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Dermal absorption

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Administrative data

Endpoint:
dermal absorption in vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Comparable to guideline study with the following acceptable restrictions: leakage of 14C-HQ from the application site due to difficulties in containing the test solution in the application device possibly resulted in contamination of urine, faeces, and cage wash either with unabsorbed 14C or with ingested test chemical. Consequently a slight overestimation of adsorption may be possible based on the study data. Study acceptable for weight of evidence approach.
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Referenceopen allclose all

Reference Type:
publication
Title:
Metabolism and disposition of hydroquinone in Fischer 344 rats after oral or dermal administration.
Author:
English JC, Deisinger PJ
Year:
2005
Bibliographic source:
Food Chem Toxicol 43, 483 - 493
Reference Type:
secondary source
Title:
Unnamed
Year:
1988

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 427 (Skin Absorption: In Vivo Method)
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Hydroquinone, HQ
- Analytical purity: > 99%
- Radiochemical purity (if radiolabelling): 98.9%
- Specific activity (if radiolabelling): 22.2 mCi/mmol
- Locations of the label (if radiolabelling): U-14C-HQ
- Expiration date of radiochemical substance (if radiolabelling): not specified
- Stability under test conditions: stability in aqueous dose solutions determined by HPLC with radiochemical detection and UV absorption was > 99% for at least 48 h at about 30 °C
- Storage condition of test material: not specified
Radiolabelling:
yes
Remarks:
[U-14C]-hydroquinone

Test animals

Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Wilmington, MA, USA
- Age at study initiation: 7-9 w
- Weight at study initiation: males 149 - 178 g, females 117 - 136 g
- Fasting period before study: no
- Individual metabolism cages: yes, glass metabolism chambers with efficient urine-faeces separator
- Diet: Agway Prolab RMH 3000 or 3200, ad libitum
- Water: ad libitum
- Acclimation period: at lest 3 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23-27
- Humidity (%): 31-70
- Air changes (per hr): dehumdified air was drawn through the glass metabolism chambers at a rate of approximately 500 mL/min
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Type of coverage:
occlusive
Vehicle:
water
Duration of exposure:
24 h
Doses:
- Nominal doses: 25, 150 mg/kg bw
- Dose volume: low dose 58 - 86 µL, high dose 338 - 497 µL as 5.4% (w/v) solution
- Rationale for dose selection: selected by Chemical Manufacturers Association (CMA) Hydroquinone Program Panel based on a previous subchronic toxicity study
No. of animals per group:
8 males and 8 females
Details on study design:
DOSE PREPARATION
- Method for preparation of dose suspensions: radiolabeled dose solutions prepared in degassed, distilled water at a concentration of 5.4% HQ (w/v) to contain nominally 20 µCi per rat
- Method of storage: no data

APPLICATION OF DOSE: with a hypodermic syringe fitted with a blunt-ended needle, through the plastic film to the skin surface inside the columnator (see below)

TEST SITE
- Preparation of test site: Dorsal skin area was shaved 24 h before administration of test substance. Immediately prior to administration, annular columnators for the test chemical administration (PTFE polymer flat rings, outer diameter 2.9 cm, inner diameter 1.6 cm, height 0.3 cm) were secured to the shaved skin with VetBond adhesive tissue. A plastic film was adhered to the upper surface of the columnator with Duro Quick Gel. After application of the test substance the plastic film was sealed with a small amount of Duro Quick Gel.
- Area of exposure: ca. 2 cm x cm
- % coverage: no data
- Type of cover / wrap if used: foil-lined Elastoplast bandaging tape

SITE PROTECTION: yes, occlusive bandage

REMOVAL OF TEST SUBSTANCE
- Removal of protecting device: Rats were transferred to a glass tray and the occlusive bandage and the plastic film. Any test chemical solution remaining at the application site was removed by aspiration with a plastic tipped pipette. Rat hair that appeared to be stained by the test chemical was clipped and reserved.
- Washing procedures and type of cleansing agent: The application site was washed sequentially with 19 cotton swabs that had been saturated with a 40% solution of pHisoDerm in warm water. If areas surrounding the application site appeared stained by the test substance, these regions were also washed. Therafter, the application site was rinsed five times by filling the columnator with distilled water and then emptying it by aspiration with a plastic-tipped pipette. The application site was then dried with a cotton swab. Columnators remained adhered until termination of the study.
- Time after start of exposure: 24 h

