Registration Dossier

Diss Factsheets

Administrative data

Link to relevant study record(s)

Referenceopen allclose all

Endpoint:
dermal absorption in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
05 June 2014 - 26 September 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 428 (Skin Absorption: In Vitro Method)
Version / remarks:
13 April 2004
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: OECD Guidance Document 28: Guidance Document For the Conduct of Skin Absorption Studies
Version / remarks:
5 March 2004
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EFSA Panel on Plant Protection Products and their Residues (PPR); Guidance on Dermal Absorption
Version / remarks:
EFSA Journal 2012; 10(4):2665
Deviations:
yes
Remarks:
according to the EFSA guidance, if standard deviations are > 25% of mean, the preferred approach should be adding the standard deviations to the mean values
GLP compliance:
yes
Specific details on test material used for the study:
RADIOLABELLING INFORMATION (if applicable)
Radiolabelled isomer 1: 2-(1,1-Dimethylpropyl)[carbonyls-14C]anthraquinone
- Radiochemical purity: 99.2%
- Specific activity: 59 mCi/mmol (211 μCi/mg), 2.18 GBq/mmol (7.78 MBq/mg)
- Locations of the label: carbonyl group

Radiolabelled isomer 2: 2-(1,2-Dimethylpropyl)[carbonyls-14C]anthraquinone
- Radiochemical purity: 99.7%
- Specific activity: 59 mCi/mmol (211 μCi/mg), 2.18 GBq/mmol (7.78 MBq/mg)
- Locations of the label: carbonyl group

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: radiolabelled samples: c.a. -20°C, cold sample: room temperature
- Solubility and stability of the test substance in the solvent/vehicle: soluble in diisobutyl carbinol (DIBC) and Caromax 20LN


TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
The test compound was applied to the skin at two concentrations, the solid pure substance as a mixture of the 2 isomers and formulation of the substance containing a mixture of the 2 isomers.
For the solid substance 498.4 mg of non-radiolabelled 2-amylanthraquinone (provided as a mixture of the of the 2 isomers) was added to 1.05 mg of [14C]-2-(1,2-dimethylpropyl)anthraquinone and 1.16 mg of [14C]-2-(1,1-dimethylpropyl)anthraquinone. The mixture was dissolved in acetonitrile and the solvent evaporated off to provide a solid racemic mixture at the desired specific activity. Following radiodilution the test compound was stored in a freezer at ca. 20ºC until the day of application to skin.
For the formulation 747.2 mg of non-radiolabelled 2-amylanthraquinone (provided as a mixture of the of the 2 isomers) was added to 1.32 mg of [14C]-2-(1,2-dimethylpropyl)anthraquinone and 1.76 mg of [14C]-2-(1,1-dimethylpropyl)anthraquinone. The mixture was dissolved in acetonitrile and the solvent evaporated off to provide a solid racemic mixture at the desired specific activity. Following radiodilution the test compound was stored in a freezer at ca. 20ºC until the day of application to skin. On the day of application to skin a 500.22 mg aliquot of the radiodiluted material was dissolved in 0.5044 g of Diisobutyl carbinol (DIBC) followed by 1.0024 g of Caromax 20LN.


