4'-tert-butyl-2',6'-dimethyl-3',5'-dinitroacetophenone

Endpoint summary

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

dermal absorption in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

secondary literature

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 427 (Skin Absorption: In Vivo Method)

GLP compliance:

not specified

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source of test material: Haarmann & Reimer GmbH, Germany

RADIOLABELLING INFORMATION (if applicable)
Samples of each compound uniformly labelled with carbon-14 in the ring were synthesised at Huntingdon Research Centre with radiochemical purities of greater than 98% and specific activities of 31.21–37.25 mCi/mmol.

Radiolabelling:

yes

Species:

rat

Strain:

other: Sprague–Dawley CD and Long Evans

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (Margate, Kent, UK)
- Age at study initiation: 6 weeks
- Weight at study initiation: 200g approximately
- Individual metabolism cages: Yes - All rats were housed individually in glass metabolism cages for the duration of the studies.
- Diet: Laboratory Diet No.1 (Spratt’s Ltd., Barking, UK) ad libitum
- Water: ad libitum

Type of coverage:

occlusive

Vehicle:

other: Mixture of ethanol and phenylethyl alcohol (minimum amount to maintain solubility — about 1%)

Duration of exposure:

6 hours (acute study)
14 days (repeated dose study)

Doses:

Acute study:
- Nominal doses: 0.5 mg/kg (11 µg/cm2 of skin)
- Dose volume: 1 mg/ml and 0.1 ml applied evenly over an area of 9 cm2 for a total dose of 0.5 mg/kg.

Repeated dose study:
- Dose volume: Area of 9 cm2 and adjusted according to daily bodyweight to provide a nominal daily dose of 0.5 mg/kg

No. of animals per group:

Acute: 16 Sprague Dawley and 5 Long Evans
Repeated dose:8 animals (strain not specified)

Control animals:

no

Details on study design:

TEST SITE
Acute: Each compound was applied to the backs of the animals, which were shaved over an area of 16 cm2. The 14C-compound was formulated in a mixture of ethanol and phenylethyl alcohol (minimum amount to maintain solubility — about 1%) at a concentration of 1 mg/ml and 0.1 ml applied evenly over an area of 9 cm2 for a total dose of 0.5 mg/kg. The treated area was covered with aluminium foil and Sleek® waterproof dressing (Smith & Nephew Pharmaceuticals, Welwyn, UK).
Repeated: As above but dosing area of 9 cm2 and adjusted according to daily bodyweight to provide a nominal daily dose of 0.5 mg/kg.

REMOVAL OF TEST SUBSTANCE
Acute: After 6 h, the dressing and foil were removed and the area of treated skin wiped with cotton wool swabs containing 1% ethanolic phenylethyl alcohol.

SAMPLE COLLECTION
Acute: After dose application animals were housed singly in glass metabowls to facilitate the separate collection of urine and faeces. Urine was collected in solid CO2 cooled containers at 0–6-, 6–24 and 24-h intervals thereafter for 5 days and faeces up to the time of sacrifice at 24-h intervals. Pairs of Sprague–Dawley rats were sacrificed by cervical dislocation at 1, 3, 6, 8, 24, 48, 96 and 120 h after dosing and a sample of blood withdrawn by cardiac puncture. The Long Evans rats were killed similarly at 6, 24, 48, 96 and 120 h. The treated area of skin and various tissues were dissected from the carcass. The bile duct of 1 Sprague–Dawley rat were cannulated with 0-0 gauge nylon cannula, under halothane:oxygen anaesthesia immediately before dosing as described above and bile and urine collected for 24 h for musk ketone.

Repeated: Urine and faeces were collected for 24-h periods after 1, 2, 3, 7, 9, 11, and 13 doses or until sacrifice. Pairs of rats were sacrificed 24 h after seven doses and 6, 24 and 48 h after 14 doses. Blood samples were obtained by cardiac puncture and after sacrifice by cervical dislocation the brain, kidneys, liver, thyroid, samples of perirenal fat and the dosed skin removed from the carcass. All samples were stored at -20°C until analysed.

SAMPLE PREPARATION
Faeces and finely minced rat carcasses, including the bones, were separately extracted once by homogenisation in methanol. After centrifugation, samples of the extract and residue were analysed. Skin samples were digested in ethanolic potassium hydroxide. Samples of urine (1 ml), plasma (0.5 ml), solvent extracts (0.5 ml), skin digests (0.5 ml), contents of expired air traps (1 ml) and cage washings were mixed with M1-31 scintillator (Packard Instrument Company, Cavesham, UK). Samples of tissue (0.05–0.5 g) and residues of extracted carcasses (0.1–0.6 g) were combusted in oxygen using an Automatic Sample Oxidiser (Model 306, Mk2, Tri-Carb®,Packard Instrument Company). Combusted products were absorbed into Carbo-Sorb™ and mixed with Permfluor®-v scintillation system. Radioactivity was measured with a Philips Liquid Scintillation analyser (Phillips, N.V., Eindhoven, Holland). Radioactivity in amounts less than twice the background was considered to be the limit of accurate measurements.

