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Evaluation of toxicity of some quaternary ammonium compounds
Journal
of Biological
Standardization
1973 1, 1 l-22
Evaluation of toxicity of some quaternary ammonium compounds* Leonida Arro and C-R. Sahstedt
The toxicity of four different ‘cationic’ quatemary ammonium compounds was tested in cultures of primary monkey kidney cells and in mice and rabbits. The results showed that the compounds were considerably toxic to tissue culturesa final dilution of 1: 40,000 being toxic everywhere. The relative toxicity 1 : 4 : 10 : 20-35 indicated that there were marked differences between the quatemaries. The titrations also showed that the toxic action was time dependent. The lethal dose in mouse tests varied from 10 to 35 mg/kg body weight. The skin test in rabbits gave almost identical level of toxicity for all compounds. Reactions were not observed with dilutions > l/8100 when the test period varied between 1 and 13 days.
INTRODUCTION Comprehensive information on the toxicology of most quaternaries is still lacking. However, a few of them have been examined carefully (Alfredson, Stiefel, Thorp, Baten & Gray, 1951; Kylin, 1949; Nelson & Lyster, 1946; Toomey & Takacs, 1945; Warren, Becker, Marsh & Shelton, 1942). In concentrations commonly used for various disinfection purposes the quaternaries are known to be relatively non-toxic (Lawrence, 1950; Sykes, 1965). In humans, however, dilutions of 1 : 5000 of several quaternaries in water have been tolerated for long periods (Sykes, 1965) but high concentrations can cause severe skin reactions and oedema (Sykes, 1965). When tested in the same system differences between the compounds are apparent and, furthermore, different systems show variations for a single compound (Bettley, 1968; Nelson & Lyster, 1946). Large doses of quatemaries have proved to be lethal for laboratory animals, the LD,, doses are approximately 6-8 mg/kg body weight by the intraperitoneal (i.p.) route, * Received for publication 17 March 1972. From The National Bacteriological 10521 Stockholm, Sweden. Requests for reprints should be addressed to L. Arro. 7
Laboratory,
87
LEONIDA
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10-30 mg/kg by the intravenous (i.v.) route, 200-300 mg/kg by the subcutaneous (s.c.) route and 250-450 mg/kg by oral administration (Alfredson et al., 1951; Kylin, 1949; Nelson & Lyster, 1946). The present investigation was made in order to study the toxicity of four ‘cationic’ quaternaries both to primary monkey kidney cells in tissue culture and to laboratory animals, such as mice and rabbits. The reason for the test was that quatemaries are often used as preservatives in vaccines and thus can exert a toxic effect in various ways. MATERIALS
AND
METHODS
Four quaternary ammonium compounds have been tested: (a) Benzethonium chloride-lO”/o solution from AB Pharmacia.
7% OCH,-CH,-OCH,-CH2-y-CH, CH,
(b) Cetylpyridinium
chloride-crystalline
compound from AB Recip.
+ H33 16 P\ lNic I c1(c) Quarton 16 ES-crystalline
compound from Liljeholmens Stearinfabriks AB.
C,H,. SO,-
(d) Quarton 14 BCl-crystalline
+ I
compound from Liljeholmens Stearinfabriks AB.
cl-
Methods for toxicity tests in tissue cultures Tissue cultures were prepared from the kidneys of Cynomolgus and Ce-rcopithecus monkeys. After outgrowth all culture tubes were examined microscopically and only those with fully developed monolayer and without visible abnormalities were accepted. Serial tenfold dilutions were prepared from the quaternaries using as diluent Parker’s medium 199, which was the same as that used as a maintenance for the tissue cultures. The culture tubes, containing 0.9 ml medium, were inoculated with O-1ml of the dilution of quaternary using 20 tubes for each dilution. At least 20 culture tubes were inoculated with diluent only as a control group and all tubes were rolled and incubated at 37°C for 7 days. 88
TOXICITY
OF
QUATERNARY
AMMONIUM
COMPOUNDS
Both test and control cultures were examined several times during the incubation period and the results of the examinations were recorded as per cent undamaged tissue according to a grading system specially established for these tests; the principles of the system are shown in Table 1. TABLE
1. Grading
system
for recording
the damage
Microscopically
None (a)
In cells
Slight
Traces (b)
differences
from
(such as thinner, etc.) Small holes in the cell sheet
Moderate (4
Severe (4
2
3
4
5
3
4
5
3
3 3
3 4
4 4
4 5
5 5
4
4
4
5
5
5
5
5
5
5
5
5
2
Large holes or non-confluent, but with large areas of cells Non-confluent and only small pieces of tissue left Completely disintegrated
Minor (4
\ Completely’ injured (0
3
1
controls
changes
1 2
0
None
observed A
f
In cell sheet
in the tissue cultures
(a) Those which could be detected only by simultaneous comparison with the controls (such aa minor irregularities in cell shape); (b) slight granularity of small parts of the cell sheet; (c) as before, but involving larger proportions of the culture or more serious changes in some areas, but the majority of the cells still looking healthy; (e) seriously damaged cells (such as granulated, rounded up, etc.) involving the majority of the cell sheet; (f) abnormalities, such as dry appearance and colour changes, indicating cellular death. The gradings can be expressed also as per cent undamaged
Grade
0 1 2 3 4 : 5
tissue.
100% 80% corresponds
to
60% undamaged tissue 20%
40%
0%
In order to obtain a more precise assessment of the degree of the cellular damage a number of test and control cultures from each experiment were tested for their sensitivity against poliovirus (Brunender strain) as follows. The cultures were washed twice with Phosphate Buffered Saline (PBS) and 0.9 ml Parker’s medium 199 added to each tube. The cultures were inoculated with 0.1 ml poliovirus suspension, containing 100 TCID,,, and incubated in a roller apparatus at 37°C for 4 days. After the incubation period the material from the tubes that were inoculated with the same dilution of quaternary compound was pooled. The pools from the same experiment were titrated simultaneously in cell cultures derived from the same cell batch. All titrations were repeated at least once and the results of the virus titrations were used as indices of the amount of undamaged tissue. Mouse test Serial twofold dilutions of the quaternaries were prepared and O-5 ml of each dilution inoculated intravenously into five mice 16-18 g in weight. The five control animals 89
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were inoculated with diluent, Parker’s medium 199 alone. The animals were observed daily for 14 days. Skin test in rabbits Serial threefold dilutions of the quatemaries were injected intracutaneously into rabbits with O-1 ml of each dilution. The diluent was Parker’s medium 199 as before. To perform a proper comparison between the compounds all corresponding dilutions, together with diluent only, were tested on the same side in the same rabbit and four rabbits were used for each dilution. The rabbits were observed daily for evidence of toxic reactions, as indicated by erythema and infiltration. To show the magnitude of the reactions, the readings were graded as : grade 0 for those without any visible reactions and grades 1, 2 and 3 for slight, moderate and considerable reactions respectively. The readings were taken on the first, second, fourth, sixth and thirteenth days. RESULTS
AND
COMMENTS
Toxicity titration in tissue cultures Com..arison of the two test methods. As previously mentioned the toxic effect was estimated by microscopical examination of the tissue culture changes and their sensitivity to poliovirus. In all 175 sets each of ten tissue cultures were tested by both methods. The individual members in each set were graded and the means calculated and recorded as p, per cent undamaged tissue. The standard deviation s, = +18 both for Cynomolgus and Cercopithecus cultures was calculated from the pooled variances between 20 and 80% values. The range of the standard deviations for different culture sets is demonstrated in Fig. 1.
