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In vivo studies

In vivo data on the toxicokinetics, metabolism and distribution of AAPBs are available for the oral and dermal route. The fate of C12 AAPB in the rat was studied in an ADME study (ADME - absorption, distribution, metabolism, excretion) equivalent to OECD Guideline 417. Two differentially labelled samples were used: (N(lauroylaminopropyl) N,N dimethyl-N carboxymethyl ammonium (inner) carboxylate) isotopically labelled with 14C in the carboxymethyl ammonium moiety ([14C]TB); the sample had a specific activity of 3.0 µCi/mg and N(lauroylaminopropyl) N,N dimethyl-N carboxymethyl ammonium (inner) carboxylate, isotopically labelled with 14C in the 1 carbon of the lauroyl moiety ([1-14C]TB); the sample had a specific radioactivity of 7.83µCi/mg.

Aqueous solutions of the test materials were administered to rats of both sexes by gavage or topically and the fate of the 14C label was followed for up to 48 hours after dosing. Whole body autoradiography (WBA) was used to study the tissue distribution of the 14C. The levels of 14C excreted were used to estimate intestinal and skin absorption.

The following dose levels were used:

- [14C]TB gavage approximately 30mg/kg (87.3µCi/kg)

- [14C]TB topical approximately 20mg/kg (60µCi/kg; approximately 0.3 mg/cm²)

- [1-14C]TB gavage approximately 10mg/kg (81.5µCi/kg)

- [1-14C]TB topical approximately 10mg/kg (81.5 µCi/kg; approximately 0.15 mg/cm²)

After gavage administration, C12 AAPB is poorly absorbed from the intestinal tract following administration in water at 30 mg/kg or 10 mg/kg bw, respectively. Within 48 hours, approximately 5% of the 14C dose was excreted in urine and < 2 % in expired air and < 2% remained in the carcass. The remainder was excreted in the faeces as unchanged parent material (as was confirmed by thin layer chromatography (TLC) analysis in the case of labelling at the carboxymethylammonium moiety). Whole body autoradiography confirmed that absorption from the gut was low and that the tissues showing detectable levels of 14C were those predominantly associated with urinary excretion (liver, kidney cortex, urinary bladder). The urine contained traces of parent and an unidentified polar metabolite. Although metabolism of absorbed material is extensive, the lauryl moiety is not extensively removed from the rest of the molecule judging by the relatively low amounts of 14CO2 produced. There was no sex difference in the overall fate following administration by oral gavage.

Dermal application (approximately 20 mg/kg (approximately 0.3 mg/cm²) of C12 AAPB 14C-labelled at the carboxymethyl ammonium or approximately 10 mg/kg (approximately 0.15 mg/cm²) of C12 AAPB 14C-labelled in the lauryl moiety in water) followed by 48 h occlusion gave similar results. After 48 hours, approximately 3.5-6% (females) and 2-3.5% (males) was absorbed. Urine was the major route of excretion for absorbed material with expired air and faeces being relatively minor routes. A further experiment with 10 minutes exposure of [14C]TB followed by rinsing and then a 48 hour occlusion resulted in less than 0.2% absorption. TLC separations were not carried out for on urine from topically treated rats.

For more detailed results see the following table, taken from the HERA report.

Table 2: Results of the ADME (Absorption, Distribution, Metabolism, Excretion) study

Test Substance

Dosage

Protocol

Excretion

Absorption

[14C]TB

30 mg/kg bw

gavage

5 m, 5 f for

excretion

TLC* examination

of the faecal 14C

Sacrifice of

animals: after 2, 4,

8, 24, 48 (2 rats at

each time point for

whole body

autoradiography)

After 24 hours:

Faeces: 75 % (f), 96 % (m)

Urine: 4.1% (f), 6.5 % (m)

Expired air: 0.75 – 0.77 % (m and f)

After 48 hours:

Faeces: 118 % (f) no data (m)

Urine: 5.5 % (m and f)

Expired air: 0.8 % (m and f)

Metabolites in faeces: only unchanged

[14C]TB

< 10 % from

intestinal tract

[14C]TB

30 mg/kg bw

gavage

3 m,3 f for

excretion,

TLC* examination

of the urinary 14C

Sacrifice of

animals: after 48 h

After 48 h:

Faeces: 86 – 92 %

Urine: 2 – 4 %

Expired air: 1 – 1.4 %

Carcass: 0.8 – 1.4 %

No sex differences

Metabolites in urine: one more polar

metabolite than [14C]TB

< 10 % from

intestinal tract

[14C]TB

20 mg/kg bw

topical, occluded

6 m, 6 f

Sacrifice of animals

after 48 h

After 48 h

Faeces: 0.2 – 0.8 % (f > m)

Urine: 1.3 – 2.7 % (f > m)

Expired air: 0.2 – 0.3 %

Carcass: 0.3 – 2.3 % (f > m)

Appr. 6 % (f), 2 %

(m)