ANALYSIS
Chromatographic analysis:
Bile and urine samples were prepared by evaporation under reduced pressure or under a stream of nitrogen at 37°C and extracting the concentrate with methanol or acetone. After centrifugation, the supernatants, which contained greater than 90% of the radioactivity, were applied directly to thin-layer plates. Samples of urine and bile were incubated at 37°C for 16 h with an equal volume of 0.1 M sodium acetate buffer (pH5) and b-glucuronidase (Type H1, Sigma). Further samples were incubated with sulphatase (Sigma) in 0.1 M sodium hydrogen phosphate buffer. Thin layer chromatography was carried out on pre-layered Kieselgel F254 plates (E. Merck A.G., Darmstadt, Germany) of layer thickness 0.25 mm using the following developing solvents chloroform: acetone:water (4:18:1, v:v) ethyl acetate: acetone (1:1, v:v). Radioactive components
on thin-layer plates were detected either by apposition autoradiography using X-ray film or with a Berthold Mark 2 radiochromatogram scanner.

Mass spectrometry
Samples of deconjugated bile were extracted twice with ethyl acetate, the extracts concentrated and applied directly to thin-layer plates. After an initial purification using chloroform:acetone:water (4:18:1, v:v) as developing solvent, metabolites were separated using hexane:ethyl acetate (6:4 v:v). Mass spectra were obtained using a VG 7070E mass spectrometer (VG Analytical, Manchester, UK). Samples were introduced by
the direct insertion probe and subjected to alternate electron impact:chemical ionisation (ACE) conditions. Electron impact spectra were obtained with an electron energy of 70 eV and trap current
of 200 µA and chemical ionisation spectra were obtained with an electron energy of 50 eV, an emission current of 500 µA and isobutane as the reactant gas.

Signs and symptoms of toxicity:

not specified

Dermal irritation:

not specified

Absorption in different matrices:

Analysis of samples from animals sacrificed at 6 h indicated that while little material was excreted in urine and faeces during this time, appreciable amounts had been absorbed as assessed by the amounts of the dose in the carcass 17.7% (Table 2). Most of the remaining material was on the surface of the treated skin and would have been removed from those animals maintained for longer than 6 h. At 8 h, 2 h after removal of the applied dose from the surface of the skin, means of 8.5% of the dose remained in the treated skin. At 120 h absorption and excretion was essentially complete. Only small amounts remained in the carcass and similar small amounts in the treated skin (2.1–3.63%). A similar pattern of excretion of absorbed material was apparent with the larger amount in faeces compared to urine with ratios of about 2.5:1 for musk ketone.

The total absorption after 120 h calculated from material excreted in urine and faeces and that retained in the carcass excluding the treated skin was 31.1% for musk ketone. Levels in the treated skin decreased steadily after removal of the dose on the surface but did not change appreciably after 48 h. Tissue concentrations of radioactivity were generally very low with peak levels occurring at 6–8 h (Figure 2). In general, fat and liver contained the highest concentrations and levels in fat were similar for all three compounds at about 0.2 µg equiv./g. Fat concentrations declined fairly rapidly until 120 h when levels were as low as, or
lower than, other tissues at around 0.005 µg equiv./g.
E
xperiments in bile duct cannulated rats resulted in 16% of the dermal dose of musk ketone being eliminated in bile during 24 h. Only 1 to 2% of the dose was excreted in the urine.

During 14 repeated daily doses of musk ketone, excretion in the faeces and urine rose from about 1.5 and 2.4% in the urine and faeces, respectively, reaching a high for musk ketone on days 9, (2.7% in the urine) and 11,
(11.7% in the faeces) (Table 4).

Tissue levels after 14 doses were approximately three-fold higher than after one dose (Table 5). For musk ketone, highest concentrations at 24 h after the last of the 14 daily doses were in the liver representing 0.53 µg equiv./g while fat concentrations were 0.15 µg equiv./g.

Total recovery:

- Total recovery: In the acute study, recovery ranged from 95.2-103% over 120 hours.