Radiolabelling:
yes
Species:
other: in vitro human skin from 4 donors
Type of coverage:
open
Vehicle:
other: mixture of Diisobutyl carbinol (DIBC) and Caromax 20LN (0.5:1 w/w)
Duration of exposure:
6 hours, with 24 hours sampling period
Doses:
- Actual doses: 4 mg solid substance with 10 µL water added to mimic sweat, or 15 µL formulation, applied to 1.77 cm2 exposed skin
Details on in vitro test system (if applicable):
SKIN PREPARATION
- Source of skin: Dermatomed human skin (359 - 540 μm, obtained from Biopredic International)
- Type of skin: dermatomed
- Preparative technique: not specified
- Thickness of skin (in mm): 359 - 540 μm
- Membrane integrity check: yes
The skin was checked for physical damage prior to being mounted in the test system. The total weight of the cell was recorded, the receptor chamber filled with receptor fluid (((0.9% saline + 5% Bovine serum albumin (BSA) + 5% 2-hydroxypropyl-β-cyclodextrin in water (w/w/w/v)) and the weight recorded again. The system was allowed to warm to approximately 32 ± 2°C prior to application of 50 μL tritiated water (10.3 μCi/mL) onto the skin of each cell. Aliquots (2 x 50 μL) were removed from the receptor fluid chamber for analysis at 0, 0.5, 1 and 2 hours after application and replaced with an equal volume of receptor fluid. Each 50 μL aliquot was mixed with 2 mL Ultima Gold XR® (Perkin Elmer) scintillant, counted on a liquid scintillation counter and the rate of penetration determined from the counts. After the last sampling time the tritiated water was desorbed by rinsing with 0.9% saline. The skin was maintained for use by filling the receptor chamber with receptor fluid and the temperature generally kept at 32 ± 2°C. The skin sample was deemed acceptable if the rate of penetration through the sample was not greater than 2 μL/cm2/h.


PRINCIPLES OF ASSAY
- Diffusion cell: no
- Receptor fluid: 0.9% saline + 5% Bovine serum albumin (BSA) + 5% 2-hydroxypropyl-β-cyclodextrin in water (w/w/w/v)
- Solubility of test substance in receptor fluid: yes
Prior to conducting the main in vitro dermal penetration experiment the solubility of 2-amylanthraquinone in selected receptor fluids was assessed. The receptor fluids were spiked with [14C]-2-amylanthraquinone at a target concentration of 0.2 mg/mL and aliquots taken for liquid scintillation counting to determine the concentration.
- Static system: yes, Franz cells with a nominal 12 ml receptor chamber volume and 1.77 cm2 exposed skin area for dosing
- Flow-through system: no
- Test temperature: Receptor fluid temperature was recorded at each sampling time point and the temperature was maintained at 31.1 – 31.9°C
- Occlusion: no
- Reference substance: yes, radiolabelled testosterone at ca. 0.1 mg/cm2

Samples were taken from the receptor fluid at 8 sampling points over a 24 hour period:
0, 0.5, 1, 2, 4, 6 (taken prior to removal of the dose formulation), 8 and 24 hours after application.
Duplicate aliquots (2 x 50 μl) were removed for sampling and replaced with an equal volume of receptor fluid (0.9% saline + 5% Bovine serum albumin (BSA) + 5% 2-hydroxypropyl-β-cyclodextrin in water w/w/w/v).
Absorption in different matrices:
- Cell wash: mean 0.58% (SD = 0.73) (solid), 26.52** (SD = 8.70) (formulation)
**The atypically high recovery of radioactivity in the cell wash is believed to be formulation/radiolabelled material remaining in the upper portion of the Franz cell (donor chamber). As the concentrations of radioactivity in the receptor fluid were low it is unlikely this is absorbed material.
- Skin wash: mean 95.66% (SD = 1.98) (solid), 18.73% (SD = 6.50) (formulation)
- Skin test site: mean 0.01% (SD 0.2) (solid), 3.35% (SD 5.12) (formulation)
- Receptor fluid, receptor chamber, donor chamber (in vitro test system): mean: below or near the limit of detection (< 0.01%) (solid), 0.14% (SD = 0.07) (formulation)
- Stratum corneum (in vitro test system): (tape strips 3-20): solid: 0.57%, formulation: 37.33% (both values calculated according to the EFSA guidance by adding the standard deviations to mean values in case SD is ≥ 25% of mean))
Total recovery:
- Total recovery: mean 97.40% (SD = 1.78) (solid), 96.17% (SD = 2.32) (formulation)
- Recovery of applied dose acceptable: yes, above 95%
- Results adjusted for incomplete recovery of the applied dose: not applicable
- Quantification of values below LOD or LOQ: radioactivity with less than twice background counts were considered to be below the limit of accurate quantification.
Key result
Time point:
24 h
Dose:
Solid substance
Parameter:
percentage
Remarks:
potentially absorbed (receptor fluid, skin, stratum corneum (strips 3-20)
Absorption:
0.25 %
Remarks on result:
other:
Remarks:
As reported in the study report
Key result
Time point:
24 h
Dose:
33.2% in formulation (calculated value)
Parameter:
percentage
Remarks:
potentially absorbed (receptor fluid, skin, stratum corneum (strips 3-20)
Absorption:
24.82 %
Remarks on result:
other:
Remarks:
As reported in the study report
Key result
Time point:
24 h
Dose:
Solid substance
Parameter:
percentage
Remarks:
Potentially absorbed (receptor fluid, skin, stratum corneum (tape strips 3-20))
Absorption:
1 %
Remarks on result:
other:
Remarks:
calculated according to the EFSA guidance, by adding standard deviations to mean values in case SD is ≥ 25% of mean and rounding the value up to one significant figure.
Key result
Time point:
24 h
Dose:
33.2% in formulation (calculated value)
Parameter:
percentage
Remarks:
potentially absorbed (receptor fluid, skin, stratum corneum (tapes 3-20)
Absorption:
51 %
Remarks on result:
other:
Remarks:
calculated according to the EFSA guidance, by adding standard deviations to mean values in case SD is ≥ 25% of mean, and rounding the value to two significant figures.