SAMPLE COLLECTION: Animals were placed in individual glass metabolism chambers during exposure (24 h) and for further 144 h (7 d in total).
- Collection of blood: N= 4 animals at 0.5, 1, 8, 24, 48, and 96 h, samples were obtained from the orbital sinus
- Collection of urine: N=4 animals at 8 and 24 h, and thereafter at 24 h intervals; urine was sampled from metabolism chambers, and rinses of the metabolism chambers and of the glass trays (used for removal of occlusive bandage). The ventral half of the occlusive bandage was also extracted using these rinsings, so that any urine absorbed to the bandage would be liberated. Final rinse of the metabolism chamber with acetone.
and faeces: ventral half of bandage
- Collection of faeces: N=4 animals at 8 and 24 h, and thereafter at 24 h intervals
- Collection of expired air: no
- Terminal procedure: sacrifice after 7 days by carbon dioxide inhalation
- Analysis of organs: liver, kidney, lungs, spleen, heart, brain, testes and ovaries; samples of dorsal skin from the treated and a remote site, bone, abdominal fat, femoral muscle, carcass
- Analysis of columnators: together with carcass if they adhered until sacrifice; detached or fragmented columnators analyzed were they appeared usually with the faeces or chamber rinsings
- Analysis of unadsorbed test substance:
Residual test chemical solution aspired in the plastic pipette tip was diluted in water and analyzed by LSS. Stained rat hair, the dorsal half of the foil-lined bandage, the plastic film and the plastic pipette tip were extracted with methanol. Cotton swabs and skin rinsings were combined and extracted with methanol. All methanol extracts were analyzed by LSS.

SAMPLE PREPARATION
- Storage procedure:
Urine: immediate analysis by LSS, frozen storage for analysis of metabolites
Blood: immediate analysis by LSS
Faeces and tissues: frozen storage
- Preparation details:
Urine and blood: direct use for LSS
Faeces: homogenization with water, oxidative combustion of aliquots, analysis of 14C by LSS with Permafluor and Carbosorb as scintillator fluid
Tissues: tissue samples and small organs were subjected to oxidative combustion as a whole; larger organs or tissues were homogenized and aliquots were subjected to oxidative combustion for analysis of 14C by LSS with Permafluor and Carbosorb as scintillator fluid
Femurs: chemically digested and dicolorized with perchloro acid and hydrogen peroxide followed by LSS
Carcass: ground in a blender with acetone and refluxed for 24 h; acetone extracts used for LSS

ANALYSIS
- Method type(s) for identification: 14C by liquid scintillation spectroscopy (LSS); metabolites in urine by HPLC-MS, HPLC-radioactivity monitor
- Liquid scintillation counting results (cpm) converted to dpm as follows:
- Validation of analytical procedure:
- Limits of detection and quantification:

CLEAVAGE OF CONJUGATES:
Urine samples were hydrolized with glucuronidase, sulfatase, or hydrochloric acid.

OTHER: Dermal washing efficiency study:
- Exposure: 5 min-exposure with conditions as in the main study. Test chemical solution remaining at the application site was removed by aspiration with a plastic tipped pipette.
- Analysis of unadsorbed test substance:
Residual test chemical solution aspired in the plastic pipette tip was diluted in water and analyzed by LSS.
- Washing procedures and type of cleansing agent: Two types of cleansing agents were compared, a 40% solution of pHisoDerm in warm water, or satured ethanol. The application site was washed sequentially with 19 cotton swabs that had been saturated with the cleansing agent. If areas surrounding the application site appeared stained by the test substance, these regions were also washed. Therafter, the application site was rinsed five times by filling the columnator with distilled water and then emptying it by aspiration with a plastic-tipped pipette. The application site was then dried with a cotton swab. Pooled rinses, the plastic tip and the cotton swab were analyzed by LSS.
- Quantitation:

Results and discussion

Signs and symptoms of toxicity:
no effects
Dermal irritation:
yes
Remarks:
slight to severe erythema after 24 h-exposure at both dose levels
Absorption in different matrices:
14C as % of applied dose for the following dose groups: 25 mg/kg: males, females; 150 mg/kg: males, females
- Skin wash (24 h): 61, 71, 67, 62 %
- Skin test site (168 h): 0.53, 0.42, 2.20, 0.14 % corresponding to 326799, 370172, 1550211, 136212 DPM/g tissue
- Skin, untreated site (168 h): 5422, 14146, 4342, 12304 DPM/g tissue
- Blood: < or near LOQ (ca. 0.01% of applied dose or 0.06 µg equivalents HQ/g blood)
- Tissues and carcass (168 h): 10, 2.5, 5.4, 13 %
- Urine * (168 h): 11.0, 11.7, 7.8, 12.8%; with 4-8 % as HQ glucuronide, < 2% as HQ sulfate, 0.4-1.5% as unchanged HQ
- Cage wash + cage wipe * (168 h): 3.8, 5.9, 8.9, 4.4%
- Faeces * (168 h): 3.7, 2.4, 1.7, 2.8%
* Due to difficulties in containing the test solution in the columnators for a protracted period of time, 14C in urine, faeces, and cage wash may have arisen from the test chemical not contained at the intended exposure site, or ingested test chemical, as well as dermally absorbed test chemical
Total recovery:
- Total recovery: 90.1 to 94.6%
- Recovery of applied dose acceptable: within limits of OECD Guideline 427
- Results adjusted for incomplete recovery of the applied dose: no
- Limit of detection (LOD): twice the background (values not specified)

Any other information on results incl. tables

Dermal washing efficiency study:

Complete recovery (98.4 to 100.9%) by either the soapy water or ethanol skin washing procedure. Soapy water was selected as washing with ethanol induced slight erythema.

Main study:

Observations and technical difficulties:

Hydroquinone solution was not always totally contained by the columnator, as evidenced by brown staining of the hair outside the exposure site. Consequently the amount of 14C recovered by washing the exposure site at 24 h varied greatly among individual animals irrespective of sex or treatment level, ranging from 98.5 to 23.1% of administered dose. In some cases the brown staining outside the exposure site became apparent only several days after the washing procedure was performed, so that the stained areas which are contaminated with the test substance were processed with the carcasses.

Blood levels:

< or near LOQ (ca. 0.01% of applied dose or 0.06 µg equivalents HQ/g blood) at all timepoints in male and female rats except for 25 mg/kg bw female rats at 0.5 hr timepoint (0.47 µg equivalents HQ/g blood) and 150 mg/kg bw female rats at 1 hr timepoint (1.13 µg equivalents HQ/g blood)

Detection of metabolites in urine:

4-8 % as HQ glucuronide, < 2% as HQ sulfate, 0.4-1.5% as unchanged HQ; occasional detection of mercapturic acid conjugate and benzoquinone

Estimation of systemic bioavailability:

Estimation based on ratio of the urinary excretion of14C after dermal and oral administration of 14C-HQ using the 72-h14C recovery data (data from same study, see separate endpoint study record in Section 7.1.1):

Dermal absorption in male rats:  (9.64/91.67) x 100% = 10.5%

Dermal absorption in female rats:  (10.31/90.00) x 100% = 11.5% Table 1: Cumulative percentage of the dermally applied radioactivity recovered from F344 rats treated with 14C-HQ for 24 h

Dose group

Sampling timepoint (h)

Recoveries (% of applied dose)

