Use of the mouse spot test to investigate the mutagenic potential of triclosan (Irgasan®DP300)

Mutation Research, 7 9 ( 1 9 8 0 ) 7 - - 1 2 © Elsevier/North-Holland Biomedical Press

USE OF THE MOUSE SPOT TEST TO INVESTIGATE THE MUTAGENIC POTENTIAL OF TRICLOSAN (IRGASAN®DP300)

L I A N E B. R U S S E L L a n d C.S. M O N T G O M E R Y

Biology Division, Oak Ridge National Laboratory, Oak Ridge, TN 37830 (U.S.A.) ( R e c e i v e d 23 A p r i l 1 9 8 0 ) (Accepted 2 May 1980)

Summary Triclosan, a chlorophenoxyphenol used in several commercial products, was tested in the mouse in vivo somatic m u t a t i o n test (spot test) by intraperitoneal injection on day 9.25 or 10.25 postconception. Although the dose range tested overlapped the toxic, the frequency of presumed somatic mutations was n o t significantly greater in the experimental groups than in the methanol-injected controls; and the results rule out with 95% confidence a spot incidence 5 or more times greater than the control incidence. These findings fail to confirm the claim by Fahrig et al. (1978) that triclosan is mutagenic in the spot test.

Irgasan®DP300, a product of CIBA--GEIGY Corp., is utilized in soaps, deodorants, and other consumer and professional products requiring a broadspectrum antimicrobial agent. Chemically, it is 5-chloro-2-(2,4-dichlorophenoxy) phenol (see Fig. 1), or triclosan. The same c o m p o u n d was one of a series tested by Fahrig et al. (1978) in an investigation of chlorinated phenols and their impurities. The compound, which was named C13-predioxin by these authors, was reported to be positive in the mouse spot test. Because this finding implied a potential risk in the use of products containing this chemical, we also tested Irgasan®DP300 in the mouse in vivo somatic m u t a t i o n test (spot test). Materials and methods

Source and purity of compound The Irgasan®DP300 employed in our experiments was obtained from CIBA-By acceptance of this article, the publisher or recipient acknowledges the U.S. G ove rnme nt ' s right to retain a nonexclusive, royalty-free license in and to any c o p y r i g h t covering the article. R e s e a r c h sponsored by the Office of Health and Envi ronme nt a l Research, U.S. D e p a r t m e n t of Energy under c o n t r a c t W-7405-eng-26 with the Union Carbide Corporation.

Fig. 1. T h e c o m p o u n d 5 - c h l o r o - 2 - ( 2 , 4 - d i c h l o r o p h e n o x y ) p h e n o l , or t r i c l o s a n , u s e d in this s t u d y is m a r k e t e d as I r g a s a n @ D P 3 0 0 ( C I B A - - G E I G Y ) . F a h r i g ( 1 9 7 8 ) r e f e r s to " I r g a s a n D P 3 0 0 @'' as " C 1 3 - p r e d i o x i n . " F a h r i g et al. ( 1 9 7 8 ) s h o w t h e s a m e f o r m u l a f o r C 1 3 - p r e d i o x i n , b u t e r r o n e o u s l y use t h e c h e m i c a l n a m e 4chloro-2-(2,4-dichlorophenoxy)phenol.

GEIGY Corp. and had a purity of 99.7% (personal communication). CIBA-GEIGY also obtained from Fahrig a sample of the Irgasan®DP300 he had used in the spot test and referred to as C13-predioxin (Fahrig et al., 1978). This sample, analyzed by titration and gas chromatography, was found to correspond in composition to other Irgasan®DP300 batches. Like the sample supplied to us, Fahrig's was 99.7% pure (CIBA--GEIGY, personal communication). It should be noted that the formula published by Fahrig et al. (1978) for "C13-predioxin" is indeed the same as that for Irgasan®DP300, but the name supplied by these authors for the f o r m u l a - 4-chloro-2-(2,4-dichlorophenoxyl)phenol- is incorrect: the first number should be 5, not 4. The name may be abbreviated to triclosan, and we shall use this term for the remainder of this paper, to refer, both, to our Irgasan®DP300 and to Fahrig et al.'s C13-predioxin.

