TOXICOLOGY AND APPLIED PHARMACOLOGY 29,3W6 (1974)

Mutagenic and Antifertility Sensitivities of Mice to

Di-2-ethylhexyl Phthalate (DEHP) and Dimethoxyethyl

Phthalate (DMEP)

A. R. SINGH, W. H. LAWRENCE AND J. AUTIAN

Materials Science Toxicology Laboratories, College of Dentistry and College of Pharmacy, University of Tennessee Medical Units, Memphis, Tennessee 38163

Received October 1,1973; accepted December 27,1973

Mutagenic and Antifertility Sensitivities of Mice to Di-2-ethylhexyl Phthalate (DEHP) and Dimethoxyethyl Phthalate (DMEP). SINGH, A. R., LAWRENCE, W. H. AND AUITAN, J. (1974). Toxicol. Appl. Pharmacol. 29,

3546. Groups of male ICR mice were treated with a single ip injection of undiluted DEHP or DMEP representing l/3,1/2, and 2/3 of the acute LD50 dose. Immediately after injection, each male mouse was placed with 2 un- treated virgin females for mating; the females were replaced on a weekly basis for a 1Zweek period. The high dose of both phthalates produced a distinct reduction in incidence of pregnancies, with lesser effects sometimes observed from the lower doses. This effect was more persistent with DEHP than with DMEP. Both phthalates produced some degree of dose- and time- dependent antifertility and mutagenic effects. There was a reduction in the number of implantations/pregnancy and of litter size, particularly in the first few weeks (postmeiotic stage) with the high dose of the compounds. Mutational effects, as expressed by an increase in early fetal deaths and reduced numbers of total implants, were seen at various weeks during the study, but most notably during the first few weeks. Thus, early fetal deaths and semisterility (decreased incidence of pregnancies, decreased number of implantations, and reduced number of offspring) constitute a spectrum of adverse reproductive and/or genetic effects noted with these 2 phthalate esters.

Numerous plastic devices and materials used for medical, dental, and paramedical applications, as well as for food collection, processing, and packaging are made of polyvinyl chloride. To provide the desirable physical characteristics, particularly clarity and flexibility, these materials contain significant quantities of plasticizers; some finished products may contain as much as 40 % of these. Presently, one commonly used group of plasticizers is the phthalic acid esters, of which DEHP is probably the most widely used. Evidence has been accumulating to indicate that small quantities of plasticizer may be leached from such materials by blood (Trimble et al., 1966; Jaeger and Rubin, 1970; Marcel and Noel, 1970; Neergaard et al., 1971), milk (Reichle and Tengler, 1968), or various other solutions (Guess et al., 1967a). Phthalic acid esters have also been identified in human and animal tissues (Jaeger and Rubin, 1972; Nazir et al., 1971). However, toxicologic implications were discounted by many, based upon the magnitude of difference between the quantities so leached and the acute toxicity of these compounds from animal feeding experiments and ip injections. Copyright 0 1974 by Academic Press, Inc. 35 All rights of reproduction in any form reserved. Printed in Great Britain

36 SINGH, LAWRENCE AND AUTIAN

Jaeger and Rubin (1970) reported finding DEHP in tissues of 2 patients who had received transfusions of blood stored in plastic bags. They found concentrations of DEHP ranging from 2.5 to 27 mg/100 g (dry weight) in the spleen, liver, lungs, and abdominal fat; the spleen contained the least, and abdominal fat the most. In vitro

experiments using the perfused rat liver indicated that this organ would accumulate DEHP but would not hydrolyze it; a related plasticizer, butyl glycolylbutyl phthalate, was hydrolyzed by this system.

Teratogenic effects of phthalic acid esters have been reported in chicks by Haberman and his co-workers (Guess et al., 1967b; Haberman et al., 1968; Bower et al., 1970) when the phthalates were injected into the yolk sac of developing embryos. Demon- stration of mammalian teratogenicity has been reported by Singh et al. (1972) from ip injections of the phthalate esters into pregnant rats.

In addition to their use in plastics for medical and food applications, some of these esters find other commercial uses, e.g., in cosmetics and insect repellent formulations, building and construction products, automobile and home furnishings, and clothing. The phthalic acid esters generally exhibit a low order of oral toxicity; however, reports indicate ingestion can cause gastrointestinal disturbances, irritation of mucous mem- branes, and even CNS depression, Toxicity studies on phthalic acid esters have been conducted by a number of investigators (Hodge, 1943; Shaffer et al., 1945; Draize et al.,

1948; Carpenter et al., 1953; Lehman, 1955; Harris et al., 1956; McLaughlin et al., 1963; Calley et al., 1966). Most of these studies involved oral feeding experiments.

In view of previous findings of embryo-fetal toxicity and teratogenic effects from some phthalic acid esters in rats (Singh et al., 1972), it seemed worthwhile to investigate the possible mammalian mutagenic and antifertility activities of DEHP and DMEP. The mouse was chosen as the experimental species because of its suitability for the dominant lethal mutation assay for mutagens (Cattanach et al., 1968). This com- munication presents the results of dominant lethal mutation and antifertility studies on DEHP and DMEP in mice.’

