Toxicology, 6 (1976) 67--76 © Elsevier/North-Holland, Amsterdam -- Printed in The Netherlands
P E R C U T A N E O U S ABSORPTION OF T R I C L O C A R B A N IN RAT AND MAN
D. HOWES and J.G. BLACK Environmental Safety Division, Unilever Research Laboratory, Colworth House, Sharnbrook, Bedford, MK44 1LQ (Great Britain) (Received November 19th, 1975) (Accepted January 17th, 1976)
SUMMARY
The route and rate of excretion b y rats of the germicide [14C]Triclo carban formerly called trichlorocarbanilide, given by parenteral injection has been investigated. Blood levels based on radioactivity and b y chemical determination after parenteral injection have been compared with those obtained after topical application of [' 4 C/Triclocarban in acetone. Absorption of [14C]Triclocarban in soaps and in dimethylformamide (DMF) through occluded rat skin has been studied. Other soaps and a hand cleanser containing [14 C/Triclocarban have been applied to rat skin witho u t occlusion and the effects of duration of contact, concentration and the use of a solubilizer have been investigated. In humans, absorption of Triclocarban through skin after bathing daily for 28 days has been investigated by chemical analysis of blood and urine. The data show that elimination by the rat is rapid and complete principally via the faeces. Blood levels after parenteral injection are low and comparison of the radioactivity and chemical determinations suggest rapid metabolism of the Triclocarban. After application to the skin, blood levels based on 4 C are very low. Absorption of [14 C]Triclocarban through occluded rat skin was greater from DMF than from soaps. With non-occluded rat skin, absorption from soaps was less and was dependent on concentration b u t independent of duration of contact. The use of a solubilizer did not increase absorption through skin. No measurable Triclocarban (< 25 ppb) was present in blood and urine samples of volunteers during or shortly after a 28-day intensive bathing regimen.
Abbreviations: ABS, alkyi benzene sulphonate; DGME, dipropyleneglycol methyl ether; DMF, dimethyl formamide; Triclocarban (TCC), 3,4,41 -trichloroearbanilide.
67
INTRODUCTION We previously studied the deposition and percutaneous absorption of [ ~4 C] TCC a germicide now called Triclocarban, which is used in toilet soaps [1]. No radioactivity was detected in blood, liver or depot fat of guinea pigs although the sensitivity of the method was equivalent to 10 ppb of [14 C]TCC. However, small amounts of radioactivity were present in the excreta of the guinea pigs and these amounts were equivalent to an absorption of less than 0.5 pg [1~C] TCC/cm ~ through the skin. Because the guinea pig skin was not protected from the grooming activity of the animals or from losses caused by rubbing against the cages, it was uncertain whether such small amounts had, in fact, been absorbed through the skin and excreted, or whether such radioactivity represented contamination of the excreta by desquamation. In the present study we examined the absorption of [14C]Triclocarban through rat skin. Rats have advantages over guinea pigs for this type of study since their skin is more permeable than guinea pigs [2] and it is simpler to protect the treated skin area. Thus more radioactivity should be present in their excreta and it can be accepted that such radioactivity had been absorbed through the skin. The absorption of [14C]Triclocarban from some organic solvents and soaps, with or without occlusion of the skin has been investigated. In addition, absorption of Triclocarban through human skin is reported; these results are based on chemical analysis of blood and urine from subjects on a repeated whole-body immersion. EXPERIMENTAL Materials Radioactive (14C_carbonyl) 3,4,4 l-trichlorocarbanilide ([14 C]Triclocarban) was prepared and purified as previously described [3]. Solutions of [14 C] Triclocarban for injection were prepared by adding known volumes of maize oil (Food Industries Ltd. Bromborough) or 50% aqueous polyethylene glycol 400 (B.D.H. Poole, Dorset) to accurately weighed samples of [ 14 C] Triclocarban. The soap solutions, 8% (w/v) containing the equivalent of 1% (w/v) [14 C]Triclocarban in the bar (0.08% w/v) or 2% (w/v) in the bar (0.16% w/v) were prepared as described by Black et al. [1] and equilibrated for at least 24 h at 40°C before use. The test soaps were a superfatted soap, an NSD bar soap containing 30% of the synthetic detergents sodium alkoyl isethionate and sulphonated fatty acids, and a coconut oil-based soap bar containing 3.5% ABS. A liquid handcleanser containing approx. 25% soap derived from coconut/tallow oils was added to an accurately weighed sample of [14C]Triclocarban and to an accurately weighed sample of [14C]Triclocarban solibilized in Dowanol (DGME, Dow Chemical Co. Ltd. L o n d o n W.1.) equivalent to 5% (v/v) in the hand cleanser.
