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Endpoint:
dermal absorption in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Remarks:
The study was conducted according to internationally accepted protocols and guidelines and followed good laboratory practice standards.
Qualifier:
according to guideline
Guideline:
other: SCCNFP in vitro assessment of percutaneous absorption guidelines (SCCNFP, 2003)
Principles of method if other than guideline:
Reference: SCCNFP (2003). The Scientific Committee on Cosmetic Products and Non-Food Products Intended for Consumers. Basic criteria for the in vitro assessment of dermal absorption of cosmetic ingredients, SCCNFP/0750/03. SCCNFP, Brussels.
GLP compliance:
yes (incl. QA statement)
Radiolabelling:
no
Species:
human
Strain:
other: not applicable
Sex:
female
Details on test animals or test system and environmental conditions:
Full-thickness human female abdominal skin was obtained from cosmetic surgery and stored at -20 °C, and was subsequently thawed at room temperature for processing. Subcutaneous fat was removed by blunt dissection and individual portions of skin were immersed in 60 °C water for 60 seconds. The epidermis, comprised of the stratum corneum and epidermis, was then gently removed from the underlying dermis. The latter was discarded and the epidermal membrane floated onto the surface of water and taken up onto aluminum foil. The membranes were thoroughly dried and stored at -20 °C until used.

On the day of use, epidermal membranes were floated onto water from the aluminum foil and placed onto 20 mm diameter filter paper supports. The membranes were then mounted onto diffusion cells. Four donors were used. All samples were abdominal skin from Caucasian females aged 49 to 65 years.
Type of coverage:
open
Vehicle:
other: 70/30% (v/v) ethanol/water
Duration of exposure:
24 hours
Doses:
A 6% solution of dimyrcetol (70/30% ethanol/water, v/v) was applied to each skin surface at a dose of 5 µL/cm^2.
No. of animals per group:
Skin samples from 4 donors was used
Control animals:
no
Details on study design:
DOSE PREPARATION
- Method for preparation of dose solutions: Dimyrcetol (0.59923 g) was directly weighed into a tared 10 mL volumetric flask and made to volume with premixed 70/30 % (v/v) ethanol/water. The mixture was tightly capped and mixed.

APPLICATION OF DOSE/REFERENCE COMPOUND
The dimyrcetol solution was applied using a Gilson M10 Microman positive displacement pipette. Twelve replicate skin samples were dosed with the dimyrcetol and 3 replicates received blank vehicle (with dimyrcetol). The diffusion cell donor chambers were not covered to mimic in-use (unoccluded) conditions.

The positive control, 0.4% benzoic acid (described below), was applied at 25 µL/cm^2 and the diffusion cell donor chambers immediately occluded with greased coverslips. The dose was equivalent to 100 µg benzoic acid/cm^2.

VEHICLE
- Amounts applied: 5 µL/cm^2. The dose applied was equivalent to 296.2 µg/cm^2 dimyrcetol comprising 127.4 µg/cm^2 dimyrcetol A and 168.8 µg/cm^2 dimyrcetol B.
- Concentration: 6%

TEST SITE
- Preparation of test site: As described above
- Area of exposure: Approximately 1.2 cm^2, with exact areas measured for each individual cell

SAMPLE COLLECTION
200 µL samples were taken from each receptor chamber for dimyrcetol-dosed cells at 0.5, 1, 2, 3, 4, 6, 8, 10, 12, 16, 20 and 24 hours after dosing. The liquid removed by each sample was replaced with fresh temperature equilibrated blank receptor medium.

SAMPLE PREPARATION
- Storage procedure: Each sample was placed into a 200 µL glass vial (gold grade, Chromacol) and a cap with PTFE-lined septum applied prior to freezing the vial at -20 °C pending analysis.

ANALYSIS
- Method: Samples were analysed by GC-MS. Separations were performed on a RTX-5MS, 0.25 mm ID, 0.25 µm film thickness, 15 m capillary column (Thames Restek, UK, Saunderton, UK). The injection volume was 1 µL (splitless). The over program was as follows: initial, 40 °C (1.2 minute hold), ramp 1; 14 °C/min to 115 °C, ramp 2; 50 °C/min to 250 °C (4 minute hold). Quantitative detection was by selective ion monitoring (SIM) on a DSQ dual stage quadrapole II: dimyrcetol A, 59 m/z; dimyrcetol B, 95 m/z.
- Limits of detection and quantification: 0.0085 and 0.0113 µg/mL for dimyrcetol A and B, respectively, in ethanol/phosphate buffered saline; 0.0127 and 0.0169 µg/mL for dimyrcetol A and B, respectively, in acetonitrile. Six level quadratic calibrations (0.02 to 4 µg/mL) were prepared. Injection reproducibility was assessed using five replicate injections at three calibration levels for each injection solvent.

MEMBRANE INTEGRITY ASSESSMENT
The integrity of each membrane was assessed prior to application of either test solutions of dimyrcetol or benzoic acid. Isotonic saline (0.9%, 1 mL) was applied to the skin surface and electrical resistance of each mounted membrane measured 10 minutes later using a Tinsley 1604 LCR databridge at 100 Hz, using the serial equivalent circuit mode. Skin surfaces were subsequently washed three times with water, carefully dried and receptor chambers emptied prior to refilling with EPBS. The cells were then allowed to re-equilibrate to the correct temperature and to allow normal hydration to be achieved.