Fig. 1. Scatter of standard deviations
on various damage levels.
The circles in this figure are mean sn values, representing four to six different culture sets. Each culture set was inoculated with poliovirus, incubated, pooled and titrated and the mean titres, Yn, expressed as -lOlog TCID*,,/O.l ml were calculated. The standard deviation sun = f0.6 was estimated from the pooled variances. To analyse the possible correlation between these two methods the corresponding results were plotted against each other in Fig. 2. The range of Yp values, which are the * Tissue Culture
90
Infective
Dose, 50%.
TOXICITY
OF
QUATERNARY
I 80
L 100
Fig. 2. culture, p.
Relationship
AMMONIUM
between
COMPOUNDS
I
I
I
I
60
40 P c%/.)
20
0
poliovirus
titre,
Y,,
and per cent undamaged
tissue
means of at least four different tests, are shown by vertical lines. As seen in Fig. 2 there exists a relationship between these two estimations which may be expressed as Y, =f(p) where
the constants
= a.a+b
a and b can be evaluated when using a linearizing transformation Y*.p = u
and u = a+b.p
as illustrated in Fig. 3. The constants a and b are computed according to the method of
. .
200 2
.
\
‘\
IOO0 100
I 80
I 60
I 40
20\
P (%)
Fig, 3. Function
in Fig. 1 as transformed
by linearizing
transformation.
91
LEONIDA
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AND
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SALENSTEDT
least squares, giving a = -136+13 b = 7.85 + 0.10 thus u = -136+7.85~
with the correlation coefficient r = O-99. The formula above can be rewritten as y,, = -136;+7.85
and is considered to be valid for > 10 TCID,,/O*l tration of poliovirus).
ml (representing the initial concen-
Evaluation of toxicity titres of the quatemaries Four different experiments were performed, two in Cynomolgus and two in Cercopithem monkey kidney cell cultures. In each of these experiments the toxicity of all four quatemaries was titrated simultaneously. The titre was expressed as the negative logarithm of that quaternary concentration which caused 50% damage in the tissue cultures as Y. = - ‘log TCDD,,/l
-0 ml
where TCDD is the tissue culture damage dose, The proportion of damaged cultures caused by the quaternary was calculated by the formula PC-PO PC
where PQ is the mean value of the recordings of undamaged tissue in the toxicity test tubes and PC is the mean value of the recordings of the tissue of control cultures. The proportion of damaged cultures was calculated for each dilution, and toxicity titres, YQ, were interpolated according to Grber’s procedure (Finney, 1952; Kirber, 1931). The results, which are the means of two experiments, are shown in Table 2(a, b) for Cynomolgus TABLE
2.
Results of toxicity titrations performed in Cynomolgzcs and Cercopithecuscultures after various incubation times
Incubation time (days)
t
(a) Cynomolgus 1 2 4 5 7
Quarton 14 BCl
Yo with various quaternaries A > Benzethonium Quarton Cetylpyridinium chloride chloride 16 ES
4.30 4.50 4.54 4.70 4.88
4.80 5.00 5.28 5.30 5.37
5.20 540 5.53 5.80 5.88
5.50 5.70 5.90 6.10 6.22
4.10 4.30 4.83 4.80 5.13
4.80 5.20 5.25 5.30 5.48
5.10 5.30 5.71 5.90 6.10
5.80 6.00 6.23 6.30 6.52
(b) Cercopithecus
1 2 4 5 7
92
TOXICITY
OF
QUATERNARY
AMMONIUM
COMPOUNDS
and Cerco$thecus cultures respectively. As seen in Table 2 the titres differ for various quaternaries and increase with increasing incubation time. This is illustrated in Fig. 4, where the mean titres, regardless of the tissue system used, are plotted against incubation time, t. The titre ranges are illustrated by vertical lines.
0
I
234567 Time (days)
Fig. 4. Comparison of four different quatemary compounds. Mean toxicity titres, Yo, as function of incubation time, t. A-A, Quarton 14 BCI; q ...0, Benzethonium Cl; O-O, Quarton 16 ES; O-O, Cetylpyridinium Cl. To examine if there are any differences between the two tissue culture systems, the means of toxicity titres of the four quaternaries were calculated separately for Cynomo&.u and Cercopithecus cultures and plotted against incubation time, t, in Fig. 5.
6.0
4’50
2I
I
3I
4I
5I
6I
7I
Time (days)
Fig. 5. Comparison of two different tissue culture systems. Mean toxicity function of incubation time, t. Cercopithecus kidney cell cultures.
+-+,
Cynomolgus
kidney
cell cultures;
titres, YO, as x - - - x ,
93
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Toxicity titres as a function of time AS seen in Figs 4 and 5 there exists a relationship between the toxicity titres and incubation time. In the time range l-7 days the relationship fits an equation of the form :
10ez = a.xb where 10sz is the quaternary dilution, x = 1+ t (where t is the incubation time in days) and a and b are the constants. By taking logarithms (to the base 10) of both sides of the equation above -Z = loga+b.logx a straight line is obtained. This equation can be rewritten as: YQ= Y,,+b.log(l+t) where -Z is replaced by YQ, just defined as toxicity titre, and log a by Yo,, representing the intercept value. The means of the toxicity titres, given in Figs 4 and 5, show very good fit to the theoretical curves, which are calculated according to the equation above. As seen in Fig. 5 there seemsto be differences between the Cynomolgus and Cercopithcus culture system. The constants a and b were estimated by ordinary statistical calculation for each combination and the lines were examined individually before further analysis. The following equations were obtained: Yc = 4*0381+ O-8642.log (1 + t) for Quarton 14 BCl Yc = 4.5279 +0*985O.log (1 + t) for Benzethonium Cl Ye = 4*8466+1*1326.log(l
+t)
for Quarton 16 ES
Yc = S-1264+ 1.1995. log (1 + t) for Cetylpyridinium
Cl
when cultures from Cynomolgus monkeys were used, and Yc = 3*.5461+ l-7193 .log (1 + t) for Quarton 14 BCl Yc = 4.5923 + O-9719.log (1 + t) for Benzethonium Cl Ya = 4.5404 + 1.7125.log (1 + t) for Quarton 16 ES Yc = 54466 + 1.1453. log (1 + t) for Cetylpyridinium
Cl
when cultures from Cercopithecus monkeys were used. All the functions showed very good correlation, the coefficients being at least 0.94. The standard errors of regression coefficients varied from 0.07 to 0.21. Estimation of relative toxicities in tissue cultures Estimation of relative toxicities and their error limits was carried out separately for CynomoZgus(A series) and Cercopithecus (B series) cultures. Examination of variance ratios showed that the deviations from parallelism were not greater than could be attributed to random fluctuations. Thus the pooled regression coefficients were computed and the functions above converted to parallel lines as follows. &Al
= 3.9237 + l-04535 .log (1 + t)
GAS = 4.4897 + 1.04535 .log (1 + t) 94
TOXICITY
OF QUATERNARY
AMMONIUM
COMPOUNDS
Y**a = 4.9017 + 1*04535. log (1 + t) YQAI = 5*2237+1*04535.log(l+t) and YQBl = 3*7557+ 1.3872O.log (1 + t) YoB, = 4.3297 + 1.38720. log (1 + t) YQBa= 4.7457 + l-38720. log (1 + t) YQB4= 5.2937 + l-38720. log (1 + t) A and B refer to A and B series and 1, 2, 3 and 4 to Quarton 14 BCl, Benzethonium Cl, Quarton 16 ES and Cetylpyridinium Cl respectively. The logarithm of the relative toxicity, log R, between two quaternaries, i.e. Benze-
where indices
thonium
Cl and Quarton
14 BCl in A series, is obtained
as
log R = YQaz- YQA~= 4.4897 - 3.9237 = 0.5660 Thus the relative Table 3.