Preparation of solutions and administration Fahrig et al. report that they dissolved triclosan in Hank's balanced salt solution (HBSS). We found that, on combining the substance with HBSS, it formed a gelatinous mass that resisted all attempts (including heating and various kinds of agitation) to dissolve or disperse it. It dissolved readily in DMSO, propylene glycol (PG), polyethylene glycol (PEG 400), ethanol and methanol. A pilot study was carried out in which altogether 148 females from our C57BL ? X T c~ crosses were exposed to these solvents, by themselves or containing various amounts of triclosan, on days 9.25 or 10.25 postconception. Because of the toxicity encountered with other solvents (see Results), methanol was chosen as the solvent for the experiment proper. Triclosan was dissolved in 100% methanol, and enough distilled water was added to achieve a level of 60% of the alcohol. (Note: at 55% methanol, triclosan starts to settle o u t of the solution.) Injected volume was a constant ratio of animal weight, namely 0.1 ml per 25-g mouse; and the exposure level was adjusted by varying the concentration of triclosan in the solution. Administration was by intraperitoneal injection.

Animals, mating and observation The m e t h o d used was the in vivo somatic mutation test, developed by Russell and Major (1957) and used in more recent years in chemical mutagenesis studies (e.g., Russell, 1977; Fahrig, 1975, 1978; Davidson and Dawson, 1977), where it is often referred to as the mammalian spot test. The meaning of the test and interpretation of the various types of spots has been discussed else~ where (Russell, 1978, 1979; Fahrig, 1978). Inbred C57BL/E females (a/a), 3--6 m o n t h s old, were mated to multiplerecessive T-stock (a/a; b/b; cehp/cChp; dse/dse; s/s) males and checked for vagi-

nal plugs on 5 successive mornings. Either 9 or 10 mornings after finding of a plug (days 9.25 or 10.25 postconception), presumed pregnant females were injected with trichlosan in 60% methanol, or with an equal volume of 60% methanol only. A female was presumed pregnant if she had gained at least 1.5 g by day 9.25, or at least 2.2 g by day 10.25. Females that gained less than these respective amounts were injected with 0.1 ml methanol only; if they turned out to have been, in fact, pregnant, observations on their offspring were added to those for the vehicle-control group (except for calculations of average littersize). Occasionally, vaginal plugs went undetected. Offspring from such matings constitute the " u n t r e a t e d control". The experiment was conducted in 6 batches between March and October 1979 in order to obtain adequate numbers of offspring for scoring. Results

Toxicity to mothers Some of the solvents were toxic by themselves. Of 24 and 21 pregnant females injected with 0.2 ml DMSO or 0.2 ml PEG-400, resp., 4 and 5 died within 9 days, i.e., prior to the expected date of delivery. When triclosan was added to some of these solvents for exposures of 5, 10 or 20 mg/kg, the fraction of females dying prior to expected date of delivery was 2/8, 5/8 and 6/6, resp., in the case of DMSO, and 1/9, 0/9 and 5/7, resp., in the case of PEG-400. The 28 females that survived these 2 series yielded only 2 litters, both at 5 mg/kg of triclosan. At doses of trichlosan of 50 or 100 mg/kg, all of 12 injected females died when the solvent was either DMSO or PG. When trying alcohols as possible solvents, we found that 0.1 ml of 95% ethanol (without triclosan) killed each of 6 females within minutes; after injection with the same volume of 50% ethanol, each of 6 females died within 5 h. However, while 0.1 ml (per 25-g mouse) of 100% methanol was toxic (11 of 13 females died before expected delivery), 60% methanol was well tolerated. Of 296 females that received 0.1 ml (per 25-g mouse), only 2 (0.7%) died before the delivery date. Triclosan when dissolved in 60% methanol killed none of 267 females at doses of 1, 2, 4 or 8 mg/kg (weighted average dose, 5.8 mg/kg); 12 of 41 at 25 mg/kg; 2 of 4 at 50 mg/kg; and 2 of 2 at 75 mg/kg. When dissolved in 60% methanol, triclosan is thus probably less toxic than when dissolved in PEG-400, and clearly less so than when dissolved in DMSO. For all results discussed in the remainder of this paper, methanol was the solvent for triclosan.

Survival o f exposed embryos Results bearing on survival of embryos are presented in Table 1. Prenatal survival was clearly reduced by 25 mg/kg triclosan, as indicated by reductions in littersize in both the 9.25- and 10.25-day treated groups. The reduction in proportion of females delivering in the latter group is largely, if not entirely, the result of toxic effects on the mothers (see above). Levels of exposure below 25 mg/kg produced no obvious effects on prenatal survival. For both days 9.25 and 10.25, postnatal survival (between birth and 12 days) was severely reduced after 25 mg/kg of triclosan, and slightly, though significantly, reduced after 8 mg/kg triclosan. An average exposure of 3.2 mg/kg

lo TABLE

1

SURVIVAL TRICLOSAN Stage (days p.c.)