Time point:

120 h

Dose:

0.5 mg/kg

Parameter:

percentage

Absorption:

31.1 %

Remarks on result:

other: Acute study

Conclusions:

In dermal absorption and disposition studies of musk ketone in rats, the total absorption after 120 h from an acute 6 hour exposure was 31.1%. In general, fat and liver contained the highest concentrations with concentrations declining fairly rapidly until 120 h when levels were at similar levels to other tissues. Excretion after 14 day repeated dosing was mainly in the faeces. In urine samples taken after 14 day repeated treatments, most of the metabolites were not simple conjugates such as glucuronides and sulphates. Analysis of bile samples did however show the presence of polar metabolites, which appeared to consist almost entirely of glucuronide conjugates. There is some bioaccumulation after repeated daily dosing, it is not likely that this would continue over longer periods of time.

Executive summary:

In dermal absorption and disposition studies of musk ketone (Hawkins and Ford, 1999), Sprague–Dawley CD and Long Evans male rats were administered nominal doses of 0.5 mg/kg bw of ring-labelled 14C-musk ketone (98%; in a mixture of phenylethyl alcohol and ethanol) for 6 hours (acute study) and monitored up to 120 hr post-dosing and 14 repeated daily doses of 0.5 mg/kg bw of ring-labelled 14C-musk ketone. Bile duct cannulation (1 rat) was also performed in the acute study and bile and urine were collected at 24 hrs. Faeces and urine were collected during both studies and blood, brain, kidneys, liver, thyroid and perirenal fat were retained for analysis. Thin layer chromatography and mass spectrometry was performed to identify metabolites.

Mean dermal absorption values for the acute treatment group for which the skin site was washed after 6 hours with sacrifice at 120 hrs (n=3), was 31.3%. In the acute disposition study, fat and liver contained the highest concentrations with concentrations declining fairly rapidly until 120 h when levels were at similar levels to other tissues. Excretion after 14 day repeated dosing was mainly in the faeces (11.7% compared to 2.7% in urine). In urine samples taken after 14 day repeated treatments, most of the metabolites were not simple conjugates such as glucuronides and sulphates but could not be further identified. Analysis of bile samples did however show the presence of polar metabolites, which appeared to consist almost entirely of glucuronide conjugates. There is some bioaccumulation after repeated daily dosing however it is not likely that this would continue over longer periods of time.

Reference 2

Endpoint:

dermal absorption in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

secondary literature

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 427 (Skin Absorption: In Vivo Method)

GLP compliance:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
Samples of the non-radiolabelled and carbon-14 uniformly ring-labelled musks were obtained as described by Hawkins and Ford (1999).

- Source of test material: Haarmann & Reimer GmbH, Germany

RADIOLABELLING INFORMATION
Samples of each compound uniformly labelled with carbon-14 in the ring were synthesised at Huntingdon Research Centre. The specific activity of the compound was musk ketone 20.8 µCi/mg.

Radiolabelling:

yes

Species:

other: Human

Sex:

male

Details on test animals or test system and environmental conditions:

Two healthy adult male volunteers of similar build were enrolled for the studies, which were conducted on three separate occasions. The subjects were in the age range 18–50 years and were within ±10% of normal weight for age and build. Within 7 days of commencement of the study, each subject was submitted to a suitable physical examination at which medical history was obtained and blood and urine samples were taken for laboratory analysis. The study was subject to review and approval by the Ethics Committee of the Institute of Clinical Pharmacology, Dublin, Ireland, where it was conducted. Within 7 days of the end of the study, a physical examination and blood and urine analysis was carried out which showed the test compound had no detectable effect on the subjects’ health. For 12 h before dosing and during the 5-day experimental period the subjects were confined to the hospital clinical pharmacology unit. Each subject fasted for 12 h before dose administration.

Type of coverage:

occlusive

Vehicle:

other: ethanol with 1% phenylethyl alcohol

Duration of exposure:

6 hours (after ethanol allowed to evaporate for 30 mins)

Doses:

- Nominal doses: 2 mg (0.028 mg/kg); 20 µg/cm2
- Dose volume: 2 ml

No. of animals per group:

2 males

Details on study design:

DOSE PREPARATION
- Method for preparation of dose suspensions: The carbon-14 labelled test compounds were dissolved in ethanol containing the minimum amount of phenylethyl alcohol (approximately 1%) to give a concentration of 1 mg/ml.

TEST SITE
Two ml of musk ketone (in ethanol/phenylethyl alcohol) was applied evenly to an area of 100 cm2 on the unshaven skin of the upper left quadrant of the chest and the ethanol allowed to evaporate for 30
min. The treated area was covered with a protective gauze held in position with adhesive tape.

REMOVAL OF TEST SUBSTANCE
At 6 h after administration, the dressing was removed and the treated skin wiped with cotton wool swabs using phenylethyl alcohol/ethanol (1.99%, v/v).

