was a concentration of 1000 ppm in water and a nominal 100 ppm in feed.
Samples of the feed containing Ni in a nominal dose of 100 ppm were analyzed
using neutron activation and found to contain 68 ± 5 ug Ni/g of feed.
The experiment to elicit the teratologic potential of Ni was begun using
these concentrations. Time-bred mice were received from the supplier and
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assigned to 3 groups of 24 to receive 1000-ppm Ni in water and 68 ug Ni/g of
feed; or 7 groups of 12 to receive 500-ppm Mi in water and ordinary feed; and
to equal groups of concurrent sham controls for each dose (to receive tap water
and ordinary feed). When received, each group was allocated for handling as
described earlier.
Analyses were conducted using analysis of variance techniques for each
variable in each group of mice. Pregnancy rates and other incidental data were
analyzed using contingency tables. Unless stated otherwise, all data were
analyzed using the litter as the experimental unit.
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SECTION 3
RESULTS
Rates of pregnancy in bred mice were affected when Mi was administered in
drinking water at concentrations of 500 or 1000 ppm on days 2-17 of gestation.
No statistical difference was observed in shams of this group; therefore, all
shams were treated as a single 0-pprn group in Table 2. The ratio of
non-pregnant to bred females in the shams of the 500-ppm group was 15:44 (34%)
and the corresponding ratio in the shams of the 1000-ppm group was 9:31 (29%),
A value of P = 0.0001 (x2 = 23.04, DF = 2) resulted from pooling of shams and
testing with contingency tables for differences in pregnancy rates across dose.
The distinct decrease in the incidence of pregnancy in the group that received
1000 ppm (21% pregnant), when compared to the more normal rate of 65-68% seen
in bred mice that received 0 or 500 ppm, was the obvious reason for this
statistical result.
The body mass measurements in bred, non-pregnant mice are shown in
Table 3. Analysis of these values revealed no statistical difference between
sham animals used as concurrent controls (0-ppm N1) and those receiving 500- or
1000-ppm Ni (F = 1.73, DF = 1, P = 0.19). However, when the sham values were
pooled (X = 29.2, SD = 2) and the analysis done on a dose basis (0, 500, or
1000 ppm), a significant decrease was seen in the body masses of bred but
non-pregnant mice (F = 4.90, DF = 2, P = 0.011). Therefore, it appears that
6

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the decrease in their mean body mass was caused by the consumption of drinking
water dosed with 1000-ppm Mi but not by the dose level of 500 ppm.
TABLE 2. PREGNANCY INCIDENCES IN TIME-BRED CD-I MICE
. GIVEN 0-, 500-, OR 1000-ppm NI IN DRINKING WATER
ON DAYS 2-17 OF GESTATION
Ni	Not Pregnant	Pregnant	Pregnant
(ppm)	(No.)	(No.)	(%)
0 24
51

68
500 14
26

65
1000 . 27
7

21
TABLE 3. BODY MASSES
GIVEN 0-, 500-,
ON DAYS
(g) OF BRED BUT NON-PREGNANT CD-I
OR 1000-ppm NI IN DRINKING WATER
2-17 OF PRESUMED GESTATION
MICE


v Ni
(ppm)



500
1000

Concurrent shams (0 ppm)
29.6 ± 1.9
N = 15
28.6 ± 2.
N = 9
1
Treated (500 or 1000 ppm)
29.7 ± 1.5
N - 14
26.5 ± 1.
N = 27
7
7

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Table 4 contains the results of counts of fetuses and fetal masses in dams
that received 0-, 500-, or 1000- ppm Ni in drinking water. The concurrent
shams of each dose (500 or 1000 ppm) were pooled based on the lack of a
statistical difference. The numbers of living, dead, or total fetuses in
litters were not significantly different in dams receiving 0, 500, or 1000 ppm.
Fetal mass per litter was significantly decreased in litters receiving 1000-ppm
Ni (P = .007, F = 5.37, DF =2).
TABLE 4. NUMBERS OF FETUSES OF PREGNANT CD-I MICE GIVEN 0-, 500-,
OR 1000-ppm NI IN DRINKING WAT&R ON DAYS 2-17 OF GESTATION
Ni
(ppm)

0
500
1000
Li tters (No.)
51
26
7
Live fetuses*
10.4
11.6
11.7

(2.6)
(2.0)
(3.3)
Dead or resorbed fetuses*
0.78
0.46
0.86

(0.92)
(0.58)
(0.90)
Total fetuses*
11.2
12.0
12.6

(2.5)
(1.8)
(2.7)
Fetal mass (g)*
1.00
0.99
0.86
(0.12)
(0.07)
(0.09)
*Means and SD (numbers in parentheses).
8

