Appendix: Evaluation of individual chemicals and future examination
This committee mainly discussed future fundamental lines and examined, among
the substances suspected of being endocrine disruptors, some substances used asmaterials for plastic food containers.

Substances being eluted from foodcontainers have high potential for intake with foods, and therefore are regulatedby the Food Sanitation Law to prevent the occurrence of hygienic problems. The
substances were discussed at the Joint Committee of Toxicity and Apparatus,
Containers and Packages under the Food Sanitation Council, which met on March13, 1998.

Current information about these substances, and the basic line offuture research, can be summarized as follows.
We refer to data that have not been published as papers. These data should be
examined in detail after the official

publication of papers.

1. Polycarbonate
Owing to its high transparency and hardness, polycarbonate is used in foodcontainers, compact discs, car light covers and OA instruments.

This plastic is

generally formed by repeated polycondensation using bisphenol A andcarbonylchloride or diphenylcarbonate as materials. Bisphenol A is suspected ofbeing an endocrine disruptor.
z

Annual food-related usage (investigated by Japan Hyginic Olefine andStyrene Plastics Association, unit: 1,000 ton)
Production: 251; export: 121; domestic sales: 130, including food-related use:
4(1) Elution of bisphenol A
Elution of bisphenol A from tableware was studied at the National Institute ofHygienic Sciences and other facilities.

As shown in Table 1, its elution volumes

were approximately below 100 ppb, and many were below the detection
limit*91*92*93*94*95*96.
N-heptan (mimicing solvent for oils, fats and liposoluble foods), 4% of acetic acid(mimicing solvent for water-soluble and acid foods), ethanol (mimicing solvent for65

alcoholic beverages) and water (other foods) were used as elution solvents. Theelution volumes from n-heptan were relatively high, but some were below thedetection limit. It seems that elution volumes are influenced by many factors,
such as temperature, extent of deterioration in tableware, conditions of tableware
use and kind of solvent.
(2) Safety of bisphenol A① Screening
・ In vitro study
Binding capacity of bisphenol A to hormone receptor, and gene activation
resulting from such binding, can be determined by in vitro study.
It has been pointed out that bisphenol A has estrogenic (female sex hormone)activity; there are reports showing the extent of such action:1) In a study to examine binding antagonism with 3H-estradiol (E2) and rat
uterine cytosolic fraction, binding affinity of bisphenol A to estrogen receptor(ER) was 0.012 assuming that of E2 as 100*97.
2) Bisphenol A that eluted from a polycarbonate flask showed estrogenic action
and its binding capacity to ER was 1/2,000 that of E2*98.
3) In a study to examine binding affinity with ER using MCF-7 (humanmammary carcinoma cell line), binding affinities of bisphenol A in themedium with or without adult human male serum were 0.01 and 0.006,
respectively, assuming the binding affinity of E2 as 100*99.
・ In vivo study
Bisphenol A (1.0 - 1,000 mg/kg, once a day for 4 days) was given subcutaneously
to ovariectomiyed B6C3F1 mice.

Increase in uterine weight and relevant

histological changes were found in parallel to the induction of heat-shockprotein*100.
(Reference data)
At present, a quick, simple and representative in vivo method for evaluating
estrogenic action of chemicals is to administer chemicals to rodents such as rats
and weigh the increase in uterine weight. Subcutaneous administration ofbisphenol A at a dose of 400 mg/kg once daily for 3 days to immature rats
resulted in increase in uterine weight*101.

66

② Repeat dose toxicity study
The two-generation reproductive study has been considered suitable forcomprehensively evaluating endocrine disrupting influence on reproductivefunction and investigating influence on subsequent generations.

In a two-generation reproductive study, a certain chemical is administered over twogenerations and, its influence on reproductive functions such as estrus, mating,conception, delivery and nursing, and neonatal growth after delivery andweaning are examined.
As regards bisphenol A, though the data from two-generation reproductive
studies are not sufficient, several animal experiments have been reported:1) bisphenol A was given to pregnant mice at doses of 2 and 20 µg/kg/day, andthe prostate weight of 6 month-old male offsprings increased by 30 to 35%*99.
2) In a 103 week chronic toxicity study in F344 rats fed a mixed diet (1,000 and2,000 ppm), rats in all subject groups lost weight, but carcinogenicity was
not observed*102.
3) In a 103-week chronic toxicity study in B6C3F1 mice fed a mixed diet (male:1,000 and 5,000 ppm; female: 5,000 and 10,000 ppm), mice in groups withthe 5,000 ppm and more lost weight, but carcinogenicity was notobserved*102.
4) In a subacute toxicity study using dogs, rats and mice, only the dogs in thehighest (9,000 ppm) group showed increase in the liver weight *102.
5) After rat dams (SD) were given drinking water containing bisphenol A (0.005,0.05, 0.5, 5 and 50 mg/L) over the period from 2 days after pregnancy to 21days after parturition, bisphenol A was detected in the dam’s milk in the 5and 50 mg/L groups. There was no estrogenic signs in the pregnant andnursing dams, although there was an increase in dams’ relative kidneyweight in the 50 mg/L group as a sign of general toxicity*103. In the same
study, bisphenol A did not affect sexual maturation of female offsprings,
esterous