METHODS

Two phthalic acid esters were included in this study, di-2-ethylhexyl phthalate (DEHP) and dimethoxyethyl [bis(2-methoxyethyl)] phthalate (DMEP).2 Test animals were adult virgin male and female mice of the Harlan/ICR albino Swiss strain.’ Both male and female mice were 8-10 weeks old and weighed approximately 25-30 g.

Acute toxicity of the phthalates was determined by administering graded doses of these specific samples ip to male mice, and observing the animals for 7 days for mortality. The acute LD50 values and 95% confidence limits were calculated by Cornfield and Mantel’s modification (1950) of Karber’s method.

Before being assigned to this study, all male mice were checked for fertility by caging them with 2 adult females for 1 week; if 1 or both females became pregnant, the male mouse was accepted for the study. The male mice were injected ip with the undiluted

1 Reference to some of the preliminary results of this study has been made by Dillingham and Autian (Environmental Health Perspectives, January, 1973, pp. 81-89) and by Autian (EnvironmentaI Health Perspectives, June, 1973, pp. 3-26).

z Matheson, Coleman, and Bell, Cincinnati, Ohio. 3 Harlan Industries, Inc., Cumberland, Indiana.

MUTAGENICITY OF PHTHALATE ESTERS 37

phthalate esters at dose levels representing l/3, l/2, and 2/3 of the acute LD50. A group of 10 injected males was used with each dose level.

Immediately following injection, each male mouse was caged with 2 virgin adult female mice. The females were replaced weekly with 2 new virgin females during the 1Zweek period. An untreated group of 10 male mice was maintained in the same manner as a control, Food4 and fresh, clean tap water were provided ad libiwm. The onset of gestation was established by the presence of vaginal plug and was designated as day 0; the following day was recorded as day 1 of the gestation period. The pregnant mice were left undisturbed throughout the experimental period, except for transferring them to clean cages. Each weekly group was composed of 20 female mice.

Between days 13 and 17 of gestation, the pregnant mice were sacrificed with an overdose of ether. The uterine horns and ovaries were surgically exposed to permit counting and recording the number of corpora lutea, total number of implantations, early and late fetal deaths, preimplantation losses, and viable fetuses (litter size). Raw data were recorded on forms designed for key-punching, and were analyzed on an IBM 360-40 computer system for mean, SD, and SE. Data were also evaluated by analysis of variance using matrix inversion techniques (Snedecor and Cochran, 1967).

A two-factor analysis of variance was employed, using least-squares methods for unequal cell frequencies. All data were tested for normality. Only the number of early fetal deaths/pregnancy was found to be non-Gaussian (p < O.Ol), and this was trans- formed into a normal distribution by using In (X + 0.1). For the highest dose of DMEP there were no pregnancies in week 1, while there were no pregnancies in week 2 for the highest dose of DEHP. The analyses, therefore, were conducted on data in which the values for weeks 1 and 2 were pooled.

To evaluate the differences among percent pregnancies, a 2-way analysis of variance

was employed using Tukey’s test (Snedecor and Cochran, 1967) for additivity (a test for interaction in factorial experiments when there is only 1 observation per cell).

The antifertility effect was measured as a reduction in incidence of pregnancies, while the mutagenic effect was determined directly from the increased number of early fetal deaths in individual mice, and indirectly by the reduced number of total im- plantations (Epstein et al., 1970).

RESULTS

The acute LD50 for DEHP was 38.35 ml/kg, and for DMEP 3.57 ml/kg, with 95 % confidence limits of 34.11 to 43.11 and 3.21 to 3.97 ml/kg, respectively.

Four male mice in the high dose group of DEHP died 2 weeks after initiation of the study, and 1 died after 10 weeks. Two male mice in the high dose group of DMEP died within 2 hr of injection and were immediately replaced. No mortalities occurred in the 2 lower dose groups of either compound.

Late fetal deaths were uncommon in all groups studied, with a frequency of l/l70 (0.59 %) for controls, 9/309 (2.91%) for the DEHP-treated groups, and 12/435 (2.76 %) for the DMEP-treated mice. Although late fetal deaths were observed more often in the phthalate-treated groups, there was no apparent relationship to the dose of DEHP or DMEP administered.

’ Purina Laboratory Chow, Ralston Purina Co., St. Louis, Missouri.

38 SINGH, LAWRENCE AND AUTIAN

DEHP and DMEP induced some degree of infertility and mutagenicity in males subsequently mated with untreated females. The parameters (percent pregnancy, implants/pregnancy, early fetal deaths/pregnancy, and litter size/pregnancy) generally showed a dose-related effect. Table 1 and 2 give the weekly means for each treated group and controls.

