Keio J. Med. 24: 19-37, 1975

TOXICITY OF IN EXPERIMENTAL ANIMALS -CONCENTRATIONS OF THE CHEMICAL IN THE ELUTION FROM DISHES OF FORMALDEHYDE RESIN IN SOME VEGETABLES

KENZABURO TSUCHIYA, YOSHIO HAYASHI, MITSUKO ONODERA and TAKAO HASEGAWA

Department of Public Health and Preventive Medicine, School of Medicine, Keio University

(Received for publication Oct. 16, 1974)

ABSTRACT

The study composes of the following experiments: 1) The first experiment was carried out in order to calculate the lethal dose (LD50) of formaldehyde and of formalin solution. 2) The second one was to examine the quantitative method and to measure the concentration of formaldehyde in solution eluted from plastic bowls made of formaldehyde resin. 3) In the final experiment formaldehyde in some foods was measured. Princinal results obtained were as follows: 1) LD50 of formaldehyde by oral administration was determined to be 500-800 my/ke for rats. 2) The maximum allowable concentration of the chemical for chronic ing was estimated to exist at the level of around 25 to 30 mg/day/50 kg for

man. 3) However, it was conceivable that various kinds of food would contain formaldehyde as high as almost 20 ppm. More elaborate studies are required on this point. 4) It was confirmed that formaldehyde is eluted from plastic dishes and bowls made from a monomer of formaldehyde from the non-detectable con centration at 40•Ž up to 20 ppm at 90•Ž, and up to almost 400 ppm in solu tion of 4% acetic acid of 90•Ž, showing only 10 ppm at 40•‹ to 50•Ž, after leaving the solutions in the bowls for 15 minutes.

Formaldehyde is used for various purposes in industry, and the manufactur ing industry of synthetic resin, deodorizer, sterilizing agent, antiseptics, dye, leather, rubber and synthetic fiber and the industry of proofing and re

19 20 Kenzaburo Tsuchiya et al

ducer are attended with the dangers of exposure to formaldehyde. The toxic action of formaldehyde was studied by Flury et al,1,2 using cats. The animals died chiefly of edema, hemorrhage and inflammation of the mucous membrane of the respiratory organs, and in cases where death did not occur, the symptoms being limited to irritation of the mucous membrane, they recovered without sequelae. The maximum allowable concentration in the atmosphere of the working environment ranges from 10 to 5 ppm1,3 but it seems unlikely up until the present that formaldehyde has posed a great problem in industrial medicine in Japan. However, since no research under actual conditions has been conducted, the real situation is uncertain.

In accordance with the recent production of various kinds of plastic table ware made from synthetic resins, it was found that formaldehyde was eluted from the tableware made of formaldehyde resin, presenting a problem in food sanitation. Noted in particular was a study4 in which the toxic action of formal dehyde was found by chronic experiments with rabbits, though the number of cases was small. It was though that this problem required full examination from a public health point of view. The chronic toxic action of a substance is a very difficult problem, and in consideration of the period, concentrations, species of animals and method of medical observation, it is thought to be almost impossible to discover the limits sufficient to induce chronic toxic action experimentally. Therefore, the maximum limit possible to perform actually would be to administer the water which was actually used for the dish or bowl made of synthetic resin to animals of 2-3 species for 1-2 years for physiological, biochemical and pathological examina tions. However, since even such an experimental study requires great expense, effort and a certain period of time, the authors decided to first calculate the lethal dose as a preliminary study for an estimation of the rough dose for chronic intoxication in humans in order to contribute it as basic material for future experiments on chronic intoxication. In addition, the amount of formaldehyde in various foods which we eat daily was measured for comparison with the amount of eluted formaldehyde from the tableware made from synthetic resin commercially available. The problem of formaldehyde in the tableware made of synthetic resin seems to have been brought forward in the United States as well, and the dispute as a political problem between the Association for Ceramics and the Association for Resin is noted in the November 1951 issue of the Journal of Modern Plastics. Although we encountered the "Formaldehyde-Its and Potential Dangers" in Supplement No. 181 of the Public Health Report for 1945 as litera Toxicity of Formaldehyde 21 ture, unfortunately, we were not able to obtain in journal concerned nor reprints in spite of all our efforts. Accordingly, it is not possible to introduce literature on chronic disturbances by the oral administration of formalin, but the present study is though to be contributory in the sense mentioned above. With this study as a basis, studies on chronic intoxication should be further developed in the future.