TABLE
toxicity
3. Summary
was calculated
of relative toxicities
Quaternary compound
Result of RL
Benzethonium
and the results tabulated
chloride
Relative toxicities to Quarton 14 BCl and their error limits fi I I Cynomolgus cultures Cercopithecuscultures 2.92
-I R RL
2.58 3.68
3.75 4.63
7.55
-I R
6.73
9.77 12.0
15.9 chloride
5.45
9.51
RU Cetylpyridinium
in
estimated from the data of Table 2
RU
Quarton 16 ES
as antilogarithms
14.2 23.8
20.0
34.5 25-l
50.1
The error limits of log R are found by direct application of the t distribution : logRL = (YQa-YQ,)-t.Q and log Ru = (Yea- YoI) + t . SE
where log R, and R, are the lower and upper limits respectively and s, is the standard error of estimate. This error of estimate was calculated from pooled variances. Homogeneity of variances is a necessary condition for validity, thus the variance ratios for the four preparations were first considered. Since there was no evidence of invalidity, the pooled variances could be calculated according to the following:
V(logR) = sa(&+&)
= sEa
where V is the variance, s is the pooled standard deviation and N, and NI are the number 95
LEONIDA
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of values. Standard error of the estimate sx was computed: SE(A)= +0*04583 and SE(B) = f0.07432
respectively for the A and B series. The 5% significance level was chosen. For 12 degrees of freedom the t-deviate is 2.179. The antilogarithms were taken and the results RL and R, tabulated in Table 3. As seen in Table 3 there are considerable differences between the quaternaries within the two tissue culture systems. A comparison between the systems shows slightly higher toxic effects in Cercopithecus cultures. The differences were exceedingly small for Benzethonium Cl and Quarton 16 ES, but almost significant for Cetylpyridinium Cl. As can be seen also in Table 3, the error limits are larger in the B series, thus the results are less reliable than in the A series, where Cynomolgus cultures were used. Mouse test The lethal doses of the quaternaries were determined in two experiments and the titres were calculated according to Kgrber (Grber, 1931; Finney, 1952). The means are recorded as LD,, values and are also expressed as mg/kg body weight, killing 50% of the animals, in Table 4. All control animals were healthy during the experiments. As can be seen from the table, Benzethonium Cl and Quarton 16 ES are less toxic to mice than the two other compounds.
TABLE 4. Lethal dose of various quaternary compounds in mice
Quaternary compound
- lolog LDbO
Benaethonium chloride Cetylpyridinium chloride Quarton 16 ES Quarton 14 BCl
2.93 346 2.96 3.20
w/k 35 10 32 18
Skin test in rabbits Altogether 24 rabbits were used in these experiments. In all, six dilutions were inoculated each into four rabbits. The test animals showed varying degree of reactions depending on the dose of the quaternaries and on the incubation time. Inoculations with the diluent (Parker’s medium 199) showed no sign of skin reactions. The reaction levels of different dilutions of the quaternaries after various incubation times are illustrated in Fig. 6. The readings were recorded according to the earlier described grading system for skin reactions and used as a qualitative measure of the toxic reaction. Without paying regard to the reaction level and only recording if there was any reaction or not, the titres of skin reactions (SR), -lOlog SRD,,, were calculated according to Kiber (1931) and recorded for each reading day (see Table 5). As seen in both Table 5 and Fig. 6, Quarton 16 ES seems to be somewhat less toxic to rabbit skin than the three other compounds. 96
TOXICITY
OF QUATERNARY
AMMONIUM
COMPOUNDS
Days after inoculation
Fig. 6. Comparison of reaction grade of quatemaries to rabbit skin on different dose levels and after various incubation times. n , Quarton 16 ES; 0, Benzethonium Cl; I, Cetylpyridinium Cl; TABLE
5. Comparison of skin toxicity titres, SRD,,, between the tested quatemaries and after various incubation times Calculated
Incubation time (days) 1 2 4 6 7 13
r Quarton 16 ES 2.24 2.47 3.19 3.19 3.19 2.72
SRDbo for various quatemaries A
Benzethonium chloride 2.36 3.43 3-19 3.19 3.19 3.43
Cetylpyridinium chloride 2.36 3.19 2.95 2.95 2.95 3.19
\
Quarton 14 BCI 2.47 3.43 ND 3.19 ND 3.19
DISCUSSION Cell cultures are commonly used in toxicity tests (Penso & Balducci, 1963; Pomerat & Leake, 1954; Verne, 1954; Willmer, 1965). They have the advantages of giving rapid
information about the degree of toxicity at the cellular level and allow the possibilities of recording the results as a function of time as well as being able to evaluate the results by statistical analysis. The limitations are obvious, such as the difficulty in reaching general conclusions and giving little information of the effect on the whole organism (Penso & Balducci, 1963; Willmer, 1965). The examination of the test cultures can be carried out in many ways. Thus in many experiments a microscopical or a metabolic inhibition test would be enough, but in other cases a more precise assessment, such as determination of protein or DNA content (Willmer, 1965), would be necessary.