TABLE

Dr-~-ETHYL PREC~NANCIES, IMPLANTS PER PREGNANCY,

PER PREGNANCY, (MEAN If:

Weeks after administration

of DEHP

Percent pregnancyb Implants per pregnancy Dosage (ml/kg) Dosage (ml/kg)

0 12.78 19.17 25.56 0 12.78 19.17 25.56

1 50 35 15 10 11.5k0.37 lO.O& 1.25 11.7+_0.88 2 55 50 45 0 11.5kO.71 11.5kO.40 11.2kO.66 3 70 35 70 16.7+ 10.7 f. 0.77 9.9 + 1.26 11.0&0.39 4 80 80 65 8.3+ 11.920.45 11.8kO.32 10.5 + 0.81 5 70 70 60 16.7+ 11.2kO.79 12.1 +0.35 12.2 + 0.64 6 65 60 70 50.0+ 11.3 + 0.38 12.9 + 0.63 11.2kO.47 7 60 60 75 25.0+ 11.3 kO.41 11.3e0.31 11.220.39 8 75 60 60 8.3+ 12.1 * 0.59 11.0 * 0.33 10.8 + 0.87 9 80 50 60 16.7+ 11.0+0.30 11.3+0.34 11.5&0.44

10 70 55 50 33.3+ 10.1 f 0.34 11.0 + 0.98 10.5 + 0.73 11 85 60 70 60.0++ 12.6 + 0.52 11.3 + 0.52 11.1 +0.39 12 90 55 65 50.0++ 11.5 If: 0.74 10.2 + 0.88 11.2kO.42

Overall mean 71 56 59 25.0 11.4kO.17 11.2kO.18 11.2kO.17

3.0 + 2.0

8.5 y2.50 10.0 * 0.01 12.0 + 1.0 10.7 + 1 .o 12.3 + 0.33 11.0*0.0 12.5 + 0.w 11.5 kO.29 11.2 + 1.3 10.0 f. 0.4 !

9.4 + 0.41)

u Males were mated sequentially over a period of 12 weeks after a single ip injection of DEHP. * All values are based on 20 females mated weekly to 10 males, except for occasional male deaths when size

of group was reduced as indicated: + = 12 females mated with 6 males; ++ = 10 females mated with 5 males.

TABLE

DIMETHOXYETHYL

PREGNANCIES, IMPLANTS PER PREGNANCY. EARLY PER PREGNANCY, (MEAN +

-

Percent prgenancyb Implants per pregnancy Weeks after Dosage (ml/kg) Dosage (ml/kg)

administration - --- -

of DMEP 0 1.19 1.79 2.38 0 1.19 1.79 2.38

1 50 15 55 0 11.5kO.37 12.Okl.O 9.5 & 0.95 -

2 55 3.5 90 20 11.5TO.71 8.4 + 1.2 11.6i0.36 8.8 + 1.25

3 70 60 85 5 10.7 + 0.77 11.3 f 0.30 10.8 + 0.60 4.0 + 0.0

4 80 80 75 5 11.9 5~ 0.45 12.6 + 0.49 11.3 + 0.56 12.0 f 0.0

5 70 80 75 35 11.2kO.79 11.5kO.47 11.5kO.52 9.9 $0.51

6 65 90 50 60 11.3 kO.38 12.1 kO.37 11.9kO.57 10.6+ 1.06

7 60 80 90 10 11.3 + 0.41 10.9 f 0.25 11.1 + 0.57 10.5 + 0.50

8 75 90 60 60 12.1 310.59 10.7 + 0.37 12.6 + 0.40 10.7 + 0.93

9 80 80 80 55 11.0k0.30 10.4+0.68 11.4kO.46 11.3kO.52

10 70 80 75 50 10.1 +0.34 10.8+0.56 11.1 +0.61 11.1 +0.43

11 85 75 75 55 12.6 + 0.52 10.7 + 0.41 10.9 f 0.32 10.7 f 0.27

12 90 80 95 70 11.5 k0.74 11.3 +0.49 11.3 kO.35 10.6kO.32

Overall mean 71 70 75 35 11.4+0.17 11.1 kO.16 11.3kO.15 9.2 f 0.25

’ Males were mated sequentially over a period of 12 weeks after a single ip injection of DMEP. b All values are based on 20 females mated weekly to 10 males.

MUTAGENICITY OF PHTHALATE ESTERS 39

Percent Pregnancy Pregnancy is defined as evidence of implantation of 1 or more fertilized ova. The

240 control female mice (20/week for 12 weeks) showed the mean incidence of preg- nancies to be 71% with a SE of 3.5 %. The highest dose level of both phthalates produced a marked reduction in incidence of pregnancies, the mean incidence being 25 % for

1 HEXYL~HTHALATE: EARLYFETALDEATHSANDLITTERSIZE SE) INFEMALEMICE'

Early deaths per pregnancy Litter size Dosage (ml/kg) Dosage (ml/kg)

0 12.78 19.17 25.56 0 12.78 19.17 25.56

0.40 + 0.22 0.36 + 0.15 d.43 +_ 0.17 0.69 + 0.28 0.21 + 0.15 0.62 + 0.18 0.25 k 0.13 0.60 f 0.16 q.50 * 0.13

8 .21 .71 & * 0.11 0.24 0.22 5 0.10 0.43 + 0.05

0.57 + 0.43 0.00 3.00 f 2.00 0.80 + 0.25 1.11 kO.39 - 0.71 f 0.29 0.64 + 0.23 5.50 f 5.50 0.50 + 0.18 0.77 * 0.34 0.00 0.71 + 0.22 0.67 + 0.26 0.50 f 0.50 0.92 + 0.23 0.57 k 0.23 0.67 + 0.33 1.08 + 0.82 0.40 + 0.16 0.67 &- 0.67 0.42 + 0.15 1.17 + 0.49 0.00 0.60 f 0.22 0.58 f 0.19 0.00 1.18 f 0.44 0.30*0.15 0.00 0.58 f 0.19 0.43 f 0.17 0.33 * 0.21 1.36 k 0.72 0.23 + 0.12 0.00 0.79 * 0.11 0.57 + 0.06 0.89 f 0.35

