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Reference
Endpoint:
basic toxicokinetics in vitro / ex vivo
Type of information:
other: Expert statement
Adequacy of study:
key study
Study period:
2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Expert statement, no study available
Qualifier:
no guideline required
Principles of method if other than guideline:
Expert statement
GLP compliance:
no
Details on absorption:
Incozol 4 was shown to rapidly hydrolyse when getting in contact with water, especially at low pH values. Therefore, bioavailability after oral exposure was assessed only for the hydrolysis products isobutyraldehyde and several “hydrolysed Incozol 4 structures”. Based on their molecular weight and physico-chemical properties, dissolution in the gastro-intestinal fluids and contact with the mucosal surface might occur and may allow direct uptake into the systemic circulation through aqueous pores or via carriage of the molecules across the membrane with the bulk passage of water.
There are no indications of bioavailability after a single administration of 5000 mg Incozol 4/kg bw as diuresis was the only clinical finding. Long-term administration of Incozol 4 revealed ambiguous findings in the 1000 mg/kg bw/day dose group. Bioavailability after repeated oral administration of high doses of Incozol 4 or rather its hydrolysis products could therefore not be excluded.
Based on the vapour pressure of approximately 0.0005 Pa at 20°C Incozol 4 is not expected to become airborne in its vapour form. However, if Incozol 4 is degraded hydrolytically, inhalation exposure of isobutyraldehyde, one of the hydrolysis products, is expected due to its high vapour pressure. If the substance reaches the lungs, absorption directly across the respiratory tract epithelium by passive diffusion is likely to occur due to its log Pow value (0.77) and water solubility (60 g/L). Systemic signs of toxicity were observed when isobutyraldehyde was administered by inhalation in a 90 day-toxicity study on rats, indicating, that the substance is bioavailable after inhalation exposure (ECHA).
Uptake of Incozol 4 into the stratum corneum is assumed due to the lipophilic properties of the substance. However, transfer into the epidermis might be limited by the low water solubility. It is general accepted that if a compound’s water solubility falls between 1-100 mg/L, absorption can be anticipated to be low to moderate. Therefore, based on the molecular weight and physico–chemical properties of Incozol 4 dermal penetration of the substance might be slow. However, results achieved from an LLNA revealed that Incozol 4 has skin sensitising properties and dermal uptake must have occurred, although it may only has been a small fraction of the applied dose. Further, hydrolytical degradation of Incozol 4 in the moisture of the skin and uptake of the hydrolysis products could not be excluded.
Taken together, physico-chemical properties and experimental data indicate a low bioavailability of Incozol 4 via oral and dermal route. Exposure via inhalation route of the non-hydrolysed product is considered to be unlikely.
Details on distribution in tissues:
Assuming that Incozol 4 or rather its hydrolysis products “hydrolysed Incozol 4 structures” and isobutyraldehyde are absorbed into the body following oral intake, they may be distributed via the blood stream over the whole organism. Extracellular concentration is expected to be higher than intracellular due to their water solubility and low log Pow values. Direct transport through aqueous pores is likely to be the main route within systemic circulation
Based on their BCF values both, the parent molecule Incozol 4 and its hydrolysis products have no potential to bioaccumulate in the human body.
Details on excretion:
As discussed above, Incozol 4 will be hydrolysed after being in contact with an aqueous solution and will probably not be excreted in its non-hydrolysed form.
The degradation product “hydrolysed Incozol 4 structures” might by excreted via faeces due to its molecular weight (>300 g/mol in rat). In case of further hydrolysis products, renal excretion would be favoured due to increasing water solubility and decreasing molecular weight.
Isobutyraldehyde is oxidised to carbon dioxide and excreted via the exhaled air (DiVincenzo and Hamilton, 1979).
Details on metabolites:
Based on the structure of the molecule, Incozol 4 degraded hydrolytically after being in contact with an aqueous solution. The first degradation product “hydrolysed Incozol 4 structures” may be further degraded hydrolytically to a certain extend to diethanolamine, 1,6-hexanediamine and carbon dioxide under basic conditions.
Isobutyraldehyde is estimated to be oxidised to isobutyric acid which was shown to be rapidly oxidised to carbon dioxide (DiVincenzo and Hamilton, 1979).
No metabolic activation of Incozol 4 and its hydrolysis products is expected as indicated by the negative in vitro genotoxicity assays in the presence of S9 mix.
Bioaccessibility (or Bioavailability) testing results:
Taken together, physico-chemical properties and experimental data indicate a low bioavailability of Incozol 4 via oral and dermal route. Exposure via inhalation route of the non-hydrolysed product is considered to be unlikely.
Conclusions:
Based on physico-chemical characteristics, particularly water solubility and octanol-water partition coefficient absorption via oral and dermal route is expected to be low. Exposure via inhalation route of the non-hydrolysed product is considered to be unlikely. Incozol 4 rapidly hydrolyses into “hydrolysed Incozol 4 structures” and isobutyraldehyde when getting in contact with an aqueous solution. Based on water solubility, extracellular concentration of the hydrolysis products is assumed to be higher than intracellular. Isobutyraldehyde is estimated to be oxidised to isobutyric acid which was shown to be rapidly oxidised to carbon dioxide. “Hydrolysed Incozol 4 structures” may be further degraded hydrolytically to a certain extend to diethanolamine, 1,6-hexanediamine and carbon dioxide under basic conditions. No metabolic activation of Incozol 4 and its hydrolysis products is expected. Excretion via faeces is assumed to be the main excretion pathway of “hydrolysed Incozol 4 structures” due to its molecular weight (>300 g/mol in rat). Based on the oxidation to carbon dioxide, isobutyraldehyde is excreted via the exhaled air. In consideration of the BCF values and the expected metabolism, bioaccumulation of Incozol 4 itself and its hydrolysis products is not likely to occur.