cycle,andpituitary

andhypothalamic

regulation

onprogesterone*104.
6) In studies using SD rats fed a mixed diet (1,000, 3,000 and 9,000 ppm;
equivalent to approx. 50, 150 and 450 mg/kg/day, respectively) for 100 daysin F0 and 90 days in F1, dose-related decrease in body weight gain wasobserved in dam and F1 infant groups given 3,000 ppm or more, but67bisphenol A did not influence on indices relevant to litter size at birth,postnatal mortality and fertility*105.
7) In studies using SD rats fed a mixed diet (100, 250, 750 and 1,000 ppm;
equivalent to approx. 5, 12.5, 37.5 and 50 mg/kg/day, respectively) for 90
days, bisphenol A had no influence other than low body weight in the maleparent at 1,000 ppm group; bisphenol A did not influence female estrous
cycle and the above reproductive indices*105.
8) In a multi-generation reproductive study in ICR mice fed a mixed diet (2,500,5,000 and 10,000 ppm; equivalent to approx. 473.5, 875 and 1,750 mg/kg/day,respectively), decrease in body weight gain, increase in the liver and kidneyweight, prolonged gestation period, low values of male accessory
reproductive organs, and reduction in sperm motility etc. were observed inF0 group at 10,000 ppm; and decreased in food intake, litter-size at birth,and numbers of survived pups per litter were observed in the groups at 5,000
ppm or more.

As for F1 groups, increase in mortality was observed at

10,000 ppm group; increase in the liver and kidney weight, and decrease inmale reproductive organ weight were seen in all groups given 2,500 ppm ormore, but decrease in litter-size at birth was not observed in any group.
Some adverse influence on reproduction was observed in such high-dosegroups, that showed systemic toxicosis*106.
9) In a teratogenic study in SD rats injected intra peritoneally for 15 days fromgestation day (GD) 1 to 15 (85 and 125 mg/kg/day, corn oil), maternal
toxicities such as decrease in pregnancy ratio etc. were observed at 125
mg/kg group; fetal toxicity such as ossification failure was seen in the groups
at 85 mg/kg or more*107.
10) In a teratogenic study in ICR mice orally administered for 10 days from
GD 6 to 15 (500, 750, 1,000 and 1,250 mg/kg/day), increase in dam mortality
was observed in all groups; and decrease in relative liver weight was seen ingroups at 500 mg/kg or more.

In 1,250 mg/kg group, decrease in uterine

weight and numbers of corpora lutea, and increase in numbers of dead andabsorbed fetuses and of fetuses with defects were observed, with associatedsevere toxicosis in dams*108.
11) In a teratogenic study in SD rats orally administered for 10 days from GD
6 to 15 (160, 320, 640 and 1,280 mg/kg/day), increase in dam mortality wasobserved at 1,280 mg/kg group; decrease in body weight gain was seen ingroups given 160 mg/kg or more.

Fetal toxicity and teratogenesis were not68

observed in groups given 640 mg/kg or less.

Because many dams at 1,280

mg/kg group died, teratological evaluation could not be done*108.
③ Pharmacodynamics
Studies on chemical accumulation and metabolite influence would provide
information whether the chemical effect in vivo was caused by the parent
compound itself or by the metabolites, as well as cues to clarify the causes of invivo effects.

Particularly in case of endocrine disruptors with suspectedconcerns of adverse effects over generations, information on the bioaccumulation has a vital importance.

In view of the influence on the fetus,

chemical concentration in the umbilical cord blood would provide one of theuseful indices.
There are not many reports on in vivo dynamics of bisphenol A.

In a single

oral dose (800 mg/kg) study on C bisphenol A in male rats, 28% and 56% of 14C
14

that has been administrated were excreted into the urine and the stool,respectively, within 8 days after administration.

It was also reported that

residual 14C was not detected in the body at 8 days after administration*109.
(3) Conclusion
Bisphenol A in vitro has approx. 10-5 to 10-4 times of ER binding capacity, comparedto that of estradiol (E2).

In vitro study results suggest that bisphenol A might

have a similar action in vivo as well, however, in vivo, multiple factors such as
metabolism,

biologicalprocess

includingexcretion,

endocrine

feedback

mechanisms and influence of blood component etc. would complicate the situation.
Therefore, it is necessary to evaluate the actual in vivo effect after carefulassessment of the in vivo testing.
In vivo studies on prostate weight and carcinogenicity have been carried out, butthere are no data showing in vivo toxicity of bisphenol A at the actual exposurelevel, by the following reasons: 1) prostate weight is not a general assessment item,
and the mechanism of its weight gain is unknown; 2) carcinogenicity and twogeneration reproductive studies were performed at the relatively high doses of
1,000 ppm and more and 100 ppm and more, respectively.

The US

Environmental Protection Agency has settled the RfD (equivalent to acceptable69

daily intake (ADI)) of bisphenol A as 0.05 mg/kg/day*102.
As indicated above, there is no scientific information that bisphenol A, at thelevels eluting from polycarbonate, seriously affects human health; therefore, itwould not be necessary to prohibit the use of polycarbonate at present.

However,

endocrine disruptors arise a new problem; some researchers claim that even small
amounts of bisphenol A can have an effect especially in infants, whose endocrine
systems are still immature, even if it is not toxic to adults, whose endocrine
feedback systems has already been established.

We should therefore conduct

further research such as two-generation reproductive studies.
(Reference data)
Recently, exposure to drinking water with very low concentrations of bisphenol A,
during gestation and lactation was conducted; it was reported that bisphenol A has
no influence on the offsprings.

However, this report was presented at an

academic meeting, and should be reviewed in detail after published as a paper*110.
(4) Future research・ A two-generation reproductive study in rats, and a Pharmaco-dynamic study inrats (bio-accumulation, transfer to the fetus and milk) etc.
・ Bisphenol A concentration research in human umbilical cord blood and breast
milk, and research for organ distribution
・ Research for elution volumes from tableware and cans70