11.1 kO.43 11.2kO.69 10.3 f 0.77 11.2kO.54 11 .o f 0.78 10.7 & 0.38 11.1 kO.43 11.5 + 0.57 10.5 f 0.30

9.9 f 0.36 11.9 kO.53 11.3 + 0.76 11.0+0.17

9.4 + 1.31 10.7 + 0.47

9.0 + 1.23 11.3 kO.38 11.6kO.43 12.0 + 0.60 10.3 +_ 0.95 10.5 + 0.36 10.7 _+ 0.42

9.8 f. 1.29 10.7 + 0.51

8.8 +_ 1.37 10.4 + 0.23

11.7 + 0.88 0.00 10.1 +0.75 - 10.4 + 0.43 3.0 + 3.0 9.7 * 0.79 10.0 * 0.0

11.5kO.67 11.5kl.50 10.6 + 0.40 9.5 +_ 1.36 10.7 k 0.47 11.7 & 0.88 9.651.16 ll.O+O.O

10.7 + 0.50 12.5 + 0.50 10.1 + 0.71 11.5 + 0.29 10.6 +_ 0.41 10.7 + 1.31 10.9 +_ 0.42 10.0 + 0.45 10.6 + 0.18 8.6 + 0.65

2

Y

ATE: ETAL DEATHS, AND LITTERSIZE

$E) INFEMALE MICE"

0

Early deaths per pregnancy Litter size Dosage (ml/kg) Dosage (ml/kg)

-

1.19 1.79 2.38 0 1.19 1.79 2.38

0.40 + 0.22 0.36iO.15 0.43 f 0.17

8 &I + 0.28 .21 * 0.15

0.62kO.18 0.25 + 0.13

!i &O&O.16 .50+0.13

0.21 50.11 0.71 f 0.24 @22 f. 0.10 Q.43 f 0.05

0.67 + 0.33 0.57 + 0.43 0.42 + 0.19 0.31 * 0.12 0.31 * 0.20 0.28 it 0.11 0.25 + 0.11 0.22 * 0.10 0.25 + 0.11 0.31 + 0.12 0.20 + 0.11 0.50 + 0.16 0.36 + 0.04

0.82 + 0.46 - 0.28 + 0.14 1.50 + 1.20 0.53 f 0.17 1.00 + 0.0 0.53 + 0.24 3.00 + 0.0 0.33 k 0.16 0.57 c 0.37 0.6oIko.34 1.50_+ 1.0 0.44 + 0.20 0.50 + 0.50 0.42 f 0.19 0.08 + 0.08 0.88 2 0.26 1.33 + 0.27 0.60 + 0.24 0.20 + 0.13 0.67 + 0.21 0.00 0.68 f 0.19 0.57 + 0.29 0.57 f 0.06 0.85 + 0.18

11.1 f 0.43 11.2kO.69 10.3 f 0.77 11.2kO.54 11.0+0.78 10.7 + 0.38 11.1 f 0.43 11.5 kO.57 10.5 + 0.30

9.9 2 0.36 11.9kO.53 11.3 + 0.76 11.0*0.17

11.3 f 1.20 7.9 * 1.01

10.8 & 0.32 12.2 5 0.54 11.2 +_ 0.48 11.7kO.39 10.7 + 0.23 10.4 + 0.43 10.1 + 0.67 10.4 + 0.59 10.5 + 0.39 10.8 * 0.46 10.7 + 0.16

8.6 C 1.18 11.3 f 0.42 10.3 f 0.66 10.7 + 0.59 11.2kO.57 11.3 _+ 0.52 10.7 2 0.55 12.2 f. 0.53 10.6 + 0.56 10.5 +_ 0.60 10.1 + 0.47 10.6 + 0.32 10.7 + 0.17

- 7.3 f 1.60 3.0 f 0.0 9.0 + 0.0 9.3 +I 0.61 9.1 _+ 1.34

10.0 _+ 0.0 10.6 + 0.92

9.9 +_ 0.62 10.8 f 0.53 10.7 f 0.27

9.9 + 0.42 8.3 + 0.29

40 SINGH, LAWRENCE AND AUTIAN

DEHP and 35% for DMEP. The 2 lower doses of DEHP also exhibited a reduced incidence of pregnancies (56 and 59 %); the 2 lower doses of DMEP yielded values comparable to the controls (70 and 75 %).

Tukey’s additivity test was significant for the DMEP data at the 0.01 level when the data were not transformed. This indicates a dose-week interaction and necessitated data transformation to analyze for the main effects. When the DMEP data were trans- formed [In (100 - X)], the results of Tukey’s additivity test were no longer significant. No transformation was required for the DEHP data.

The analyses indicated that there was a significant effect of time and dosage upon percent pregnancies for both DEHP and DMEP.