DETERMINATION OF SKIN DISTRIBUTION
After 24 hours, excess dimyrcetol solution was removed from the diffusion cell donor chambers and placed into tightly capped glass vials for subsequent analysis. The skin samples were removed from the diffusion cells and placed onto small discs of plastic held in place with cyanoacrylate glue. Remaining test material on the skin samples was removed by gently wiping with dry cotton buds and these placed into 20 mL vials with 2 mL of acetonitrile. Each membrane sample was tape stripped 10 times using adhesive tape (D-Squame, CuDerm Inc., Dallas, TX USA). The tape strips were grouped by placing into the same vial as follows: strip 1, strips 2-3, strips 4-6 and strips 7-10. Acetonitrile (2 mL) was added to each group. Diffusion cell chambers were wiped with cotton buds and these placed into 20 mL vials. Donor chambers were then washed with 15 mL of acetonitrile to recover any condensed test material. All vials were capped and stored at -20 °C pending analysis.

ASSESSMENT OF EVAPORATIVE LOSSES
The potential loss due to evaporation was determined by assembling 5 diffusion cells and replacing skin membranes with thin PTFE sheeting. Receptor chambers were filled with water. Diffusion cells were immersed in the constant temperature water bath maintained at 37 °C throughout the experiment. The 6% dimyrcetol solution was applied to the PFTE membranes in the same manner as with skin membranes. One PTFE diffusion cell was dismantled at 1, 2, 6, 12 and 24 hours. During the dismantling, PTFE sheets were removed and washed with 10 mL, then 5 mL of acetonitrile. The washings were combined in 20 mL vials. The diffusion chambers were washed with 15 mL of acetonitrile and the solution transferred to 20 mL vials. Vials were stored at -20 °C pending analysis.

DATA CALCULATIONS
Data calculations were performed for each dimyrcetol component (A and B) and also for the combined. According to the SCCNFP guidelines, levels of dimyrcetol in the epidermis, plus any remaining stratum corneum after tape stripping, filter paper supports and receptor fluids were combined to produce a total absorbed dose.
Details on in vitro test system (if applicable):
PRINCIPLES OF ASSAY
- Diffusion cell: Horizontal, Franz-type
- Receptor fluid: Phosphate buffered saline (PBS, pH 7.4). 25/75% (v/v) ethanol/PBS (EPBS) was prepared by mixing 62.5 mL ethanol and 187.5 mL PBS in a 250 mL flask.
- Solubility of test substance in receptor fluid: The solubility of dimyrcetol in EPBS was found to be 342-400 µg/mL prior to performing the skin penetration studies.
- Static system: Yes
- Test temperature: 32 +/- 1 °C
- Occlusion: No
- Reference substance: Benzoic acid (min. 99.5%) obtained from Sigma (Poole, UK). Prepared at 4 mg/mL in 50/50% (v/v) ethanol/water and applied to skin samples in the same manner as the test substance. Diffusion cell donor chambers were immediately occluded using greased coverslips.
Signs and symptoms of toxicity:
not examined
Dermal irritation:
not examined
Total recovery:
See table 2.
Key result
Time point:
24 h
Dose:
6% solution
Parameter:
percentage
Absorption:
2.5 %
Remarks on result:
other:
Remarks:
Dimyrcetol A (2,6-dimethyloct-7-en-2-yl), applied dose: 127 µg/cm^2
Key result
Time point:
24 h
Dose:
6% solution
Parameter:
percentage
Absorption:
0.662 %
Remarks on result:
other:
Remarks:
Dimyrcetol B (2,6-dimethyloct-7-en-2-yl formate ester), applied dose: 169 µg/cm^2
Key result
Time point:
24 h
Dose:
6% solution
Parameter:
percentage
Absorption:
1.45 %
Remarks on result:
other:
Remarks:
Dimyrcetol, combined

Table 2: Skin absorption of dimyrcetol (% of applied dose, mean±SE, n=12) at 24 hours


 














































































Compartment



dimyrcetol A,


dose: 127 µg/cm2



dimyrcetol B,


dose: 169 µg/cm2



combined,


dose: 296 µg/cm2



 



 



 



 



Wipe



0.166±0.013



0.096±0.007



0.126±0.010



Donor chamber



0.333±0.029



0.891±0.276



0.651±0.163



Strip 1



0.065±0.015



0.031±0.006



0.045±0.010



Strips 2-3



0.069±0.011



0.033±0.004



0.048±0.007



Strips 4-6



0.034±0.004



0.015±0.002



0.023±0.003



Strips 7-10



0.014±0.003



0.011±0.002



0.012±0.002



Epidermis



0.066±0.017



0.028±0.008



0.045±0.012



Filter paper



0.068±0.006



0.138±0.017



0.108±0.013



Permeated



2.36±0.19



0.496±0.034



1.30±0.10



Overall recovery



3.18±0.21



1.74±0.29



2.36±0.24



 


Stability of dimyrcetol solutions:


 


Dimyrcetol A appeared to permeate to a greater degree than dimyrcetol B. Approximately 20% of dimyrcetol B was observed to hydrolyze to dimyrcetol A in 24 hours from a solution containing 4 µg/mL (determined in a separate experiment). The rate of hydrolysis was not sufficient to fully explain the differences in permeation values.