68
Solutions of [14 C]Triclocarban were prepared by addition of known volumes of DMF, (GPR grade Hopkin and Williams) or acetone (laboratory reagent, Fisons) to accurately weighed samples of [14 C] Triclocarban. Animals and treatment Female Colworth Wistar rats {115--120 g} were used throughout and were treated as described by Black and Howes [4]. Both occlusive [5] and nonocclusive [4] protection of the skin were used. Analysis for Carbon-14 All samples were counted in a Tri-Carb 4322 liquid scintillation spectrometer (Packard Instruments Ltd., Downers Grove, Ill. USA) and counting efficiencies were determined by a channels ratio technique or by the addition of an internal standard. Aqueous samples were counted in an NE 260 liquid scintillator (Nuclear Enterprises Ltd. Sighthill, Edinburgh). The protective patches from the animals were extracted with methanol and aliquots of the extracts counted in NE 260. Skin samples were counted by solubilisation in " S o l u e n e " (Packard Instruments Ltd.) as described previously [4]. All other biological samples were prepared for counting using a Tri-Carb 305 sample oxidizer (Packard Instruments Ltd.). Determination o f Triclocarban by GLC Triclocarban was extracted from blood or freeze-dried urine with acetone, separated by thin-layer chromatography on silica gel, converted to its trimethyl silyl derivative and analysed by gas liquid chromatography (GLC) using an electron capture detector [6]. Human studies The soap used in this study contained 10% (w/w) sodium alkoyl isethio:late and 2% (w/w) non-radioactive Triclocarban. The bathing regimen was to immerse the b o d y up to the neck when lying in a domestic bath for 5 min in water at 40°C. A thorough lathering, by applying the soap bar directly to the b o d y for 2--3 min to produce a stiff foam over the entire b o d y while standing in the bath, was followed by a 5-min immersion in the bath. The lathering and immersion were repeated, adding hot water to maintain the bath temperature at 40°C. The total bath time was not less than 25 min. The b o d y was dried with a towel w i t h o u t showering. The regimen was followed daily for 28 days and blood samples were taken on the morning of days 0, 14 and 28 and stored at --20°C in 10-ml plastic vials containing lithium sequesterene (Stayne Laboratories Ltd., High Wycombe, Bucks, England) until analysed. Urine was collected on days 0, 7, 14, 21 and 28 and concentrated by freeze-drying. A total of 12 volunteers (6 male and 6 female) participated in the trial. In 1 male, blood and urine were collected on day 7 after the bathing regimen ceased. The weight of soap used by each individual was recorded.
69
RESULTS
Turnover of intraperitoneally injected Triclocarban T h e recoveries o f r a d i o a c t i v i t y in t h e urine and faeces o f rats during 5 d a y s are p r e s e n t e d in T a b l e I. It can be seen t h a t m o s t o f t h e r a d i o a c t i v i t y a p p e a r s in t h e faeces and t h a t t h e b u l k o f r a d i o a c t i v i t y is e l i m i n a t e d f r o m t h e b o d y in 2 days. T h e r e c o v e r y o f t h e d o s e is essentially c o m p l e t e b y 5 days. D u r i n g t h e first 2 d a y s 93.2% o f t h e d o s e was e x c r e t e d a n d this figure was used t o c o r r e c t t h e r e c o v e r i e s f r o m rats t r e a t e d t o p i c a l l y w i t h [ 14 C] T r i c l o c a r b a n . T h e levels o f r a d i o a c t i v i t y and o f T r i c l o c a r b a n in t h e b l o o d as d e t e r m i n e d c h e m i c a l l y f o l l o w i n g i n t r a p e r i t o n e a l and s u b c u t a n e o u s injections ( T a b l e II) s h o w t h a t t h e r a d i o a c t i v i t y in t h e b l o o d e x p r e s s e d as equivalents o f Tricloc a r b a n is always m u c h greater t h a n t h e a m o u n t o f T r i c l o c a r b a n as d e t e r m i n e d c h e m i c a l l y . In fact, t h e level o f T r i c l o c a r b a n never rises a b o v e t h e sensitivity limit o f t h e m e t h o d . T h u s t h e r a d i o a c t i v i t y m u s t be p r e s e n t as m e t a b o l i t e ( s ) . T h e m a x i m u m level o f r a d i o a c t i v i t y o c c u r s earlier a f t e r intrap e r i t o n e a l injection t h a n a f t e r s u b c u t a n e o u s injection.