EXPERIMENTAL METHODS

1) Calculation of the lethal dose.

About 400 male rats were used for the experiments. Their body weight ranged from 100 g to 200 g, and animals of the same body weight and age were used in an experiment as long as possible. The rats were of the Wister strain, but because of the large number required they were purchased from animal dealers whose reliability was not necessarily high. The number of rats purchased each time was about 50, and those not showing a certain increase in the body weight or those with a poor increase rate were not included. The animals were raised in metal cages in groups of 5 each, fed on solid feed and placed in an animal room which maintained a temperature of 18•‹ to 20•Ž. Formaldehyde was administered in single doses through sonde, and included in the calculation were rats which died after one week of the administration. As will be stated later, most animals died within 24 hours of the administration, a few dying on the 3rd day. Those which survived lived until the experiments were finished. The LD5O was calculated from the formaldehyde solution prepared from the special-grade paraformaldehyde and the commercial special-grade formalin (in cludes methyl alcohol of more than 10%) according to body weight (age) and concentration (2% and 4%). The routinely adopted Litchfield's method was used for the calculation.

2) Measurement of formaldehyde in the eluate of plastic bowl of formaldehyde resin.

As will be mentioned later, the method by Schiff's reagent with fuchsine or rosaniline added was examined in various ways, and the calibration curve was prepared every time. Combined was the method•‹ by chromotropic acid which is said to react specifically with formaldehyde. Using the dilute solution of formal dehyde of a known amount measured by iodimetry, recovery test was performed to confirm the advantages, disadvantages and confidence limits of the above-men tioned three methods (the details of this methodology will not be included here).

Observed also was the relationship between the degree of coloration of the Rimini 22 Kenzaburo Tsuchiya et al reaction, qualitative reaction, and the value of quantitative analysis. The method for eluting plastic bowl will be found in the data to follow and will not be included here.

3) Measurement of formaldehyde in foods.

Formaldehyde in foods was measured according to the literature mentioned below.7 However, some part still requires examination, and therefore, only a part of it will be reported as the results of a preliminary experiment. It was found necessary to examine the analytical method in the future so that formaldehyde should be measured for various kinds of food.8

EXPERIMENTAL RESULTS

1) Lethal dose.

As mentioned previously, male rats were used for the measurement of the

lethal dose. Table 1 and Fig. 1 shows the relationship between the dose admin

istered orally of 2% of the commercial special-grade formalin and the mortality

rate. The body weight was 184 g in average, and the variation was large

(standard deviation= 31.7 g). Table 2 and Fig. 2 demonstrates the results of the administration of 2% solution of formaldehyde not containing methyl alcohol. The rats in this case weighed less than those in Table 1, being 103.4 g •} 13.5 g,

Fig. 1 LD50 for 2% solution of for Fig. 2 LD50 for 2% solution of for malin. maldehyde. LD50 and 95% confidence lim LD50 and 95% confidence lim its: 460 (330-650) mgm/kgm its: 550 (423-715) mgm/kgm S and 95% confidence limits: S and 95% confidence limits: 1.52 (1.04-2.42) 1.6 (1.12-2.29) Toxicity of Formaldehyde 23

Table 1 Oral administration of 2% formalin solution

LD50 460(330-650)mgm/kgm Mean of total body weight: 180.9g•}31.7

Table 2 Oral administration of 2% formaldehyde solution

LD50 550(420-715)mgm/kgm

Mean of total body weight: 103.4g•}13.5 q/, ,

Fig. 3 LD50 for 4% solution of for Fig. 4 LD50 for 4% solution of for maldehyde. maldehyde. Lll50 and 95% confidence lim LD50 and 19/20 confidence lim its: 590 (770-460) mgm/kgm its: 675 (803-560) mgm/kgm S and 95% confidence limits: S and 19/20 confidence limits: 1.65 (2.56-1.06) 1.36 (1.57-1.17) 24 Kenzaburo Tsuchiya et al