Description of key information

Based on physico-chemical characteristics, particularly water solubility and octanol-water partition coefficient absorption via oral and dermal route is expected to be low. Exposure via inhalation route of the non-hydrolysed product is considered to be unlikely. Incozol 4 rapidly hydrolyses into “hydrolysed Incozol 4 structures” and isobutyraldehyde when getting in contact with an aqueous solution. Based on water solubility, extracellular concentration of the hydrolysis products is assumed to be higher than intracellular. Isobutyraldehyde is estimated to be oxidised to isobutyric acid which was shown to be rapidly oxidised to carbon dioxide. “Hydrolysed Incozol 4 structures” may be further degraded hydrolytically to a certain extend to diethanolamine, 1,6-hexanediamine and carbon dioxide under basic conditions. No metabolic activation of Incozol 4 and its hydrolysis products is expected. Excretion via faeces is assumed to be the main excretion pathway of “hydrolysed Incozol 4 structures” due to its molecular weight (>300 g/mol in rat). Based on the oxidation to carbon dioxide, isobutyraldehyde is excreted via the exhaled air. In consideration of the BCF values and the expected metabolism, bioaccumulation of Incozol 4 itself and its hydrolysis products is not likely to occur.

Key value for chemical safety assessment

Bioaccumulation potential:
no bioaccumulation potential

Additional information

Toxicological profile of Incozol 4

An acute oral toxicity study conducted with Incozol 4 using rats revealed a LD50 value of greater the 5000 mg/kg bw. Diuresis was observed in all animals during the observation period. No mortality and no systemic signs of toxicity occurred in an acute dermal toxicity study with the test item. The LD50 was therefore determined to be greater than 2000 mg/kg bw. Slight dermal irritation symptoms were observed in all animals up to Day 6 of the observation period. No acute inhalation toxicity study was performed with Incozol 4 as inhalation exposure is considered negligible due to its low vapour pressure.

In an in vitro skin irritation study conducted with Incozol 4 no skin irritation was observed when applied on reconstructed human skin epidermis model (EPISKIN, Toxi Coop, 2012). This result was supported by an in vivo skin irritation study on rabbits (Bayer 1978). In an Isolated Chicken Eye Test (ICET) the test item did not cause ocular corrosion or severe irritation (Toxi Coop 2012). However, an eye irritation test performed Incozol 4 on rabbits showed that the substance caused slight to severe conjunctival and slight to moderate corneal irritant effects, fully reversible within 2 weeks (Toxi Coop 2012). A local lymph node assay revealed that Incozol 4 has skin sensitising properties.

Incozol 4 induced reverse mutations in a bacterial reverse mutation test (Ames test) with four Salmonella typhimurium strains and one Escherichia coli strain (2009). The mutagenic response was depended on the bacterial strain and on the presence of a metabolic activation system (S9 liver homogenate). The chemical caused an increase in the reverse mutation frequency in the tester strains TA 100 in the presence and in the absence and in the E.coli WP2 uvrA strain in the absence of metabolic activation. In a second Ames test conducted with four Salmonella typhimurium strains (Bayer, 1988), no biologically relevant increase of the mutant count compared to the negative control was observed. This negative result was supported by an in vitro mammalian cell gene mutation assay (mouse lymphoma assay, Toxi Coop 2012) as well as by an in vitro chromosome aberration assay both in the absence and presence of metabolic activation. In conclusion, Incozol 4 is considered to be non-mutagenic.

A 28 day repeated dose toxicity study with Incozol 4 was performed in male and female Wistar rats ( Korea Testing & Research Institute, 2012). The chemical was administered orally (by gavage) once a day for a total of 28 days at 0 (vehicle control), 98, 312 and 1000 mg/kg bw/day. A 14-day recovery period (5 animals per sex per group) was set for the control and 1000 mg/kg bw/day groups.

