Disodium 4,4'-bis[(4-anilino-6-morpholino-1,3,5-triazin-2-yl)amino]stilbene-2,2'-disulphonate

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Test procedures cannot be subsumed under a testing guideline, nevertheless are well documented and scientifically acceptable.

Data source

Reference

Reference Type:

publication

Title:

Percutaneous absorption and disposition of TINOPAL EMS.

Author:

Black J.G, Moule R.C. and Philip J.

Year:

1977

Bibliographic source:

Toxicology, 8 (1977) 33-42 © Elsevier/North-Holland Scientific Publishers, Ltd.

Materials and methods

Objective of study:

absorption

excretion

Principles of method if other than guideline:

A cotton-substantive, anionic, fluorescent whitening agent manufactured by several suppliers under various trade names e.g. Tinopal EMS, has been synthesized in radioactive form. Test substance was administrated by some different routs: oral, dermal, subcutaneous and interperitoneal.

GLP compliance:

no

Remarks:

pre-GLP study

Test material

Test material information

Constituent 1

Reference substance name:

Disodium 4,4'-bis[(4-anilino-6-morpholino-1,3,5-triazin-2-yl)amino]stilbene-2,2'-disulphonate

EC Number:

240-245-2

EC Name:

Disodium 4,4'-bis[(4-anilino-6-morpholino-1,3,5-triazin-2-yl)amino]stilbene-2,2'-disulphonate

Cas Number:

16090-02-1

Molecular formula:

C40H38N12Na2O8S2

IUPAC Name:

disodium 2,2'-ethene-1,2-diylbis{5-[(4-anilino-6-morpholin-4-yl-1,3,5-triazin-2-yl)amino]benzenesulfonate}

Radiolabelling:

yes

Remarks:

tritiated

Test animals

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: Colworth-Wistar strain.
- Weight at study initiation: approx. 100 g body weight).
- Housing: rats were placed in individual metabolism cages.
- Diet: ad libitum, a pelleted diet.
- Water: ad libitum

Administration / exposure

Route of administration:

other: oral, dermal, intraperitoneal and subcutaneous.

Results and discussion

Toxicokinetic / pharmacokinetic studies

Details on absorption:

Very small amounts in detergent were absorbed through rat skin in occlusion test. Small amounts were absorbed through skin when applied in ethanol. It is concluded that the possibility of systemic toxic effects in man as a result of percutaneous absorption is remote.

Details on excretion:

Most of the dose was excreted rapidly in the faeces. After parenteral administration to rats, the radioactivity was rapidly excreted in the faeces with small amounts remaining in tissues and organs.

Metabolite characterisation studies

Metabolites identified:

not measured

Any other information on results incl. tables

A count rate of twice background was accepted as the sensitivity limit, from which the limits of detection as equivalents of [3H]DADM were: blood (4 ppb), liver (4 ppb), kidney (3 ppb), heart (7 ppb), brain (3 ppb), spleen (8 ppb), bladder (35 ppb), testes (3 ppb), depot fat (3 ppb), skeletal muscle (3 ppb), urine 12 ng/day, faeces 32 ng/day. The term ppb is defined as parts per 109 (w/v) or (w/w) where applicable.

ORAL

The bulk of the radioactivity was excreted by both groups of rats intubated with [3H]DADM in the faeces, mostly during the first 24 h. Thereafter progressively much smaller amounts were excreted in the faeces. The urine from both intubated groups contained a very small amount of radioactivity which was limited to the first 24-h collection.

In the rats intubated with [3H]DADM in detergent, the urine contained slightly more tritium which was statistically significantly greater (P = 0.05, Student's t-test) than the amount present in the urine of rats intubated with [3H]DADM in water. At 24 h residual tritium was very low (less than the equivalent of 0.1 µg DADM) in the intestinal tract, liver, kidneys, brain and testes from both groups. The levels of tritium in these organs were not significantly different from both groups of rats and no measurable tritium was present in blood, lungs, heart, bladder, spleen, renal depot fat or skeletal (thigh) muscle.