For DEHP, there was a significant reduction in percent pregnancies of all treated groups throughout this time period (1 through 12 weeks). The Newman-Keul’s a posteriori test (Snedecor and Cochran, 1967) showed that the highest dose of DEHP produced the lowest incidence of pregnancies. While the 2 lower doses were not different from each other, both were significantly lower than the controls. For DMEP, using the logarithmic transformation, the a posteriori test revealed that the incidences of preg- nancy for the 2 lower doses were not different from the controls, but the high dose produced a significant reduction for the 12-week period.

Implants/Pregnancy

Total implants (including living fetuses and early and late fetal deaths, when present) were recorded for each mouse. Tables 1 and 2 present the mean and SE for each group determined on a weekly basis. Preimplantation losses were presumably reflected by a reduction in total number of implants/pregnancy. For the 170 pregnant control mice, the mean number of implants/pregnancy ranged between 10.1 and 12.6 for the various weeks, with an overall mean of 11.4 and a SE of 0.17.

Examination of Tables 1 and 2 shows there was a marked reduction in number of implantations during the first 3 weeks in the groups receiving the high dose of DEHP and DMEP. For these high doses, the mean number of implants/pregnancy for the 1Zweek study was also lower than the controls. This was not observed in the IZweek means for the lower dose levels.

It may be noted in Table 3 that analysis of all data (1 through 12 weeks) revealed significant differences with DEHP for certain criteria. Significance was seen in the analysis of weeks and weeks versus dosage. When the data were subdivided into post- meiotic (l-3 weeks) and premeiotic (4-8 weeks) stages of the sperm at the time of DEHP injection, the postmeiotic stage showed a significant (p < 0.01) reduction in _, implants which was time-dependent with these 3 weeks for the high dose level. How- ever, no drug response was observed upon implantations with the 2 lower doses, pos- sibly because these doses are subthreshold for this effect. Neither of the other time intervals (4-8 or 9-12 weeks) revealed significant effects. !

Examination of Table 3 shows that analysis of all data (1 through 12 weeks) indicates some areas of significance for DMEP. When the data were subdivided into the afore- mentioned time periods, a significant effect of dosage was observed during the post- ’ meiotic stage (1-3 weeks), and suggested a dose-week interaction (significant atp < 0. IO) % No consistent effects were observed during the last 9 weeks of the study.

1 c

TABL

E 3

SUM

MAR

YOFA

NAL

YSIS

OFV

ARIA

NC

E~

Impl

ants

pe

r Ea

rly

feta

l de

aths

Li

tter

size

per

preg

nanc

y pe

r pr

egna

ncyb

p~

gnna

cy

F-R

atio

(d

f’)

F-R

atio

(d

f”)

F-R

atio

(d

f’)

Cat

egor

ies

DEH

P D

MEP

D

EHP

DM

EP

DEH

P D

MEP

Wee

ks:

1-12

with

co

ntro

ls

Dos

age

(adj

uste

d)

2.26

’ (3

) W

eeks

(a

djus

ted)

4.

82”

(3)

2.50

6 (3

) 3.

02”

(3)

4.23

/ (3

) 7.

23f

(3)

3.68

f (1

0)

2.66

” (1

0)

1.50

(1

0)

2.06

’ (1

0)

5.42

f (1

0)

2.6Y

(1

0)

Dos

e x

week

1.

95f

(30)

1.

55’

(30)

0.

87

(30)

1.

37

(30)

2.

60f

(30)

1.

51”

(30)

W

ithin

su

bcla

sses

-

(435

) -

(561

) -

(435

) -

(561

) -

(435

) -

(561

) W

eeks

: l-3

w

ithou

t co

ntro

ls

Dos

age

(adj

uste

d)

10.0

9f (

2)

5.01

f (2

) 1.

86

(2)

0.96

(2

) 25

.18f

(2)

5.

39y

(2)

Wee

ks

(adj

uste

d)

2.51

(1

) 0.

97

(1)

0.16

(1

) 0.

30

(1)

0.38

(1

) 0.

47

(1)

Dos

e x

week

3.

57=

(2)

2.78

d (2

) 0.

20

(2)

0.36

(2

) 1.

35

(2)

2.15

(2

) W

ithin

su

bcla

sses

-

(48)

-

(67)

-

(48)

-

(67)

-

(48)

-

(67)

W

eeks

: 4-

8 w

ithou

t co

ntro

ls

Dos

age

(adj

uste

d)

2.04

(2

) 1.

20

(2)

0.48

(2

) 2.

75d

(2)

1.41

(2

) 3.

26’

(2)

Wee

ks

(adj

uste

d)

0.81

(4

) 0.

66

(4)

0.58

(4

) 1.

90

(4)

0.48

(4

) 0.

30

(4)

Dos

e x

week

0.

89

(8)

1.23

(8

) 0.

40

(8)

0.97

(8

) 0.

86

(8)

1.44

(8

) W

ithin

su

bcla

sses

-

(130

) -

(173

) -

(130

) -

(173

) -

(130

) -

(173

) W

eeks

: 9-

12 w

ithou

t co

ntro

ls

Dos

age

(adj

uste

d)

0.17

(2

) 0.

60

(2)

5.55

f (2

) 2.

96

(2)

1.31

(2

) 0.

