D-gluconic acid, compound with N,N''-bis(4-chlorophenyl)-3,12-diimino-2,4,11,13-tetraazatetradecanediamidine (2:1)

Endpoint summary

Additional information

Toxicokinetic studies have been performed for the oral and the dermal route of application in several species including humans. Studies with other Chlorhexidine salts like diacetate were used (see read across statement under chapter 13.2 Other assessment reports.)

Oral Route

Independent from the dose applied and the species (including rats, dogs, marmosets, rhesus monkeys and humans), about 0.2 - 1.3 % of the dose are excreted with via urine. For mice conflicting results have been reported: One study gave 3.7 - 7.2 % of the dose, while another study gave only 0.46 - 1.25 %. The majority of the applied dose (in most studies > 90 %) is recovered from faeces, which is considered to represent unabsorbed material. This is confirmed by data from a study with rats, were only 0.2 % of the orally applied dose was found in bile. In studies with dogs and rats investigating the distribution of chlorhexidine digluconate, chlorhexidine was also detected in the liver.

Based on the observations in rats, where excretion via the urine and biliary excretion have been quantified, possibly equal amounts are excreted in the urine and bile.

Overall, the absorption in different species therefore may account for up to 2.6 % of the dose, if the one study with mice with higher excretion via urine is not taken into consideration.

 

Excretion of 14C-radioactivity (as % of dose ± SE) following oral administration of
14C‑chlorhexidine digluconate to different species              

Species Strain	Dose* (mg/kg b.w.)	^14C-Label^1	Urine	Faeces	Days
Rat^4 n.r.	5 50^2 50^3 5 4.4	R R R C C	0.4 0.4 0.7 0.2 0.7	99.5 93.2 97.5 92.2 100.8	7 10 10 7 3
Rat	4			Bile 0.2	3
Mouse^4 n.r.	27 8	R R	7.2 3.7	91.9 101.6	7 7
Mouse C57B1/10J	25 50 100 200	R	0.52 0.46 0.57 1.25	70.3 77.5 75.7 85.9	7 7 7 7
Dog^4 n.r.	5 0.05 4.9	R R C	0.8 0.9 0.2	102.0 100.0 100.3	7 7 7
Marmoset^4	7.3 6.6	R R	1.3 0.4	89.4 106.7	5 7
Rhesus monkey^4	5.5	R	1.2	102.6	7
Human	0.07	R	0.3	81.9	6

*: reported as chlorhexidine base; n.r. not reported
1: R: ring-, C: chain-labelled
2: male, after 6 months daily dosing
3: female, after 6 months daily dosing
4: no. of animals not reported but probably n=1 for each data set.

 

 

Distribution

Data from an autoradiographic study in female mice (distribution of chlorhexidine from 14C-labelled chlorhexidine digluconate and diacetate) showed that the radiolabelled chlorhexidine (or a metabolite containing the 14C-activity) had a high affinity to the epithelia of the gastrointestinal and the respiratory tract and also could be detected in the liver and in the kidney.

The distribution of chlorhexidine was also measured after repeated (chronic) oral exposure for up to one year in dogs and two years in rats. Limited data are also available for mice. The concentration of chlorhexidine in blood and various organs of rats was dose- and time-dependent and highest concentrations in blood were reached after one year and generally did not increase further or declined during the second half of the study. The highest concentrations were found in kidneys and in mesenteric lymph nodes, while concentration in other organs were at least one order of magnitude lower.

In dogs, detectable levels of chlorhexidine in blood were only found in the highest dose group. Blood chlorhexidine increased with increasing exposure time and the observed decline from 6 – 9 months can be explained by the dose reduction after 28 weeks (40à25 mg/kg bw/d). Highest concentrations were found in liver, followed by kidney and mesenteric lymph nodes, while other organs contained much lower concentrations.

 

The data for the organs showing the highest concentrations of chlorhexidine are summarised in the following Table. In all species, the concentration of chlorhexidine was dose-dependent and the effect was more pronounced in dogs than in rats. The most marked increase is seen in the liver of dogs. Species-specific differences are evident when the concentration of chlorhexidine in this organ is compared between dogs and rats. These toxicokinetic differences are reflected by differences in the target organ toxicity between both species.

 

 

Concentration of chlorhexidine in blood and organs of various species after repeated oral or dermal
administration of chlorhexidine digluconate

Species	Exposure duration, route, dose (mg chlorhexidine/kg · d)	Chlorhexidine content (µg/g wet weight)
		Blood	Mesenteric lymph node	Kidney	Liver
Dog	1 a, oral (bolus), 0.5	n.d.	~ 0.6	~ 3	~ 2
	1 a, oral (bolus), 5	n.d.	~ 4	~ 50	~ 60
	1 a, oral (bolus), 40/25*	0.1 ± 0.04	~ 93	~ 300	~ 600
Rat	2 a, oral (drinking water), 5	not detectable	6.7±6.0	11.9±4.7	0.24±0.11
	2 a, oral (drinking water), 25	0.09±0.09	26.5±30.6	51.0±19.9	0.84±0.53
	2 a, oral (drinking water), 50	0.26 ± 0.14	48.2 ± 36.3	124.2 ± 83.6	1.81 ± 1.08
	2 a, oral (food), 5	0.06 + 0.03	22.5 ± 2.3	9.8 ± 0.9	0.12 ± 0.05
	2 a, oral (food), 25	0.47 ± 0.06	39.5 ± 5.7	27.2 ± 4.1	0.70 ± 0.09
	2 a, oral (food), 50	0.83 ± 0.07	43.7 ± 6.3	37.1 ± 4.8	0.73 ± 0.08
Mouse	52 weeks, oral (food), 100	n.d.	n.d.	22 ± 3	n.d.
	65 weeks, oral (food), 100	n.d.	n.d.	62 ± 6	n.d.
	52 weeks, oral (food), 200	n.d.	n.d.	83 ± 3	n.d.
	65 weeks, oral (food), 200	n.d.	n.d.	169 ± 21	n.d.
	52 weeks, oral (food), 400	n.d.	n.d.	229 ± 8	n.d.
	65 weeks, oral (food), 400	n.d.	n.d.	388 ± 27	n.d.
Rhesus monkey	13 weeks, dermal (5 min wash), 400	0- 0.011^1	n.d.	0.018-0.044	0-0.017^1(bile: 0)
Humans	3 weeks, 5 d/week, 5 times/d, dermal, 2x5 ml 4 % (2x3 min wash)	not detectable^2	n.d.	n.d.	n.d.

