T National Cancer Institute -H I CARCINOGENESIS Technical Report Series = NO. 159 £ 1979 I O D m BIOASSAY OF OFFICIAL NTP FILE COPY DO NOT REMOVE FROM THIS OFFICE PHTHALIC ANHYDRIDE FOR POSSIBLE CARCINOGENICITY CAS No. 85-44-9 NCI-CG-TR-159 z o 6 o H 30 Ul CO U.S. DEPARTMENT OF HEALTH, EDUCATION, AND WELFARE Public Health Service National Institutes of Health (O >g CO
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T National Cancer Institute
-H I CARCINOGENESIS
Technical Report Series = NO. 159 £ 1979
I
O
D m
BIOASSAY OF
OFFICIAL NTP FILE COPY
DO NOT REMOVE FROM THIS OFFICE
PHTHALIC ANHYDRIDE
FOR POSSIBLE CARCINOGENICITY
CAS No. 85-44-9
NCI-CG-TR-159
z o 6 o H 30
Ul CO
U.S. DEPARTMENT OF HEALTH, EDUCATION, AND WELFARE Public Health Service National Institutes of Health
(O>g CO
BIOASSAY OF
PHTHALIC ANHYDRIDE
FOR POSSIBLE CARCINOGENICITY
Carcinogenesis Testing Program Division of Cancer Cause and Prevention
National Cancer Institute National Institutes of Health Bethesda, Maryland 20014
U.S. DEPARTMENT OF HEALTH, EDUCATION, AND WELFARE Public Health Service
National Institutes of Health
DHEW Publication No. (NIH) 79-1715
ii
BIOASSAY OF PHTHALIC ANHYDRIDE
FOR POSSIBLE CARCINOGENICITY
Carcinogenesis Testing Program Division of Cancer Cause and Prevention
National Cancer Institute National Institutes of Health
FOREWORD; This report presents the results of the bioassay of phthalic anhydride conducted for the Carcinogenesis Testing Program, Division of Cancer Cause and Prevention, National Cancer Institute (NCI), National Institutes of Health, Bethesda, Maryland. This is one of a series of experiments designed to determine whether selected chemicals have the capacity to produce cancer in animals. Negative results, in which the test animals do not have a greater incidence of cancer than control animals, do not necessarily mean that the test chemical is not a carcinogen, inasmuch as the experiments are conducted under a limited set of circumstances. Positive results demonstrate that the test chemical is carcinogenic for animals under the conditions of the test and indicate that exposure to the chemical is a potential risk to man. The actual determination of the risk to man from chemicals found to be carcinogenic in animals requires a wider analysis.
CONTRIBUTORS: This bioassay of phthalic anhydride was conducted by the NCI Frederick Cancer Research Center (FCRC) (1), Frederick, Maryland, operated for NCI (2) by Litton Bionetics, Inc.
The manager of the bioassay at FCRC was Dr. B. Ulland, the toxicologist was Dr. E. Gordon, and Drs. R. Cardy and D. Creasia compiled the data. Ms. S. Toms was responsible for management of data, Mr. D. Cameron for management of histopathology, Mr. L. Callahan for management of the computer branch, and Mr. R. Cypher for the management of the facilities. Mr. A. Butler performed the computer services. The histopathology of early deaths was performed by Drs. B. Ulland, R. Schueler, R. Ball, and R. Cardy. The lesions of the rats and mice were reviewed by Dr. D. G. Fairchild (D? and the diagnoses included in this report represent his interpretations.
Animal pathology tables and survival tables were compiled at EG&G Mason Research Institute (3). Statistical analyses were
ill
performed by Dr. J. R. Joiner (4) and Ms. P. L. Yong (4), using methods selected for the bioassay program by Dr. J. J. Gart (5). The chemicals used in this bioassay were analyzed at FCRC by Dr. W. Zielinsky (1). The chemical analyses were reviewed and approved by Dr. W. Lijinski (1).
This report was prepared by Tracer Jitco (4) under the direction of NCI. Those responsible for the report at Tracer Jitco were Dr. C. R. Angel, Acting Director of the Bioassay Program; Dr. S. S. Olin, Deputy Director for Science; Dr. J. F. Robens, toxicologist; Dr. R. L. Schueler, pathologist; Dr. G. L. Miller, Ms. L. A. Waitz, Ms. M. S. King, and Mr. W. D. Reichardt, bioscience writers; and Dr. E. W. Gunberg, technical editor, assisted by Ms. Y. E. Presley.
The following scientists at NCI (2) were responsible for evaluating the bioassay experiment, interpreting the results, and reporting the findings: Dr. Kenneth C. Chu, Dr. Cipriano Cueto, Jr., Dr. J. Fielding Douglas, Dr. Richard A. Griesemer, Dr. Thomas E. Hamm, Dr. William V. Hartwell, Dr. Morton H. Levitt, Dr. Harry A. Milman, Dr. Thomas W. Orme, Dr. Sherman F. Stinson, Dr. Jerrold M. Ward, and Dr. Carrie E. Whitmire.
(1) Frederick Cancer Research Center, P.O. Box B, Frederick, Maryland.
(2) Carcinogenesis Testing Program, Division of Cancer Cause and Prevention, National Cancer Institute, National Institutes of Health, Bethesda, Maryland.
(3) EG&G Mason Research Institute, 1530 East Jefferson Street, Rockville, Maryland.
(4) Tracer Jitco, Inc., 1776 East Jefferson Street, Rockville, Maryland.
(5) Mathematical Statistics and Applied Mathematics Section, Biometry Branch, Field Studies and Statistics, Division of Cancer Cause and Prevention, National Cancer Institute, National Institutes of Health, Bethesda, Maryland.
iv
SUMMARY
A bioassay of phthalic anhydride for possible carcinogenicity was conducted by administering the test chemical in feed to F344 rats and B6C3F1 mice.