08

(2)

Wee

ks

(adj

uste

d)

1.25

(3

) 0.

23

(3)

0.22

(3

) 3.

77”

(3)

0.89

(3

) 0.

28

(3)

Dos

e x

week

0.

49

(6)

0.49

(6

) 0.

63

(6)

2.47

’ (6

) 0.

70

(6)

0.55

(6

) W

ithin

su

bcla

sses

-

(98)

-

(162

) -

(98)

-

(162

) -

(9%

-

(162

)

r” Ana

lyses

of v

aria

nce

for

week

s l-3

,4-8

, an

d 9-

12,

with

con

trols

, we

re a

lso

calc

ulat

ed. R

esul

ts o

f the

se a

nalys

es a

dded

no

new

info

rmat

ion,

and

thus

are

om

itted

from

the

tabl

e to

con

serv

e sp

ace.

b

Loga

rithm

ic t

rans

form

atio

n [in

(dat

a +

O.l)

] wa

s pe

rform

ed o

n da

ta p

rior

to a

naly

sis.

’ D

egre

es o

f fre

edom

. d S

igni

fican

t atp

< 0

.10.

e S

igni

fican

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42 SINGH, LAWRENCE AND AUTIAN

Early Fetal Deaths

Tables 1 and 2 present the mean number of early fetal deaths and SE by weeks. It may be noted that several of the phthalate-treated groups had a higher incidence of early fetal deaths than the corresponding controls; this is especially true during the first 3 weeks for the high dose group of DEHP.

Since anaIysis of variance assumes normality of distribution and a test of early fetal death data revealed an asymmetric, Poisson distribution, it was necessary to analyze the

Dn-2-Ethylhexyl Phthalate IDEHP:

- Control

o-------i) Low Dose

l ---------• Mlddle Dose

a---c Hfgh Dose

*No pregnancies occurred dut this week

Ocmethovyethyl Phtholote (DMEP)

2 80

2 70- 5 E 9 60-

E 50- : F 40

a 30-

20 t

10

0 t

i

Weeks of Motmg After Treatment

FIG. 1. Percent of pregnant mice showing two or more early fetal deaths.

data after logarithmic transformation [In (data + O.l)] to correct for the skewness of this distribution (Snedecor and Cochran, 1967). In addition, data from weeks 1 and 2 were combined and averaged for the analysis, since 0 y0 pregnancies were observed with the high dose of one compound during week 1 and with the other during week 2.

When the DEHP data were analyzed (Table 3) for effect of dosage, time (weeks), or dose-week interaction (i.e., without controls), no significance was observed for any of these factors during the first 8 weeks. Failure to detect a statistically significant effect upon early fetal deaths for one or more of these factors during the early part of the

MUTAGENICITY OF PHTHALATE ESTERS 43

study might be due to the small sample sizes (for the high dose levels) and large variances in data, particularly in the high dose level. A significant (p < 0.01) effect of dosage was revealed for the 9- to 1Zweek period.

For the DMEP-treated groups, analysis of variance during the 9- to 1Zweek period (with controls) revealed an effect of time (F= 3.47) and for dose-week interaction (F= 2.39), both of which are significant at p < 0.05. When the controls were deleted and the analyses repeated, these remained significant at the same level (Table 3).

The relative distribution of early fetal deaths among pregnancies is shown in Fig. 1. This depicts the percent of pregnant mice with 2 or more early fetal deaths as a function of the phthalate ester injected in the male, dose of phthalate employed, and time of mating following injection.

Litter Size

Litter size, defined as the number of live fetuses/pregnancy, showed a significant (p < 0.01) dose-related reduction during the first 3 weeks with both phthalates. This effect apparently persisted for DMEP in the 4- to 8-week period, but not for DEHP (Table 3).

DISCUSSION

The spermatogenic cycle in the mouse is approximately 6 weeks, which may be subdivided as follows: spermatogonial mitosis, 6 days; spermatocytes, 14 days; spermatids, 9 days; testicular sperm, 5.5 days; and epididymal sperm, 7.5 days (Bate- man, 1958). Other workers (Oakberg, 1956; Partington and Jackson, 1963; Ehling ef al., 1968) have suggested the complete spermatogenic cycle in the mouse may require up to 8 weeks, and in mutagenic studies, many investigators employ the 4- to 8-week period to represent the premeiotic stage. Thus, with the experimental procedure used, pregnancies which occurred during the first 3 weeks after injection of the test substance should be due to sperm that were in the postmeiotic stage at the time of phthalate injection; those occurring during weeks 4 to 8 represent sperm in the premeiotic stage at the time of injection; and those occurring during weeks 9 through 12 represent a second spermatogenic cycle.

The single ip injection of phthalate ester prior to mating was found, generally, to elicit a dose-related, time-dependent trend of antifertility and mutagenicity, the specific pattern being a function of the compound injected. Although early fetal deaths during the first 3 weeks did not yield statistically significant Fvalues as discussed previously, the high dose of DEHP resulted in 100 % of the implants terminating in early deaths during the first week and 65 % during the third week (there were no implantations during the second week), while the high dose of DMEP produced 17% during the second week and 25 % during the third week (there were no implantations during the first week). This compares to values of 3.1 to 4.0% for the controls during this time period. All 3 dose levels of DEHP and the high dose of DMEP exhibited some degree of antifertility (as reflected by reduced incidences of pregnancy) throughout the study.