The solubility of [14C]-2-amylanthraquinone in the selected receptor fluid 0.9% saline + 5% Bovine serum albumin (BSA) + 5% 2-hydroxypropyl-β-cyclodextrin in water (w/w/w/v) was determined. The percentage variation in concentration before and after centrifugation was less than or equal to 7.1% and therefore the solubility of [14C]-2-amylanthraquinone in the receptor fluid was considered acceptable.

To determine the skin preparation integrity tritiated water was applied to the test vessels as described in section 2.3.2. Skin samples were deemed viable if the rate of penetration was not greater than 2 μL/cm2/h. The observed rate of penetration was in the range of 0.460 to 1.82 μL/cm2/h.

During this study the reference standard ([14C]-testosterone) produced a mean penetration rate of 0.0187 μg/cm2/h. Radioactivity recovered was greater than 97%. These results indicate the test system was functional throughout the duration of the study.

Conclusions:
Dermal absoprtion of 2-amylanthraquinone in the form of a solid substance and a 33.2% formulation was studied in an in vitro dermal absortion test. The potentially absorbed amounts were calculated to be 1% and 51% for a solid substance and a formulation, respectively.
Executive summary:

In a GLP-compliant OECD Guideline 428 study, dermal penetration of 2 -amylanthraquinone was studied in an in vitro dermal absorption test using dermatomized human skin from 4 volunteers. The substance was applied undiluted (with 10 µL water added to mimic sweat), or as a formulation in a mixture of Diisobutyl carbinol (DIBC) and Caromax 20LN (0.5:1 w/w) (calculated concentration 33.2% w/w). The experiment was conducted in Franz cells, representing a static cell design, with a nominal 12 mL receptor chamber volume and 1.77 cm2 exposed skin area for dosing, with each skin sample tested in duplicate. Integrity of the human skin used in each test vessel was demonstrated to be suitable. Functionality of the test system was demonstrated with a  reference compound. After 6 hours exposure the skin was washed, and dermal penetration was determined after 24 hours sampling time. Skin fractionation was performed by tape stripping. The total radioactivity recovery was above 95% in both experiments. The potentially absorbed doses (based on the radioactivity in the receptor fluid, skin and stratum corneum (tapes 3 -20)) were reported to be 0.25% for the solid substance and 24.82% for the formulation. However, as standard deviations in most cases exceeded the 25% of the mean values, the dermal absorption values were recalculated by adding standard deviations to the mean values in accordance with the EFSA Guidance on dermal absorption (2012). This resulted in the potentially absorbed doses of 0.6% for the solid substance and 51.35% for the formulation, which were rounded up to 1 and 2 significant figures, respectively, in accordance with the EFSA Guidance on dermal absorption (2012). This resulted in dermal absoprtion values of 1% for pure substance and 51% for the formulation.