SAMPLE COLLECTION
Blood samples (10 ml) were withdrawn into heparinised tubes at pre-dose and at 0.25, 0.5 and 0.75, 1, 2, 4, 6, 10, 12, 16, 24, 36, 48, 72, 96 and 120 h after dosing. A portion (1 ml) was retained and plasma
separated from the remainder. Urine was collected at 0–2, 2–4, 4–6, 6–8, 8–10, 10–12, 12– 24, 24–48, 48–72, 72–96 and 96–120 h. Faeces were collected separately at 24-h intervals and all samples stored at −20 °C. At 120 h a part (approximately 12.5 cm2) of the treated skin was stripped with 10 successive applications of transparent adhesive tape and the strips retained for measurement of radioactivity.

SAMPLE PREPARATION AND ANALYSIS
Faeces were extracted once by homogenisation in methanol. After centrifugation, samples of the separated extract and residue were measured for radioactivity. The gauze, cotton wool swabs and adhesive tape strips were separately extracted with acetone in a Soxhlet apparatus for 3 h. The extraction was repeated and radioactivity measured in both extracts. Samples of urine (4 ml) and plasma (0.5 ml) were mixed with MI-31 scintillator (Packard Instrument Company, Caversham Caversham, UK). Samples of extracted faeces and cotton wool were combusted in oxygen using an Automatic Sample Oxidiser (Model 306, Mk2, Tri-Carb®, Packard Instrument Company, Caversham, UK). The combusted products in oxygen were absorbed into CarboSorb™ and mixed with Permafluor®-V scintillator system. Radioactivity was measured with a Philips Liquid Scintillation analyser (Philips N.V., Eindhoven, Holland). Radioactivity in amounts less than twice background was considered to be below the limit of accurate measurement.

Urine samples were extracted with ethyl acetate before and after incubation with beta-glucuronidase. For enzyme incubation, aliquots of urine were mixed with equal volumes of 0.1 M sodium acetate buffer (pH 5), beta-glucuronidase (Type H1, Sigma) added and incubated for 16 h at 37 °C. Extracts were analysed using thin layer chromatography on pre-layered Kieselgel F254 plates (Merck, Darmstadt, Germany) of layer thickness 0.25 mm using solvent systems hexane/ethyl acetate (6:4, v/v) and ethyl acetate/acetone (1:1, v/v). Radioactive components on thin-layer plates were detected either by opposition autoradiography using Singul XIRP X-ray film (Blishen, London, UK) or with a Berthold Mark II radiochromatogram scanner (Model LB 2722). Chromatograms of human urine metabolites were compared with those for rat bile metabolites (Hawkins and Ford, 1999).

Signs and symptoms of toxicity:

not specified

Dermal irritation:

not specified

Absorption in different matrices:

The systemic availability as determined based on the excretion of radioactivity in urine and faeces after 120 hours was very low for both subjects (0.34 and 0.49% of the dose in urine and 0.09 and 0.02% in faeces, respectively) (Table 1). Most of the applied material was recovered from the surface of the skin (66.8-75.7%) as well as from the protective gauze at 6 h (10.3-19.8%). A mean of 0.5% musk ketone were absorbed based on the amounts excreted in urine and faeces during 5 days.

The maximum rate of excretion occurred during days 2–3. During day 5, only 0.02% of the doses were excreted. No radioactivity was detected in any plasma or sample with limits of detection of 0.0004% (4×10−5) dose/ml (musk ketone), which is consistent with the low absorption. Similarly, no radioactivity was detected in any blood samples at a limit of detection of 0.0001% dose/ml indicating that there was no material selectively bound to blood cells. Additionally, no radioactivity was detected (<0.02% dose) in the skin strips taken at 120 h.

Total recovery:

- Total recovery: The recovery from both subjects was 86.6 adn 86.9% respectivley.

Time point:

120 h

Dose:

0.028 mg/kg

Parameter:

percentage

Absorption:

0.5 %

Although concentrations of radioactivity in human urine samples were very low, some information on the nature of metabolites was obtained by analysis of solvent extracts of urine. The amounts of radioactivity extracted were greatly enhanced after treatment with β-glucuronidase (60%). The parent compounds were well separated from metabolites in the chromatographic systems developed and none were detected in any urine extracts Musk ketone human samples contained one major component and a chromatographically similar minor component, which corresponded to a major rat bile metabolite.

Conclusions:

In a dermal absorption and disposition study of musk ketone in humans, a mean of 0.5% was absorbed based on the amounts excreted in urine and faeces during 5 days. Most of the material was excreted in the urine with a negligible amount found in faeces. No radioactivity was detected in any plasma sample, consistent with low absorption, and no radioactivity was detected (<0.02% dose) in skin strips taken at 120 h. Analysis of urine samples indicated excretion as mainly single glucuronide conjugates.