RECOVERY OF TRITIUM AFTER INTUBATION INTO RATS OF [3H] DADM

Results are the mean  ± standard deviation for the number of animals given in parenthesis. The specific activity of the [3H]DADM was

137 µCi/mg and the dose was given in 0.5 ml of vehicle

Intubation	Sample	Recovery of tritium as [3H]DADM (µg)
		0-24 h	24-48 h	48-72 h
78.729 ± 0.413 µg in detergent	Urine	0.041 ± 0.003 (7)	<0.012 (6)	<0.012 (3)
	Faeces	70.712 ± 4.123 (7)	1.934 ± 0.843 (4)	0.091 (1)
	Total	70.753 ± 4.124 (7)	1.934 ± 0.843 (4)	0.091
79.246 ± 1.229 µg in water	Urine	0.029 ± 0.006 (9)	<0.012 (6)	<0.012 (3)
	Faeces	76.903 ± 1.478 (9)	1.928 ± 0.707 (6)	0.065 ± 0.026 (3)
	Total	76.932 ± 1.480 (9)	1.928 ± 0.707 (6)	0.065 ± 0.026 (3)

(x): N. of animals.

INTREPERITONEAL

After intraperitoneal injection of [3H]DADM into rats the faeces of the first 24 h contained most of the excreted tritium. Thereafter the faecal content of tritium decreased rapidly. The urine contained a very low proportion of the dose during the first 24 h and thereafter a constant, extremely small amount was excreted daily. During the first 2 days the excreta contained 93.2% of the dose. The content of tritium in organs and tissues at 24 h was very low. The liver contained the equivalent of 0.056 + 0.006(3) µg DADM

and the kidneys contained 0.258 + 0.020(3) µg. These amounts decreased during 96 h to 0.011 + 0.004(3) and 0.115 + 0.040(3) respectively. The testes, bladder, spleen and lung all contained less than the equivalent of 0.01 µg DADM and no measurable tritium was present in blood, brain, heart, renal depot fat or skeletal muscle.

Rats were injected intraperitoneally with 35.3 ± 0.8 µg [3H]DADM with a specific activity of 107 µCi/mg, in 0.5 ml 1 % (w/v) detergent. Results are the mean ±standard deviation for the number of animals given in parenthesis.

Day	Recovery of tritium as [3H] DADM (µg)
	Urine	Faeces
1	0.215 ± 0.080 (12)	30.646 ± 3.882 (12)
2	0.027 ± 0.015 (9)	2.008 ± 1.308 (9)
3	0.024 ± 0.009 (6)	0.107 ± 0.042 (6)
4	0.020 ± 0.003 (3)	0.013 ± 0.068 (3)

SUBCUTANEOUS

After subcutaneous injection of [3H]DADM into rats most of the tritium was excreted in the faeces during the first 48 h, after which time the content of tritium in the faeces decreased dramatically. The urine contained a very low proportion of the dose which decreased steadily during the 4 days. The content of tritium in organs and tissues in a second group of rats, 2 of which were killed at 24, 48 and 96 h after injection of 44.26 ± 1.38 µg [3H]DADM showed that the liver contained a mean of the equivalent of 0.572 µg DADM and both kidneys contained a mean of 0.311 µg DADM. These amounts decreased during 96 h to less than 0.01 µg in the

liver and to 0.209 µg in the kidneys. Also at 24 h, the blood contained the equivalent of 0.015 µg/ml, whereas, both testes, heart and spleen each contained less than 0.004 µg. The levels of tritium in the bladder, brain, renal depot fat and skeletal muscle were all below the limits of detection at 24 h after injection.

Rats were injected subcutaneously with 57.1 ± 1.4 µg [3H]DADM with a specific activity of 107 µCi/mg, in 0.5 ml 1 % (w/v) detergent. Results are the mean ±standard deviation for the number of animals given in parenthesis.

Day	Recovery of tritium as [3H] DADM (µg)
	Urine	Faeces
1	0.312 ± 0.056 (3)	31.083 ± 3.619 (3)
2	0.118 ± 0.058 (3)	22.824 ± 3.664 (3)
3	0.077 ± 0.032 (3)	1.996 ± 0.452 (3)
4	0.043 ± 0.018 (3)	0.323 ± 0.178 (3)

PERCUTANEOUS

A series of experiments were done to investigate the extent of percutaneous absorption of [3H]DADM. Thus three concentrations in 1% detergent solution of between 0.007 % (w/v) and 0.009 % (w/v) [3H]DADM were applied in separate experiments to 8 cm² of clipped rat dorsal skin, with or without rinsing, and the site protected with an occlusive patch. Scintillation counting of the treated skin at 24 h revealed a deposition of 0.2 to 0.4 1g/cm² for rinsed skins and of approx. 0.5 to 1.0 µg/cm² for unrinsed skins.