9.25

10.25

AND SPOT FREQUENCY IN DISSOLVED IN METHANOL

Triclosan in 60% methanol (mg/kg)

Number of females injected a

HETEROZYGOUS

MICE

TREATED

IN

UTERO

Proportion with litter a (%)

Average litter size a

Number born b

Survival to 12 days b (%)

RS c (%)

WMVS c (%)

0 d 1, 2, 4 (av. 2.8) 8 25

28 e 76

46.4 e 52.6

6.5 e 5.9

117 241

94.0 94.6

1.8 0

6.4 3.9

27 8

51.9 50.0

7.4 4.8

104 19

87.5 57.9

0 0

3.3 9.1

0 d 2, 4 (av. 3.2) 8 25

29 f 35

65.5 f 62.9

5.6 f 5.4

287 121

95.1 86.0

0 1.9

11.0 10.6

125 31

60.8 25.8

5.6 4.3

427 34

85.7 50.0

0.8 0

15.3 47.1

untreated

--

5.1

118

94.9

0

--(23)

WITH

5.4

a Based only on females that met criteria for sufficient weight gain between plug and treatment days, namely at least 1.5 g for day 9.25, and at least 2.2 g for day 10.25. b B a s e d o n all b i r t h s , i n c l u d i n g t h o s e f r o m f e m a l e s t h a t h a d g a i n e d less t h a n t h e a m o u n t s h o w n i n f o o t note a. c Based on number of animals alive at 12 days. d 60% methanol only. e Results for additional females with insufficient weight gain: of 42 females injected, 16.7% bore litters; average littersize, 4.6. f Results for additional females with insufficient weight gain: of 197 females injected, 19.3% bore litters; average littersize, 4.8.

also caused a significant reduction in postnatal survival (P ~ 0.01) when treatm e n t was on day 10.25. After 50 mg/kg triclosan, only one of 4 injected females delivered (2 died, see above); she had a litter of 3, all of whom died before day 12.

Morphology One newborn in the 9.25-day--1 mg/kg group had a tail kink. Because of the absence of tail kinks and of other externally recognizable abnormalities in the higher dose groups, it is unlikely that this single occurrence was the result of triclosan.

Spots The last 2 columns of Table 1 list the frequencies of animals exhibiting either of 2 types of spots, namely, recessive spots (RS) and midventral white spots (WMVS). With respect to the latter spots, which are interpreted to be the result of cell killing (Russell and Major, 1957; Russell, 1979), it can be seen that, within the 9.25-day set of results, the incidence is not increased by triclosan (the result for the small 25 mg/kg group not differing significantly from control). Within the 10.25-day set, which overall yields somewhat higher WMVS frequencies, triclosan has a clear effect at 25 mg/kg (P ~ 0.0001 in comparison with the 10.25-day-methanol control). A few animals with RS were found in the methanol control and in the 10.25-

11

TABLE 2 OVERALL

FREQUENCY

OF RECESSIVE SPOTS (RS)

Contemp. untreated control 60% methanol control T r i c l o s a n , d a y 9 . 2 5 , 4.9 m g ] k g (av.) T r i c l o s a n , d a y 1 0 . 2 5 , 7.7 m g ] k g (av.)

Number offspring obs.

RS (%)

112 383 330 487

0 0.52 0 1.0

day triclosan groups, where the incidence appeared not to be dose-related. The RS frequencies are summarized in Table 2. There were no cases of RS among 330 animals that had been treated with triclosan as 9.25-day embryos. Among 487 treated on day 10.25, there were 5 (1.0%). Of 383 methanol controls, 2 had RS (0.5%). The RS frequency in the 10.25-day triclosan group does not differ significantly from that in the methanol control (P = 0.7). Discussion

The spot test was carried out in a dose range that overlaps the toxic. The bulk of the data for day 10.25, for example, comes from 8 mg/kg treatment, which produces a significant reduction in postnatal survival and an increase in cell killing (as indicated by WMVS incidence); and the upper end of the dose range in which spot observations were made, 25 mg/kg, was markedly toxic both to the mothers and to the animals exposed in utero. In spite of being administered in the near-toxic dose range, however, triclosan, dissolved in methanol and injected on day 10.25 or day 9.25 p.c., did not produce an RS incidence that differed significantly from that found after methanol alone. For either of the treatment days, the results rule out, with 95% confidence (using Fisher's exact test), an RS incidence 5 or more times greater than the control incidence. This is true for the sum of all doses on either day, or for the 8 mg/kg dose alone on day 10.25. The day-9.25 results also rule out an RS incidence 2 or more times greater than control incidence. It should be noted that the 9.25day set was included in our experiment for 2 reasons: (a) in case triclosan transport to the embryo requires some time; and ( b ) t o provide another possible comparison with the results of Fahrig et al. whose " t e n t h day of fetal developm e n t " probably corresponds to day-9.25 postconception (Fahrig, 1978). A comparison with the experiment of Fahrig et al. (1978) becomes difficult, because these authors report results for 50 mg/kg triclosan dissolved in HBSS. Since, in our experience, the substance appears to be insoluble in HBSS, it is possible that the fluid injected by these investigators contained little or no triclosan in solution, accounting for the fairly good production and survival of offspring from pregnant females injected at a supposed level of 50 mg/kg. When triclosan is truly in solution, the toxicity at 50 mg/kg turns out to be so severe as to make it impossible to obtain adequate numbers of surviving offspring. The 2.4% RS frequency reported by Fahrig et al. appears puzzling if, as one must conclude, little, if any, triclosan was in solution in their experiment.