Table 3 Oral administration of 4% formaldehyde solution

LD50 590(460-770)mgm/kgm Mean of total body weight: 112.1g•}15

Table 4 Oral administration of 4% formaldehyde solution

LD50 675(567-803)mam/kgm Mean of total body weight: 105.1g•}10

Fig. 5 LD50 for 4% formaldehyde so Fig. 6 LD50 for 4% formalin solution. tion. LD50 and confidence limits: 832 LD50 and 95% confidence lim (965-617) mgm/kgm its: 640 (742-551) mgm/kgm S and 95% confidence limits: S and 95% confidence limits: 1.35 (1.59-1.14) 1.45 (1.18-1.77) Toxicity of Formaldehyde 25

Table 5 (Addition of Table 3 and Table 4) Oral administration of 4% formaldehyde solution

LD50 640(551-742)mgm/kgm Mean of total body weight: 109.1g

Table 6 Oral administration of 4% formalin solution

LDS50 832(617-965)mwm/kem Mean of total body weight: 109.4•}11.7 and the LD50 was 550 mg/kg. The means of LD5O of both cases were within the confidence limits, and no conclusion as to which is larger can be made. In the case of 2% solution, the dose sometimes exceeded several millilitters in volume, and since this was thought to give an excessive load to the stomachs of the rats, producing some effects, the concentration was doubled (4%) for experiments. The result by 4% formaldehyde was LD50+590 mg/kg for the first time (Table 3 and Fig. 3) and 675 mg/kg for the second (Table 4 and Fig. 4), the addition of them amounting to 640 mg/kg (Table 5 and Fig. 5). The average body weight in two experiments was nearly the same, as shown in Table 3 and 4. The LD50 of 4% formalin in nearly the same body weight was 832 mg/kg (Table 6 and Fig. 6) and the confidence limits of 90% ranged from 965 to 617 mg which over laps the confidence limits of formaldehyde. Therefore, in order to observe differ ences in the LD5o by body weight, an experiment was performed on rats weighing 176.6 -L 19.8, and a value, LD5o=710 mg/kg (Table 7 and Fig. 7). It was found from the above results that even though some differences were observed according to the rat or its body weight, there was no extreme difference 26 Kenzaburo Tsuchiya et al

Fig. 7 LD50 for 2% formaldehyde solu tion. LD5o and 95% confidence lim its 710 (910-550) mgm/kgm S and 95% confidence limits: 1.47 (2.01-1.05)

Table 7 Oral administration of 2% formaldehyde solution

LD50 710(550-910)mgm/kgm Mean of total body weight: 176.6•}19.8

in the LD50. There was no significant difference between formaldehyde and the methyl alcohol containing formalin, nor any significant difference according to concentration. However, since in the case of low concentrations, volume of the administration becomes large, it can not be said that the maximum amount pro duced no effects on LD50. The results of several experiments led to the conclusion that the LD50 of formaldehyde exists at the LD50 around 600-700 mg/kg, as shown in Table 8, the comprehensive results in those experiments. Toxicity of Formaldehyde 27

Table 8 Mean values of LD60 and slope of regression curve with confidence limits (comprehensive results)

2) The amount of formaldehyde in the eluate of plastic bowls.