Executive summary:

In dermal absorption and disposition studies in humans (Hawkins et al., 2002), 2 male subjects were administered nominal doses of 0.028 mg/kg (20 µg/cm2) of ring-labelled 14C-musk ketone (in a mixture of ethanol with 1% phenylethyl alcohol) to the unshaven skin of the chest for 6 hours and monitored up to 120 hrs post-dosing. Blood, faeces, urine and skin strips were collected. Thin layer chromatography was performed on β-glucuronidase-treated urine samples to identify metabolites.

A mean of 0.5% was absorbed based on the amounts excreted in urine and faeces during 5 days. Most of the material was excreted in the urine with a negligible amount found in faeces. No radioactivity was detected in any plasma sample, consistent with low absorption, and no radioactivity was detected (<0.02% dose) in skin strips taken at 120 h. Analysis of urine samples indicated excretion as mainly single glucuronide conjugates.

Description of key information

A toxicokinetic assessment was conducted in accordance with REACH Annex VIII 8.8.1. The substance musk ketone is an organic light yellow mono-constituent powder with a purity of >99% (w/w) to <100% (w/w), with a typical concentration of 99.9% (w/w).

There are no data available on the toxicokinetics of musk ketone after oral and inhalation exposure. Two dermal absorption and disposition studies of musk ketone in rats (Hawkins and Ford, 1999) and humans (Hawkins et al., 2002) are available. The toxicokinetic analysis is based on these publications, physicochemical and in vivo toxicological data. In vivo studies covering the oral route (acute oral toxicity study in rats, pre-natal developmental toxicity study in rats), dermal route (90 day repeated dose toxicity study in rats, skin sensitisation in guinea pigs) and inhalational route (acute inhalational toxicity study in rats) are available. Further details on endpoints are available in the IUCLID 6 registration dossier.

Based on the publications, physicochemical data and available in vivo toxicological data, absorption of musk ketone is expected to be moderate in the rat (oral), moderate in the rat and very low in humans (dermal) and low in the rat (inhalation). Musk ketone is likely to be widely distributed in rats and bioaccumulation is unlikely over long periods of time. Glucuronide conjugates are mainly excreted in the faeces. In humans, most of any absorbed material is excreted in the urine as mainly single glucuronide conjugates.

The absorption rates of 50% (oral), 50% (dermal) and 100% (inhalation) are accepted for chemical risk assessment purposes.

Key value for chemical safety assessment

Bioaccumulation potential:

no bioaccumulation potential

Absorption rate - oral (%):

50

Absorption rate - dermal (%):

50

Absorption rate - inhalation (%):

100

Additional information

1. Physicochemical properties

In accordance with the ECHA Guidance on Information Requirements and Chemical Safety Assessment, Chapter R.7C Section R.7.12 (Endpoint Specific Guidance), the physicochemical properties can provide an insight into the potential behaviour of musk ketone in the body.

Absorption - oral

The molecular weight of musk ketone (294.3 g/mol) is in the range for favourable oral absorption (<500 g/mol). It is moderately lipophilic (log Pow 4.24 at 25°C) and it is poorly soluble in water (<0.1 mg/L at 20°C). Oral absorption is expected to be low but if absorption does occur, it is likely via the lymphatic system through micellular solubilisation, based on the lipophilicity and poor water solubility.

Absorption – dermal

As musk ketone is poorly soluble in water, low dermal uptake is expected. The moderate log Pow value indicates slow transfer to the epidermis and entry into systemic circulation. Overall the molecular weight, log Pow and poor water solubility indicate that dermal absorption of musk ketone is likely to be low.  

Absorption – inhalation

The particle size distribution report for musk ketone indicates a range of 0.324μm – 44.69μm (D10: 2.061 μm, D50: 10.82μm, D90: 23.84 μm). This indicates that 100% of the particles are available in the inhalable fractions of air (<100 μm). Particles with aerodynamic diameters of above 1-5 μm have the greatest probability of settling in the nasopharyngeal region whereas particles with aerodynamic diameters below 1-5 μm are most likely to settle in the tracheo-bronchial or pulmonary regions. As there are fractions in both these categories, the substance is likely to be distributed throughout the respiratory tract upon inhalation. Greater than 50% of particles will be able to penetrate the alveolar region of the respiratory tract (<15 µm). As a poorly soluble dust, musk ketone could be coughed or sneezed out of the body or swallowed (refer to oral absorption). Based on the poor water (0.1 mg/L at 20°C) and lipophilicity (4.24), it is likely that any absorption will be low and occur via micellular solubilisation and the lymphatic system. However, due to the very low vapour pressure of Musk ketone (6.68×10-4Pa at 25°C), exposure via the inhalation route is expected to be negligible.

