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

Dermal absorption

Administrative data

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

Data source

Reference

Reference Type:

publication

Title:

Dermal absorption and disposition of musk ambrette, musk ketone and musk xylene in rats.

Author:

Hawkins and Ford

Year:

1999

Bibliographic source:

Toxicology Letters 111, 95–103

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 427 (Skin Absorption: In Vivo Method)

GLP compliance:

not specified

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

201-328-9

EC Name:

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

Cas Number:

81-14-1

Molecular formula:

C14H18N2O5

IUPAC Name:

1-(4-tert-butyl-2,6-dimethyl-3,5-dinitrophenyl)ethanone

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

Test animals

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

Administration / exposure

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.

Results and discussion

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.

Percutaneous absorption

Time point:

120 h

Dose:

0.5 mg/kg

Parameter:

percentage

Absorption:

31.1 %

Remarks on result:

other: Acute study

Applicant's summary and conclusion

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.