Application of Triclocarban with occlusion Most o f t h e [14 C] T r i c l o c a r b a n applied to r a t skin in D M F and in t h e t w o s o a p p r e p a r a t i o n s is r e c o v e r e d in t h e occlusive and p r o t e c t i v e p a t c h m a t e r i a l and o n l y small a m o u n t s r e m a i n in t h e skin ( T a b l e I I I ) . T h e b l o o d levels o f r a d i o a c t i v i t y , e x p r e s s e d as e q u i v a l e n t s o f T r i c l o c a r b a n , are low. T h e a m o u n t in b l o o d a f t e r t h e a p p l i c a t i o n to skin in D M F is significantly higher t h a n t h a t a f t e r a p p l i c a t i o n to skin in t h e soaps. T h e b l o o d levels a f t e r t h e t w o s o a p t r e a t m e n t s are n o t significantly d i f f e r e n t . A b s o r p t i o n t h r o u g h t h e skin is TABLE I EXCRETION OF 14C BY RATS INJECTED INTRAPERITONEALLY WITH [14 C]TRICLOCARBAN Six rats were injected with 1.70 + 0.11 pCi [14C]Triclocarban specific activity 1.50 pCi/mg in polyethylene glycol (0.5 ml) and placed in separate metabolism cages. Urine and faeces were collected daily for 6 days. Results are mean + S.D. No measurable radioactivity was excreted on the 6th day. (Limits of sensitivity were 0.001 pCi/24-h sample of urine and 0.002 pCi/24-h sample of faeces). Day
Mean daily excretion (pCi) Urine
1 2 3 4 5 Total
70
0.118 0.015 0.003
P E R C U T A N E O U S ABSORPTION OF T R I C L O C A R B A N IN RAT AND MAN
D. HOWES and J.G. BLACK Environmental Safety Division, Unilever Research Laboratory, Colworth House, Sharnbrook, Bedford, MK44 1LQ (Great Britain) (Received November 19th, 1975) (Accepted January 17th, 1976)
SUMMARY
The route and rate of excretion b y rats of the germicide [14C]Triclo carban formerly called trichlorocarbanilide, given by parenteral injection has been investigated. Blood levels based on radioactivity and b y chemical determination after parenteral injection have been compared with those obtained after topical application of [' 4 C/Triclocarban in acetone. Absorption of [14C]Triclocarban in soaps and in dimethylformamide (DMF) through occluded rat skin has been studied. Other soaps and a hand cleanser containing [14 C/Triclocarban have been applied to rat skin witho u t occlusion and the effects of duration of contact, concentration and the use of a solubilizer have been investigated. In humans, absorption of Triclocarban through skin after bathing daily for 28 days has been investigated by chemical analysis of blood and urine. The data show that elimination by the rat is rapid and complete principally via the faeces. Blood levels after parenteral injection are low and comparison of the radioactivity and chemical determinations suggest rapid metabolism of the Triclocarban. After application to the skin, blood levels based on 4 C are very low. Absorption of [14 C]Triclocarban through occluded rat skin was greater from DMF than from soaps. With non-occluded rat skin, absorption from soaps was less and was dependent on concentration b u t independent of duration of contact. The use of a solubilizer did not increase absorption through skin. No measurable Triclocarban (< 25 ppb) was present in blood and urine samples of volunteers during or shortly after a 28-day intensive bathing regimen.
Abbreviations: ABS, alkyi benzene sulphonate; DGME, dipropyleneglycol methyl ether; DMF, dimethyl formamide; Triclocarban (TCC), 3,4,41 -trichloroearbanilide.