2. Other data in the literature

Two dermal absorption and disposition studies of musk ketone in rats (Hawkins and Ford, 1999) and humans (Hawkins et al., 2002) are available.

In a dermal absorption and disposition study (Equivalent or similar to OECD 427), Sprague–Dawley CD and Long Evans male rats were administered nominal doses of 0.5 mg/kg bw of ring-labelled 14C-musk ketone (98%; in a mixture of phenylethyl alcohol and ethanol) for 6 hours (acute study) and monitored up to 120 hr post-dosing and 14 repeated daily doses of 0.5 mg/kg bw of ring-labelled 14C-musk ketone. Bile duct cannulation (1 rat) was also performed in the acute study and bile and urine were collected at 24 hrs. Faeces and urine were collected during both studies and blood, brain, kidneys, liver, thyroid and perirenal fat were retained for analysis. Thin layer chromatography and mass spectrometry was performed to identify metabolites. Mean dermal absorption values for the acute treatment group for which the skin site was washed after 6 hours with sacrifice at 120 hrs (n=3), was 31.3%. In the acute disposition study, fat and liver contained the highest concentrations with concentrations declining fairly rapidly until 120 h when levels were at similar levels to other tissues. Excretion after 14 day repeated dosing was mainly in the faeces (11.7% compared to 2.7% in urine). In urine samples taken after 14 day repeated treatments, most of the metabolites were not simple conjugates such as glucuronides and sulphates but could not be further identified. Analysis of bile samples did however show the presence of polar metabolites, which appeared to consist almost entirely of glucuronide conjugates. There is some bioaccumulation after repeated daily dosing however it is not likely that this would continue over longer periods of time.

In a dermal absorption and disposition study in humans (Equivalent or similar to OECD 427), 2 male subjects were administered nominal doses of 0.028 mg/kg (20 µg/cm2) of ring-labelled 14C-musk ketone (in a mixture of ethanol with 1% phenylethyl alcohol) to the unshaven skin of the chest for 6 hours and monitored up to 120 hrs post-dosing. Blood, faeces, urine and skin strips were collected. Thin layer chromatography was performed on β-glucuronidase-treated urine samples to identify metabolites. A mean of 0.5% was absorbed based on the amounts excreted in urine and faeces during 5 days. Most of the material was excreted in the urine with a negligible amount found in faeces. No radioactivity was detected in any plasma sample, consistent with low absorption, and no radioactivity was detected (<0.02% dose) in skin strips taken at 120 h. Analysis of urine samples indicated excretion as mainly single glucuronide conjugates.

Taken together, the results from these studies demonstrate that dermal absorption was 60-fold lower in humans compared to rats, resulting in a correspondingly lower systemic exposure for equivalent doses. The primary metabolism of musk ketone is probably similar for human and rat in that phase I oxidation is involved but with different hydroxylated metabolites being formed. The major human metabolites were different from those in the rat but the polarity indicated they could be monohydroxylated analogues (via methyl hydroxylation of the t-butyl group). It therefore seems highly probable that the major human urinary metabolites are formed by this pathway, these metabolites being conjugated with glucuronic acid and excreted in urine. The glucuronides of the hydroxylated rat metabolites are however eliminated in bile and a consequence is that they are available for further metabolism, including reduction of the nitro groups to potentially toxic arylamines, which can be reabsorbed. Biliary excretion is usually the main contributing reason for extensive faecal excretion in the rat.

3. Information from other studies in the dossier

Absorption – oral

In an acute oral toxicity study in male and female Sprague-Dawley rats (equivalent or similar to 401), musk ketone in corn oil did not cause mortality at a dose level of 5000 mg/kg bw in any animal. All animals exhibited sedation and/or piloerection at 1 hour following intubation, and tremors, some sedation, and respiratory abnormalities (depression and short, rapid respiration) at 3 and 6 hours following intubation. Test animals showed no toxic signs on day 1, and continued to remain normal during the remainder of the 14-day observation period. Body weights at days 0 and 14 were within a normal range for both male and female animals. A single animal had an enlarged spleen; otherwise, no gross pathological organ changes were observed in any animals. The LD50 (male/female) was >5000 mg/kg bw.

