5-amino-o-cresol

Endpoint summary

Description of key information

According to the result of the key studies, the registered substance 4 -amino-2 -hydroxytoluene was assessed in different test in order to define the differents toxicokinetic parameters (Absorption, Distribution, Metabolism, Excretion in in vivo studies performed on rats and complemented by in vitro studies for metabolism and dermal penetration) for different routes of exposure.

In oral exposure route, the absorption rate was defined as 95% of the total administered amount. Additionnaly, the test item was showed an high permeability across gastro intestinal tract.After absorption, it was readily distributed into all organs (mainly in thyroid, bladder, liver, skin and gastro intestinal tract). The test substance was mainly excreted in urine (89%) and faeces (11%) where metabolites were found as glucuronide, sulfate and N-acetyl of the test susbtance. The substance can be metabolized in liver in four metabolites : – AHT glucuronide, AHT O-sulfate, N-acetyl-AHT and N-Acetyl AHT-sulfate. However, it was mainly metabolized by sulfate conjugation in liver. Furthermore, there is no significant differences between metabolic pathway of the test item in rats, mice or human liver.

By dermal exposure route, the absorption rat was defined as 53.0% of the total administered amount. The test item was distributed in carcass, liver and blood at 14.6%. The substance was mainly excreted by urine (39%) in which three metabolites were found : glucuronide, sulfate and N-acetyl of 4-amino-2-hydroxytoluene. The skin can metabolize the test item, (HaCaT cells), extensively N acetylation which is the most aboundant metabolic pathway. Some in vitro dermal absorption studies showed a absorption rate of 3.995 µg/cm2 in pig skin samples (with a 2.5% formulation) and 25.6µg/cm2 on human skin (with a 5% test item in fomulation). In presence of a reaction partner, the test item can be considered as biologically available.

Intravenously, the test item was readily distributed into all organs, extensively metabolized and excreted via urine (94%). The test substance was metabolized to a) sulphated (41%) b) N-acetylated and N-acetylated and sulphated (37%) and c) glucuronidated (15%) forms of test substance

Key value for chemical safety assessment

Bioaccumulation potential:

low bioaccumulation potential

Absorption rate - oral (%):

95

Absorption rate - dermal (%):

53

Additional information

To assess the ADME and toxicokinetics parameters of the registered substance 4 -amino-2 -hydroxytoluene, 3 in vivo key studies and 16 in vitro key studies were availables:

- One in vivo study was performed in order to determine toxicokinetics parameters in rats exposed by oral routes (Klimisch 1, GLP compliant, OECD 427). Female Wistar rats  were used in study. Animals were treated with following doses (dose volume: 10 mL/ kg bw):12.5 mg/kg bw (Groups 1 and 3); 500 mg/kg bw (Groups 2 and 4) and they received radioactive dose of 9.556, 10.256, 9.737 and 10.327 MBq/ kg bw, respectively. They were observed for mortality, clinical signs and body weight during the study at different time intervals and thereafter were sacrificed by euthanasia. Metabolite products and toxicokinetics parameters were determined. The maximum oral absorption rate was measured at 95% with urine data and 40% with blood data. The average total remaining radioactivity in blood, carcass and tissues was between 0.4 and 0.9% of the administered dose in the oral groups, indicating no major accumulation of radioactivity after 96 hours. These tissues were thyroid, bladder, liver, carcass, skin and GI. Urine was the major route of excretion of the test substance, accounting for 89% and fecal excretion was only a minor route of excretion after oral administration and accounted for 11%. Based on above, after oral administration, 4-amino-2-hydroxytoluene was extensively absorbed, readily distributed into all organs, extensively metabolized and excreted via the urine. Oral absorption was high, regardless of the dose (84 - 95% of the applied dose). Three major metabolites (glucuronide, sulfate and N-acetyl of the test substance) were detected in the urine. Following oral administration, the most abundant metabolic pathway was sulfatation. (Wenker MAM, 2005)