Groups of 50 rats of each sex were administered phthalic anhydride at one of two doses, either 7,500 or 15,000 ppm, for 105 weeks. Matched controls consisted of 20 untreated rats of each sex. All surviving rats were killed at the end of the period of administration of the test chemical.
Groups of 50 mice of each sex were administered the test chemical at one of two doses, initially either 25,000 or 50,000 ppm, for 32 weeks. Because of excessive depressions in the amount of body weight gained in the dosed groups, the doses for the males were then reduced to 12,500 and 25,000 ppm, respectively, and the doses for the females were reduced to 6,250 and 12,500 ppm. Administration of the test chemical at the lowered doses was continued for 72 weeks. The time-weighted average doses for the males were either 16,346 or 32,692 ppm, and those for the females were either 12,019 or 24,038 ppm. Matched controls consisted of 20 untreated mice of each sex. All surviving mice were killed at the end of the period of administration of the test chemical.
Mean body weights of the high-dose male rats and of the low- and high-dose mice of each sex were lower than those of the corresponding controls; mean body weights of the low-dose male rats and of both the low- and high-dose female rats were essentially unaffected by administration of the test chemical. Depressions in the amount of body weight gained in the male and female mice were dose related throughout the bioassay. Survivals of the rats and mice were not affected by administration of the test chemical.
No tumors occurred in the rats or mice of either sex at incidences that could be clearly related to the administration of the test chemical.
It is concluded that under the conditions of this bioassay, phthalic anhydride was not carcinogenic for F344 rats or B6C3F1 mice of either sex.
v
vi
TABLE OF CONTENTS
Page
I. Introduction 1
II. Materials and Methods 5
A. Chemical 5 B. Dietary Preparation 5 C. Animals 6 D. Animal Maintenance 7 E. Subchronic Studies 9 F. Chronic Studies 12 G. Clinical and Pathologic Examinations 12 H. Data Recording and Statistical Analyses 15
III. Resul ts - Rats 21
A. Body Weights and Clinical Signs (Rats) 21 B. Survival (Rats) 21 C. Pathology (Rats) 24 D. Statistical Analyses of Results (Rats) 25
IV. Results - Mice 29
A. Body Weights and Clinical Signs (Mice) 29 B. Survival (Mice) .' 29 C. Pathology (Mice) 32 D. Statistical Analyses of Results (Mice) 33
V. Discussion 35
VI. Bibliography 37
APPENDIXES
Appendix A Summary of the Incidence of Neoplasms in Rats Administered Phthalic Anhydride in the Diet 39
Table Al Summary of the Incidence of Neoplasms in Male Rats Administered Phthalic Anhydride in the Diet 41
vii
Page
Table A2 Summary of the Incidence of Neoplasms in Female Rats Administered Phthalic Anhydride in the Diet 45
Appendix B Summary of the Incidence of Neoplasms in Mice Administered Phthalic Anhydride in the Diet .. 49
Table Bl Summary of the Incidence of Neoplasms in Male Mice Administered Phthalic Anhydride in the Diet 51
Table B2 Summary of the Incidence of Neoplasms in Female Mice Administered Phthalic Anhydride in the Diet 54
Appendix C Summary of the Incidence of Nonneoplastic Lesions in Rats Administered Phthalic Anhydride in the Diet 59
Table Cl Summary of the Incidence of Nonneoplastic Lesions in Male Rats Administered Phthalic Anhydride in the Diet 61
Table C2 Summary of the Incidence of Nonneoplastic Lesions in Female Rats Administered Phthalic Anhydride in the Diet 68
Appendix D Summary of the Incidence of Nonneoplastic Lesions in Mice Administered Phthalic Anhydride in the Diet 75
Table Dl Summary of the Incidence of Nonneoplastic Lesions in Male Mice Administered Phthalic Anhydride in the Diet 77
Table D2 Summary of the Incidence of Nonneoplastic Lesions in Female Mice Administered Phthalic Anhydride in the Diet 81
Appendix E Analyses of the Incidence of Primary Tumors in Rats Administered Phthalic Anhydride in the Diet 87
viii
Page
Table El Analyses of the Incidence of Primary Tumors in Male Rats Administered Phthalic Anhydride in the Diet 89
Table E2 Analyses of the Incidence of Primary Tumors in Female Rats Administered Phthalic Anhydride in the Diet 94
Appendix F Analyses of the Incidence of Primary Tumors in Mice Administered Phthalic Anhydride in the Diet 99
Table Fl Analyses of the Incidence of Primary Tumors in Male Mice Administered Phthalic Anhydride in the Diet 101
Table F2 Analyses of the Incidence of Primary Tumors in Female Mice Administered Phthalic Anhydride in the Diet 104
TABLES
Table 1 Phthalic Anhydride Subchronic Feeding Studies in Rats and Mice 10
Table 2 Phthalic Anhydride Chronic Feeding Studies in Rats 13
Table 3 Phthalic Anhydride Chronic Feeding Studies in Mice 14
FIGURES
Figure 1 Growth Curves for Rats Administered Phthalic Anhydride in the Diet 22
Figure 2 Survival Curves for Rats Administered Phthalic Anhydride in the Diet 23
Figure 3 Growth Curves for Mice Administered Phthalic Anhydride in the Diet 30
IX
Page
Figure 4 Survival Curves for Mice Administered Phthalic Anhydride in the Diet 31
x
I. INTRODUCTION
Phthalic anhydride (CAS
85-44-9; NCI C03601) is an
important chemical intermediate
in the plastics industry. From
it are derived numerous
Phthalic anhydride phthalate esters that function
as plasticizers in synthetic
resins (Knuth, 1973; Noller, 1966). Phthalic anhydride itself is
f used as a monomer for synthetic resins such as glyptal, the alkyd
resins, and the polyester resins (Noller, 1966). Phthalic
anhydride is a precursor of anthraquinone, phthalein, rhodamine,
phthalocyanine, fluorescein, and xanthene dyes (Towle et al.,
1968; Noller, 1966). Reaction of phthalic anhydride with ammonia
yields phthalimide, a useful reagent in the synthesis of primary
amines, the agricultural fungicide phaltan, and thalidomide
(Noller, 1966). Other reactions yield phenolphthalein, benzoic
(cerebrum and cerebellum), and all tissue masses. Peripheral
blood smears also were made for all animals, whenever possible.