67
INTRODUCTION We previously studied the deposition and percutaneous absorption of [ ~4 C] TCC a germicide now called Triclocarban, which is used in toilet soaps [1]. No radioactivity was detected in blood, liver or depot fat of guinea pigs although the sensitivity of the method was equivalent to 10 ppb of [14 C]TCC. However, small amounts of radioactivity were present in the excreta of the guinea pigs and these amounts were equivalent to an absorption of less than 0.5 pg [1~C] TCC/cm ~ through the skin. Because the guinea pig skin was not protected from the grooming activity of the animals or from losses caused by rubbing against the cages, it was uncertain whether such small amounts had, in fact, been absorbed through the skin and excreted, or whether such radioactivity represented contamination of the excreta by desquamation. In the present study we examined the absorption of [14C]Triclocarban through rat skin. Rats have advantages over guinea pigs for this type of study since their skin is more permeable than guinea pigs [2] and it is simpler to protect the treated skin area. Thus more radioactivity should be present in their excreta and it can be accepted that such radioactivity had been absorbed through the skin. The absorption of [14C]Triclocarban from some organic solvents and soaps, with or without occlusion of the skin has been investigated. In addition, absorption of Triclocarban through human skin is reported; these results are based on chemical analysis of blood and urine from subjects on a repeated whole-body immersion. EXPERIMENTAL Materials Radioactive (14C_carbonyl) 3,4,4 l-trichlorocarbanilide ([14 C]Triclocarban) was prepared and purified as previously described [3]. Solutions of [14 C] Triclocarban for injection were prepared by adding known volumes of maize oil (Food Industries Ltd. Bromborough) or 50% aqueous polyethylene glycol 400 (B.D.H. Poole, Dorset) to accurately weighed samples of [ 14 C] Triclocarban. The soap solutions, 8% (w/v) containing the equivalent of 1% (w/v) [14 C]Triclocarban in the bar (0.08% w/v) or 2% (w/v) in the bar (0.16% w/v) were prepared as described by Black et al. [1] and equilibrated for at least 24 h at 40°C before use. The test soaps were a superfatted soap, an NSD bar soap containing 30% of the synthetic detergents sodium alkoyl isethionate and sulphonated fatty acids, and a coconut oil-based soap bar containing 3.5% ABS. A liquid handcleanser containing approx. 25% soap derived from coconut/tallow oils was added to an accurately weighed sample of [14C]Triclocarban and to an accurately weighed sample of [14C]Triclocarban solibilized in Dowanol (DGME, Dow Chemical Co. Ltd. L o n d o n W.1.) equivalent to 5% (v/v) in the hand cleanser.
68
Solutions of [14 C]Triclocarban were prepared by addition of known volumes of DMF, (GPR grade Hopkin and Williams) or acetone (laboratory reagent, Fisons) to accurately weighed samples of [14 C] Triclocarban. Animals and treatment Female Colworth Wistar rats {115--120 g} were used throughout and were treated as described by Black and Howes [4]. Both occlusive [5] and nonocclusive [4] protection of the skin were used. Analysis for Carbon-14 All samples were counted in a Tri-Carb 4322 liquid scintillation spectrometer (Packard Instruments Ltd., Downers Grove, Ill. USA) and counting efficiencies were determined by a channels ratio technique or by the addition of an internal standard. Aqueous samples were counted in an NE 260 liquid scintillator (Nuclear Enterprises Ltd. Sighthill, Edinburgh). The protective patches from the animals were extracted with methanol and aliquots of the extracts counted in NE 260. Skin samples were counted by solubilisation in " S o l u e n e " (Packard Instruments Ltd.) as described previously [4]. All other biological samples were prepared for counting using a Tri-Carb 305 sample oxidizer (Packard Instruments Ltd.). Determination o f Triclocarban by GLC Triclocarban was extracted from blood or freeze-dried urine with acetone, separated by thin-layer chromatography on silica gel, converted to its trimethyl silyl derivative and analysed by gas liquid chromatography (GLC) using an electron capture detector [6]. Human studies The soap used in this study contained 10% (w/w) sodium alkoyl isethio:late and 2% (w/w) non-radioactive Triclocarban. The bathing regimen was to immerse the b o d y up to the neck when lying in a domestic bath for 5 min in water at 40°C. A thorough lathering, by applying the soap bar directly to the b o d y for 2--3 min to produce a stiff foam over the entire b o d y while standing in the bath, was followed by a 5-min immersion in the bath. The lathering and immersion were repeated, adding hot water to maintain the bath temperature at 40°C. The total bath time was not less than 25 min. The b o d y was dried with a towel w i t h o u t showering. The regimen was followed daily for 28 days and blood samples were taken on the morning of days 0, 14 and 28 and stored at --20°C in 10-ml plastic vials containing lithium sequesterene (Stayne Laboratories Ltd., High Wycombe, Bucks, England) until analysed. Urine was collected on days 0, 7, 14, 21 and 28 and concentrated by freeze-drying. A total of 12 volunteers (6 male and 6 female) participated in the trial. In 1 male, blood and urine were collected on day 7 after the bathing regimen ceased. The weight of soap used by each individual was recorded.