- One in vivo study was performed in order to determine toxicokinetics parameters in rats exposed by dermal routes (Klimisch 1, GLP compliant, OECD 427 & 417). Female Wistar rats were used in study. Animals were treated dermally with "O" rings with following doses: 0.15 mg/cm2 (12.5 mg/ kg bw) for group 1 and 3, and 0.45 mg/ cm2 (37.5 mg/ kg bw) for group 2 and 4. Animals were observed for mortality, clinical signs and body weight during the study period and thenafter were sacrificed by euthanasia. The radiopurity (after dosing) in Groups 1, 2, 3 and 4 formulations was 98.7, 98.9, 91.9 and 93.2%, respectively. Blood was sampled on groups 3 and 4  and treated and untreated skin (shaved skin from the abdominal region) was removed for radioactive analysis. The amount of test substance excreted through urine in Groups 1 and 2 was 39% and 18%, respectively. The average total recovery of radioactivity in Groups 1 and 2 was 89.97% and 88.627% of the administered dose, respectively. Occlusive administration of 4-amino-2-hydroxytoluene (in ethanol) for 24 hours resulted in high dermal absorption values with saturation phenomena observed for the high dose group (0.173 mg/cm2). Three major metabolites (glucuronide, sulfate and N-acetyl of 4-amino-2-hydroxytoluene) were detected in the urine. Following dermal application,N-acetylation was the most abundant metabolic pathway. (Wenker MAM, 2005)

-One in vivo study was performed in order to determine toxicokinetics parameters in rats exposed by intravenous route (Klimisch 1, GLP compliant, OECD 427).Female Wistar rats were used in this study. Animals were treated intravenously with following doses 12.5 mg/kg bw and received radioactive doses of 9.991 and 10.251 MBq/Kg bw for group 1 and 2 respectively. Animals were observed for mortality, clinical signs and body weight during the study period and thenafter were sacrificed by euthanasia. Blood, urine, feces, tissues were sampled. At the termination of the study the average total remaining radioactivity in blood and carcass and tissues was between 0.4 and 0.9% of the administered dose in Group 1 (mass balance), indicating no major accumulation after 96 hours. A number of tissues from animals in the intravenous dose groups had relatively high residual concentrations, 2 fold or higher, compared to the concentration in blood. Blood concentrations in Group 1 (mass balance) were 2.2 to 2.8 times higher than plasma concentrations, indicating some distribution of the test substance into the red blood cells. Excretion via urine was the major route of elimination. 94% of test substance was excreted through urine. The highest excretion was observed in the first 6 hours after test substance administration. Excretion via faeces was only a minor route of excretion. 6% of the test substance was excreted through faeces. 4-amino-2-hydroxytoluene when administered intravenously was readily distributed into all organs, extensively metabolized and excreted via urine. The test substance was metabolized to a) sulphated (41%) b) N-acetylated and N-acetylated and sulphated (37%) and c) glucuronidated (15%) forms of test substance. (Wenker MAM, 2005)

In order to assess the metabolic pathway of the registered substance, in vitro studies were performed on liver cells (4 studies) and human keratinocytes cells (2 studies):

- Primary human hepatocytes were used in three studies. Two were GLP compliant and quoted as Klimisch 1 in which a comparative metabolic stability and metabolite profile of 4-amino-2-hydroxytoluene was investigated by incubating the test substance with cryopreserved primary human, rat, and mouse hepatocytes in suspension culture. The incubate samples were collected at different time intervals and analyzed for metabolite formation by LC-MS/MS . 10 μM of the test substance were incubated with a period of 4 hours. The test substance was rapidly metabolized in human, rat and mouse hepatocytes. In the first study, exposure of rodent (mice, rats) and human hepatocytes to 4-Amino-2-hydroxytoluene under identical test conditions suggests no significant differences in the metabolic rate/capacity or the metabolic profile. Under the conditions applied in this study, test substance was extensively metabolized by sulfate conjugation. The second study provided that the 5-amino-2-methylphenol was extensively metabolized by sulfate conjugation by human, rat and mice hepatocytes. The others studies were performed on human hepatocytes (Klimisch 2, scientificaly robust method, Non GLP), The findings indicate that the hepatic metabolism of 4-amino-2-hydroxytoluene was similar in rodents and humans and includes predominantly Phase II conjugation (sulfatation and N-acetylation of test substance) even if formation of four metabolites– AHT glucuronide, AHT O-sulfate, N-acetyl-AHT and N-Acetyl AHT-sulfate was possible by liver.(Powrie, 2005) (Obringer CM, 2011) (Krebsfanger N, 2003)

- Two studies were performed on human derived keratinocytes cell line (Klimisch 2, Non GLP compliant,followed scientific standard). They were conducted to assess the metabolism of test substance (radiolabelled in the second study) following incubation with human keratinocytes (HaCaT cells) for 24 hours. The above results conclude that the capacity of human keratinocytes (HaCaT) to N-acetylate 4-Amino-2-hydroxytoluene in 4-Acetylamino-2-Hydroxytoluene was shown for the first study and the incubation of 14C- 5-amino-2-methyl phenol (14C-AHT) with HaCaT cells resulted in formation of monoacetylated AHT as a single metabolic biotransformation product. (Obringer CM, 2009) (Beck 2005)