A few tissues from some animals were not examined, particularly
from those animals that may have died early, been missing, or
been in advanced states of cannibalization or autolysis. Thus,
the number of animals from which particular organs or tissues
were examined microscopically varies and does not necessarily
represent the number of animals that were placed on study in each
group.
H. Data Recording and Statistical Analyses
Pertinent data on this experiment have been recorded in an
automatic data processing system, the Carcinogenesis Bioassay
15
Data System (Linhart et al., 1974). The data elements include
descriptive information on the chemicals, animals, experimental
design, clinical observations, survival, body weight, and
individual pathologic results, as recommended by the
International Union Against Cancer (Berenblum, 1969). Data
tables were generated for verification of data transcription and
for statistical review.
These data were analyzed using the appropriate statistical
techniques described in this section. Those analyses of the
experimental results that bear on the possibility of
carcinogenicity are discussed in the statistical narrative
sections.
Probabilities of survival were estimated by the product-limit
procedure of Kaplan and Meier (1958) and are presented in this
report in the form of graphs. Animals were statistically
censored as of the time that they died of other than natural
causes or were found to be missing; animals dying from natural
causes were not statistically censored. Statistical analyses for
a possible dose-related effect on survival used the method of Cox
(1972) for testing two groups for equality and Tarone's (1975)
extensions of Cox methods for testing for a dose-related trend.
One-tailed P values have been reported for all tests except the
16
departure from linearity test, which is only reported when its
two-tailed P value is less than 0.05.
The incidence of neoplastic or nonneoplastic lesions has been
given as the ratio of the number of animals bearing such lesions
at a specific anatomic site (numerator) to the number of animals
in which that site is examined (denominator). In most instances,
the denominators included only those animals for which that site
was examined histologically. However, when macroscopic
examination was required to detect lesions prior to histologic
sampling (e.g., skin or mammary tumors), or when lesions could
have appeared at multiple sites (e.g. , lymphomas), the
denominators consist of the numbers of animals necropsied. The
purpose of the statistical analyses of tumor incidence is to
determine whether animals receiving the test chemical developed a
significantly higher proportion of tumors than did the control
animals. As a part of these analyses, the one-tailed Fisher
exact test (Cox, 1970) was used to compare the tumor incidence of
a control group with that of a group of dosed animals at each
dose level. When results for a number of dosed groups (k) are
compared simultaneously with those for a control group, a
correction to ensure an overall significance level of 0.05 may be
made. The Bonferroni inequality (Miller, 1966) requires that the
P value for any comparison be less than or equal to 0.05/k. In
17
cases where this correction was used, it is discussed in the
narrative section. It is not, however, presented in the tables,
where the Fisher exact P values are shown.
The Cochran-Armitage test for linear trend in proportions, with
continuity correction (Armitage, 1971), was also used. Under the
assumption of a linear trend, this test determines if the slope
of the dose-response curve is different from zero at the one-
tailed 0.05 level of significance. Unless otherwise noted, the
direction of the significant trend is a positive dose relation
ship. This method also provides a two-tailed test of departure
from linear trend.
A time-adjusted analysis was applied when numerous early deaths
resulted from causes that were not associated with the formation
of tumors. In this analysis, deaths that occurred before the
first tumor was observed were excluded by basing the statistical
tests on animals that survived at least 52 weeks, unless a tumor
was found at the anatomic site of interest before week 52. When
such an early tumor was found, comparisons were based exclusively
on animals that survived at least as long as the animal in which
the first tumor was found. Once this reduced set of data was
obtained, the standard procedures for analyses of the incidence
18
of tumors (Fisher exact tests, Cochran-Armitage tests, etc.) were
followed.
When appropriate, life-table methods were used to analyze the
incidence of tumors. Curves of the proportions surviving without
an observed tumor were computed as in Saffiotti et al. (1972).
The week during which an animal died naturally or was sacrificed
was entered as the time point of tumor observation. Cox's methods
of comparing these curves were used for two groups; Tarone's
extension to testing for linear trend was used for three groups.
The statistical tests for the incidence of tumors which used
life-table methods were one-tailed and, unless otherwise noted,
in the direction of a positive dose relationship. Significant
departures from linearity (P less than 0.05, two-tailed test)
were also noted.
The approximate 95 percent confidence interval for the relative
risk of each dosed group compared with its control was calculated
from the exact interval on the odds ratio (Gart, 1971). The
relative risk is defined as pf/p where p is the true
binomial probability of the incidence of a specific type of tumor
in a dosed group of animals and p is the true probability of
the spontaneous incidence of the same type of tumor in a control
group. The hypothesis of equality between the true proportion of
19
a specific tumor in a dosed group and the proportion in a control
group corresponds to a relative risk of unity. Values in excess
of unity represent the condition of a larger proportion in the
dosed group than in the control.