Endpoint:
basic toxicokinetics, other
Type of information:
other: theoretical approach
Adequacy of study:
key study
Study period:
27-01-2011
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study is based on expert judgment.
Principles of method if other than guideline:
A theoretical approach of the toxicokinetic properties of the substance based on the available physico-chemical properties and toxicological data.
GLP compliance:
no
Type:
absorption
Results:
Oral and dermal: 50%; inhalation: 100%.
Conclusions:
Interpretation of results (migrated information): bioaccumulation potential cannot be judged based on study results
For risk assessment purposes the following absorption factors were derived:
oral absorption factor: 50%
inhalation absorption factor: 100%
Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23 February 2016-28 June 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to same study
Objective of study:
metabolism
Qualifier:
according to guideline
Guideline:
other:
Version / remarks:
Study performed as a part of a 90-day repeated dose toxicity study, conducted according to OECD Guideline 408 (May 2008)
Deviations:
no
Principles of method if other than guideline:
The metabolite identification was performed as part of a 90-day repeated dose toxicity study with male and female Wistar rats, exposed to 0 (concurrent vehicle controls), 7.5, 25 and 75 mg 2-amylanthraquinone/kg bw/day. The metabolites were identified in the pooled urine samples collected overnight at the end of the study from 5 control and 5 high-dose (75 mg/kg bw/day) rats using LC-PDA-MS method, with detection in the positive ionization mode. More than 60 m/z values of possible metabolites were detected in the pooled urine samples of high-dose males. The identification of such large number of metabolites was not withint the scope of the study and therefore only major metabolites, based on the UV signal, were selected for identification purposes.
GLP compliance:
yes
Remarks:
However, analytical part of metabolite identification in urine was not performed under GLP.
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature protected from light
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: Stability for at least 24 hours at room temperature and for at least 3 weeks in freezer is confirmed over the concentration range 1 to 200 mg/mL.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: On some occasions it was considered necessary to heat the test item prior to weighing to a maximum temperature of 91.1°C for maximally 82 minutes.
Radiolabelling:
no
Species:
rat
Strain:
Wistar
Remarks:
Crl:(WI) BR
Details on species / strain selection:
Recognized by international guidelines as the recommended test system for repeated dose toxicity studies (e.g. EPA, FDA, OECD and EC).
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: not applicable
- Age at study initiation: ca. 6 weeks
- Weight at study initiation: males/females, mean (SD): controls 144 (10.0)/128 (6.3), low dose 142 (8.3)/128 (7.2), mid-dose 144 (5.8)/126 (4.9), high dose 143 (7.0)/127 (5.5)
- Fasting period before study: no
- Housing: Group housing of 5 animals per sex in Macrolon cages (MIV type, height 18 cm) with sterilized sawdust as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) and paper as cage-enrichment
(Enviro-dri, Wm. Lilico & Son (Wonham Mill Ltd), Surrey, United Kingdom). During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage (Ancare corp., USA; dimensions: 48.3 x 26.7 x 20.3 cm) without cage-enrichment, bedding material, food and water.
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany). During motor activity measurements, animals did not have access to food for a maximum of 2 hours.
- Water: Free access to tap water
- Acclimation period: at least 5 days

DETAILS OF FOOD AND WATER QUALITY:
Diet and water evaluation for contaminants and/or nutrients was performed according to facility standard procedures. There were no findings that could interfere with the study.

ENVIRONMENTAL CONDITIONS (set to maintain)
- Temperature (°C): 18-24
- Humidity (%): 40-70
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 23 February 2016 To: 28 June 2016
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Days 1-14: Formulations (w/w) were prepared daily within 6 hours prior to dosing, and were homogenized to visually acceptable levels.
Days 15-92: Formulations (w/w) were prepared daily within 3 weeks prior to dosing and were homogenized to a visually acceptable level.