 


Evaporative losses:


Very rapid evaporative losses were recorded, with <10% recovered for both components after 1 hour form diffusion cells equipped with PTFE discs. No dimyrcetol was detectable in these cells after 6 hours. Dimyrcetol B evaporated more rapidly than dimyrcetol A (8.97% recovery at 1 hour), but neither component was detectable on PTFE surfaces at 2 hours.


 


Benzoic acid permeation:


The reference compound, benzoic acid, displayed a mean maximal absorption rate of approximately 29.8 µg/cm^2/h. This measured value is within the range of measurements reported in a multi-centre study (Van de Sandt et al., 2004).


 

Conclusions:
The total absorbed dose of dimyrcetol was 2.50 +/- 0.20% and 0.662 +/- 0.040% for components A and B, respectively, and a combined total of 1.45 +/- 0.10%. Overall recoveries of dimyrcetol were low with values 3.18 +/- 0.21%, 1.74 +/- 0.29% and 2.36 +/- 0.24% of the applied dose for dimyrcetol A, B and combined, respectively. Evaporative losses were rapid from the application surfaces with <10% recovered from PTFE surfaces after 1 hour. Evaporative losses explain the low recoveries reported.
Executive summary:

The in vitro percutaneous absorption of dimyrcetol under in-use (unoccluded) conditions at an application concentration of 6% (w/v) in 70/30% (v/v) ethanol/water was determined in the current study. The absorption of each component, dimyrcetol A (2,6-dimethyl-7-octen-2-ol) and dimyrcetol B (2,6-dimethyl-7-octen-2-ol formate ester) were separately measured and combined for total dimyrcetol absorption.


 


Component A was observed to more rapidly permeate human skin than component B. In addition, component B was found to more rapidly evaporate than component A. Hydrolysis of dimyrcetol B to A was also an issue, and may have contributed to the overall observed absorption values.


 


The levels of dimyrcetol in the epidermis (with any remaining in stratum corneum after tape stripping), filter paper membrane supports and receptor fluids combined gave total absorbed doses of 2.50 +/- 0.20% and 0.662 +/- 0.040% for components A and B, respectively, and a combined value of 1.45 +/- 0.10%. Overall recoveries of dimyrcetol were low with values 3.18 +/- 0.21%, 1.74 +/- 0.29% and 2.36 +/- 0.24% of the applied dose for dimyrcetol A, B and combined, respectively. Evaporative losses were responsible for the low recoveries recorded.

Endpoint:
dermal absorption in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Remarks:
The study was conducted according to internationally accepted protocols and guidelines and followed good laboratory practice standards.
Qualifier:
according to guideline
Guideline:
other: SCCNFP in vitro assessment of percutaneous absorption guidelines (SCCNFP, 2003)
Principles of method if other than guideline:
Reference: SCCNFP (2003). The Scientific Committee on Cosmetic Products and Non-Food Products Intended for Consumers. Basic criteria for the in vitro assessment of dermal absorption of cosmetic ingredients, SCCNFP/0750/03. SCCNFP, Brussels.
GLP compliance:
yes (incl. QA statement)
Radiolabelling:
no
Species:
human
Strain:
other: not applicable
Sex:
female
Details on test animals or test system and environmental conditions:
Full-thickness human female abdominal skin was obtained from cosmetic surgery and stored at -20 °C, and was subsequently thawed at room temperature for processing. Subcutaneous fat was removed by blunt dissection and individual portions of skin were immersed in 60 °C water for 60 seconds. The epidermis, comprised of the stratum corneum and epidermis, was then gently removed from the underlying dermis. The latter was discarded and the epidermal membrane floated onto the surface of water and taken up onto aluminum foil. The membranes were thoroughly dried and stored at -20 °C until used.

On the day of use, epidermal membranes were floated onto water from the aluminum foil and placed onto 20 mm diameter filter paper supports. The membranes were then mounted onto diffusion cells. Four donors were used. All samples were abdominal skin from Caucasian females aged 49 to 65 years.
Type of coverage:
occlusive
Vehicle:
other: 70/30% (v/v) ethanol/water
Duration of exposure:
24 hours
Doses:
A 6% solution of dimyrcetol (70/30% ethanol/water, v/v) was applied to each skin surface at a dose of 5 µL/cm^2. Donor chambers were occluded using greased (high vacuum grease, Dow Corning) glass coverslips immediately following application of the dose.
No. of animals per group:
Skin samples from 4 donors were used.
Control animals:
no
Details on study design:
DOSE PREPARATION
- Method for preparation of dose solutions: Dimyrcetol (0.59923 g) was directly weighed into a tared 10 mL volumetric flask and made to volume with premixed 70/30% (v/v) ethanol/water. The mixture was tightly capped and mixed.

APPLICATION OF DOSE/REFERENCE COMPOUND
The dimyrcetol solution was applied using a Gilson M10 Microman positive displacement pipette. Twelve replicate skin samples were dosed with the dimyrcetol and 3 replicates received blank vehicle (with dimyrcetol). The diffusion cell donor chambers were occluded using greased (high vacuum, Dow Corning) glass coverslips immediately following application of the dose.