The lower and upper limits of the confidence interval of the
relative risk have been included in the tables of statistical
analyses. The interpretation of the limits is that in
approximately 95% of a large number of identical experiments, the
true ratio of the risk in a dosed group of animals to that in a
control group would be within the interval calculated from the
experiment. When the lower limit of the confidence interval is
greater than one, it can be inferred that a statistically
significant result (P less than 0.025 one-tailed test when the
control incidence is not zero, P less than 0.050 when the control
incidence is zero) has occurred. When the lower limit is less
than unity, but the upper limit is a greater than unity, the
lower limit indicates the absence of a significant result while
the upper limit indicates that there is a theoretical possibility
of the induction of tumors by the test chemical, which could not
be detected under the conditions of this test.
20
III. RESULTS - RATS
A. Body Weights and Clinical Signs (Rats)
The mean body weights of the high-dose male rats were lower than
those of the corresponding controls from week 13 to the end of
the bioassay; mean body weights of the low-dose males and both
the low- and high-dose females were essentially unaffected by
administration of the test chemical (figure 1). Arched back,
rough hair coat, ulceration, and corneal opacity occurred only in
dosed groups, but at low incidences. Wasting and tissue masses
were common to the dosed and control groups. Fluctuation in the
growth curves may be due to mortality; as the size of a group
diminishes, the mean body weight may be subject to variation.
B. Survival (Rats)
The Kaplan and Meier curves estimating the probabilities of
survival for male and female rats administered phthalic anhydride
in the diet at the doses of this bioassay, together with those of
the matched controls, are shown in figure 2. The result of the
Tarone test for dose-related trend in mortality is not significant
21
D 9 8
8 « B 5 8 @ i ° A
A A A A A
O
Io u!
300
^ Q O m z 200
e 1001 I
D
O
A
MALE RATS
MATCHED CONTROL
LOW DOSE
HIGH DOSE
40 50 60 70
TIME ON STUDY (WEEKS)
S3 UI
D ffl
> a B o m B 8 8
ilBi
FEMALE RATS
D MATCHED CONTROL
O LOW DOSE
A HIGH DOSE
TIME ON STUDY (WEEKS)
Figure 1. Growth Curves for Rats Administered Phthalic Anhydride in the Diet
22
^
0.90 •
0.80
_! 0.70 •
U_ 0
0.50
t
5 CO o ccp^
000
0.90
_l °'7° "
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PR
OB
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3 O
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MALE RA1 •s
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^ HIGH DOSE
fee" -••-••-• •-••-..
FEMALE RX vrs
3 MATCH ED CO
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1 HIGH DOSE
A-"" < 3o, )Gj
A-..;
|
45 60 75
TIME ON STUDY (WEEKS)
4 •••OrV"""* |^^f *
"rW&JW
Foi
TIME ON STUDY (WEEKS)
Figure 2. Survival Curves for Rats Administered Phthalic Anhydride in the Diet
23
in either sex. In male rats, an indicated departure from linear
trend (P = 0.037) is observed, due to the earlier mortality of
the control group when compared with that of either the high- or
low-dose group. The results of the Cox test applied to any two
of the three groups show no statistically significant difference
between groups of any pair.
In male rats, 36/50 (72%) of the high-dose group, 44/50 (88%) of
the low-dose group, and 14/20 (70%) of the control group lived to
the end of the bioassay. In females, 41/50 (82%) of the
high-dose group, 42/50 (84%) of the low-dose group, and 17/20
(85%) of the control group lived to the end of the bioassay.
Sufficient numbers of rats of each sex were at risk for the
development of late-appearing tumors.
C. Pathology (Rats)
Histopathologic findings on neoplasms in rats are summarized in
Appendix A, tables Al and A2; findings on nonneoplastic lesions
are summarized in Appendix C, tables Cl and C2.
24
By inspection, there appeared to be no difference between the
dosed and control groups in frequency or distribution of
neoplasms, except for malignant lymphoma in the female rats. The
incidence of malignant lymphoma in the control females was 1/20;
in low-dose females, 11/50; in high-dose females, 4/50. Due to
the high and fluctuating incidence of this type of malignant
lymphoma in control F344 rats, the apparent differences in
incidences of the tumor in the dosed and control groups were not
considered to be compound related.
Severe chronic inflammatory, degenerative, or proliferative
lesions frequently seen in aged rats occurred with approximately
equal frequency and severity in the dosed and control groups of
animals.
Based on the histopathologic examination, there was no conclusive
evidence for the carcinogenicity of phthalic anhydride in F344
rats under the conditions of this bioassay.
D. Statistical Analyses of Results (Rats)
Tables El and E2 in Appendix E contain the statistical analyses
of the incidences of those primary tumors that occurred in at
25
least two animals of one group and at an incidence of at least 5%
in one or more than one group.
In female rats, the result of the Cochran-Armitage test for
positive dose-related trend in the incidence of alveolar/
bronchiolar adenomas is significant (P = 0.020), but the results
of the Fisher exact test are not significant. The results of the
statistical tests on the incidences of alveolar/bronchiolar
carcinomas and of alveolar/bronchiolar adenomas or carcinomas are
not significant. In male rats, the results of the statistical
tests on the incidences of lung tumors are not significant.
A departure from linear trend (P = 0.019) is found in the
incidence of lymphoma in female rats, due to the relatively large
proportion of 11/50 (22%) in the low-dose group compared with
4/50 (8%) in the high-dose group and 1/20 (5%) in the control
group. The results of the Fisher exact test are not
significant. Current historical records at this laboratory
indicate an incidence of lymphoma in female rats of 14/285
(4.9%), and, although the majority of the control groups had
incidences of less than 5%, one control grout) was observed to
have an incidence as high as 4/20 (20%). Since the results of
the Fisher exact test were not significant and since the
historical data concerning lymphoma indicates the possibility of
26
an occasional high spontaneous rate of lymphoma, the evidence of
association of the lymphomas in the dosed group of female rats
with the chemical is questionable.
A significant dose-related trend (P = 0.037) in the negative
direction is observed in the incidence of pheochromocytomas of
the adrenal in male rats.