Formulations were heated to a maximum temperature of 65°C for maximally 2 hours to obtain visual homogeneity. Formulations were released for dosing when they obtained a temperature of 40°C or lower. Adjustment was made for specific gravity/density of the test item and vehicle. No correction was made for the purity/composition of the test item.
On some occasions it was considered necessary to heat the test item prior to weighing to a maximum temperature of 91.1°C for maximally 82 minutes. Formulations for all dosing days were be heated to a maximum temperature of 47.5°C for maximally
38 minutes to obtain visual homogeneity. Formulations were released for dosing when they obtained a temperature of 40°C or lower. Adjustment was made for specific gravity/density of the test item and vehicle. No correction was made for the
purity/composition of the test item.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at Charles River Den Bosch and on information from the sponsor.
- Concentration in vehicle: 1.5, 5 and 15 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg bw
Duration and frequency of treatment / exposure:
Once daily, 7 days/week, for 13 weeks
Dose / conc.:
0 mg/kg bw/day
Remarks:
vehicle controls
Dose / conc.:
75 mg/kg bw/day
No. of animals per sex per dose / concentration:
5 males/dose (for urine sampling for (non-GLP) metabolite investigation
Control animals:
yes, concurrent vehicle
Positive control reference chemical:
Not used.
Details on study design:
- Dose selection rationale:
Details on dosing and sampling:
METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled: urine. Urine was collected into a specimen vial, using a metabolism cage. After sampling the urine was pooled per dose level and stored at -80 °C until further analysis. Constant fractions of urine from the males per dose group were combined and homogenized in order to obtain one urine sample per dose (control and high-dose group).
The urine sample of the high-dose group 4 and the (blank) urine sample of concurrent vehicle controls were homogenized and centrifuged for 5 min at 16000 g at room temperature. For analysis, the urine samples were injected undiluted.
- Time and frequency of sampling: once overnight (approximately 15-20 hrs) at the end of treatment
- From how many animals: first 5 control males and first 5 high-dose males, pooled. During the sampling period animals were deprived of food but water was provided.
- Method type for identification: LC-PDA-MS, with detection in the positive ionization mode. The test substance is known to be a mixture, with 2-(1,1-dimethylpropyl)anthraquinone and 2-(1,2-dimethylpropyl)anthraquinone being the major components (together 92%) and therefore two major peaks were observed in the chromatograms. More than 60 m/z values of possible metabolites were detected in the pooled urine samples of high-dose males. The identification of such large number of metabolites was not withint the scope of the study and therefore only major metabolites, based on the UV signal, were selected for identification purposes.

- Other: Structural identification:
Using PDA chromatograms and accurate MS data, the investigation of metabolites in the sample was evaluated according to the following approach:
1. The spectra obtained from the pooled urine (high-dose group) sample were screened for m/z values that were absent in the blank urine sample.
2. For identification purposes, additional MSn analyses were performed if necessary to generate MS2 and MS3 spectra.
3. The spectra obtained were interpreted to determine the molecular weight and (partial) identity of the major metabolites.
For screening, software MetWorks 1.2.0 (Thermo Fisher Scientific Inc.) was used.

MS detection:
LTQ Orbitrap mass spectrometer: electrospray probe position x = 0.075 inch, y = 0, z = C; capillary temperature 400°C. The MS was operated in positive ion mode with 2 scan events. Mass accuracy was determined from external calibration.
Scan event:
(1) Full FTMS scan m/z 100-1000 at resolution of 30000 (at m/z 400).
(2) Data dependent MS2 of the 3 most intense ions at unit resolution.
Additional analysis to generate MSn were performed using:
Scan event:
(1) Full FTMS scan m/z 100-1000 at resolution of 15000 (at m/z 400).
(2) Data dependent MS2 at resolution of 7500 (at m/z 400)
(3) Data dependent MS3 at resolution of 7500 (at m/z 400)
For MS2 and MS3 a normalized collision energy of 25% and 35%, respectively, with an isolation width of 1.0 were used. A tune file optimized for test item 2-amylanthraquinone was used.