The method used in our investigations was designed in order to study the effect on cells of various quaternary compounds as a function of the exposure time. The degree of 97
LEONIDA
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damage was analysed by microscopical examination of the cultures and the results were recorded according to an arbitrarily chosen grading system. It was found that correlating with another analytical method, the sensitivity testing against poliovirus was very good. However, as seen in Fig. 2, the relationship is non-linear and the drop of virus titre is much larger than the decrease of undamaged tissue. This could be explained by the mode of action of quaternary compounds in general (Lawrence, 1950). They react with cell membranes and thus possibly hamper the attachment or ingestion of the virion. Obviously the quaternaries exert a deleterious effect to the cell membranes without visible microscopical signs. On the other hand, the results of the microscopical examinations should never be looked upon as true estimates of the cell condition, but only as giving relative information. Despite the theoretical shortcomings, the graded recordings proved to be valuable in the estimations of the degree of damage to tissue cultures as a function of time, giving statistically reproducible results. The investigations showed that there were considerable differences between the quatemaries. Thus the relative toxicity can be expressed approximately as follows: 1 : 4 : 10 : 20 in tissue cultures of Cynomolgus origin, 1 : 4 : 10 : 35 in tissue cultures of Cercopithecus origin, 2 : 1 : 1 : 3 in mouse test and 3 : 3 : 1 : 2 in rabbit skin test for Quarton 14 BCl, Benzethonium Cl, Quarton 16 ES and Cetylpyridinium Cl respectively. The differences between the two tissue culture systems are negligible, except for the last compound, where the difference seems to reach the significance level. There were considerable differences, however, between the toxicity levels in the in vitro and in vivo tests. The differences between the compounds seemed to be less in the in vivo than in the in vitro system. This may perhaps reflect better possibilities to neutralize harmful substances in the whole body. Remarkable also is the varying order of the compounds with regard to toxicity, Quarton 16 ES being ten times more toxic than Quarton 14 BCl in tissue cultures but less toxic in animal tests. There are other reports (Bettley, 1968; Nelson & Lyster, 1946; Verne, 1954) showing the same phenomenon. The estimation of toxicity of four different quaternary compounds showed that all the compounds were toxic to monkey kidney cell cultures, the TCDD,, dose varying between O-7 and 25 pg/ml. Direct comparisons with other investigations cannot be done, because of differences in the test systems or complete lack of data. Thus there is much information available concerning Cetylpyridinium Cl and Benzethonium Cl (Klein & Stevens, 1945; Lawrence, 1950; Merck & Co. Inc., 1960; Nelson & Lyster, 1946; Swan, 1944; Toomey, 1945; Warren et al., 1942) but nothing at all about the two Quartons. However, when compared with the accumulated information (Pomerat & Leake, 1954) of the effect of other chemical substances and drugs on chicken embryo tissue cultures, the toxicity of the quaternaries, as reported in this paper, seemed to be quite high. This is of practical importance since quaternary compounds are often used as preservatives in vaccines in a dilution of 1 : 40,000 or less. The present investigations showed that the quaternaries diluted 1 : 40,000 exert a strong toxic effect on monkey kidney tissue cultures, which can interfere with the safety testing of the vaccines. This problem will be discussed in a later report. In vivo tests showed less differences in toxicity than the in vitro tests. The range of lethal doses in mice was 10-35 mg/kg body weight. The results were in the same order as earlier reported for Cetylpyridinium Cl and other cationic quaternaries (Merck & Co. Inc., 1960; Nelson & Lyster, 1946; Warren et al., 1942). The skin testing in rabbits 98
TOXICITY
OF
QUATERNARY
AMMONIUM
COMPOUNDS
seemed to be the least sensitive method because almost the same reaction level was obtained with all compounds, possibly somewhat lower with Quarton 16 ES. The last method may be inappropriate also in the sense that the rabbit, by itching and scratching itself, can interfere with the true result. REFERENCES Alfredson, B. V., Stiefel, J. R., Thorp, F. Jr, Baten, W. D. & Gray, M. L. (1951). Toxicity studies on alkyldimethylbenzylammonium chloride in rats and dogs. Journal of American Pharmaceutical Association 40, 263-267. Bettley, F. R. (1968). The toxicity of soaps and detergents. BritishJournal of Dermatology 80, 635642. Finney, D. J. (1952). Statistical Method in BioZogicaZ Assay, pp. 370-375 & pp. 524-530. London: Charles Griflin & Co. mrber, G. (1931). Beitrag zur kollektiven Behandhmg pharmakologischer Reihenversuche. Archiv fiir experimentelle Pathologie und Pharmakologie 162, 480-487. Klein, M. & Stevens, D. A. (1945). The in vitro and in wivo activity of synthetic detergents against influenza A virus. Journal of Immunology 50, 265-273. Kylin, 0. (1949). Studies of the toxicity of some surface active quaternary ammonium compounds. Reprint XIIth International Dairy Congress, Stockholm, section IIIa, 100-107. Lawrence, A. (1950). Surface-active Quaternary Ammonium Germicides, pp. 113-126. New York: Academic Press. Merck & Co. Inc. (1960). The Merck Index of Chemicals and Drugs, 7th ed., pp. 130-227. Rahway, New Jersey: Merck & Co. Inc. Nelson, J. W. & Lyster, S. C. (1946). The toxicity of myristyl-gamma-picolinium chloride. Journal of American Pharmaceutical Association 35, 89-94. Paul, J. (1965). Cell and Tissue Culture, 3rd ed., pp. 336-341. Edinburgh and London: E. & S. Livingstone. Penso, G. & Balducci, D. (1963). Tissue Cultures in Biological Research, pp. 360-366. Amsterdam, London & New York: Elsevier Publishing Co. Pomerat, C. M. & Leake, C. D. (1954). Short term cultures for drug assays: general considerations. Annals of the New York Academy of Sciences 58, 1110-1128. Swan, K. C. (1944). Reactivity of the ocular tissues to wetting agents. AmericanJournal of OphthaZmoZogy 27, 1118-l 122. Sykes, G. (1965). Disinfection and Sterilisation, 2nd ed., pp. 368-369. London: E. & F. N. Spon Ltd. Toomey, J. A. & Takacs, W. S. (1945). Effect of cationic detergents in cotton rats: Neutralizing effect of cetamium against poliomyelitis virus. Archives of Pediatrics 62, 337-339. Verne, J. (1954). Cellular sensitivity to drug action in short-term tissue cultures: in vitro correlations with sensitivity in wivo. Annals of the New York Academy of Sciences 58, 1195-1201. Warren, M. R., Becker, T. J., Marsh, D. G. & Shelton, R. S. (1942). Pharmacological and toxicological studies on cetylpyridinium chloride, a new germicide. Journal of Pharmacology and Experimental Therapeutics 74, 401408. Willmer, E. N. (1966) Cells and tissues in culture methods. Biology and Physiology, Vol. 3, pp. 3.51-396. London & New York: Academic Press.