In each of the 95% confidence intervals for relative risk, shown
in the tables, one is included; this indicates the absence of
significant positive results. It should also be noted that each
of the intervals has an upper limit greater than one, indicating
the theoretical possibility of the induction of tumors by
phthalic anhydride, which could not be detected under the
conditions of this test.
27
28
IV. RESULTS - MICE
A. Body Weights and Clinical Signs (Mice)
Mean body weights of dosed male and female mice were lower than
those of corresponding controls throughout the bioassay, and
depressions in the amount of body weight gained were dose related
(figure 3). Tissue masses were observed at low incidences and
were common to the dosed and control groups. Fluctuation in the
growth curves may be due to mortality; as the size of a group
diminishes, the mean body weight may be subject to variation.
B. Survival (Mice)
The Kaplan and Meier curves estimating the probabilities of
survival for male and female mice administered phthalic anhydride
in the diet at the doses of this bioassay, together with those of
the matched controls, are shown in figure 4. The result of the
Tarone test for dose-related trend in mortality is not
significant in either sex.
j In male mice, 47/50 (94%) of the high-dose group, 37/50 (74%) of
29
O GO O O
g >.Q O m
30 nB8°AA
HQ5A
00
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A
o A
A
A AA . AA AA
5 20- i
MALE MICE
D MATCHED CONTROL
O LOW DOSE
A HIGH DOSE
10 20 30 40 50 60 70 90 100
TIME ON STUDY (WEEKS)
O % O 00
A :oDocP AA
o of °°aP
<
FEMALE MICE
D MATCHED CONTROL
O LOW DOSE
A HIGH DOSE
10 20 30 10 60 70 80 90 100 110
TIME ON STUDY (WEEKS)
Figure 3. Growth Curves for Mice Administered Phthalic Anhydride in the Diet
30
110
PR
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the low-dose group, and 17/20 (85%) of the control group survived
to the end of the bioassay. In females, 40/50 (80%) of the
high-dose group, 45/50 (90%) of the low-dose group, and 16/20
(80%) of the control group survived to the end of the bioassay.
Sufficient numbers of mice of each sex were at risk for the
development of late-appearing tumors.
C. Pathology (Mice)
Histopathologic findings on neoplasms in mice are summarized in
Appendix B, tables Bl and B2; findings on nonneoplastic lesions
are summarized in Appendix D, tables Dl and D2.
Several chronic inflammatory, degenerative, or proliferative
lesions frequently seen in aged laboratory mice occurred with
approximately equal frequency and severity in the dosed and
control groups of animals.
Based on the histopathologic examinations, the nature, incidence,
or severity of the lesions observed provided no clear evidence of
carcinogenic effect of the phthalic anhydride on B6C3F1 mice
under the conditions of this bioassay.
32
D. Statistical Analyses of Results (Mice)
Tables Fl and F2 in Appendix F contain the statistical analyses
of the incidences of those primary tumors that occurred in at
least two animals of one group and at an incidence of at least 5%
in one or more than one group.
The results of the Cochran-Armitage test for positive dose-
related trend in incidences of tumors and those of the Fisher
exact test comparing the incidence of tumors in the control group
with that in each dosed group in the positive direction are not
significant in either sex.
In male mice negative results are observed in the incidence of
alveolar/bronchiolar carcinomas. A significant dose-related
trend in the negative direction (P = 0.025) is also observed in
the incidence of adenomas of the thyroid in the female mice.
In each of the 95% confidence intervals for relative risk, shown
in the tables, the value of one or less than one is included;
this indicates the absence of significant positive results. It
should also be noted that each of the intervals (except for that
of the incidence of alveolar/bronchiolar carcinomas of the lung
in low-dose male mice) has an upper limit greater than one,
33
indicating the theoretical possibility of the induction of tumors
by phthalic anhydride, which could not be detected under the
conditions of this test.
34
V. DISCUSSION
Mean body weights of the high-dose male rats and of the low- and
high-dose mice of each sex were lower than those of the
corresponding controls; mean body weights of the low-dose male
rats and of both the low- and high-dose female rats were
essentially unaffected by administration of the test chemical.
Depressions in the amount of body weight gained in the male and
female mice were dose related throughout the bioassay. Other
clinical signs were common to dosed and control groups of the
rats and mice or occurred only at low incidences. Survivals of
the rats and mice were not affected by administration of the test
chemical. Assays of the dosed feed mixtures indicated that they
may have been unstable under the conditions of use.
In the female rats, alveolar/bronchiolar adenomas occurred at
incidences that were dose related in the positive direction (P =
0.020), but, in direct comparisons, were not significantly higher
in either of the dosed groups than in the control group (controls
0/20, low-dose 0/50, high-dose 5/50). Neither these adenomas in
the high-dose female rats nor any tumors in the dosed groups of
male rats or male or female mice can be clearly related to
administration of the test chemical.
35
It is concluded that under the conditions of this bioassay,
phthalic anhydride was not carcinogenic for F344 rats or B6C3F1
mice of either sex.
36
VI. BIBLIOGRAPHY
Armitage, P., Statistical Methods in Medical Research, John Wiley & Sons, Inc., New York, 1971, pp. 362-365.
Baader, E. W., Erbrankungen durch Phthalsaure und ihre Verbindungen. Arch. Gewerbepathol. Gewerbehyg. L3:419-453, 1955.
Berenblum, I., ed., Carcinogenicity Testing! A Report o£ the Panel on Careinogenicity of_ the Cancer Research Commission of the UICC, Vol. 2_. International Union Against Cancer, Geneva, 1969.
Cox, D.R., Regression models and life tables. J_. R. Statist. Soc. B 34_:187-220, 1972.
Cox, D. R., Analysis £f_ Binary Data, Methuen & Co., Ltd. London, 1970, pp. 48-52.