The positive control, 0.4% benzoic acid (described below), was applied at 25 µL/cm^2 and the diffusion cell donor chambers immediately occluded with greased coverslips. The dose was equivalent to 100 µg benzoic acid/cm^2.

VEHICLE
- Amounts applied: 5 µL/cm^2. The dose applied was equivalent to 296.2 µg/cm^2 dimyrcetol comprising 127.4 µg/cm^2 dimyrcetol A and 168.8 µg/cm^2 dimyrcetol B.
- Concentration: 6%

TEST SITE
- Preparation of test site: As described above
- Area of exposure: Approximately 1.2 cm^2, with exact areas measured for each individual cell

SAMPLE COLLECTION
200 µL samples were taken from each receptor chamber for dimyrcetol-dosed cells at 0.5, 1, 2, 3, 4, 6, 8, 10, 12, 16, 20 and 24 hours after dosing. The liquid removed by each sample was replaced with fresh temperature equilibrated blank receptor medium.

SAMPLE PREPARATION
- Storage procedure: Each sample was placed into a 200 µL glass vial (gold grade, Chromacol) and a cap with PTFE-lined septum applied prior to freezing the vial at -20 °C pending analysis.

ANALYSIS
- Method: Samples were analysed by GC-MS. Separations were performed on a RTX-5MS, 0.25 mm ID, 0.25 µm film thickness, 15 m capillary column (Thames Restek, UK, Saunderton, UK). The injection volume was 1 µL (splitless). The over program was as follows: initial, 40 °C (1.2 minute hold), ramp 1; 14 °C/min to 115 °C, ramp 2; 50 °C/min to 250 °C (4 minute hold). Quantitative detection was by selective ion monitoring (SIM) on a DSQ dual stage quadrapole II: dimyrcetol A, 59 m/z; dimyrcetol B, 95 m/z.
- Limits of detection and quantification: 0.0085 and 0.0113 µg/mL for dimyrcetol A and B, respectively, in ethanol/phosphate buffered saline; 0.0127 and 0.0169 micrograms/mL for dimyrcetol A and B, respectively, in acetonitrile. Six level quadratic calibrations (0.02 to 4 µg/mL) were prepared. Injection reproducibility was assessed using five replicate injections at three calibration levels for each injection solvent.

MEMBRANE INTEGRITY ASSESSMENT
The integrity of each membrane was assessed prior to application of either test solutions of dimyrcetol or benzoic acid. Isotonic saline (0.9%, 1 mL) was applied to the skin surface and electrical resistance of each mounted membrane measured 10 minutes later using a Tinsley 1604 LCR databridge at 100 Hz, using the serial equivalent circuit mode. Skin surfaces were subsequently washed three times with water, carefully dried and receptor chambers emptied prior to refilling with EPBS. The cells were then allowed to re-equilibrate to the correct temperature and to allow normal hydration to be achieved.

DETERMINATION OF SKIN DISTRIBUTION
After 24 hours, excess dimyrcetol solution was removed from the diffusion cell donor chambers and placed into tightly capped glass vials for subsequent analysis. The skin samples were removed from the diffusion cells and placed onto small discs of plastic held in place with cyanoacrylate glue. Remaining test material on the skin samples was removed by gently wiping with dry cotton buds and these placed into 20 mL vials with 2 mL of acetonitrile. Each membrane sample was tape stripped 10 times using adhesive tape (D-Squame, CuDerm Inc., Dallas, TX USA). The tape strips were grouped by placing into the same vial as follows: strip 1, strips 2-3, strips 4-6 and strips 7-10. Acetonitrile (2 mL) was added to each group. Diffusion cell chambers were wiped with cotton buds to remove sealing grease and the wipes and coverslips placed into vials. Donor chambers were then washed with 15mL of acetonitrile to recover any condensed test material. The donor chamber wash was added to the vial containing the donor chamber wipes. All vials were capped and stored at -20 °C pending analysis.

ASSESSMENT OF EVAPORATIVE LOSSES
The potential loss due to evaporation was not determined in this study. Although sealed coverslips has proven a reliable method for prevention of volatile materials, some compounds may permeate through the sealing grease and be lost by evaporation. This has been observed in a similar studies conducted with linalool (R03/13a,b,c/05 and R03/15c/07).