Late fetal deaths did not occur frequently in any group, but did show a mean inci- dence 5 to 6 times higher in the phthalate-treated groups than in the controls. It is generally recognized (Epstein et al., 1970; Bateman, 1958) that nongenetic factors contribute to, or are responsible for, late fetal deaths.

44 SINGH, LAWRENCE AND AUTIAN

During the 12 weeks of this study, 56% of the female mice exposed to males treated with the low dose of DEHP became pregnant, while for the middle dose, the value was 59%. This compares to a value of 71% for the untreated controls. The reduction in incidence of pregnancies was most pronounced during the first 2 or 3 weeks, and although fluctuating from week to week, tended to remain below control values for most time periods. The high dose of DEHP, however, showed a rather persistent anti- fertility effect throughout the 1Zweek period; in only 1 of the 12 time-periods did the pregnancy rate exceed 50%. The mean incidence of pregnancies for this group was 25 %.

The high dose of DMEP showed a reduced incidence of pregnancies which was most pronounced during the first 5 weeks; these weekly values ranged from 0 to 35 %. From weeks 6 through 12, the incidences ranged from 50 to 70x, except for week 7, in which it was only 10%. For the 2 lower doses of DMEP, there seems to be a slight early antifertility effect which disappeared quite rapidly. The overall mean values for the low and middle doses, respectively, were 70 and 75x, compared to 71% for the controls.

Both of the high dose, phthalate-treated groups demonstrated a noticeable reduction in the number of implants/pregnancy during the first 3 weeks of the study; thereafter, the values were essentially comparable to the controls.

The litter size of the females bred to males given the high dose of DEHP was mar- kedly reduced for the first 3 weeks after administration. In the first week, only 10% of the exposed females became pregnant, with a mean of 3 (5 + 1 + 2) implantations/ pregnancy, and all of these terminated in early fetal deaths. During the second week, none of the exposed females became pregnant, thus the litter size was again 0. In the third week, 16.7 % of the exposed females became pregnant, with a mean litter of 3.0. By the fourth week, although the incidence of pregnancies was still greatly reduced, the litter size/pregnancy was essentially normal (10.0 for this group as compared to an overall mean of 11 .O for the controls). It tended to remain in this range throughout the rest of the study. DMEP also produced a reduction in litter size during the first 3 weeks of the study.

Analysis of variance indicated that there was a dose-related effect upon early fetal deaths 9 to 12 weeks after ip injection of DEHP. For this Cweek period, the untreated control mice showed a mean of 0.41 early fetal deaths/pregnancy; for the low dose of DEHP, the mean was 0.93, for the middle dose 0.385, and for the high dose it was 0.0825. This would suggest that the lower dose affects spermatogenesis in some manner to increase early fetal deaths, while the effect of the highest dose was to decrease early fetal deaths. Table 1 shows that in only one of the last 5 weeks (the 1 lth) of the study were early fetal deaths observed in the group which received the high dose of DEHP, a unique feature in this study. Although pregnancy rate for the high dose of DEHP was still substantially less than controls (means of 33.7 y0 versus 80%) during this period, the number of implants/pregnancy and litter sizes were almost identical. Furthermore, the sperm producing fertilization at this time would have been in the very early pre- meiotic stage at the time of DEHP injection, or else arose from a subsequent spermato- genie cycle. Since no apparent pattern for fertility or infertility of a particular male was seen, the cause of this observation is speculative. The groups administered DMEP did not exhibit a similar pattern.

MUTAGENICITYOFPHTHALATE ESTERS 45

ACKNOWLEDGMENTS

The authors are grateful to Mrs. Arm McEachran for her guidance and assistance with statistical analysis of the data. This work was supported in part by Research Grant DE-02944-05 from the National Institute of Dental Research, Bethesda, Maryland 20014, and computational services were provided through USPHS Grant HL-09495.

REFERENCES

BATEMAN, A. J. (1958). Mutagenic sensitivity of maturing germ cells in the male mouse. Heredity 12,213-232.

BOWER, R. K., HABERMAN, S. AND MINTON, P. D. (1970). Teratogenic effects in the chick embryo caused by esters of phthalic acid. J. Pharmacol. Exp. Ther. 171, 314-324.

CALLEY, D., AUTIAN, J. AND GUESS, W. L. (1966). Toxicology of a series of phthalate esters. J. Pharm. Sci. 55,158-162.

CARPENTER, C. P., WEIL, C. S. AND SMYTH, H. F., JR. (1953). Chronic oral toxicity of di(2- ethylhexyl) phthalate for rats, guinea pigs, and dogs. Arch. Znd. Hyg. Occup. Med. 8,219-226.

CATTANACH, B. M., POLLARD, C. E. AND ISAACSON, J. H. (1968). Ethyl methanesulfonate- induced chromosome breakage in the mouse. Mutation Res. 6,297-307.

CORNFIELD, J. AND MANTEL, N. (1950). Some new aspects of the application of maximum likelihood to the calculation of the dosage response curve. Amer. Stat. Assoc. J. 45,181-210.