Fassett, D. W., Organic acids, anhydrides, lactones, acid halides and amides, thioacids. In: Patty, F. A., ed., Industrial Hygiene and Toxicology, Vol. II, Interscience Publishers, New York, 1963T p. 1824-1825.
Gart, J. J., The comparison of proportions: a review of significance tests, confidence limits and adjustments for stratification. Rev. Int. Stat. Inst. 39:148-169, 1971.
Giam, C. S., Chan, H. S., Neff, G. S., and Atlas, E. L., Phthalate ester plasticizers: a new class of marine pollutant. Science 199:419-421, 1978.
Guess, W. L., Jacob, J., and Autian, J., A study of polyvinyl chloride - blood bag assemblies. Drug Intelligence 1^:120-121,125-127, 1967.
Kaplan, E. L. and Meier, P., Nonparametric estimation from incomplete observations. J_. Am. Statist. Assoc. 5_3_:457-481, 1958.
Knuth, C. J., Plasticizers. In: Encyclopedia £f_ Chemistry, Hampel, C. A. and Hawley, G. G., eds., Van Nostrand Reinhold Co., New York, 1973, pp. 863-865.
Linhart, M. S., Cooper, J. A., Martin, R. L., Page, N. P., and Peters, J. A., Carcinogenesis bioassay data system. Comp. and Biomed. Res. 7:230-248, 1974.
37
Merlevede, E. and Elskens, J., Les intoxications dues a 1" anhydride phtalique, 1' anhydride maleique et aux phtalates. Arch. Bel. Med. Soc. 1M10):445-457, 1957.
Miller, R. G., Jr., Simultaneous Statistical Inference, McGraw-Hill Book Co., New York, 1966, pp. 6-10.
National Institute for Occupational Safety and Health, Suspected Carcinogens - A Subfile of the Registry of Toxic Effectsof Chemical Substances, National Institute for Occupational Safety and Health, Cincinnati, Ohio, 1976, p. 136.
Noller, C. R., Aromatic carboxylic acids and their derivatives. In: Chemistry of Organic Compounds, W. B. Saunders Co., Philadelphia, 196T", pp. 602-605.
Policard, A., Gauthier, G., Hugonnier, R., and Roche, L., L'Intoxication par I1anhydride phtalique. Arch. Mai. Profess. 10:1, 1949.
Saffiotti, U., Montesano, R., Sellakumar, A. R., Cefis, F., and Kaufman, D. G., Respiratory tract carcinogenesis in hamsters induced by different numbers of administrations of benzo(a) pyrene and ferric oxide. Cancer Res. 32/1073 -1081, 1972.
Tarone, R. E., Tests for trend in life table analysis. Biometrika 62; 679-682, 1975.
Towle, P. H., Baldwin, R. H., and Meyer, D. H., Phthalic acids and other benzenepolycarboxylic acids. In: Kirk-Othmer Encyclopedia of Chemical Technology, Vol. 15, Mark, H. P., McKetta, J. J,, Jr., Othmer, D. F., eds., John Wiley & Sons, Inc., New York, 1968, pp. 444-487.
United States International Trade Commission, Synthetic Organic Chemicals - United States Production and Sales,1976,USITC Publication 833, United States International Trade Commission, Washington, B.C., 1977a.
United States International Trade Commission, Imports of Benezoid Chemicals and Products, 1976. USITC Publication 8"28,United States International Trade Commission, Washington, D.C., 1977b.
Zhilova, N. A. and Kasparov, A. A., Phthalic anhydride and N-nitrosodiphenylamine (Vulcalent A). Chem. Abstr. 71:280, 1969.
38
APPENDIX A
SUMMARY OF THE INCIDENCE OF NEOPLASMS IN
RATS ADMINISTERED PHTHALIC ANHYDRIDE IN THE DIET
39
40
TABLE A1.
SUMMARY OF THE INCIDENCE OF NEOPLASMS IN MALE RATS ADMINISTERED PHTHALIC ANHYDRIDE IN THE DIET
MATCHED CONTROL LOW DOSE HIGH DOSE
ANIHAIS INITIALLY IN STUDY 20 50 50 ANIBA1S KiCECESIEC 20 50 50 ANIBJLS EXAMINED HISTOPATHOIOGICALLY 20 50 50
*MAHMABY GLAND ADENOMA, NCS CYSTADENOBA, NOS FIBBCADENCMA
*PBEPUIIAL GLAND CABCINOKA,NCS
*CLIIOBAL GLAND ACENCHA, NCS
J01EBUS ENDCBETEIAL STROMALCAECINOSABCOMA
8£EVCUS SYSTEM
tEEAIN
(47)
(49) 219
(49) 1
3
(50) 3
(50) 1
6
(50)
(50)
(50) 61
(50) 1CARCINOMA, NOS, INVASIVE
MIDULLCBLASTCMA 1 (2%)
SPECIAL SENSE CBGANS
NCNE
f NUKEEB OF JNIMALS HITH TISSUE EXAEINED HICRCSCCPICAL1Y * NUBBEE OF ANIMALS NECBOFSIED
(4%) (39%)
(2%)
(6%)
(6%)
(2%)
(12%)
(12%) (2%)
(2%)
46
TABLE A2. FEMALE RATS: NEOPLASMS (CONTINUED)
MATCHED CONTROL LOW DOSE HIGH DOSE
M U S C U I C S K E I E I A I S Y S I E K
N C K E
B C E Y C A V I T I E S
N O N E
A L L C T H E K S Y S T E M S