DATA CALCULATIONS
Data calculations were performed for each dimyrcetol component (A and B) and also for the combined. According to the SCCNFP guidelines, levels of dimyrcetol in the epidermis, plus any remaining stratum corneum after tape stripping, filter paper supports and receptor fluids were combined to produce a total absorbed dose.
Details on in vitro test system (if applicable):
PRINCIPLES OF ASSAY
- Diffusion cell: Horizontal, Franz-type
- Receptor fluid: Phosphate buffered saline (PBS, pH 7.4). 25/75% (v/v) ethanol/PBS (EPBS) was prepared by mixing 62.5 mL ethanol and 187.5 mL PBS in a 250 mL flask.
- Solubility of test substance in receptor fluid: The solubility of dimyrcetol in EPBS was found to be 342-400 µg/mL prior to performing the skin penetration studies.
- Static system: Yes
- Test temperature: 32 +/- 1 °C
- Occlusion: Yes
- Reference substance: Benzoic acid (min. 99.5%) obtained from Sigma (Poole, UK). Prepared at 4 mg/mL in 50/50% (v/v) ethanol/water and applied to skin samples in the same manner as the test substance. Diffusion cell donor chambers were immediately occluded using greased coverslips.
Signs and symptoms of toxicity:
not examined
Dermal irritation:
not examined
Total recovery:
See table 2.
Key result
Time point:
24 h
Dose:
6% solution
Parameter:
percentage
Absorption:
10.4 %
Remarks on result:
other:
Remarks:
Dimyrcetol A (2,6-dimethyloct-7-en-2-yl), applied dose: 127 µg/cm^2
Key result
Time point:
24 h
Dose:
6% solution
Parameter:
percentage
Absorption:
2.13 %
Remarks on result:
other:
Remarks:
Dimyrcetol B (2,6-dimethyloct-7-en-2-yl formate ester), applied dose: 169 µg/cm^2
Key result
Time point:
24 h
Dose:
6% solution
Parameter:
percentage
Absorption:
5.67 %
Remarks on result:
other:
Remarks:
Dimyrcetol, combined

  Table 2: Skin absorption of dimyrcetol (% of applied dose, mean±SE, n=12) at 24 hours


 














































































Compartment



dimyrcetol A,


dose: 127 µg/cm2



dimyrcetol B,


dose: 169 µg/cm2



combined,


dose: 296 µg/cm2



 



 



 



 



Wipe



1.02±0.019



0.757±0.108



0.871±0.138



Donor chamber



14.5±1.6



17.0±1.7



15.9±1.6



Strip 1



0.020±0.009



0.014±0.006



0.017±0.007



Strips 2-3



0.012±0.005



0.010±0.003



0.011±0.004



Strips 4-6



0.008±0.004



0.007±0.003



0.007±0.003



Strips 7-10



0.005±0.002



0.007±0.002



0.006±0.002



Epidermis



0.229±0.041



0.089±0.011



0.149±0.023



Filter paper



0.468±0.051



0.450±0.041



0.458±0.044



Permeated



9.66±1.08



1.59±0.11



5.06±0.53



Overall recovery



25.9±2.6



19.9±1.8



22.5±2.1



 


 


Recoveries:


Low overall recoveries were achieved: 25.9 +/- 2.6, 19.9 +/- 1.8 and 22.5 +/- 2.1 % of the applied for dimyrcetol A, B and combined, respectively. Low values were likely due to evaporative loss.


 


Benzoic acid permeation:


The reference compound, benzoic acid, displayed a mean maximal absorption rate of approximately 29.8 µg/cm^2/h. This measured value is withing the range of measurements reported in a multi-centre study (Van de Sandtet al., 2004).

Conclusions:
Total absorbed doses of 10.4 +/- 1.2, 2.13 +/- 0.15 and 5.67 +/- 0.58% were recorded for dimyrcetol A, B and combined, respectively. Overall recoveries of dimyrcetol were low with values 25.9 +/- 2.6%, 19.9 +/- 1.8% and 22.5 +/- 2.1% of the applied dose for dimyrcetol A, B and combined, respectively. Evaporative losses explain the low recoveries reported.
Executive summary:

The in vitro percutaneous absorption of dimyrcetol under occluded conditions at an application concentration of 6% (w/v) in 70/30% (v/v) ethanol/water was determined in the current study. The absorption of each component, dimyrcetol A (2,6-dimethyl-7-octen-2-ol) and dimyrcetol B (2,6-dimethyl-7-octen-2-ol formate ester) were separately measured and combined for total dimyrcetol absorption.


 


Component A was observed to more rapidly permeate human skin than component B. Hydrolysis of dimyrcetol B to A (determined in a separate study) was also an issue, and may have contributed to the overall observed absorption values.


 


The levels of dimyrcetol in the epidermis (with any remaining in stratum corneum after tape stripping), filter paper membrane supports and receptor fluids combined gave total absorbed doses of 10.4 +/- 1.2, 2.13 +/- 0.15 and 5.67 +/- 0.58% for dimyrcetol A, B and combined, respectively. Overall recoveries of dimyrcetol were low with values 25.9 +/- 2.6%, 19.9 +/- 1.8% and 22.5 +/- 2.1% of the applied dose for dimyrcetol A, B and combined, respectively. Evaporative losses were responsible for the low recoveries recorded.

Endpoint:
basic toxicokinetics, other
Remarks:
Expert statement
Type of information:
other: Expert statement based on physicochemical and toxicity data.
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Expert statement based on physicochemical and toxicity data
Conclusions:
Based on physicochemical characteristics, absorption by the oral and dermal routes is likely. This assumption is supported by the results of toxicokinetic studies with the analogue substances linalool (oral absorption) and dimyrcetol (dermal absorption) as well as by the results of the subacute and subchronic oral toxicity studies, the latter revealing a NOAEL of 30 mg/kg bw/day. Bioaccumulation of the substance is rather unlikely. After being absorbed into the body, 2,6-dimethyloct-7-en-2-ol is most likely distributed into the interior part of cells due to its lipophilic properties (log Pow = 3.25) and in turn, the intracellular concentration may be higher than extracellular concentration, particularly in adipose tissues. Due to the effects observed in the subchronic oral toxicity study, 2,6-dimethyloct-7-en-2-ol or its metabolites may target the liver, kidneys and the bone marrow. Ambiguous results on potential metabolic transformation to more toxic metabolites were obtained from the genotoxicity studies but results from the read-across substance linalool, however, indicate conjugation to non-toxic metabolites. On the basis of the molecular substance characteristics as well as of the results from toxicokinetic studies with the analogue substance linalool, excretion via urine is favoured.
Executive summary:

2,6-dimethyloct-7-en-2-ol is a mono-constituent substance. It is a colourless viscous liquid at room temperature with a molecular weight of 156.27 g/moL. The substance shows a good solubility in water (939 mg/L at 20 °C). The log Pow was determined to be 3.25. The test substance has a vapour pressure of 20 Pa at 25 °C.


 


Absorption


According to the ECHA Guidance on Information Requirements and Chemical Safety Assessment, Chapter R.7c (Endpoint specific guidance), oral absorption is favoured for molecules with molecular weights of less than 500 g/moL. The substance is assumed to readily dissolve in gastrointestinal fluid due to its good water solubility of 939 mg/L and the log Pow of 3.25 favours absorption by passive diffusion. Taken together, the physiochemical properties indicate that 2,6-dimethyloct-7-en-2-ol becomes bioavailable following the oral route. This assumption is confirmed by read-across to toxicokinetics studies with linalool (CAS 201-134-4), an analogue substance, which showed a rapid excretion of the substance, after intragastric administration to rats, suggesting also a rapid absorption from the gut. Also the subacute oral toxicity study with 2,6-dimethyloct-7-en-2-ol  and the subchronic oral toxicity study with the read-across substance dimyrcetol (mixture of 2,6-dimethyloct-7-en-2-yl and 2,6-dimethyloct-7-en-2-yl formate ester, CAS 18479-58-8), where a dose-dependent decrease in body weights was noted (both in the subacute and subchronic study). Furthermore, an increase in relative liver and kidney weights as well as hematological changes, adipose infiltration of bone marrow and diuresis were observed in the subchronic toxicity study.


 


Due to the low volatility (vapour pressure of 20 Pa at 25 °C and high boiling point of 193 °C) of 2,6-dimethyloct-7-en-2-ol, it is unlikely that the substance will be available as a vapour. As the substance is a liquid, no particle formation is expected. Hydrophilic substances are likely to be retained within the mucus and thus, because of the good water solubility of the substance, penetration may be reduced. Although the logPow of 3.25 would favour uptake through the respiratory tract epithelium by passive diffusion, it is very unlikely that the substance reaches the respiratory tract due to the previously mentioned properties.


 


Dermal uptake is favoured by a liquid state of the substance, a molecular weight of less than 100 g/moL and a water solubility between 100-10,000 mg/L. 2,6-dimethyloct-7-en-2-ol is a non-volatile liquid with a relatively low molecular weight of 156.27 g/moL, a logPow of 3.25 and good water solubility of 939 mg/L, thus dermal absorption may be a potential uptake route. Especially the high water solubility enables a partition of the substance from the stratum corneum into the epidermis. This assumption is supported by the fact that the substance is classified for skin and eye irritation (category 2), indicating that the substance is absorbed to a certain extend. Furthermore, two in vitro dermal absorption studies with the analogue substance dimyrcetol using human skin, showed a dermal absorption of 2,6-dimethyloct-7-en-2-ol of up to 2.5±0.2 % (unoccluded conditions) or up to 10.4 % (occluded conditions).


 


 


Distribution


As mentioned above, the physicochemical properties of 2,6-dimethyloct-7-en-2-ol favour absorption by the oral and dermal route. The systemic absorption and distribution within the body is also demonstrated by the dose-dependent effects on body weight (subchronic and subacute study) and by effects on absolute and relative liver and kidney weights, the presence of adipose infiltration of bone marrow, haematological changes and diuresis effects in the subchronic study using the read-across substance. After being absorbed into the body, 2,6-dimethyloct-7-en-2-ol is most likely widely distributed due to its relatively low molecular weight and may enter into the interior part of cells due to its lipophilic properties (log Pow 3.25) and in turn, the intracellular concentration may be higher than extracellular concentration, particularly in adipose tissues. Due to effects on liver/kidneys/bone marrow observed in the subchronic repeated-dose toxicity study, it can be assumed that liver, kidneys and bone marrow may be target organs of the substance. In toxicokinetic studies with the analogue substance linalool, linalool was found in liver, gut, skin and skeletal muscles. Enterohepatic circulation may play a role as, after intragastric administration of the analogue substance linalool, 40% of the biliary conjugates were hydrolyzed and reabsorbed in the first pass. 


 


Although the log Pow of 2,6-dimethyloct-7-en-2-ol indicates a weak potential bioaccumulation potential, the rapid excretion observed for the analogue substance linalool suggests no tissue accumulation.


 


 


Metabolism


There is no direct experimental data to characterize the metabolism of the test substance. Instead, the anticipated metabolism is derived from expert judgement and reapplication of metabolic properties of related substances.


 


As some liver effects were observed in the subchronic repeated dose oral toxicity study, the substance is principally able to reach the liver and its metabolic capacities.