The method for determination of formaldehyde in the eluate of plastic bowls made of formaldehyde resin must be established. First, detailed examinations were attempted on the methods of colorimetric determination by iodimetry and Schiff's reagent (fuchsine, rosaniline) and that by chromotropic acid. a) Examination of the reagent or the quantitative determination. Full examination of the method by iodimetry revealed that samples with the amount of formaldehyde ranging from several to several ten milligrams were accurately measurable, and therefore, it will not be detailed here. Dilute con centrations up to 5 mg can be measured by the method of Schiff's reagent, for which fuchsine or rosaniline are used. Although fuchsine and rosaniline are equal in the chemical term, the reagent from a Japanese company was labelled as "fuchsin" and that of Merck said "rosalinine ." Thus, the two reagents were used separately for the following experiments. Since these coloring matters were found to change after preparation, changes in the extinction according to the number of days elapsed were obtained (Tables 9 and 10 and Figs. 8 and 9). As observed in the tables and figures, the extinction increases as a whole according to the number of days elapsed. It also changes according to the temperature and other factors every time. This increase is less in rosaniline than in fuchsine, suggesting that the letter is more stable (Tables 9 and 10, Figs. 8 and 9). How ever, for the measurement of samples the standard curve should be made every time. In the second step the effects of solvent on coloration should be examined when samples are eluted by such substances as acetic acid. As observed in Table 11 and Fig. 10, the degree of coloration, i.e., the extinction, in the case of acetic 28 Kenzaburo Tsuchiya et al

Fig. 8 Changes in the extinction by Fig. 9 Changes of values of optical the number days after pre density the number of days paration of reagent (Schiff's after preparation of reagent reagent-rosaniline). (Schiff's reagent fuchsine).

Table 9 Changes in the extinction by the number of days elapsed after preparation (by the use of rosaniline of Merck for the Schiff's reagent) Toxicity of Formaldehyde 29

Fig. 10 Comparison of the extinction Fig. 11 Amount of formaldehyde between distilled water and eluted from pasticl bowls. 4% acetic acid used as solvent (Schiff's reagent-rosaniline).

Table 10 Changes in the extinction by the number of days elapsed after preparation (by the use of fuchsine from a Japanese company for the Schiff's reagent) 80 Kenzaburo Tsuchiya et al

Table 11 Comparison of the extinction between distilled water and acetic acid used as solvent (Schiff's reagent rosaniline)

Table 12 Changes in the extinction after preparation of reagent when 4% acetic acid was used as solvent (Schiff's reagent-rosaniline)

acid is different from that of water. Since the changes were linear, quantitative determination was found to be possible if acetic acid water (the same one as that used for elution) was used for preparing the standard curve. Since the standard curve differs similarly to that of water as noted in Table 12, it is needless to say that it should be determined separately each day. The method by chromotropic acid is more stable than that by Schiff's re agent (Table 13), having the advantage of always producing the identical stand ard curve but is slightly dangerous and inconvenient since it requires a large Toxicity of Formaldehyde 31 amount of sulfuric acid and many measuring flasks. Table 14 presents com parison of the results of measurements by fuchsine with those by chromotropic acid, in which they agree well. Table 15 shows the relationship between the formaldehyde content and the color tone of Rimini reaction and the reaction of

Table 13 Changes in the extinction by chromotropic acid

Table 14 Comparison between Schiff's reagent (fuchsine) and chromotropie acid

Table 15 Content of formaldehyde and color tone of Rimini reaction and egg white-iron reaction

The purple blue color of 2 ƒÊg/ml fluid in the egg white-iron reaction is almost close to the light blue color of OƒÊg/ml. The larger the content of formaldehyde, the darker the color tone of purple in the light purple color in 5 ƒÊg/ml-40ƒÊg/ml. 32 Kenzaburo Tsuchiya et al

Table 16 Recovery test (Schiff's reagent-fuchsine)

Mean of recovery rate 94.5%

egg white with iron. Table 16 presents the results of recovery test for which 5 ƒÊg was added to the preliminarily measured formaldehyde in the eluate. After having confirmed the advantages, disadvantages and the precautions to be taken in each measuring method, formaldehyde eluting from the plastic bowls made of formaldehyde resin was measured. b) The amount of eluted formaldehyde in plastic bowls.