N C N I
A N I M A L D I S P O S I T I O N S U P f l A E Y
A N I K A L S I N I T I A L L Y IK S T U D Y 20 50 50 N A T U B A L D £ A T H 5 > 2 6 2 M C E I E U N D SACHIFICE 1 2 7 S C H E E U L E C SACfi lFICE A C C I D E N T A L L Y K I L L E D T E R M I N A L S A C B I F I C E 17 42 41 A K I K A L K I S S I N G
31 I N C L D D E S A U T C L Y Z E D A N I M A L S
# N U B B E E C F A N I M A L S W I T H T I S S U E E X A M I N E D HICBOSCOPICALLY * N U M E E B O F A N I M A I S N E C E O F S I E D
47
TABLE A2. FEMALE RATS: NEOPLASMS (CONTINUED)
MATCHED CONTROL LOW DOSE HIGH DOSE
T U H O B S O B B A B Y
TC1AL A K J H A I S HUH EEIBABY 1UHOES* 13 37 36 TCTAL P B I B A B Y TUBOtS 18 58 53
TOTAL A N I H A I S KITH B E N I G N TDHCBS 12 27 32 TOTAL E E S I G N TUHOJ5S 15 38 44
TOTAL A N I M A L S HUH M A L I G N A N T TDHOES 3 16 8 ICT6L M A L I G N A N T TDHOES 3 20 9
A S I B A L S INITIALLY IN STUDY 20 50 50 N A T U R A L DiATHi HOBIBDND SACBIFICE SCHEDULED SACBIFICE ACCIEENTAILY KILLED 6 1EBHINAL SACRIFICE 17 37 47 AKIBAL MISSING 1
TOTAL ANIHAIS KITH BENIGN TUMOBS 2 6 5 TOTAL BBNI6N TUHOBS 2 6 6
TC1AL ASIBALS WITH BAIIGNANT TUHOBS 10 16 15 TOTAL MALIGNANT 1UMOES 11 20 15
TOTAL ANIHALS WITH SECONDARY TUMOBSt 1 1 TOTAL SfCCNDABY TUHOBS 1 1
TOTAL AKIBALS WITH TUMOBS UNCEETAINBENIGN OB MALIGNANT
TOTAL UNCEBTAIN 1UBCBS
TOTAL ANIHALS WITH 1UHOBS UNCEBTAINPBIBABY CB HE1ASTA1IC TOTAL UNCEBTAIN TUHOBS
* PBIHABY 1U«OBS: ALI TUHORS EXCEPT SECONDARY TUHOBS f SECCNCABY 1UMOBS: HETASTATIC TUHOBS OB TOHORS INVASIVE INTO AN ADJACENT ORGAN
53
TABLE B2.
SUMMARY OF THE INCIDENCE OF NEOPLASMS IN FEMALE MICE ADMINISTERED PHTHALIC ANHYDRIDE IN THE DIET
A N I M A L S I M T I A L L Y I N £1UDY A N I M A L S M I S S I N G A N I M A L S N E C B C F S I E C A N I H A L S E X A M I N E D H I S T O P A T H O L O G I C A L L Y
I N l i G C H I N I A f i Y S Y S I E M
*SUBCU1 TISSUE N E D B I L E M C M A , M A L I G N A N T
B E S E I F A T C B Y S Y S T E M
t t iUNG A I V E C L A B / E B C N C H I O I A B A D E N O M A A I V E C L A B / E B O N C H I O L A B C A E C I N O M A C S T E C S A E C C M A , M E I A S T A T I C
H E B A T C P C I H T 1 C SYS1EH
*HUL1IPIE O E G A N S M A L I G N A N T L Y M P H C K A , N O S M A L I G . L Y M P H O M A , HISTICCYTIC T Y P E PLASMA-CELL 1QHOE
*BLCCD L E U K E M I A , NOS
*HEflATOPOIETIC SYSTEM H A L I G l i A N T L Y B F H O M A , N O S
ISPLEEN H E H A N G I O M A H E M A N G I C S A B C O M A
IHESBNTEEIC L. NODE M A L I G N A N T L Y H P H O M A , N C S H S L I G . L Y M P H O M A , HISTIOCYTIC 1YPE
*LI VEE B A L I G . L Y E E H O M A , HISTIOCJTIC TYPE
>£> (b) Number of tumor-bearing animals/number of animals examined at site (percent).
(c) Beneath the incidence of tumors in the control group is the probability level for the Cochran-Armitage test when P is less than 0.05, otherwise, not significant (N.S.) is indicated. Beneath the incidence of tumors in a dosed group is the probability level for the Fisher exact test for the comparison of that dosed group with the matched-control group when P is less than 0.05; otherwise not significant (N.S.) is indicated.
(d) A negative trend (N) indicates a lower incidence in a dosed group than in a control group.
(e) The probability level for departure from linear trend is given when P is less than 0.05 for any comparison.
(f) The 95% confidence interval of the relative risk between each dosed group and the control group.
Table E2. Analyses of the Incidence of Primary Tumors in Female Rats Administered Phthalic Anhydride in the Diet (a)
Topography: Morphology
Lung: Alveolar/Bronchiolar Adenoma (b)
P Values (c,d)
Relative Risk (f) Lower Limit Upper Limit
Weeks to First Observed Tumor
Lung: Alveolar/Bronchiolar Carcinoma (b)
P Values (c,d)
Relative Risk (f) Lower Limit Upper Limit
Weeks to First Observed Tumor
Matched Control
0/20 (0)
P = 0.020
—
1/20 (5)
N.S.
105
Low Dose
0/50 (0)
.
— — —
—
3/50 (6)
N.S.
1.200 0.106 61.724
101
High Dose
5/50 (10)
N.S.
Infinite 0.525 Infinite
105
1/50 (2)
N.S.