No mortality was observed through this study. Body weight gain and food consumption were not influenced by the test substance. No test item related changes were observed in hematology and clinical chemistry parameters throughout the dosing period. Statistically significant changes (decrease in neutrophil (%) and increase in lymphocyte (%) in male animals and decrease in neutrophil, lymphocyte, eosinophil, Mean Corpuscular Volume (MCV) and Mean Corpuscular Hemoglobin (MCH) in female animals) were found in male and female animals after the recovery period. Blood urea nitrogen was increased and inorganic phosphate was decreased in male and female animals of the recovery group, respectively. There was a significant decrease in the absolute testis weight compared to control in male animals of the 1000 mg/kg bw/day dose group and in absolute seminal vesicle weight in the corresponding recovery group. In female animals of the high dose recovery group a significant increase of the absolute and relative kidney and ovary weight was noted. In the absence of any macroscopic or microscopic organ and tissue alterations the observations described above were considered to be non-adverse. The respective NOAEL was set to 1000 mg/kg bw/day for male and female animals.

In a 90 day repeated dose toxicity study the test item was administered orally (by gavage) to male and female Hsd.Han: Wistar rats (n=15 animals/sex in the control and high dose groups, n= 10 animals/sex in the low and middle dose groups) once a day at 0 (vehicle control), 100, 300 and 1000 mg/kg bw/day doses, applied in a dose volume of 5 mL/kg bw for 90 or 91 days. Five animals per sex in the control and high dose groups were assigned to the recovery groups and were treated identically up to Day 89. Then they were observed without administration for four weeks (recovery observations).

No animals died during the course of the study. No signs of toxicity related to the test item were detected at any dose level (1000, 300 or 100 mg/kg bw/day) at the daily and detailed weekly clinical observations or in the course of the functional observation battery. The behavior and physical condition of animals were normal during the entire observation period. A 1000 mg/kg bw/day dose of Incozol 4 induced salivation (male and female), changes in body weight development (male and female), and food consumption (male and female) after consecutive 90-day oral (gavage) administration to Hsd.Han:Wistar rats. These changes were fully reversible in male and female animals up to the end of the four weeks post-treatment period. Changes in some red blood cell parameters (red blood cell count, hemoglobin concentration or hematocrit; male or female), in biochemical parameters (calcium, albumin or total protein and urea; male or female) or organ weights (liver or kidneys; male or female) might be indicative of a possible test item influence however without any structural (histopathological) alterations to substantiate their toxicological relevance. The slight changes in some of the above mentioned parameters (hematology, clinical chemistry, organ weights) at 300 or 100 mg/kg bw/day remained well within or were marginal to the range of historical control and without supporting changes in related parameters therefore were judged to be toxicologically not significant. The NOAEL was set to 300 mg/kg bw/d.

 

Toxicokinetic analysis of Incozol 4

Incozol 4 is a yellowish liquid at room temperature with a molecular weight of 486.6452 g/mol. The calculated water solubility of the substance is 28.24 mg/L at 20 °C. The log Pow of Incozol 4 was calculated to be 2.92. Based on this log Pow, a BCF of 39.23 L/kg wet-wt was calculated. The vapour pressure of Incozol 4 is approximately 0.0005 Pa at 20 °C. In an aqueous solution, Incozol 4 is rapidly degraded hydrolytically to isobutyraldehyde and several partially or fully hydrolysed structures of Incozol 4. The half-lives of Incozol 4 in aqueous solution at pH = 4 and 7 were considered to be <2 min and at pH 9 < 18 hours.

All hydrolysis substances have a lower log Pow value than Incozol 4 itself (approximately 1.03 as worst case for hydrolysed Incozol 4 strucutresand 0.77 for isobutyraldehyde). Also the BCF values are lower as compared to Incozol 4 (70.97 as worst case value for "hydrolysed Incozol 4 structures" and 3.162 L/kg wet-wt for both hydrolysis products, respectively). Water solubility of hydrolysis products is higher than of Incozol 4 itself (8.4 g/L for “hydrolysed Incozol 4 structures” and 60 g/L for isobutyraldehyde). The vapour pressure of isobutyraldehyde is above the value of Incozol 4 (189 hPa). The calculated vapour pressure of “hydrolysed Incozol 4 structures” is 3.99E-12 Pa.

 

Absorption

Incozol 4 was shown to rapidly hydrolyse when getting in contact with water, especially at low pH values. Therefore, bioavailability after oral exposure was assessed only for the hydrolysis products isobutyraldehyde and “hydrolysed Incozol 4 strucutres”. Based on their molecular weight and physico-chemical properties, dissolution in the gastro-intestinal fluids and contact with the mucosal surface might occur and may allow direct uptake into the systemic circulation through aqueous pores or via carriage of the molecules across the membrane with the bulk passage of water. 