Table 17 and Fig. 11 show the concentrations of formaldehyde (ƒÊg/ml)

eluted from plastic bowls, filled with 200 cc of distilled water, for 15 minutes at

intervals of 10•Ž starting with 30•Ž. As evident from the figure, formaldehyde

increases linearly with the rise in temperature. As Table 18 and Fig. 12 demon

strate, when a similar elution was performed with 4% acetic acid water for 15 minutes from 20•Ž to 90•Ž, the increase of formaldehyde became similar to an exponential curve. Therefore, the amount of formaldehyde eluted in water and

Table 17 The amount of formaldehyde eluted from plastic bowls (when extracted using 200 cc of distilled water) (the figures in the table indicate ƒÊg/ml of formaldehyde)

When eluted at 30•Ž, the value cannot be obtained from the calibration curve due to a weak degree of coloration. Toxicity of Formaldehyde 33

Fig. 12 Concentration of formal dehyde eluted from plastic bowls (when extracted by 4% acetic acid).

Table 18

(when extracted using 200 cc of 4% acetic acid) (the figures in the table indicate ƒÊg/ml of formaldehyde)

even in acetic acid water at normal temperature is ignorable.

3) Formaldehyde in foods.

As shown in Table 19, it seems that a considerable amount of formaldehyde is contained in vegetables and fruits. Since, as indicated in the table, there are 34 Kenzaburo Tsuchiya et al

Table 19 Formaldehyde in vegetables

some errors according to the method of measurement, and the method of treating foods is thought to be called into question, it is necessary to perform more de tailed studies in the future.

DISCUSSION

It is very difficult to determine the toxicity of a substance for the living body since many conditions must be taken into consideration. The chief condi tions are :

1) acute, subacute, chronic 2) the route of intake of the toxic substance (ordinarily, via the mouth, skin and respiratory tract) 3) species, sex, age, individual differences, physiological biochemical and histo pathological conditions of animals.

The toxicity of a substance is not clarified until all these minimum conditions are met. Since the toxicity on the human body cannot be studied experimentally except for special cases, we cannot help but refer to the results of animal experi ments. Therefore, strictly speaking, it would be impossible to determine the amount of a substance exerting the minimum toxic action on man. The logical conclusion of this is that it is desirable to remove totally any substance with the least toxic action from the human environment. However, this is almost impos sible in reality, and for instance, its enforcement in industry would mean suspen sion of industry. Nevertheless, it is necessary in spite of various difficulties to establish a maximum allowable concentration which would not cause trouble in the human body, and environmental toxic substance should be controled at least to this degree. However, even this maximum allowable concentration is not abso- Toxicity of Formaldehyde 35 lute and is not free of constant changes according to the results of new experi ments or experiences.

It has been known for many years that formaldehyde possesses toxicity for organisms and, as stated in the beginning poses problems in industrial health. The maximum allowable concentration in the atmosphere has been regulated to

5 ppm-10 ppm in industrial hygiene. Irritation of the skin and the mucous mem brane of the respiratory tract has been the chief toxic effect of atmospheric formaldehyde and the action on the human body after absorption (precluding pulmonary edema and pneumonia, etc.) has not been clarified. The maximum allowable concentration of 5 ppm in the atmosphere results roughly in 6ƒÊg/1.

Estimating the respiratory volume of an adult at light work to be about 600-1000 lit. per hour, and in 8-hour work the volume amounts to 4800-8000 lit. If it is assumed that all the formaldehyde is incorporated into the body, approximately

30-50 mg enters the body. It is unknown how the above-mentioned maximum allowable concentration was obtained, and no literature is found on the oral maximum allowable con centration. Since, as stated previously, establishment of the maximum allowable concentration in the case of long-term administration in humans requires a long time and considerable expense, we hastened to obtain the LD50. The experi mental results showed a considerable fluctuation in the LD50 even in rats accord ing to their conditions (body weight, species, etc.). However, a indicated in the literature,9 it seems to be correct that the LD50 is present in the vicinity of 800 mg/kg. In our experiments, this LD50 was nearly in the confidence limits in the latter half (Tables 4-7) but was slightly lower than this value in the former half (Tables 1-3), showed a higher level of toxicity. Although we expected some late effects of formaldehyde prior to the experiment, they were not observed by single administration as referred to previously, and animals surviving more than 3 days started to show recovery in the body weight in several days after the administration and survived until the experiments were finished. As for the concentration, animals administered with a large dose of 2% tended to show a slightly stronger toxicity than those with a higher concentra tion of 4% due probably to the accompaniment of mechanical action (acute oaatric dilatation). In any case, it would be justifiable to estimate the LD50 conservatively to be 500 mg/kg-800 mg/kg. If chronic effects are to be inferred here, the minimum amount for possible manifestation of chronic toxication is presumed to be ob tained by multiplying those values by 1/1000, i.e., the individual difference is 1/10 in the routinely performed practice, 1/10 in case of the human body and 1/10 because it is chronic. If we are further moderate to estimate 0.5 mg/kg/day, 36 Kenzaburo Tsuchiya et al