The rinse water contained 79% of the applied [3H]DADM. The exact amount retained by the nylon patch was difficult to measure because combustion was unsatisfactory and extraction by solvents was inconsistent.

Autoradiography of the skin at 24 h after treatment showed a low to moderate grain density over the stratum comeum and around the mouth of the hair follicles but revealed no evidence of transepidermal penetration. No meastlrable radioactivity was excreted. Enzyme separated dermis contained very small quantities of radioactivity, equivalent to 3 -33 ng/cm² from rinsed animals and 8-50 ng/cm² from unrinsed animals. Some or all of this tritium might be within the residual hair follicles. At 0.012% (w/v) [3H]DADM in detergent solution, application to 15 cm² of clipped rat dorsal skin without rinsing and with the treated area protected with a nylon occlusive patch, resulted in a deposition of approx. 1.6 µg/cm². Small amounts of tritium were detected in the excreta. During the first 2 days the amount of tritium excreted was the equivalent of 0.249 µg [3H]DADM which, on correction for a 93.2% recovery of an intraperitoneal dose, represents an absorption through rat skin of 0.018 µg/cm². At 24 h autoradiography of the treated skin revealed a low to moderate grain density over the stratum corneum and around the mouth of the hair follicles. At 48 and 72 h however, there was evidence of penetration through the wall of the hair follicle into the lower dermis.

When measurable absorption through skin occurred, tissue levels were very low. At 24 h after application, the intestinal tract contained the equivalent of approx. 0.07 µg [3H]DADM, the liver, kidneys, brain, testes, heart and spleen each contained less than the equivalent of 0.007 µg [3H]DADM, but no measurable radioactivity was present in blood, bladder or renal depot fat. Skeletal muscle contained the equivalent of 20 -50 ppb [3H]DADM. At 48 and 72 h after application the tissue levels decreased to below the limits of detection. Thus there was no accumulation or persistence of tritium in the kidneys of topically-treated rats. From the 2 rats treated topically with [3H]DADM in ethanol, measurable amounts of tritium were present in faeces, in the large and small intestines and their contents and also in the contents of the stomach. The total amount of tritium in excreta and tissues was equivalent to 0.182 µg [3H]DADM. The liver, kidneys, bladder and heart of one rat contained small amounts of tritium, each less than the equivalent of 0.01 µg [3H]DADM. Samples which contained no measurable radioactivity were urine, blood, stomach, testes, spleen, brain, skeletal muscle and depot fat.

Rats were treated topically with 60.46 ± 1.95 µg [3H]DADM with a specific activity of 107 µCi/mg in 1 % aqueous detergent, over 15 cm² clipped dorsal skin. Results are the mean ± standard deviation for the number of animals given in parenthesis.

Day	Recovery of tritium as [3H] DADM (µg)
	Urine	Faeces
1	0.023 ± 0.020 (9)	0.199 ± 0.294(9)
2	<0.012(6)	0.027 ± 0.029(6)
3	<0.012(3)	0.016 ± 0.027(3)

DISCUSSION

The intubation studies showed that most of the tritium was excreted quickly and almost completely in the faeces, in agreement with the data of Muecke et al.^[1]. Only small amounts, of the order of 0.1% of the dose, were absorbed and excreted in the urine. The amount of radioactivity excreted in the urine of the rats intubated with [3H]DADM in detergent is slightly, but significantly greater (P = 0.05, Student's t-test) than the amount of radioactivity excreted by rats intubated with [3H]DADM in water. When dosed parenterally, rats rapidly and completely excreted radioactivity from [3H]DADM, principally in the faeces.

The rate of excretion was more rapid by the rats injected intraperitoneally than by those injected subcutaneously. At 24, 48 and 96 h after injection by both routes, the tissue levels were low but there appeared to be a relatively slow loss of radioactivity from the kidneys suggesting perhaps a degree of persistance or localization of [3H]DADM or its metabolite(s) in this organ.