DRAIZE, J. H., ALVAREZ, E., WHITESELL, M. F., WOODARD, G., HAGAN, E. C. AND NELSON, A. A. (1948). Toxicological investigations of compounds proposed for use as insect repellents. J. Pharmacol. Exp. Ther. 93,26-39.

EHLING, U. H., CUMMING, R. B. AND MALLING, H. V. (1968). Induction of dominant lethal mutations by alkylating agents in male mice. Mutation Res. 5,417--428.

EPSTEIN, S. S., ARNOLD, E., STEINBERG, K., MACKINTOSH, D., SHAFNER, H. AND BISHOP, Y. (1970). Mutagenic and antifertility effects of TEPA and METEPA in mice. Toxic01 Appl. Pharmacol. 17,23-40.

GUESS, W. L., JACOB, J. and AUTIAN, J. (1967a). A study of polyvinyl chloride blood bag assemblies. I. Alteration or contamination of ACD solutions. Drug Intel. 1, 120-127.

GUESS, W. L., HABERMAN, S., ROWAN, D. F., BOWER, R. K. AND AU~IAN, J. (1967b). Charac- terization of subtle toxicity of certain plastic compounds used in manufacturing of the polyvinyls. Amer. J. Hosp. Pharm. 24,494501.

HABERMAN, S., Gurss, W. L., ROWAN, D. F., BOWMAN, R. 0. AND BOWER, R. K. (1968). Effects of plastics and their additives on human serum proteins, antibodies and developing chick embryos. SPE (Sot. Plastics Eng.) J. 24, 62-69.

HARRIS, R. S., HODGE, H. C., MAYNARD, E. A. AND BLANCHET, H. J. (1956). Chronic oral toxicity of 2ethylhexyl phthalate in rats and dogs. Arch. Znd. Health 13, 259-264.

HODGE, H. C. (1943). Acute toxicity for rats and mice of 2-ethylhexanol and 2-ethylhexyl phthalate. Proc. Sot. Exp. Biol. Med. 53,20-23.

JAEGERR. J. andRusn$ R. J., (1970). Plasticizers from plastic devices: Extraction, metabolism, and accumulation by biological systems. Science 170,46@-462.

JAEGER, R. J. AND RUBIN, R. J. (1972). Migration of a phthalate ester plasticizer from poly- vinyl chloride blood bags into stored human blood and its localization in human tissues. N. Engl. J. Med. 287,1114-l 118.

LEHMAN, A. J. (1955). Insect repellents. Ass. Food Drug O$ic. U.S. Quart. Bull. 19,87-99. MARCEL, Y. L. AND NOEL, S. P. (1970). Contamination of blood stored in plastic packs.

Lancet 1, 35-36. MCLAUGHLIN, J., JR., MARLIAE, J. P., VERREIT, M. J., MIJTCHLER, M. K. AND FITZHUGH,

0. G. (1963). The injection of chemicals into the yolk sac of fertile eggs prior to incubation as a toxicity test. Toxieol. Appl. PZzarmacol. 5760-771.

NAZIR, D. J., ALCARAZ, A. P., BIERL, B. A., BEROZA, M. and NAIR, P. P. (1971). Isolation, identification, and specific localization of di-2-ethylhexyl phthalate in bovine heart muscle mitochondria. Biochemistry 10,4228-4232.

46 SINGH, LAWRENCE AND AUTIAN

NEERGAARD, J., NIELSEN, B., FAURBY, V., CHRISTENSEN, D. H. AND NIELSEN, 0. F. (1971). Plasticizers in PVC and the occurrence of hepatitis in a hemodialysis unit. Band. J. Ural. Nephrol. 5, 141-145.

OAKBERG, E. F. (1956). Duration of spermatogenesis in the mouse and timing of stages of the cycle of the seminiferous epithelium. Am. J. Anat. 99,507-516.

PARTINGTON, M. AND JACKSON, H. (1963). The induction of dominant lethal mutations in rats by alkane sulphonic esters. Genet. Res. 4, 333-345.

REICHLE, A. AND TENGLER, H. (1968). Methods for determination of plasticizer migration from synthetic materials into food. IV. Migration of bis(2-ethylhexyl) phthalate and masamoll from synthetic rubber into milk. Deut. Lebensm.-Rundsch. 64,142-145.

SHAFFER, C. B., CARPENTER, C. P. AND SMYTH, H. F., JR. (1945). Acute and subacute toxicity of di(Zethylhexy1) phthalate with note upon its metabolism. J. Znd. Hyg. Toxicol. 27, 130- 135.

SINGH, A. R., LAWRENCE, W. H. AND AUTIAN, J. (1972). Teratogenicity of phthalate esters in rats. J. Pharm. Sci. 61, 51-55.

SNEDECOR, rJ. W. AND COCHRAN, W. G. (1967). Statistical Methods, 6th ed. pp. 273-275, 299-338. Iowa State Univ. Press, Ames, Iowa.

TRIMBLE, A. S., GOLDMAN, B. S., YAO, J. K., KOVATS, L. K. AND BIGELOW, W. G. (1966). Plastics-a source of chemical contamination in surgical research. Surgery 59, 857-859.