In a developmental toxicity study (equivalent or similar to OECD 414/GLP) musk ketone was administered to 25 female CrI:CD BR VAF/Plus (Sprague-Dawley)/dose in corn oil by gavage at dose levels of 0, 15, 45, 150 mg/kg bw/day from days 7 through 17 of gestation. No abortions, premature deliveries or deaths occurred during the study. All rats survived until scheduled sacrifice. Increased incidences of dried feces and perioral substance occurred in the 45 mg/kg bw/day dosage group; these two adverse clinical observations, as well as urine-stained abdominal fur, excessive salivation, dehydration, red substance on forepaws and tremors occurred in significantly increased numbers (p≤O.01) in the 150 mg/kg bw/day dosage group rats, and one or two 150 mg/kg bw/day dosage group rats also had chromorhinorrhea or chromodacryorrhea.  Effects were first observed on gestation days (DGs) 13 and 7 in the 45 and 150 mg/kg bw/day dosage groups, respectively.  Urine stained abdominal fur, perioral substance, dehydration and dried feces continued to occur in a few 150 mg/kg bw/day dosage group rats during the postdosage period (DGs 18 to 20).

Dosage-dependent, statistically significant (p≤O.05 to p≤0.01) reductions in weight gains and absolute (g/day) and relative (g/kg/day) feed consumption values for the entire dosage period (calculated as DGs 7 to 18) occurred in the 45 and 150 mg/kg bw/day dosage groups.  These effects of the test article were most severe on DGs 7 to 10, when signi6cant weight loss (p≤O.01) occurred in the 150 mg/kg bw/day dosage group.  These two dosage groups had significant increases (p≤O 05 to p≤O.01) in body weight gains and absolute and relative feed consumption values after completion of the dosage period (DGs 18 to 20), rebound phenomena that commonly occur in these types of studies. Despite these rebound phenomena, body weight gains and absolute and relative feed consumption values for the entire period after initiation of dosage (DGs 7 to 20) and for the entire pregnancy (DGs 0 to 20) were reduced or significantly reduced (p≤0.05 to p≤0.01) in the 45 and 150 mg/kg bw/day dosage groups.  Body weights were generally significantly reduced (p≤O.05 to p≤0.01)  on DGs 8 through 20 in the 45 and 150 mg/kg bw/day dosage groups.

Pregnancy incidences were comparable in the four dosage groups. The 150 mg/kg bw/day dosage was associated with increased postimplantation loss [evident as significant increases (p≤O.05) in the litter averages for total and early resorptions, a tendency for increased late resorptions and percentage of resorbed conceptuses per litter, and increased numbers of dams with any resorptions or with all conceptuses dead or resorbed] and significantly reduced (p≤0.01) fetal body weight.  The values for the various parameters identifying postimplantation loss generally exceeded the historical ranges of the Testing Facility but were not sufficiently severe to result in statistically significant or biologically important differences in live litter size.  No dosage-dependent, statistically significant or biologically important differences occurred in the litter averages for corpora lutea, implantations or percent male fetuses. Two 150 mg/kg bw/day dams had litters consisting of only resorbed conceptuses.  There were no dead fetuses.  All placentae appeared normal except those of a 150 mg/kg bw/day dosage group dam that had peripartum bleeding, which appeared pale. All gross external, soft tissue and skeletal malformations and variations in the fetuses were considered unrelated to the test article.

The maternal NOAEL is 15 mg/kg bw/day.  The 45 and 150 mg/kg bw/day dosages were toxic to the dams resulting in adverse clinical observations and reductions in maternal body weight gains, body weights and absolute and relative feed consumption values. The developmental NOAEL is 45 mg/kg/day.  The 150 mg/kg bw/day dosage was associated with increased post-implantation loss (total and early resorptions) and reduced fetal body weight. Based on these data, musk ketone is not selectively toxic to development. Adverse effects on embryo-fetal development (post-implantation loss and reduced fetal body weight) occurred only at the higher of two dosages that were toxic to the dams.

Together with the physiochemical data, this data indicates that moderate oral absorption may be expected. For chemical safety assessment purposes, based on the physicochemical properties and information in the dossier, an oral absorption rate of 50% is accepted.