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SECTION 4
DISCUSSION
The rate of water consumption in mice was 160 ml/kg body mass/24 h
estimated by observations of bred mice gang-caged from the 14th through the
18th day of gestation. During 24 h of this study, the consumption of Ni at
concentrations of 500 and 1000 ppm in water was estimated to be 80 and
160 mg/kg body mass, respectively. For the period of administration (2nd
through 17th day of gestation), the total consumption of Ni at concentrations
of 500 and 1000 ppm was estimated to be 1280 and 2550 mg/kg body mass,
respectively.
Only the higher consumption of Ni caused any toxic response. Only 7 of
the 27 mice that received 1000 ppm of Ni in their water were pregnant. Fetuses
from the surviving pregnancies had a 15% loss of mass (P = 0.009). The decline
in pregnancy rate and fetal mass appeared to be accompanied by maternal
toxicity, as seen in the loss of mass in non-pregnant bred mice from the same
high-dose group (Table 3). Manifestations of anomalies due to Ni consumption
were not seen.
In the mouse, the LD50 to a single IP or oral dose of nickel acetate is 32
or 420 mg/kg body mass, respectively (Nickel, 1975). In this ratio the oral
route is 13 times less effective for producing Ni toxicity. Lu et al. (1979)
9

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observed a wide range and high incidence of terata in mice fetuses administered
4.6 mg of NiCI2/kg body mass IP once between the 7th and 11th day of gestation.
On the basis of the ratio of effectiveness of an oral vs. an injected dose, the
4.6-mg IP dose used by Lu would be equivalent to a 60-mg oral dose.
In our study, we could not induce the level of fetal response, i.e., the
wide range and incidence of terata, seen by Lu after we administered Ni in
160 mg/kg orally daily (1000 ppm in drinking water). Only decreased fetal body
mass was observed. Any higher dose was so toxic for both dam and conceptuses
that the frequency did not continue. Therefore, our 160-mg/kg dose is the
closest response to Lu1s 4.6 mg/kg IP that we could achieve. As the calculated
oral equivalent of 4.6 mg/kg IP is 60 mg/kg, our 1000-ppm dose is 2.7 times
less effective. Therefore, such calculations do not adequately account for the
observed differences.
The variation in results may depend on some other factor in the route of
administration which decreases the effectiveness of low but continuous oral
intake compared to high single parenteral route. It appears, therefore, that
the fetal effects of Ni poisoning may be overestimated by experiments using
parenteral methods.
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REFERENCES
Lu, C., N. Matsumoto, and S. Iijima. 1979. Teratogenic effects of nickel
chloride on embryonic mice and its transfer to embryonic mice.
Teratology, 19:137-142.
Panel on Nickel, Sunderman, F.W., Chairman. 1975. Medical and Biological
Effects of Environmental Pollutants: Nickel. National Academy of
Sciences, Washington, DC.
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1, REPORT NO, 2-
EPA-600/1-83-007
3. RECIPIENT'S ACCESSION NO.
PBS J 22538 3
«*, TITLE anO SUBTITLE
Fetotoxic Effects of Nickel in Drinking Water
in Mice
S. REPORT DATE
June 1983
6. PERFORMING ORGANIZATION code
7. AUTHORIS)
Ezra Berman and Blair Rehnberg
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME ANO ADDRESS
Experimental Bioloay Division, HERL
U.S. EPA
Research Triangle Park, N.C. 27711
10. PROGRAM ELEMENT NO.
ACGF1A
11. CONTRACT/GRANT NO.
12. SPONSORING AGENCY NAME ANO ADDRESS
Office of Research and Development
Health Effects, Research Laboratory
US Environmental Protection Agency
Research Triangle Park, NC 27711*
13. TYPE OF REPORT AND PERIOD COVERED
14. SPONSORING AGENCY CODE
EPA/600/11
19. SUPPLEMENTARY NOTES
\ :
. \ !'
TECHNICAL REPORT DATA
(Please read Jnsirucn&ns on the reverse before compiermg/
16. ABSTRACT
Nickel chloride was administered in drinking water to pregnant nice from
the 2nd through the 17th day of gestation at nickel doses of 0, BOD, or 1000 pom.
Fetal or maternal toxicity was not seen after administration of 500 ppm of
nickel. However, the higher dose caused spontaneous abortions, loss of fetal
mass in survivors, and loss of maternal mass. The oral route of administration
via drinking water was at least 2.7 tines less effective than parenteral routes
in producing fetal effects, i
\
7.
key words anc document analysis
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