The nature of the radioactivity in tissues and excreta was not identified and the tritium might be present either as the intact parent molecule or as metabolite(s). However, the similarity in the excretion patterns of tritium suggests little or no loss of tritium from [3H]DADM by metabolism. Such a conclusion is supported by the data of Muecke et al.^[1] who showed that for DADM labelled with 14C in the triazine rings, only cis- and transisomers were present in the faeces of rats intubated with [¹⁴C]DADM.

In order to demonstrate absorption through skin the concentration of [3H]-DADM in detergent was increased beyond the expected user concentration and the treated area of skin was doubled. Under these conditions the penetration, corrected for a 93% recovery in 48 h of an intraperitoneally injected dose, was only 0.018 µg/cm². These conditions represent an exaggeration of 6 (for concentration), 3 (for occlusion ^[2]) and 5 (for difference between rat and human skin permeability, based on the minimum difference observed by Howes and Black^[2] or 90 times more than the average expected absorption through human skin. At 24, 48 and 96 h after application to the skin, the tissue levels were all very low. The kidney, in particular did not show either a relatively high retention of radioactivity or any tendency for the radioactivity to persist. Thus the amounts of DADM, the active ingredient in the fluorescent whitening agents such as Tinopal EMS, likely to be absorbed through human skin are very small, estimated to be of the order of

0.0002 µg/cm², and the possibility of systemic toxic effects is remote. In contrast to the findings from [3H]DADM in detergent, small amounts of [3H]DADM applied in alcohol were absorbed into the blood stream with consequent distribution throughout the body. However in many tissues the amounts of tritium, expressed as equivalents of [3H]DADM, were below the level of sensitivity of the method. Based on the measurable counts of tissues and excreta the absorption of [3H]DADM through rat skin was equivalent to 0.01 µg/cm² during the 2 days of the experiment. In order to achieve this degree of absorption through skin, a concentration of 0.04% (w/v) [3H]-DADM in alcohol was required, or 20 times the expected user concentration, together with the use of an occlusive patch. The data in this report emphasizethe very low or negligible extent of percutaneous absorption of DADM in contrast to our findings with the germicides hexachlorophene ^[3], Triclosan ^{[4 -5]} and Triclorcarban ^[2,6], and with some surface-active

agents ^[15].

REFERENCE

^[1] W. Muecke, G. Dupuis and H.O. Esser, in R. Anliker and G. Muller (Eds.), Fluorescent Whitening Agents, Environmental Quality and Safety, Suppl. Vol. IV, Georg Thieme, Stuttgart, Chap. VI/5. (Inluded into the current IUCLID section).

^[2] D. Howes and J.G. Black, Toxicology, 6 (1976) 67.

^[3] J.G. Black, W.E. Sprott, D. Howes and T. Rutherford, Toxicology, 2 (1974) 127.

^[4] J.G. Black and D. Howes, J. Soc. Cosmet. Chem., 26 (1975) 305.

^[5] J.G. Black, D. Howes and T. Rutherford, Tbxicology, 3 (1975) 33.

^[6] J.G. Black, D. Howes and T. Rutherford, Toxicology, 3 (1975) 253.

^[7] D. Howes, J. Soc. Cosmet. Chem., 26 (1975) 47.

Applicant's summary and conclusion

Conclusions:

Most of the dose was excreted rapidly in the faeces. After parental administration to rats, the radioactivity was rapidly excreted in the faeces with small amounts remaining in tissues and organs. It is concluded that the possibility of systemic toxic effects in man as a result of percutaneous absorption is remote.

Executive summary:

Method

A cotton-substantive, anionic, fluorescent whitening agent manufactured by several suppliers under various trade names e.g. Tinopal EMS, has been synthesized in radioactive form. Test substance was administrated by some different routs: oral, dermal, subcutaneous and interperitoneal.

Results

Resulted in little absorption from the intestinal tract as evidenced by low radioactivity in the urine and tissues. Most of the dose was excreted rapidly in the faeces. After parenteral administration to rats, the radioactivity was rapidly excreted in the faeces with small amounts remaining in tissues and organs. There was slight evidence of retention of radioactivity in the kidneys. Very small amounts of Tinopal EMS in detergent were absorbed through rat skin, but only when concentrations greater than those normally used by the consumer, together with occlusion of the skin were employed. Small amounts were absorbed through skin when applied in ethanol. It is concluded that the possibility of systemic toxic effects in man as a result of percutaneous absorption is remote.