*: dose reduction after 28 weeks; n.d.: not determined; 1: chlorhexidine could be detected only in 1 of 6 animals;
2: limit of detection (LOD) in the order of 0.01-0.05 µg/ml. 3: LOD 3 ng/ml.

 

Few data are available concerning the metabolism of chlorhexidine digluconate. Para-chloroaniline (pCA), a known chemical breakdown product of chlorhexidine (may be present in concentrations up to 500 ppm as an impurity in the marketed chlorhexidine digluconate), is known to be rapidly excreted via urine after oral uptake. The substance could not be detected in urine samples from mouse, dog, rat, marmoset and humans following oral administration of 14C-labelled chlorhexidine. Further data show that ring- and chain 14C-labelled chlorhexidine produced the same metabolic pattern with respect to excretion in urine and faeces, indicating that cleavage of the molecule seems to be minimal. Analysis of faeces extracts from orally dosed animals revealed only chlorhexidine.

In summary, the available data indicate that the small amounts of chlorhexidine which may be absorbed following oral administration are not metabolised to a measurable extent.

 

 

Dermal route

 

Detailed information concerning excretion after dermal exposure to chlorhexidine digluconate is summarised in the following Table. While in rats some absorption was found, all studies with humans showed only very low absorption. Only in one study minor amounts could be detected in faeces. In 1 of 2 subjects treated with aqueous solution and in 1 of 3 subjects treated with hand wash solutions detectable levels were found in faeces (LOD: 0.006 % of the dose). In urine the detection limit is higher than in faeces; therefore, a reliable estimation of the amounts absorbed is not possible. If one assumes as for the oral studies, that similar amounts will be excreted in urine and in faeces, than dermal absorption in humans will amount to a maximum of about 0.2 % of the dose under different exposure conditions.

 

 

Excretion of¹⁴C-radioactivity (as % of dose ± SE) following dermal administration
of¹⁴C‑chlor­hexidine digluconate to different species

Species Strain	Chlorhexidine solution Concentration Amount Exposure duration	Radio-label	Exposure condition	Urine	Faeces	Days
Rat	0.04 % 300 µl 24 h Occlusive patch	C	intact skin	0.1	4.2	7
			damaged skin	0.3	3.5
Humans	50 cm² 3 h non-occlusive		5 % aqueous solution	< 0.02	< 0.009	10
			4 % hand wash	< 0.02	< 0.009	10
	1.4 aqueous dilution of skin cleanser 3h non occlusive			< 0.1	n.d.	1
	Hand scrubbing 3 min with 5 ml 5x /day, 5 days/week, 3 weeks			Not detectable	Not detectable

 

 

 

Additional data (summary of dermal uptake of chlorhexidine without specifying the chlorhexidine salt) are reported by EMEA (Committee for Veterinary Medicinal Products). No chlorhexidine could be detected in blood of neonatal Rhesus monkeys (except for one animal), which were bathed for 90 days in a cleanser containing 8 % chlorhexidine digluconate, and only trace amounts of chlorhexidine could be detected in fat, kidney, liver. Only the skin showed an appreciable amount of chlorhexidine, while no chlorhexidine could be detected in bile. In humans, no chlorhexidine could be detected in the blood of infants washed in a 4 % chlorhexidine solution. Similarly, chlorhexidine could not be detected in the blood of adults after a single topical application of a 5 % solution of 14C- labelled chlorhexidine to 50 cm² skin (LOD for blood: 0.005 µg/ml [0.02 % of applied dose]) or after repeated daily use in a pre-operative “scrub” over a period of 6 months (LOD for blood: 0.01 µg/ml).

Some additional information may be gathered from a comparison of the acute lethal toxicity of chlorhexidine for different routes of administration. The acute LD50 for i.v. administration is about two orders of magnitude lower than the LD50 for oral and the ratio is even higher for the (non-lethal) concentrations applied to the skin. This large differences in acute lethal toxicity between a route where the substance directly enters the body and routes where absorption through the gastrointestinal tract or the skin is necessary to elicit systemic toxic effects further supports the conclusion that oral and – even more – dermal absorption of chlorhexidine is low.

 

 

Conclusion

 

Chlorhexidine – as is expected because of its cationic nature – is strongly bound to mucosa and skin. The chlorhexidine content in the organs of animals after repeated oral administration show that some chlorhexidine is absorbed from the gastrointestinal tract. Kinetic studies indicate that the oral absorption is low (ca. 2 % of the dose, based on data of urinary and biliary excretion).

Dermal absorption is very low and considerably lower than oral absorption. Experimental data for humans show that uptake is too low to be measurable even with 14C-labelled chlorhexidine. Taking into account the limit of detection, a value of 0.02 % is considered as an upper bound estimate for percutaneous absorption.