TREATMENT FOR CLEAVAGE OF CONJUGATES: not applicable
Statistics:
Not used in the metabolite determination
Preliminary studies:
Not applicable
Type:
metabolism
Results:
Major metabolites were result of combinations of one or two oxidations with ketone formation, sulfonation and glucuronice acid conjugation
Details on absorption:
Not applicable
Details on distribution in tissues:
Not applicable
Details on excretion:
Not applicable
Metabolites identified:
yes
Details on metabolites:
An overview of some of the major metabolites detected in the pooled urine sample of high-dose males is presented in the section "Any other information on results incl. tables". These metabolites are the result of combinations of one or two
oxidations with ketone formation, sulfonation and glucuronic acid conjugation. In addition, the parent compound 2-amylanthraquinone was detected in the pooled urine sample but at very low level.
Bioaccessibility (or Bioavailability) testing results:
Not applicable.

Table 1. Characterized metabolites of 2 -amylanthraquinone detected in the pooled urine sample of high dose males.

m/z of [M+H]+ion

Retention times1(min)

Mass shift2(Da)

Possible metabolic reaction

279.138

22.2

22.4

0

= 2-amylanthraquinone = 2-(1,1-dimethylpropyl)anthraquinone+ 2-(1,2-dimethylpropyl)anthraquinone

309.112

13.0

13.7

15.7

16.1

+29.974

Hydroxylation and ketone on aliphatic chain

325.107

12.1

14.0

14.5

+45.969

Two hydroxylations and ketone on aliphatic chain

375.090

24.7

+95.952

Hydroxylation and sulfonation on aliphatic chain

471.165

11.9

+192.027

Hydroxylation and glucuronic acid conjugation on aliphatic chain

1) Retention time in MS chromatograms

2) Mass shift compared to parent compound

Conclusions:
A number of metabolites were identified in the pooled urine samples of male rats who were exposed for 13 weeks to 75 mg/kg bw/day 2-amylanthraquinone. These metabolites are the result of combinations of one or two oxidations with ketone formation, sulfonation and glucuronic acid conjugation. In addition, the parent compound 2-amylanthraquinone was detected in the pooled urine sample but at very low level.
Executive summary:

The metabolite identification in the pooled urine samples from high-dose males was conducted as a part of a GLP-compliant OECD guideline 408 study with Wistar rats. In the study, groups of male and female rats received 0 (concurrent vehicle controls), 7.5, 25 and 75 mg/kg bw/day test substance once a day, 7 days/week for 13 weeks. The urine samples for metabolite identification were collected at the end of the study overnight in metabolite cages from the first five control and high-dose male rats. An LC-PDA-MS method was developed for the analysis of the samples, with MS detection in the positive ionization mode. The accurate mass spectral data were screened for m/z values of possible metabolites. More than 60 m/z values were detected in the pooled urine samples, and only major metabolites, based on the UV signal, were selected for identification purposes. A number of major metabolites were identified, which were the the result of combinations of one or two oxidations of the aliphatic chain with ketone formation, sulfonation and glucuronic acid conjugation. In addition, the parent compound 2-amylanthraquinone was detected in the pooled urine sample but at very low level. This indicates that the parental substance is readily metabolized, undergoing primarily the oxidation of the aliphatic chain, followed by subsequent sulphonation and/or glucuronic acid conjugation.

Description of key information

Based on Toxicokinetic Assessment report according to Guidance for the implementation of REACH. Guidance on information requirements and chemical safety assessment. Chapter R.7c: Endpoint specific guidance. European Chemical Agency, May 2008, absorption factors for oral and respiratory routes of exposure have been derived.

Dermal absorption values of 1% for pure substance and 50% for the substance in a formulation (calculated concentration 33.2% w/w) were derived from a reliable in vitro dermal absorption study.