99
of Biological
Standardization
1973 1, 1 l-22
Evaluation of toxicity of some quaternary ammonium compounds* Leonida Arro and C-R. Sahstedt
The toxicity of four different ‘cationic’ quatemary ammonium compounds was tested in cultures of primary monkey kidney cells and in mice and rabbits. The results showed that the compounds were considerably toxic to tissue culturesa final dilution of 1: 40,000 being toxic everywhere. The relative toxicity 1 : 4 : 10 : 20-35 indicated that there were marked differences between the quatemaries. The titrations also showed that the toxic action was time dependent. The lethal dose in mouse tests varied from 10 to 35 mg/kg body weight. The skin test in rabbits gave almost identical level of toxicity for all compounds. Reactions were not observed with dilutions > l/8100 when the test period varied between 1 and 13 days.
INTRODUCTION Comprehensive information on the toxicology of most quaternaries is still lacking. However, a few of them have been examined carefully (Alfredson, Stiefel, Thorp, Baten & Gray, 1951; Kylin, 1949; Nelson & Lyster, 1946; Toomey & Takacs, 1945; Warren, Becker, Marsh & Shelton, 1942). In concentrations commonly used for various disinfection purposes the quaternaries are known to be relatively non-toxic (Lawrence, 1950; Sykes, 1965). In humans, however, dilutions of 1 : 5000 of several quaternaries in water have been tolerated for long periods (Sykes, 1965) but high concentrations can cause severe skin reactions and oedema (Sykes, 1965). When tested in the same system differences between the compounds are apparent and, furthermore, different systems show variations for a single compound (Bettley, 1968; Nelson & Lyster, 1946). Large doses of quatemaries have proved to be lethal for laboratory animals, the LD,, doses are approximately 6-8 mg/kg body weight by the intraperitoneal (i.p.) route, * Received for publication 17 March 1972. From The National Bacteriological 10521 Stockholm, Sweden. Requests for reprints should be addressed to L. Arro. 7
Laboratory,
87
LEONIDA
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SALENSTEDT
10-30 mg/kg by the intravenous (i.v.) route, 200-300 mg/kg by the subcutaneous (s.c.) route and 250-450 mg/kg by oral administration (Alfredson et al., 1951; Kylin, 1949; Nelson & Lyster, 1946). The present investigation was made in order to study the toxicity of four ‘cationic’ quaternaries both to primary monkey kidney cells in tissue culture and to laboratory animals, such as mice and rabbits. The reason for the test was that quatemaries are often used as preservatives in vaccines and thus can exert a toxic effect in various ways. MATERIALS
AND
METHODS
Four quaternary ammonium compounds have been tested: (a) Benzethonium chloride-lO”/o solution from AB Pharmacia.
7% OCH,-CH,-OCH,-CH2-y-CH, CH,
(b) Cetylpyridinium
chloride-crystalline
compound from AB Recip.
+ H33 16 P\ lNic I c1(c) Quarton 16 ES-crystalline
compound from Liljeholmens Stearinfabriks AB.
C,H,. SO,-
(d) Quarton 14 BCl-crystalline
+ I
compound from Liljeholmens Stearinfabriks AB.
cl-
Methods for toxicity tests in tissue cultures Tissue cultures were prepared from the kidneys of Cynomolgus and Ce-rcopithecus monkeys. After outgrowth all culture tubes were examined microscopically and only those with fully developed monolayer and without visible abnormalities were accepted. Serial tenfold dilutions were prepared from the quaternaries using as diluent Parker’s medium 199, which was the same as that used as a maintenance for the tissue cultures. The culture tubes, containing 0.9 ml medium, were inoculated with O-1ml of the dilution of quaternary using 20 tubes for each dilution. At least 20 culture tubes were inoculated with diluent only as a control group and all tubes were rolled and incubated at 37°C for 7 days. 88
TOXICITY
OF
QUATERNARY
AMMONIUM
COMPOUNDS
Both test and control cultures were examined several times during the incubation period and the results of the examinations were recorded as per cent undamaged tissue according to a grading system specially established for these tests; the principles of the system are shown in Table 1. TABLE
1. Grading
system
for recording
the damage
Microscopically
None (a)
In cells
Slight
Traces (b)
differences
from
(such as thinner, etc.) Small holes in the cell sheet
Moderate (4
Severe (4
2
3
4
5
3
4
5
3
3 3
3 4
4 4
4 5
5 5
4
4
4
5
5
5
5
5
5
5
5
5
2
Large holes or non-confluent, but with large areas of cells Non-confluent and only small pieces of tissue left Completely disintegrated
Minor (4
\ Completely’ injured (0
3
1
controls
changes
1 2
0
None
observed A
f
In cell sheet
in the tissue cultures
(a) Those which could be detected only by simultaneous comparison with the controls (such aa minor irregularities in cell shape); (b) slight granularity of small parts of the cell sheet; (c) as before, but involving larger proportions of the culture or more serious changes in some areas, but the majority of the cells still looking healthy; (e) seriously damaged cells (such as granulated, rounded up, etc.) involving the majority of the cell sheet; (f) abnormalities, such as dry appearance and colour changes, indicating cellular death. The gradings can be expressed also as per cent undamaged
Grade
0 1 2 3 4 : 5
tissue.
100% 80% corresponds
to
60% undamaged tissue 20%
40%
0%
In order to obtain a more precise assessment of the degree of the cellular damage a number of test and control cultures from each experiment were tested for their sensitivity against poliovirus (Brunender strain) as follows. The cultures were washed twice with Phosphate Buffered Saline (PBS) and 0.9 ml Parker’s medium 199 added to each tube. The cultures were inoculated with 0.1 ml poliovirus suspension, containing 100 TCID,,, and incubated in a roller apparatus at 37°C for 4 days. After the incubation period the material from the tubes that were inoculated with the same dilution of quaternary compound was pooled. The pools from the same experiment were titrated simultaneously in cell cultures derived from the same cell batch. All titrations were repeated at least once and the results of the virus titrations were used as indices of the amount of undamaged tissue. Mouse test Serial twofold dilutions of the quaternaries were prepared and O-5 ml of each dilution inoculated intravenously into five mice 16-18 g in weight. The five control animals 89
LEONIDA
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were inoculated with diluent, Parker’s medium 199 alone. The animals were observed daily for 14 days. Skin test in rabbits Serial threefold dilutions of the quatemaries were injected intracutaneously into rabbits with O-1 ml of each dilution. The diluent was Parker’s medium 199 as before. To perform a proper comparison between the compounds all corresponding dilutions, together with diluent only, were tested on the same side in the same rabbit and four rabbits were used for each dilution. The rabbits were observed daily for evidence of toxic reactions, as indicated by erythema and infiltration. To show the magnitude of the reactions, the readings were graded as : grade 0 for those without any visible reactions and grades 1, 2 and 3 for slight, moderate and considerable reactions respectively. The readings were taken on the first, second, fourth, sixth and thirteenth days. RESULTS
AND
COMMENTS
Toxicity titration in tissue cultures Com..arison of the two test methods. As previously mentioned the toxic effect was estimated by microscopical examination of the tissue culture changes and their sensitivity to poliovirus. In all 175 sets each of ten tissue cultures were tested by both methods. The individual members in each set were graded and the means calculated and recorded as p, per cent undamaged tissue. The standard deviation s, = +18 both for Cynomolgus and Cercopithecus cultures was calculated from the pooled variances between 20 and 80% values. The range of the standard deviations for different culture sets is demonstrated in Fig. 1.