 


The genotoxicity studies indicated no remarkable differences in genotoxicity between treatments with or without metabolic activation. Higher cytotoxicity was observed in the presence of metabolic activation systems (Ames test and in vitro mammalian chromosome aberration test) but the opposite effect was observed in an in vitro mammalian cell gene mutation test. Therefore, no clear statement can be made on the metabolic (de)toxification of the substance but metabolism seems to occur to a certain extent.


 


This assumption is supported by toxicokinetic studies with the analogue substance linalool which showed increased microsomal cytochrome P-450 enzymes and metabolism of linalool mainly into 8-hydroxy-linalool and 8-carboxy-linalool. This suggests also conjugation of 2,6-dimethyloct-7-en-2-ol to non-toxic metabolites in the liver.


 


 


Excretion


The physicochemical properties of the substance favour excretion via urine (low molecular weight and high water solubility). In two toxicokinetic studies with the analogue substance linalool, its metabolites 8-hydroxy-linalool and 8-carboxy-linalool were excreted via urine (approx. 58% of the applied dose) whereas 23% were found to be exhaled in form of CO2 and up to 15% were eliminated by fecal excretion after enterohepatic circulation, suggesting a similar excretion pattern for 2,6-dimethyloct-7-en-2-ol.

Description of key information

Based on physicochemical characteristics, absorption by the oral and dermal routes is likely. This assumption is supported by the results of toxicokinetic studies with the analogue substances linalool (oral absorption) and dimyrcetol (dermal absorption) as well as by the results of the subacute and subchronic oral toxicity studies, the latter revealing a NOAEL of 30 mg/kg bw/day. Bioaccumulation of the substance is rather unlikely. After being absorbed into the body, 2,6-dimethyloct-7-en-2-ol is most likely distributed into the interior part of cells due to its lipophilic properties (log Pow = 3.25) and in turn, the intracellular concentration may be higher than extracellular concentration, particularly in adipose tissues. Due to the effects observed in the subchronic oral toxicity study, 2,6-dimethyloct-7-en-2-ol or its metabolites may target the liver, kidneys and the bone marrow. Ambiguous results on potential metabolic transformation to more toxic metabolites were obtained from the genotoxicity studies but results from the read-across substance linalool, however, indicate conjugation to non-toxic metabolites. On the basis of the molecular substance characteristics as well as of the results from toxicokinetic studies with the analogue substance linalool, excretion via urine is favoured.

Key value for chemical safety assessment

Bioaccumulation potential:
low bioaccumulation potential

Additional information

Dermal absorption in vitro (unoccluded), RL1


The in vitro dermal absorption of the analogue material, dimyrcetol, was determined under in-use (non-occluded) conditions using epidermal skin membranes from female cosmetic surgery donors. At a maximum use concentration of 6% in 70/30 (v/v) ethanol/water, 2.50 +/- 0.20% of dimyrcetol A (2,6-dimethyloct-7-en-2-ol) and 0.662 +/- 0.040% of dimyrcetol B (2,6-dimethyloct-7-en-2-ol formate), respectively, were absorbed in 24 hours. These values include material that had permeated to receptor fluid as well as that present in the epidermis as determined from tape stripping. Overall dimyrcetol dermal absorption under non-occluded, in-use conditions was low as well as total recoveries of material under these conditions (2.36 +/- 0.24%).


 


Dermal absorption in vitro (occluded), RL1


The in vitro dermal absorption of the analogue material, dimyrcetol, was determined under occluded conditions using epidermal skin membranes from female cosmetic surgery donors. Overall 24 -hour absorption was increased to 10.4 +/- 1.2% and 2.13 +/- 0.15% for dimyrcetol A and B, respectively, under occluded conditions. Overall recovery under occluded conditions increased also compared with non-occluded conditions with a value of 22.5 +/- 2.1% obtained. Significant losses due to evaporation were recorded under both non-occluded and occluded conditions. These losses due to evaporation are not inconsistent with a material with a low measured vapor pressure given that under the conditions employed in the current studies, only a sparingly small amount of material was applied as a thin film, and given a slow rate of percutaneous absorption, the majority of material was lost before skin absorption could occur. It is anticipated that such losses will also occur under in use conditions. Also, the increased dermal absorption for dimyrcetol component A may have been due in part to hydrolytic conversion from the formate ester.


 


Please refer to Section 13 of this IUCLID file for read-across documentation and rationale for the selection of representative analogue chemicals.


 


Expert statement, RL2


2,6-dimethyloct-7-en-2-ol is a mono-constituent substance. It is a colourless viscous liquid at room temperature with a molecular weight of 156.27 g/moL. The substance shows a good solubility in water (939 mg/L at 20 °C). The log Pow was determined to be 3.25. The test substance has a vapour pressure of 20 Pa at 25 °C.