Key value for chemical safety assessment

Absorption rate - oral (%):
50
Absorption rate - dermal (%):
1
Absorption rate - inhalation (%):
100

Additional information

The water solubility of 2-amylanthraquinone is very low (0.15 mg/L). Since in general a compound needs to be dissolved before it can be taken up from the gastro-intestinal tract, it is unlikely that 2-amylanthraquinone will show a high systemic exposure after oral administration. However, its lipophilic character (log Pow of 5.9 and 6.1 of the major compounds and log Pow of 3.6 of the minor component) indicates that uptake by micellular solubilisation may be of particular importance. For risk assessment purposes oral absorption of 2-amylanthraquinone is set at 50%. The results of the toxicity studies do not provide reasons to deviate from this proposed oral absorption factor.

 

Once absorbed, distribution of the test substance throughout the body will be limited due to its low water solubility. Based on its lipophilic character, intracellular concentration is expected to be higher than extracellular concentration. Absorbed 2-amylanthraquinone might undergo conjugation. The biotransformation products will either be excreted via the bile (high molecular weight compounds) or the urine (low molecular weight compounds).

The identification of main metabolites of 2 -amylanthraquinone in urine was performed as a part of a 90 -day repeated dose toxicity study. The major metabolites were identified using LC-PDA-MSA analysis with positive ionization mode in pooled urine samples, collected overnight at the end of the study from 5 high-dose males that were exposed to 75 mg/kg bw/day test substance by oral gavage daily for 13 weeks. More than 60 m/z values of possible metabolites were detected in the urine sample, and the major metabolites based on the UV signal were selected for identification purposes. These metabolites were the result of combinations of one or two oxidations with ketone formation, sulfonation and glucuronic acid conjugation. In addition, the parent compound 2-amylanthraquinone was detected in the pooled urine sample but at very low level. This indicates that 2 -amylanthraquinone is readily metabolized in the body, undergoing primarily an oxidation of the aliphatic chain, followed by subsequent sulfonation and glucuronic acid conjugation, followed by excretion in urine.

 

Due to the high boiling point (423°C) and low vapour pressure (2.24 x 10-4Pa) it is not to be expected that 2-amylanthraquinone will reach the nasopharyncheal region or subsequently the tracheobronchial or pulmonary region. However, if 2-amylanthraquinone reaches the tracheobronchial region, uptake by means of micelluar solubilisation may be of particular importance, due to the highly lipophilic properties of this substance, as indicated by the log Pow >4 of the main components and poor water solubility (less than 1 mg/L, namely 0.15 mg/L). Overall, although it is unlikely that 2-amylanthraquinone will be available to a high extent after inhalation via the lungs due to the low vapour pressure, for risk assessment purposes the inhalation absorption of 2-amylanthraquinone is set at 100%.

 

Dermal penetration of 2 -amylanthraquinone was studied in a GLP-compliant guideline in vitro dermal absorption test using dermatomized human skin from 4 volunteers. The substance was applied undiluted (with 10 µL water added to mimic sweat), or as a formulation in a mixture of Diisobutyl carbinol (DIBC) and Caromax 20LN (0.5:1 w/w) (calculated concentration 33.2% w/w). After 6 hours exposure the skin was washed, and dermal penetration was determined after 24 hours sampling time. Skin fractionation was performed by tape stripping. The total radioactivity recovery was above 95% in both experiments. The potentially absorbed doses (based on the radioactivity in the receptor fluid, skin and stratum corneum (tapes 3 -20)) were reported to be 0.25% for the substance and 24.82% for the substance in the formulation. However, as standard deviations in most cases exceeded the 25% of the mean values, the dermal absorption values were recalculated by adding standard deviations to the mean values in accordance with the EFSA Guidance on dermal absorption (2012). This resulted in the potentially absorbed doses of 1% for the substance and 51% for the substance in the formulation (rounded up to 1 and 2 significant values in accordance with the EFSA Guidance on dermal absorption (2012)). However, as dermal absorption normally does not exceed oral absorption, dermal absorption value of 50% for the absorption of the substance from formulation can be set for 2 -amylanthraquinone for risk assessment purposes.

Based on the present available data, no additional conclusions can be drawn on the distribution, metabolism and excretion of 2-amylanthraquinone after dermal absorption.