0.400 0.005 30.802
102
Table E2. Analyses of the Incidence of Primary Tumors in Female Rats Administered Phthalic Anhydride in the Diet (a)
(b) Number of tumor-bearing animals/number of animals examined at site (percent). oo
(c) Beneath the incidence of tumors in the control group is the probability level for the Cochran-Armitage test when P is less than 0.05, otherwise, not significant (N.S.) is indicated. Beneath the incidence of tumors in a dosed group is the probability level for the Fisher exact test for the comparison of that dosed group with the matched-control group when P is less than 0.05; otherwise not significant (N.S.) is indicated.
(d) A negative trend (N) indicates a lower incidence in a dosed group than in a control group.
(e) The probability level for departure from linear trend is given when P is less than 0.05 for any comparison.
(f) The 95% confidence interval of the relative risk between each dosed group and the control group.
APPENDIX F
ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS
IN MICE ADMINISTERED PHTHALIC ANHYDRIDE IN THE DIET
99
100
Table Fl. Analyses of the Incidence of Primary Tumors in Male Mice Administered Phthalic Anhydride in the Diet (a)
Table Fl. Analyses of the Incidence of Primary Tumors in Male Mice Administered Phthalic Anhydride in the Diet (a)
(continued)
(a) Dosed groups received time-weighted average doses of 16,346 or 32,692 ppm.
(b) Number of tumor-bearing animals/number of animals examined at site (percent).
(c) Beneath the incidence of tumors in the control group is the probability level for the Cochran-Armitage test when P is less than 0.05, otherwise, not significant (N.S.) is indicated. Beneath the incidence of tumors in a dosed group is the probability level for the Fisher exact test for the comparison of that dosed group with the matched-control group when P is less than 0.05; otherwise not significant (N.S.) is indicated.
(d) A negative trend (N) indicates a lower incidence in a dosed group than in a control group.
(e) The probability level for departure from linear trend is given when P is less than 0.05 for any comparison.
(f) The 95% confidence interval of the relative risk between each dosed group and the control group.
Table F2. Analyses of the Incidence of Primary Tumors in Female Mice Administered Phthalic Anhydride in the Diet (a)
Topography: Morphology
Lung: Alveolar/Bronchiolar Carcinoma (b)
P Values (c,d)
Relative Risk (f) Lower Limit Upper Limit
Weeks to First Observed Tumor
Lung: Alveolar/Bronchiolar Carcinoma or Adenoma (b)
P Values (c,d)
Relative Risk (f) Lower Limit Upper Limit
Weeks to First Observed Tumor
Matched Low Control Dose
1/20 (5) 3/49 (6)
N.S. N.S.
1.224 0.108 62.958
99 44
1/20 (5) 6/49 (12)
N.S. N.S.
2.449 0.332
110.166
99 44
High Dose
1/48 (2)
N.S.
0.417 0.006 32.058
104
2/48 (4)
N.S.
0.833 0.047 48.155
104
Table F2. Analyses of the Incidence of Primary Tumors in Female Mice Administered Phthalic Anhydride in the Diet (a)
(a) Dosed groups received time-weighted average doses of 12,019 or 24,038 ppm.
(b) Number of tumor-bearing animals/number of animals examined at site (percent).
(c) Beneath the incidence of tumors in the control group is the probability level for the Cochran-Armitage test when P is less than 0.05, otherwise, not significant (N.S.) is indicated. Beneath the incidence of tumors in a dosed group is the probability level for the Fisher exact test for the comparison of that dosed group with the matched-control group when P is less than 0.05; otherwise not significant (N.S.) is indicated.
(d) A negative trend (N) indicates a lower incidence in a dosed group than in a control group.
(e) The probability level for departure from linear trend is given when P is less than 0.05 for any comparison.
(f) The 95% confidence interval of the relative risk between each dosed group and the control group.
Review of the Bioassay of Phthalic Anhydride* for Carcinogenicity by the Data Evaluation/Risk Assessment Subgroup of the Clearinghouse on Environmental Carcinogens
December 13, 1978
The Clearinghouse on Environmental Carcinogens was established in May, 1976, in compliance with DREW Committee Regulations and the Provisions of the Federal Advisory Committee Act. The purpose of the Clearinghouse is to advise the Director of the National Cancer Institute on the Institute's bioassay program to identify and evaluate chemical carcinogens in the environment to which humans may be exposed. The members of the Clearinghouse have been drawn from academia, industry, organized labor, public interest groups, and State health officials. Members have been selected on the basis of their experience in carcinogenesis or related fields and, collectively, provide expertise in chemistry, biochemistry, biostatistics, toxicology, pathology, and epidemiology. Representatives of various Governmental agencies participate as ad hoc members. The Data Evaluation/Risk Assessment Subgroup of the Clearinghouse is charged with the responsibility of providing a peer review of reports prepared on NCI-sponsored bioassays of chemicals studied for carcinogenicity. It is in this context that the below critique is given on the bioassay of Phthalic Anhydride.
The reviewer for the report on the bioassay of Phthalic Anhydride agreed with the conclusion that the compound was not carcinogenic under the conditions of test. After a brief description of the experimental design, he said that both the dose levels tested and the animal survival were adequate. There was no objection to the reviewer's motion that the report on the bioassay of Phthalic Anhydride be accepted as written.
Clearinghouse Members Present:
Arnold L. Brown (Chairman), University of Wisconsin Medical School Joseph Highland, Environmental Defense Fund William Lijinsky, Frederick Cancer Research Center Henry Pitot, University of Wisconsin Medical Center Verne A. Ray, Pfizer Medical Research Laboratory Verald K. Rowe, Dow Chemical USA Michael Shimkin, University of California at San Diego Louise Strong, University of Texas Health Sciences Center Kenneth Wilcox, Michigan State Health Department
Subsequent to this review, changes may have been made in the bioassay report either as a result of the review or other reasons. Thus, certain comments and criticisms reflected in the review may no longer be appropriate.
107
JJ.U.S. GOVERNMENT P R I N T I N G OFFICE: 1979-281-217/3012