69
RESULTS
Turnover of intraperitoneally injected Triclocarban T h e recoveries o f r a d i o a c t i v i t y in t h e urine and faeces o f rats during 5 d a y s are p r e s e n t e d in T a b l e I. It can be seen t h a t m o s t o f t h e r a d i o a c t i v i t y a p p e a r s in t h e faeces and t h a t t h e b u l k o f r a d i o a c t i v i t y is e l i m i n a t e d f r o m t h e b o d y in 2 days. T h e r e c o v e r y o f t h e d o s e is essentially c o m p l e t e b y 5 days. D u r i n g t h e first 2 d a y s 93.2% o f t h e d o s e was e x c r e t e d a n d this figure was used t o c o r r e c t t h e r e c o v e r i e s f r o m rats t r e a t e d t o p i c a l l y w i t h [ 14 C] T r i c l o c a r b a n . T h e levels o f r a d i o a c t i v i t y and o f T r i c l o c a r b a n in t h e b l o o d as d e t e r m i n e d c h e m i c a l l y f o l l o w i n g i n t r a p e r i t o n e a l and s u b c u t a n e o u s injections ( T a b l e II) s h o w t h a t t h e r a d i o a c t i v i t y in t h e b l o o d e x p r e s s e d as equivalents o f Tricloc a r b a n is always m u c h greater t h a n t h e a m o u n t o f T r i c l o c a r b a n as d e t e r m i n e d c h e m i c a l l y . In fact, t h e level o f T r i c l o c a r b a n never rises a b o v e t h e sensitivity limit o f t h e m e t h o d . T h u s t h e r a d i o a c t i v i t y m u s t be p r e s e n t as m e t a b o l i t e ( s ) . T h e m a x i m u m level o f r a d i o a c t i v i t y o c c u r s earlier a f t e r intrap e r i t o n e a l injection t h a n a f t e r s u b c u t a n e o u s injection.
Application of Triclocarban with occlusion Most o f t h e [14 C] T r i c l o c a r b a n applied to r a t skin in D M F and in t h e t w o s o a p p r e p a r a t i o n s is r e c o v e r e d in t h e occlusive and p r o t e c t i v e p a t c h m a t e r i a l and o n l y small a m o u n t s r e m a i n in t h e skin ( T a b l e I I I ) . T h e b l o o d levels o f r a d i o a c t i v i t y , e x p r e s s e d as e q u i v a l e n t s o f T r i c l o c a r b a n , are low. T h e a m o u n t in b l o o d a f t e r t h e a p p l i c a t i o n to skin in D M F is significantly higher t h a n t h a t a f t e r a p p l i c a t i o n to skin in t h e soaps. T h e b l o o d levels a f t e r t h e t w o s o a p t r e a t m e n t s are n o t significantly d i f f e r e n t . A b s o r p t i o n t h r o u g h t h e skin is TABLE I EXCRETION OF 14C BY RATS INJECTED INTRAPERITONEALLY WITH [14 C]TRICLOCARBAN Six rats were injected with 1.70 + 0.11 pCi [14C]Triclocarban specific activity 1.50 pCi/mg in polyethylene glycol (0.5 ml) and placed in separate metabolism cages. Urine and faeces were collected daily for 6 days. Results are mean + S.D. No measurable radioactivity was excreted on the 6th day. (Limits of sensitivity were 0.001 pCi/24-h sample of urine and 0.002 pCi/24-h sample of faeces). Day
Mean daily excretion (pCi) Urine
1 2 3 4 5 Total
70
0.118 0.015 0.003