Two in vitro studies were available to assess the potential permeability of the test substance across the gastro intestinal tract :

- The study (Klimisch 2, No GLP compliant, followed scientific standard) was performed to estimate the bioavailability of the test substance across the intestinal barrier using the A-B Permeability assay with human intestinal epithelial cell line (TC-7). The uptake of the test substance from apical to basolateral cavity was analyzed by LC-MS/MS.  A relative percent absorption of the test substance was indicated by the comparison with reference material. The mean apparent permeability coefficient (Papp) of 4-amino-2-hydroxytoluene was 103.8 x 10 (-6) cm/s (high permeability) in a A-B (apical-basolateral) permeability assay, performed by using TC-7 cell line. As the absorption from the gastro-intestinal tract is likely to be permeability limited, the high permeability observed in this assay indicates a good absorption of 4-Amino-2-hydroxytoluene after oral administration. (Jager, 2008)

-The in vitro intestinal absorption study of 4-Amino-2-Hydroxytoluene by using an in vitro cell culture barrier system was conducted to determine the uptake of the test substance across the cell monolayers (Klimisch 1, no GLP compliant) . Absorption of 4-Amino-2-Hydroxytoluene across intestinal in the apical to basolateral transport direction using CaCo-2 model was evaluated in two independent experiments. CaCo-2 cells are a human intestinal epithelial cell line. The concentration of test substance used in the study was 50 µM corresponding to 6.16 µg/mL. The following permeability coefficients (Papp) of 4-Amino-2-Hydroxytoluene were comparable in both experiments: Experiment 1(HV1): Papp = 17.8 x 10(-6) cm/s ; Experiment 2 (HV2): Papp = 38.5 x 10(-6) cm/s. 4-Amino-2-Hydroxytoluene at a concentration of 50 µM corresponding to 6.16 µg/mL showed high permeability across the intestinal barrier using an in vitro cell culture barrier system (based on the comparison with the reference substances). (Kennedy J, 2004)

Some studies were performed to determine the percutaneous penetration rate of the registration substance :

Five studies were performed on pig skin samples according to the OECD guideline 428 method with test substance, or with test substance and reaction partner (3 studies):

- Pig skin samples were used as the test system. 400 mg of the formulation (100 mg/cm2; for 4 cm2), containing 1.5% test substance (i.e. 1.5 mg/cm2) was applied to the skin samples for 30 minutes, in a typical cream formulation for oxidative hair dyes. Diffusion chambers were used. Under the test conditions of the first study, a skin penetration rate of 2.761 μg/cm2 (amount of test substance found in the receptor fluid and in the lower skin) was observed when 1.5 mg/cm2 of 4-amino-2-hydroxytoluene was applied under oxidative conditions in a typical hair dye formulation to pig skin samples, in an in-vitro skin absorption assay. On the second study, under the test conditions, a maximum amount of 2.59 ± 1.21 µg/cm2 (0.19%) of4-Amino-2-hydroxytoluene was considered as biologically available when 400 mg (100 mg/cm2) of formulation containing 1.5% of test material was applied to pig skin.

In three studies, the test item was formulated with reaction partner and applicated in pig skin samples, and it was considered as biologically available. (Sieber TP, 2007)

Two in vitro studies were performed on human skin (OECD 428 method followed, GLP compliant, Klimisch 1)The test substance was applied to the surface of skin samples in a standard hair dye formulation containing 1.5% or 5.0% test substance at a rate of 100 mg/cm2. After 60 minutes, the skin surface was washed to remove the excess test substance and the skin samples were incubated in diffusion cells for 24 hours. The amounts of test substance and its acetylated metabolite absorbed into the receptor fluid and the disposition in the skin layers following a 24 -hour incubation period were determined.The total amount of 4-amino-2-hydroxytoluene considered absorbed (amounts of radioactivity measured in epidermis, dermis, heat separation water and receptor fluid) over 24 h from the standard hair dye formulation was 25.6 ± 11.8 μg/cm² (1.72 ± 0.789 % of the applied dose) when applied to skin for 60 minutes for the study which used 5.0% test item formulation and for the 1.5% formulation, the mean total absorption in 4 donors was 0.59 ± 0.28% (9.05 ± 4.29 μg/cm2).The mean total recovery of all 4 donors was 101.36 ± 1.47% (1548.69 ± 29.87 μg/cm2). (Mass WSM, 2011) (Koganti A, 2006)