Fig. 1. Scatter of standard deviations
on various damage levels.
The circles in this figure are mean sn values, representing four to six different culture sets. Each culture set was inoculated with poliovirus, incubated, pooled and titrated and the mean titres, Yn, expressed as -lOlog TCID*,,/O.l ml were calculated. The standard deviation sun = f0.6 was estimated from the pooled variances. To analyse the possible correlation between these two methods the corresponding results were plotted against each other in Fig. 2. The range of Yp values, which are the * Tissue Culture
90
Infective
Dose, 50%.
TOXICITY
OF
QUATERNARY
I 80
L 100
Fig. 2. culture, p.
Relationship
AMMONIUM
between
COMPOUNDS
I
I
I
I
60
40 P c%/.)
20
0
poliovirus
titre,
Y,,
and per cent undamaged
tissue
means of at least four different tests, are shown by vertical lines. As seen in Fig. 2 there exists a relationship between these two estimations which may be expressed as Y, =f(p) where
the constants
= a.a+b
a and b can be evaluated when using a linearizing transformation Y*.p = u
and u = a+b.p
as illustrated in Fig. 3. The constants a and b are computed according to the method of
. .
200 2
.
\
‘\
IOO0 100
I 80
I 60
I 40
20\
P (%)
Fig, 3. Function
in Fig. 1 as transformed
by linearizing
transformation.
91
LEONIDA
ARRO
AND
C-R.
SALENSTEDT
least squares, giving a = -136+13 b = 7.85 + 0.10 thus u = -136+7.85~
with the correlation coefficient r = O-99. The formula above can be rewritten as y,, = -136;+7.85
and is considered to be valid for > 10 TCID,,/O*l tration of poliovirus).
ml (representing the initial concen-
Evaluation of toxicity titres of the quatemaries Four different experiments were performed, two in Cynomolgus and two in Cercopithem monkey kidney cell cultures. In each of these experiments the toxicity of all four quatemaries was titrated simultaneously. The titre was expressed as the negative logarithm of that quaternary concentration which caused 50% damage in the tissue cultures as Y. = - ‘log TCDD,,/l
-0 ml
where TCDD is the tissue culture damage dose, The proportion of damaged cultures caused by the quaternary was calculated by the formula PC-PO PC
where PQ is the mean value of the recordings of undamaged tissue in the toxicity test tubes and PC is the mean value of the recordings of the tissue of control cultures. The proportion of damaged cultures was calculated for each dilution, and toxicity titres, YQ, were interpolated according to Grber’s procedure (Finney, 1952; Kirber, 1931). The results, which are the means of two experiments, are shown in Table 2(a, b) for Cynomolgus TABLE
2.
Results of toxicity titrations performed in Cynomolgzcs and Cercopithecuscultures after various incubation times
Incubation time (days)
t
(a) Cynomolgus 1 2 4 5 7
Quarton 14 BCl
Yo with various quaternaries A > Benzethonium Quarton Cetylpyridinium chloride chloride 16 ES
4.30 4.50 4.54 4.70 4.88
4.80 5.00 5.28 5.30 5.37
5.20 540 5.53 5.80 5.88
5.50 5.70 5.90 6.10 6.22
4.10 4.30 4.83 4.80 5.13
4.80 5.20 5.25 5.30 5.48
5.10 5.30 5.71 5.90 6.10
5.80 6.00 6.23 6.30 6.52
(b) Cercopithecus
1 2 4 5 7
92
TOXICITY
OF
QUATERNARY
AMMONIUM
COMPOUNDS
and Cerco$thecus cultures respectively. As seen in Table 2 the titres differ for various quaternaries and increase with increasing incubation time. This is illustrated in Fig. 4, where the mean titres, regardless of the tissue system used, are plotted against incubation time, t. The titre ranges are illustrated by vertical lines.
0
I
234567 Time (days)
Fig. 4. Comparison of four different quatemary compounds. Mean toxicity titres, Yo, as function of incubation time, t. A-A, Quarton 14 BCI; q ...0, Benzethonium Cl; O-O, Quarton 16 ES; O-O, Cetylpyridinium Cl. To examine if there are any differences between the two tissue culture systems, the means of toxicity titres of the four quaternaries were calculated separately for Cynomo&.u and Cercopithecus cultures and plotted against incubation time, t, in Fig. 5.
6.0
4’50
2I
I
3I
4I
5I
6I
7I
Time (days)
Fig. 5. Comparison of two different tissue culture systems. Mean toxicity function of incubation time, t. Cercopithecus kidney cell cultures.
+-+,
Cynomolgus
kidney
cell cultures;
titres, YO, as x - - - x ,
93
LEONIDA
ARRO
AND
C-R.