There are no indications of bioavailability after a single administration of 5000 mg Incozol 4/kg bw as diuresis was the only clinical finding. Long-term administration (90 d study) of Incozol 4 revealed clear findings in the high dose group. Bioavailability after repeated oral administration of high doses of Incozol 4 or rather its hydrolysis products could therefore not be excluded.

Based on the vapour pressure of approximately 0.0005 Pa at 20°C Incozol 4 is not expected to become airborne in its vapour form. However, if Incozol 4 is degraded hydrolytically, inhalation exposure of isobutyraldehyde, one of the hydrolysis products, is expected due to its high vapour pressure. If the substance reaches the lungs, absorption directly across the respiratory tract epithelium by passive diffusion is likely to occur due to its log Pow value (0.77) and water solubility (60 g/L). Systemic signs of toxicity were observed when isobutyraldehyde was administered by inhalation in a 90 day-toxicity study on rats, indicating, that the substance is bioavailable after inhalation exposure (ECHA).

Uptake of Incozol 4 into the stratum corneum is assumed due to the lipophilic properties of the substance. However, transfer into the epidermis might be limited by the low water solubility. It is general accepted that if a compound’s water solubility falls between 1-100 mg/L, absorption can be anticipated to be low to moderate. Therefore, based on the molecular weight and physico–chemical properties of Incozol 4 dermal penetration of the substance might be slow. However, results achieved from an LLNA revealed that Incozol 4 has skin sensitising properties and dermal uptake must have occurred, although it may only has been a small fraction of the applied dose. Further, hydrolytical degradation of Incozol 4 in the moisture of the skin and uptake of the hydrolysis products could not be excluded.

 

Taken together, physico-chemical properties and experimental data indicate a low bioavailability of Incozol 4 via oral and dermal route. Exposure via inhalation route of the non-hydrolysed product is considered to be unlikely.

 

Distribution

Assuming that Incozol 4 or rather its hydrolysis products “hydrolysed Incozol 4 structures” and isobutyraldehyde are absorbed into the body following oral intake, they may be distributed via the blood stream over the whole organism. Extracellular concentration is expected to be higher than intracellular due to their water solubility and low log Pow values. Direct transport through aqueous pores is likely to be the main route within systemic circulation.

Based on their BCF values both, the parent molecule Incozol 4 and its hydrolysis products have no potential to bioaccumulate in the human body.

 

Metabolism

Based on the structure of the molecule, Incozol 4 degraded hydrolytically after being in contact with an aqueous solution. The first degradation products “hydrolysed Incozol 4 strucutres” may be further degraded hydrolytically to a certain extend to diethanolamine, 1,6-hexanediamine and carbon dioxide under basic conditions.

Isobutyraldehyde is estimated to be oxidised to isobutyric acid which was shown to be rapidly oxidised to carbon dioxide (DiVincenzo and Hamilton, 1979).

No metabolic activation of Incozol 4 and its hydrolysis products is expected as indicated by the negative in vitro genotoxicity assays in the presence of S9 mix.

 

Excretion

As discussed above, Incozol 4 will be hydrolysed after being in contact with an aqueous solution and will probably not be excreted in its non-hydrolysed form.

The degradation product “hydrolysed Incozol 4 strucutres” might by excreted via faeces due to its molecular weight (>300 g/mol in rat). In case of further hydrolysis products, renal excretion would be favoured due to increasing water solubility and decreasing molecular weight.

Isobutyraldehyde is oxidised to carbon dioxide and excreted via the exhaled air (DiVincenzo and Hamilton, 1979).

 

Summary

Based on physico-chemical characteristics, particularly water solubility and octanol-water partition coefficient absorption via oral and dermal route is expected to be low. Exposure via inhalation route of the non-hydrolysed product is considered to be unlikely. Incozol 4 rapidly hydrolyses into several “hydrolysed Incozol 4 structures” and isobutyraldehyde when getting in contact with an aqueous solution. Based on water solubility, extracellular concentration of the hydrolysis products is assumed to be higher than intracellular. Isobutyraldehyde is estimated to be oxidised to isobutyric acid which was shown to be rapidly oxidised to carbon dioxide. “Hydrolysed Incozol 4 structures” may be further degraded hydrolytically to a certain extend to diethanolamine, 1,6 -hexanediamine and carbon dioxide under basic conditions. No metabolic activation of Incozol 4 and its hydrolysis products is expected. Excretion via faeces is assumed to be the main excretion pathway of “Hydrolysed Incozol 4 structures” due to its molecular weight (>300 g/mol in rat). Based on the oxidation to carbon dioxide, isobutyraldehyde is excreted via the exhaled air. In consideration of the BCF values and the expected metabolism, bioaccumulation of Incozol 4 itself and its hydrolysis products is not likely to occur.

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