[14C]TB

20 mg/kg bw

topical, unoccluded

3 m, 3 f

Rinsed after 10

minutes

After 48 h

Faeces: 0.005 – 0.02 %

Urine: 0.02 – 0.06 %

Expired air: 0.0 – 0.02 %

Carcass: 0.04 – 0.07 %

< 0.2 % (f and m)

[1-14C]TB

10 mg/kg bw

gavage

3 m, 3 f

Sacrifice of animals

after 48 h

TLC* examination

of the urinary and

faecal 14C

After 24 h:

Faeces: 80 %

Urine: < 5 %

After 48 h:

Faeces: 79 – 90 %

Urine: 3.7 – 4.9 %

Expired air: 1 – 1.9 %

Carcass: 1 – 1.8 %

No sex differences

Metabolites in faeces:

unchanged [1-14C]TB

Metabolites in urine: mainly one polar

metabolite, traces of unchanged [1-14C]TB

< 10 % from

intestinal tract

[1-14C]TB

10 mg/kg bw

topical, occluded

3 m, 3 f

Sacrifice of animals

after 48 h

After 48 h:

Faeces: 0.4 – 0.5 %

Urine: 1 – 1.5 %

Expired air: 0.3 – 0.6 %

Carcass: 0.4 – 1.7 %

3.5 %

* TLC - thin layer chromatography

In vitro Studies

In a reliable in vitro study according to OECD guideline 428, dermal permeation and penetration of Coco AAPB was investigated using human skin. Test item was applied for 24 h to human skin biopsy samples from three female donors using Franz diffusion cells and test item amount reaching the receptor medium was measured. At the end of this permeation experiment, the remaining test item on the skin surface was collected with cotton swabs (= skin wash). After removing residual formulation, the concentration of the test item in the skin, in the Stratum corneum and deeper skin layers, was quantified. The upper corneous layer of the skin was stripped off. In total 20 tape strips were performed per each skin biopsy. The first two strips were separately analysed due to potential contaminations by residual test item on the surface of the skin. To measure penetration in deeper skin layers, cryo-sections of epidermis and dermis were prepared.

The test item did not penetrate the skin. Amounts in epidermis, dermis and receptor medium were below the detection limit of the validated analytical method. This low absorption was confirmed through the mass recovery calculation, where 98.55 % (mean value for 6 Franz cells) of test item were determined in the test solution which remained at the skin surface after 24 hours. The mean recovery in the two first tape strips was 0.17 % during all performed experiments. In the further 18 tape strips a mean recovery of 0.07 % was documented. Mean total recovery rates were 95.00 to 100.39%.

The mean absorbed dose of the test item, sum of the amounts found in the viable epidermis, dermis and receptor medium was 0 %.

Conclusion

After gavage administration, C12 AAPB is poorly absorbed from the intestinal tract. Within 48 hours, approximately 5% of the14C dose was excreted in urine and < 2 % in expired air and < 2% remained in the carcass. The remainder was excreted in the faeces as unchanged parent material. Whole body autoradiography confirmed that absorption from the gut was low and that the tissues showing detectable levels of 14C were those predominantly associated with urinary excretion (liver, kidney cortex, urinary bladder). The urine contained traces of parent and an unidentified polar metabolite. Although metabolism of absorbed material is extensive, the lauryl moiety is not extensively removed from the rest of the molecule judging by the relatively low amounts of 14CO2 produced. There was no sex difference in the overall fate following administration by oral gavage.

Dermal application followed by 48 h occlusion gave similar results. After 48 hours, approximately 3.5-6% (females) and 2-3.5% (males) was absorbed. Urine was the major route of excretion for absorbed material with expired air and faeces being relatively minor routes. A further experiment with 10 minutes exposure followed by rinsing and then a 48 hour occlusion resulted in less than 0.2% absorption.

In an in vitro experiment performed on human skin biopsy samples, a dermal absorption was not measurable.

For DNEL calculation, as default values an each 10 % absorption is assumed after oral and dermal administration of the test substance. Particularly for the dermal route, which is due to the use profile of the substance the most important exposure route to humans, this is a very conservative worst case assumption, since a reliable in vitro study on human skin showed a dermal resorption rate of zero.

Inhalatory absorption data are missing. Absorption after oral or dermal exposure in the described reliable experimental study on rats reached a maximum of 10%. In an reliable in vitro study on dermal resorption on human skin, the resorption rate for Coco AAPB was even zero.

The amounts of inhalative absorption deduced from the physico-chemical properties and from the results of oral and dermal absorption studies are expected to be low. Further on, AAPBs are amphoterics and the inhalative absorption of ionic substances is generally low. Taken together, based on physico-chemical properties of the respective AAPBs, a low bioavailability could be anticipated. As a worst case assumption for DNEL calculation, twice the measured values for oral and dermal absorption are assumed for absorption after inhalative exposure in absence of any experimental data on inhalative exposure.