SALENSTEDT
Toxicity titres as a function of time AS seen in Figs 4 and 5 there exists a relationship between the toxicity titres and incubation time. In the time range l-7 days the relationship fits an equation of the form :
10ez = a.xb where 10sz is the quaternary dilution, x = 1+ t (where t is the incubation time in days) and a and b are the constants. By taking logarithms (to the base 10) of both sides of the equation above -Z = loga+b.logx a straight line is obtained. This equation can be rewritten as: YQ= Y,,+b.log(l+t) where -Z is replaced by YQ, just defined as toxicity titre, and log a by Yo,, representing the intercept value. The means of the toxicity titres, given in Figs 4 and 5, show very good fit to the theoretical curves, which are calculated according to the equation above. As seen in Fig. 5 there seemsto be differences between the Cynomolgus and Cercopithcus culture system. The constants a and b were estimated by ordinary statistical calculation for each combination and the lines were examined individually before further analysis. The following equations were obtained: Yc = 4*0381+ O-8642.log (1 + t) for Quarton 14 BCl Yc = 4.5279 +0*985O.log (1 + t) for Benzethonium Cl Ye = 4*8466+1*1326.log(l
+t)
for Quarton 16 ES
Yc = S-1264+ 1.1995. log (1 + t) for Cetylpyridinium
Cl
when cultures from Cynomolgus monkeys were used, and Yc = 3*.5461+ l-7193 .log (1 + t) for Quarton 14 BCl Yc = 4.5923 + O-9719.log (1 + t) for Benzethonium Cl Ya = 4.5404 + 1.7125.log (1 + t) for Quarton 16 ES Yc = 54466 + 1.1453. log (1 + t) for Cetylpyridinium
Cl
when cultures from Cercopithecus monkeys were used. All the functions showed very good correlation, the coefficients being at least 0.94. The standard errors of regression coefficients varied from 0.07 to 0.21. Estimation of relative toxicities in tissue cultures Estimation of relative toxicities and their error limits was carried out separately for CynomoZgus(A series) and Cercopithecus (B series) cultures. Examination of variance ratios showed that the deviations from parallelism were not greater than could be attributed to random fluctuations. Thus the pooled regression coefficients were computed and the functions above converted to parallel lines as follows. &Al
= 3.9237 + l-04535 .log (1 + t)
GAS = 4.4897 + 1.04535 .log (1 + t) 94
TOXICITY
OF QUATERNARY
AMMONIUM
COMPOUNDS
Y**a = 4.9017 + 1*04535. log (1 + t) YQAI = 5*2237+1*04535.log(l+t) and YQBl = 3*7557+ 1.3872O.log (1 + t) YoB, = 4.3297 + 1.38720. log (1 + t) YQBa= 4.7457 + l-38720. log (1 + t) YQB4= 5.2937 + l-38720. log (1 + t) A and B refer to A and B series and 1, 2, 3 and 4 to Quarton 14 BCl, Benzethonium Cl, Quarton 16 ES and Cetylpyridinium Cl respectively. The logarithm of the relative toxicity, log R, between two quaternaries, i.e. Benze-
where indices
thonium
Cl and Quarton
14 BCl in A series, is obtained
as
log R = YQaz- YQA~= 4.4897 - 3.9237 = 0.5660 Thus the relative Table 3.
TABLE
toxicity
3. Summary
was calculated
of relative toxicities
Quaternary compound
Result of RL
Benzethonium
and the results tabulated
chloride
Relative toxicities to Quarton 14 BCl and their error limits fi I I Cynomolgus cultures Cercopithecuscultures 2.92
-I R RL
2.58 3.68
3.75 4.63
7.55
-I R
6.73
9.77 12.0
15.9 chloride
5.45
9.51
RU Cetylpyridinium
in
estimated from the data of Table 2
RU
Quarton 16 ES
as antilogarithms
14.2 23.8
20.0
34.5 25-l
50.1
The error limits of log R are found by direct application of the t distribution : logRL = (YQa-YQ,)-t.Q and log Ru = (Yea- YoI) + t . SE
where log R, and R, are the lower and upper limits respectively and s, is the standard error of estimate. This error of estimate was calculated from pooled variances. Homogeneity of variances is a necessary condition for validity, thus the variance ratios for the four preparations were first considered. Since there was no evidence of invalidity, the pooled variances could be calculated according to the following:
V(logR) = sa(&+&)
= sEa
where V is the variance, s is the pooled standard deviation and N, and NI are the number 95
LEONIDA
ARRO
AND
C-R.
SALENSTEDT
of values. Standard error of the estimate sx was computed: SE(A)= +0*04583 and SE(B) = f0.07432
respectively for the A and B series. The 5% significance level was chosen. For 12 degrees of freedom the t-deviate is 2.179. The antilogarithms were taken and the results RL and R, tabulated in Table 3. As seen in Table 3 there are considerable differences between the quaternaries within the two tissue culture systems. A comparison between the systems shows slightly higher toxic effects in Cercopithecus cultures. The differences were exceedingly small for Benzethonium Cl and Quarton 16 ES, but almost significant for Cetylpyridinium Cl. As can be seen also in Table 3, the error limits are larger in the B series, thus the results are less reliable than in the A series, where Cynomolgus cultures were used. Mouse test The lethal doses of the quaternaries were determined in two experiments and the titres were calculated according to Kgrber (Grber, 1931; Finney, 1952). The means are recorded as LD,, values and are also expressed as mg/kg body weight, killing 50% of the animals, in Table 4. All control animals were healthy during the experiments. As can be seen from the table, Benzethonium Cl and Quarton 16 ES are less toxic to mice than the two other compounds.
TABLE 4. Lethal dose of various quaternary compounds in mice
Quaternary compound
- lolog LDbO
Benaethonium chloride Cetylpyridinium chloride Quarton 16 ES Quarton 14 BCl
2.93 346 2.96 3.20
w/k 35 10 32 18
Skin test in rabbits Altogether 24 rabbits were used in these experiments. In all, six dilutions were inoculated each into four rabbits. The test animals showed varying degree of reactions depending on the dose of the quaternaries and on the incubation time. Inoculations with the diluent (Parker’s medium 199) showed no sign of skin reactions. The reaction levels of different dilutions of the quaternaries after various incubation times are illustrated in Fig. 6. The readings were recorded according to the earlier described grading system for skin reactions and used as a qualitative measure of the toxic reaction. Without paying regard to the reaction level and only recording if there was any reaction or not, the titres of skin reactions (SR), -lOlog SRD,,, were calculated according to Kiber (1931) and recorded for each reading day (see Table 5). As seen in both Table 5 and Fig. 6, Quarton 16 ES seems to be somewhat less toxic to rabbit skin than the three other compounds. 96
TOXICITY
OF QUATERNARY
AMMONIUM
COMPOUNDS
Days after inoculation
Fig. 6. Comparison of reaction grade of quatemaries to rabbit skin on different dose levels and after various incubation times. n , Quarton 16 ES; 0, Benzethonium Cl; I, Cetylpyridinium Cl; TABLE
5. Comparison of skin toxicity titres, SRD,,, between the tested quatemaries and after various incubation times Calculated
Incubation time (days) 1 2 4 6 7 13
r Quarton 16 ES 2.24 2.47 3.19 3.19 3.19 2.72
SRDbo for various quatemaries A
Benzethonium chloride 2.36 3.43 3-19 3.19 3.19 3.43
Cetylpyridinium chloride 2.36 3.19 2.95 2.95 2.95 3.19
\
Quarton 14 BCI 2.47 3.43 ND 3.19 ND 3.19
DISCUSSION Cell cultures are commonly used in toxicity tests (Penso & Balducci, 1963; Pomerat & Leake, 1954; Verne, 1954; Willmer, 1965). They have the advantages of giving rapid
information about the degree of toxicity at the cellular level and allow the possibilities of recording the results as a function of time as well as being able to evaluate the results by statistical analysis. The limitations are obvious, such as the difficulty in reaching general conclusions and giving little information of the effect on the whole organism (Penso & Balducci, 1963; Willmer, 1965). The examination of the test cultures can be carried out in many ways. Thus in many experiments a microscopical or a metabolic inhibition test would be enough, but in other cases a more precise assessment, such as determination of protein or DNA content (Willmer, 1965), would be necessary.
The method used in our investigations was designed in order to study the effect on cells of various quaternary compounds as a function of the exposure time. The degree of 97
LEONIDA
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AND
C-R.