for a person weighing 50 kg, it will be 25 mg/day . If it is 25 mg/day when water of 1000 cc containing formaldehyde is taken, the maximum allowable concentra tion would be around 25/1000 mg per 1 ml, i.e., 25 ƒÊg. Although discussion of the method for measuring formaldehyde will be omitted here since it was detailed previously, when it amounts to some micro grams in 1 ml, quantitative determination should be conducted with the afore mentioned precautions strictly observed. Consequently, the values of the formal dehyde in foods should not be fully depended upon until sufficient experiments are accumulated. It is known that a certain amount of formaldehyde is contained in foods, and it is a fact that this substance is produced in the metabolic process of organisms. Accordingly, by determining the total daily amount of this chemical ingested from foods, it may be possible to estimate the maximum allowable con centration for exposures over a long period of time. Although we were able to estimate roughly the minimum toxic effect (in ferring chronic disturbances from acute ones) from the above-mentioned experi ments, we cannot state immediately the maximum allowable concentration for chronic cases on the basis or only this experiment. We believe that the establish ment of a more reliable maximum allowable concentration is not impossible if chronic experiments are performed with the present study as an aid for the careful observation of physiological, biochemical and histopathological findings, and if the values obtained by the detailed analysis of formaldehyde in daily foods are fererred to.

CONCLUSION

1) Performing several experiments with the use of about fifty rats for each experiment, the LD50 of formaldehyde was determined to be 500 mg/kg-800 mg/k.g. 2) In reference to the value of LD5o the acceptable limit in humans via the oral route is inferred to be roughly 25-30 mg/day person in a conservative estimate. However, the value is only a tentative suggestion and further studies on chronic of the chemical and epidemiological studies are needed to draw a final conclusion on the acceptable limit value. 3) It is believed that formaldehyde is contained in many foods in amounts close to this maximum allowable concentration. Therefore, by performing experiments for chronic intoxication in reference to this concentration, the genuine limits for chronic action in drinking water and foods should be obtained in the future. This study was performed and the paper was written in the period of 1957 Toxicity of Formaldehyde 37

1958, but for some reason the publication has been postponed until 1974.

REFERENCES

1. Patty, F. A.: Industrial Hygiene and Toxicology. pp. 933, 1949 2. Flury, F. and Zernik, F.: Schadliche Gase. Springer, Berlin, 1931 3. Threshold limit value for 1955. Adopted at the 18th Annual Meeting of the Amer ican Conference of Governmental Industrial Hygienist, Buffalo, April 24-28, 1955. Arch. Ind. Health. 11: 522. 1955 4. Report of Achievements of the Institute of Public Health, Niigata Pref. No. 81, June 1957 5. Elkins, H.: Chemistry of Industrial Toxicology 316. John Wiley & Sons, Inc., 1950 6. Bricker, C. E. and Hilding, R. T.: Spectro Photometric Method for Determining Formaldehyde. I.E.C. anal. ed. 17: 400, 1945 7. Yanagisawa, F.: Food Sanitation. 8. Ki nig, J. and Schreiber, W.: Die Fltichtigen Stoffe der Nahrungsmittel. 184: 105, 1927 9. Smyth, H. F. and others: The single dose toxicity of some glycols and derivatives. J. Ind. Hyg. Toxicol. 23: 259, 1941