Absorption – dermal

In a dermal sensitization study (OECD 406/GLP) with musk ketone (≥98%) in acetone/olive oil, young adult Dunkin-Hartley albino guinea pigs were tested in a maximisation test. For induction, 3% w/v musk ketone in acetone/olive oil (intradermal injections) and 75% w/v musk ketone in acetone/olive oil (topical application) was used. For challenge, 7.5, 25 and 75% w/v musk ketone in acetone/olive oil was used for topical application. The evaluation of skin reactions after challenge was carried out at 24 and 48 hrs. The positive control was hexylcinnamaldehyde. The body weight of animals increased during the study and was not affected by the treatment. The positive control, hexylcinnamaldehyde, gave the appropriate response. Following challenge of previously induced guinea pigs with the 75% w/v preparation of the test substance in acetone/olive oil, scattered mild redness was observed in 5 of the 20 animals and on 1 of the 10 control animals. The net percentage was calculated to be 15%. Following challenge of previously induced guinea pigs with the 25% w/v preparation of the test substance in acetone/olive oil, scattered mild redness was observed in 3 of the 20 animals. There was no erythematous response in any of the control animals. The net percentage was calculated to be 15%. Following challenge of previously induced guinea pigs with the 7.5% w/v preparation of the test substance in acetone/olive oil, scattered mild redness was observed in 3 of the 20 animals. There was no erythematous response in any of the control animals. The net percentage was calculated to be 15%. Based on these results, the substance is not sensitising.

In a repeat-dose dermal toxicity study (Equivalent or similar to OECD 411), musk ketone (98%) was applied to the clipped unabraded skin of 15 Sprague-Dawley Crl:CDR(SD) rats/sex/dose at dose levels of 0, 24, 75, 240  mg/kg bw/day, 7 days/week during a 90-day period. One female in the vehicle control group, one male given 24 mg/kg bw/day musk ketone and one male given 240 mg/kg bw/day musk ketone died or were killed before the end of dosing. Apart from variable desquamation, and occasional atony of the skin, there were no significant dermatological changes at the treatment site that could be attributed to the test substanc. Throughout the study, the body weights of males and females given the high dose of musk ketone and of the females given 75 mg/kg/day were significantly lower than those of the vehicle controls, whereas food consumption was similar to or greater than that of the controls. No effects of musk ketone on body weight or food consumption were observed at other doses.

The males and females in the high-dose musk ketone groups showed variations in haematological parameters, notably decreases in haemoglobin, haematocrit, RBC counts and mean corpuscular volume. However, all of the values fell within historical ranges for this strain of rat and were not considered to be of any biological significance. There was no correlation with the observed deaths. There were no effects on clinical chemistry parameters. The livers of females exposed to the high dose of musk ketone were increased in weight, compared with vehicle controls, but this was not associated with any pathological findings. There were no gross changes in any of the organs examined, including the testes, which could be attributed to treatment. In musk ketone groups, microscopic lesions were variable and generally non-specific; there was no correlation with the observed deaths.

Neither during nor at the end of the study were specific treatment-related functional impairments observed that were attributable to musk ketone. There was no evidence of neurotoxicity in any of the rats exposed to musk ketone although detailed neuropathological examination of specially prepared sections of the central and peripheral nervous systems was carried out. The NOAEL for musk ketone in males and females was 75 mg/kg bw/day, based on the relative liver weight changes in females.

Together with the physiochemical data and publications, this data indicates that absorption via the dermal route is very low in humans and low in rats. The ECHA guidance criteria (Chapter R.7C) state that 10% dermal absorption is used when the molecular weight of the substance is >500 and the log Pow is <-1 or >4, otherwise 100% dermal absorption is used. In general, dermal absorption will not be higher than oral absorption, so for chemical safety assessment purposes a dermal absorption rate of 50% is accepted.

Absorption – inhalation

In an acute inhalation toxicity study (16073.417.084.17), a group of young adult Wistar Hannover strain rats (5/sex) were exposed to a test atmosphere of musk ketone (99.87%) for 4 hours (nose only) at a mean actual concentration of 2.99 ± 0.120 mg/L (maximum attainable concentration). Animals were then observed for 14 days. The MMAD was 2.53 - 3.39 μm and GSD was 2.67 - 3.01, indicating that at least 50% of the mass collected from the aerosol generated was within the respirable diameter (< 4 μm).  No compound-related death was registered in this study. No compound-related clinical sign was observed in this study. The mean body weight increased for both sexes in all post-exposure weighing, except in the first post-exposure weighing for the females. All animals exceeded their initial body weight at the end of the observation period of 14 days. No compound-related macroscopic finding was registered in this study. Histopathology was not considered necessary, due to the absence of compound-related clinical sign and macroscopic findings. The LC50 male/female was > 2.99 ± 0.120 mg/L.

Together with the physiochemical data, this data indicates that absorption via inhalation is likely to be low. For chemical safety assessment purposes, an inhalation absorption rate of 100% is accepted, using a conservative approach.

Distribution/Metabolism/Excretion

Based on the information provided from the publications, physicochemical data and in vivo studies, musk ketone is likely to be widely distributed in rats and bioaccumulation is unlikely over long periods of time. Glucuronide conjugates are mainly excreted in the faeces. In humans, most of any absorbed material is excreted in the urine as mainly single glucuronide conjugates.