SALENSTEDT
damage was analysed by microscopical examination of the cultures and the results were recorded according to an arbitrarily chosen grading system. It was found that correlating with another analytical method, the sensitivity testing against poliovirus was very good. However, as seen in Fig. 2, the relationship is non-linear and the drop of virus titre is much larger than the decrease of undamaged tissue. This could be explained by the mode of action of quaternary compounds in general (Lawrence, 1950). They react with cell membranes and thus possibly hamper the attachment or ingestion of the virion. Obviously the quaternaries exert a deleterious effect to the cell membranes without visible microscopical signs. On the other hand, the results of the microscopical examinations should never be looked upon as true estimates of the cell condition, but only as giving relative information. Despite the theoretical shortcomings, the graded recordings proved to be valuable in the estimations of the degree of damage to tissue cultures as a function of time, giving statistically reproducible results. The investigations showed that there were considerable differences between the quatemaries. Thus the relative toxicity can be expressed approximately as follows: 1 : 4 : 10 : 20 in tissue cultures of Cynomolgus origin, 1 : 4 : 10 : 35 in tissue cultures of Cercopithecus origin, 2 : 1 : 1 : 3 in mouse test and 3 : 3 : 1 : 2 in rabbit skin test for Quarton 14 BCl, Benzethonium Cl, Quarton 16 ES and Cetylpyridinium Cl respectively. The differences between the two tissue culture systems are negligible, except for the last compound, where the difference seems to reach the significance level. There were considerable differences, however, between the toxicity levels in the in vitro and in vivo tests. The differences between the compounds seemed to be less in the in vivo than in the in vitro system. This may perhaps reflect better possibilities to neutralize harmful substances in the whole body. Remarkable also is the varying order of the compounds with regard to toxicity, Quarton 16 ES being ten times more toxic than Quarton 14 BCl in tissue cultures but less toxic in animal tests. There are other reports (Bettley, 1968; Nelson & Lyster, 1946; Verne, 1954) showing the same phenomenon. The estimation of toxicity of four different quaternary compounds showed that all the compounds were toxic to monkey kidney cell cultures, the TCDD,, dose varying between O-7 and 25 pg/ml. Direct comparisons with other investigations cannot be done, because of differences in the test systems or complete lack of data. Thus there is much information available concerning Cetylpyridinium Cl and Benzethonium Cl (Klein & Stevens, 1945; Lawrence, 1950; Merck & Co. Inc., 1960; Nelson & Lyster, 1946; Swan, 1944; Toomey, 1945; Warren et al., 1942) but nothing at all about the two Quartons. However, when compared with the accumulated information (Pomerat & Leake, 1954) of the effect of other chemical substances and drugs on chicken embryo tissue cultures, the toxicity of the quaternaries, as reported in this paper, seemed to be quite high. This is of practical importance since quaternary compounds are often used as preservatives in vaccines in a dilution of 1 : 40,000 or less. The present investigations showed that the quaternaries diluted 1 : 40,000 exert a strong toxic effect on monkey kidney tissue cultures, which can interfere with the safety testing of the vaccines. This problem will be discussed in a later report. In vivo tests showed less differences in toxicity than the in vitro tests. The range of lethal doses in mice was 10-35 mg/kg body weight. The results were in the same order as earlier reported for Cetylpyridinium Cl and other cationic quaternaries (Merck & Co. Inc., 1960; Nelson & Lyster, 1946; Warren et al., 1942). The skin testing in rabbits 98
TOXICITY
OF
QUATERNARY
AMMONIUM
COMPOUNDS
seemed to be the least sensitive method because almost the same reaction level was obtained with all compounds, possibly somewhat lower with Quarton 16 ES. The last method may be inappropriate also in the sense that the rabbit, by itching and scratching itself, can interfere with the true result. REFERENCES Alfredson, B. V., Stiefel, J. R., Thorp, F. Jr, Baten, W. D. & Gray, M. L. (1951). Toxicity studies on alkyldimethylbenzylammonium chloride in rats and dogs. Journal of American Pharmaceutical Association 40, 263-267. Bettley, F. R. (1968). The toxicity of soaps and detergents. BritishJournal of Dermatology 80, 635642. Finney, D. J. (1952). Statistical Method in BioZogicaZ Assay, pp. 370-375 & pp. 524-530. London: Charles Griflin & Co. mrber, G. (1931). Beitrag zur kollektiven Behandhmg pharmakologischer Reihenversuche. Archiv fiir experimentelle Pathologie und Pharmakologie 162, 480-487. Klein, M. & Stevens, D. A. (1945). The in vitro and in wivo activity of synthetic detergents against influenza A virus. Journal of Immunology 50, 265-273. Kylin, 0. (1949). Studies of the toxicity of some surface active quaternary ammonium compounds. Reprint XIIth International Dairy Congress, Stockholm, section IIIa, 100-107. Lawrence, A. (1950). Surface-active Quaternary Ammonium Germicides, pp. 113-126. New York: Academic Press. Merck & Co. Inc. (1960). The Merck Index of Chemicals and Drugs, 7th ed., pp. 130-227. Rahway, New Jersey: Merck & Co. Inc. Nelson, J. W. & Lyster, S. C. (1946). The toxicity of myristyl-gamma-picolinium chloride. Journal of American Pharmaceutical Association 35, 89-94. Paul, J. (1965). Cell and Tissue Culture, 3rd ed., pp. 336-341. Edinburgh and London: E. & S. Livingstone. Penso, G. & Balducci, D. (1963). Tissue Cultures in Biological Research, pp. 360-366. Amsterdam, London & New York: Elsevier Publishing Co. Pomerat, C. M. & Leake, C. D. (1954). Short term cultures for drug assays: general considerations. Annals of the New York Academy of Sciences 58, 1110-1128. Swan, K. C. (1944). Reactivity of the ocular tissues to wetting agents. AmericanJournal of OphthaZmoZogy 27, 1118-l 122. Sykes, G. (1965). Disinfection and Sterilisation, 2nd ed., pp. 368-369. London: E. & F. N. Spon Ltd. Toomey, J. A. & Takacs, W. S. (1945). Effect of cationic detergents in cotton rats: Neutralizing effect of cetamium against poliomyelitis virus. Archives of Pediatrics 62, 337-339. Verne, J. (1954). Cellular sensitivity to drug action in short-term tissue cultures: in vitro correlations with sensitivity in wivo. Annals of the New York Academy of Sciences 58, 1195-1201. Warren, M. R., Becker, T. J., Marsh, D. G. & Shelton, R. S. (1942). Pharmacological and toxicological studies on cetylpyridinium chloride, a new germicide. Journal of Pharmacology and Experimental Therapeutics 74, 401408. Willmer, E. N. (1966) Cells and tissues in culture methods. Biology and Physiology, Vol. 3, pp. 3.51-396. London & New York: Academic Press.
99