12

Fahrig et al. did not include a vehicle control in their experiment. Their control figure of 1 spot "of genetic relevance" (i.e., RS by our terminology} in 967 observations (0.1%) represents the historical untreated control for Fahrig's laboratory. In our laboratory, too, the RS frequency in the historical, untreated control is very low, namely 0 in 843. However, the HBSS control, summed from several of our experiments, yielded an RS frequency of 2 in 298 (0.7%), not unlike that of the 60% methanol control in the experiment here reported (0.5%). We conclude that Irgasan®DP300 produces readily detectable toxic effects in embryos of dams receiving a single i.p. injection of 8 mg/kg on day 9.25 or 10.25 postconception, and slight toxic effects even at a weighted-average exposure of 3.2 mg/kg; but that there is n o evidence for induced mutagenicity at 8 mg/kg. Because of survival problems, it is impractible to study mutagenicity at higher exposure levels. References D a v i d s o n , G.E., a n d G.W.P. D a w s o n ( 1 9 7 7 ) T h e i n d u c t i o n o f s o m a t i c m u t a t i o n s in m o u s e e m b r y o s b y b e n z o [ a ] p y r e n e , Arch. Toxicol., 38, 99--103. F a h r i g , R. ( 1 9 7 5 ) A m a m m a l i a n s p o t t e s t : I n d u c t i o n o f g e n e t i c a l t e r a t i o n s in p i g m e n t cells o f m o u s e e m b r y o s w i t h X - r a y s a n d c h e m i c a l m u t a g e n s , Mol. G e n . G e n e t . , 138, 3 0 9 - - 3 1 4 . F a h r i g , R. ( 1 9 7 8 ) T h e m a m m a l i a n s p o t t e s t : A s e n s i t i v e in v i v o m e t h o d f o r t h e d e t e c t i o n o f g e n e t i c altera t i o n s in s o m a t i c cells o f m i c e , i n : A. H o n a c n d e r a n d F . J . d e S e r r e s (Eds.), C h e m i c a l M u t a g e n s , Vol. 5, Plenum, N e w York, pp. 1 5 1 - - 1 7 6 . F a h r i g , R., C.A. N i l s s o n a n d C. R a p p e ( 1 9 7 8 ) G e n e t i c a c t i v i t y o f c h l o r o p h e n o l s a n d c h l o r o p h e n o l i m p u r i ties, i n : K . R . R a o ( E d . ) , P e n t a c h l o r o p h e n o l : C h e m i s t r y , P h a r m a c o l o g y a n d E n v i r o n m e n t a l T o x i c o l o g y , P l e n u m , N e w Y o r k , pp. 3 2 5 - - 3 3 8 . Russell, L.B. ( 1 9 7 7 ) V a l i d a t i o n o f t h e in v i v o s o m a t i c m u t a t i o n m e t h o d in the m o u s e as a p r e s c r e e n f o r g e r m i n a l p o i n t m u t a t i o n s , A r c h . T o x i c o l . , 38, 7 5 - - 8 5 . Russell, L.B. ( 1 9 7 8 ) S o m a t i c cells as i n d i c a t o r s o f g e r m i n a l m u t a t i o n s in the m o u s e , E n v i r o n . H e a l t h Pers p e c t . 24, 1 1 3 - - 1 1 6 . R u s s e l l , L.B. ( 1 9 7 9 ) In v i v o s o m a t i c m u t a t i o n s y s t e m s in t h e m o u s e , G e n e t i c s , 92, s 1 5 3 - - 1 6 3 . Russell, L.B., a n d M.H. M a j o r ( 1 9 5 7 ) R a d i a t i o n - i n d u c e d p r e s u m e d s o m a t i c m u t a t i o n s in t h e h o u s e m o u s e , Genetics, 42, 1 6 1 - - 1 7 5 .