 


Absorption


According to the ECHA Guidance on Information Requirements and Chemical Safety Assessment, Chapter R.7c (Endpoint specific guidance), oral absorption is favoured for molecules with molecular weights of less than 500 g/moL. The substance is assumed to readily dissolve in gastrointestinal fluid due to its good water solubility of 939 mg/L and the log Pow of 3.25 favours absorption by passive diffusion. Taken together, the physiochemical properties indicate that 2,6-dimethyloct-7-en-2-ol becomes bioavailable following the oral route. This assumption is confirmed by read-across to toxicokinetics studies with linalool (CAS 201-134-4), an analogue substance, which showed a rapid excretion of the substance, after intragastric administration to rats, suggesting also a rapid absorption from the gut. Also the subacute oral toxicity study with 2,6-dimethyloct-7-en-2-ol  and the subchronic oral toxicity study with the read-across substance dimyrcetol (mixture of 2,6-dimethyloct-7-en-2-yl and 2,6-dimethyloct-7-en-2-yl formate ester, CAS 18479-58-8), where a dose-dependent decrease in body weights was noted (both in the subacute and subchronic study). Furthermore, an increase in relative liver and kidney weights as well as hematological changes, adipose infiltration of bone marrow and diuresis were observed in the subchronic toxicity study.


 


Due to the low volatility (vapour pressure of 20 Pa at 25 °C and high boiling point of 193 °C) of 2,6-dimethyloct-7-en-2-ol, it is unlikely that the substance will be available as a vapour. As the substance is a liquid, no particle formation is expected. Hydrophilic substances are likely to be retained within the mucus and thus, because of the good water solubility of the substance, penetration may be reduced. Although the logPow of 3.25 would favour uptake through the respiratory tract epithelium by passive diffusion, it is very unlikely that the substance reaches the respiratory tract due to the previously mentioned properties.


 


Dermal uptake is favoured by a liquid state of the substance, a molecular weight of less than 100 g/moL and a water solubility between 100-10,000 mg/L. 2,6-dimethyloct-7-en-2-ol is a non-volatile liquid with a relatively low molecular weight of 156.27 g/moL, a logPow of 3.25 and good water solubility of 939 mg/L, thus dermal absorption may be a potential uptake route. Especially the high water solubility enables a partition of the substance from the stratum corneum into the epidermis. This assumption is supported by the fact that the substance is classified for skin and eye irritation (category 2), indicating that the substance is absorbed to a certain extend. Furthermore, two in vitro dermal absorption studies with the analogue substance dimyrcetol using human skin, showed a dermal absorption of 2,6-dimethyloct-7-en-2-ol of up to 2.5±0.2 % (unoccluded conditions) or up to 10.4 % (occluded conditions).


 


 


Distribution


As mentioned above, the physicochemical properties of 2,6-dimethyloct-7-en-2-ol favour absorption by the oral and dermal route. The systemic absorption and distribution within the body is also demonstrated by the dose-dependent effects on body weight (subchronic and subacute study) and by effects on absolute and relative liver and kidney weights, the presence of adipose infiltration of bone marrow, haematological changes and diuresis effects in the subchronic study using the read-across substance. After being absorbed into the body, 2,6-dimethyloct-7-en-2-ol is most likely widely distributed due to its relatively low molecular weight and may enter into the interior part of cells due to its lipophilic properties (log Pow 3.25) and in turn, the intracellular concentration may be higher than extracellular concentration, particularly in adipose tissues. Due to effects on liver/kidneys/bone marrow observed in the subchronic repeated-dose toxicity study, it can be assumed that liver, kidneys and bone marrow may be target organs of the substance. In toxicokinetic studies with the analogue substance linalool, linalool was found in liver, gut, skin and skeletal muscles. Enterohepatic circulation may play a role as, after intragastric administration of the analogue substance linalool, 40% of the biliary conjugates were hydrolyzed and reabsorbed in the first pass. 


 


Although the log Pow of 2,6-dimethyloct-7-en-2-ol indicates a weak potential bioaccumulation potential, the rapid excretion observed for the analogue substance linalool suggests no tissue accumulation.


 


 


Metabolism


There is no direct experimental data to characterize the metabolism of the test substance. Instead, the anticipated metabolism is derived from expert judgement and reapplication of metabolic properties of related substances.


 


As some liver effects were observed in the subchronic repeated dose oral toxicity study, the substance is principally able to reach the liver and its metabolic capacities.


 


The genotoxicity studies indicated no remarkable differences in genotoxicity between treatments with or without metabolic activation. Higher cytotoxicity was observed in the presence of metabolic activation systems (Ames test and in vitro mammalian chromosome aberration test) but the opposite effect was observed in an in vitro mammalian cell gene mutation test. Therefore, no clear statement can be made on the metabolic (de)toxification of the substance but metabolism seems to occur to a certain extent.


 


This assumption is supported by toxicokinetic studies with the analogue substance linalool which showed increased microsomal cytochrome P-450 enzymes and metabolism of linalool mainly into 8-hydroxy-linalool and 8-carboxy-linalool. This suggests also conjugation of 2,6-dimethyloct-7-en-2-ol to non-toxic metabolites in the liver.


 


 


Excretion


The physicochemical properties of the substance favour excretion via urine (low molecular weight and high water solubility). In two toxicokinetic studies with the analogue substance linalool, its metabolites 8-hydroxy-linalool and 8-carboxy-linalool were excreted via urine (approx. 58% of the applied dose) whereas 23% were found to be exhaled in form of CO2 and up to 15% were eliminated by fecal excretion after enterohepatic circulation, suggesting a similar excretion pattern for 2,6-dimethyloct-7-en-2-ol.