24 h skin wash

Cage rinsesd

Urine

Faeces

Exposed skin site

Other tissues and carcass

Total

25 mg/kg – malesa

8

0.62 ± 0.13

1.56 ± 0.48

0.01 ± 0.01

24

60.75 ± 17.71

1.72 ± 0.29

3.93 ± 0.48

0.17 ± 0.19

48

2.48 ± 0.30

8.38 ± 2.70

1.11 ± 0.33

72

2.94 ± 0.42

9.64 ± 3.41

2.11± 0.95

120

3.53 ± 0.47

10.72 ±  3.99

3.40  ± 1.78

168

3.80 ± 0.55

10.98 ± 4.05

3.73 ± 1.97

0.53 ± 0.22

10.27 ± 6.64

90.06 ± 6.92

25 mg/kg – femalesb

8

1.62 ± 0.91

2.69 ± 1.03

0.08 ± 0.06

24

71.05 ± 11.43

4.52 ± 2.86

8.35 ± 3.29

0.26 ± 0.08

48

5.04 ± 2.91

10.28 ± 3.41

0.69 ± 0.27

72

5.48 ± 2.91

10.94 ± 3.24

1.17 ± 0.72

120

5.78 ± 2.91

11.52 ± 3.10

2.03 ± 1.32

168

5.93 ±  2.88

11.72 ± 3.07

2.35 ± 1.47

0.42 ± 0.29

2.57 ± 1.95

94.04 ± 5.67

150 mg/kg – malesa

8

1.13 ± 1.16

0.66 ± 0.41

0.02 ± 0.03

24

66.52 ± 5.15

5.90 ± 3.11

2.34 ± 0.81

0.17 ± 0.09

48

7.36 ± 2.50

5.44 ± 2.73

0.54 ± 0.16

72

7.93 ± 2.40

6.25 ± 3.00

0.86 ± 0.24

120

8.42 ± 2.37

6.99 ±  3.55

1.24 ± 0.31

168

8.86 ± 2.40

7.76 ± 4.01

1.69 ± 0.36

2.20 ± 1.28

5.42 ± 1.49

92.46 ± 2.65

150 mg/kg – femalesa

8

0.88 ± 0.61

1.89 ± 0.63

0.12 ± 0.14

24

61.53 ± 23.27

2.48 ± 0.55

5.42 ± 1.56

0.33 ± 0.13

48

3.06 ± 0.56

9.72 ± 2.03

0.86± 0.42

72

3.44 ± 0.75

10.67 ± 2.54

1.22 ± 0.53

120

3.90 ± 0.91

12.08 ± 2.78

2.19 ± 0.94

168

4.44 ± 1.25

12.76 ± 3.21

2.84 ± 1.37

0.14 ± 0.04

12.86 ± 15.82c

94.56 ± 3.24

Animal numbers evaluable:a N=4 each, b N=3 each

c without levels of ovaries, muscle and fat samples, which were lost during an oxidizer malfunction. Due to the low levels of radioactivity in similar tissues, the effect of these losses on radioactivity recovery is believed to be negligible.

d including tray rinses from removal of occlusive bandage; assumed not to have derived principally from urinary radioactitvity

Table 2:  Radioactivity in selected tissues d and the carcass of F344 rats at 168 h after a 24-hr dermal exposure (mean ± SD)

Tissue

Dose groups

25 mg/kg – malesa

25 mg/kg – femalesb

150 mg/kg – malesa

150 mg/kg – femalesa

% of dose

DPM/g tissue

% of dose

DPM/g tissue

% of dose

DPM/g tissue

% of dose

DPM/g tissue

Skin, test site

0.53 ± 0.22

326799

0.42 ± 0.29

370172

2.2 ± 1.3

1550211

0.14 ± 0.038

136212

Skin, sample

n.a.

5422

n.a.

14146

n.a.

4342

n.a.

12304

Carcass

10 ± 6.6

35234

2.5 ± 1.9

11768

5.4 ± 1.5

17885

13 ± 16

60041

Fat, sample

n.a.

7576

n.a.

4260

n.a.

5891

-

-c

Liver

0.049 ± 0.070

2148

0.018 ± 0.0058

1188

0.012 ± 0.012

649

0.023 ± 0.011

1568

Kidneys

0.0061 ± 0.0012

1650

0.0074 ± 0.0020

2431

0.0049 ± 0.0037

1439

0.0070 ± 0.0032

2459

Spleen

0.00071 ± 0.00083

908

0.00047 ± 0.00012

594

-

< LOQ

0.0018 ± 0.0025

2905

Lungs

0.0016 ± 0.0015

797

0.0013 ± 0.00040

801

-

< LOQ

0.00084 ± 0.00025

522

Heart

0.00094 ± 0.00056

665

0.00076 ± 0.00013

721

-

< LOQ

-

< LOQ

Bone, sample

n.a.

593

n.a.

629

n.a.

857

n.a.

1879

Muscle, sample

n.a.

523

n.a.

857

n.a.

417

-

-c

Brain

0.0012 ± 0.00024

380

0.0023 ± 0.0012

942

0.00082 ± 0.00028

266

0.0020 ± 0.00098

683

Testes

-

< LOQ

-

< LOQ

-

-

Ovaries

-

-

0.00023 ± 0.000074

863

-

-

-

-c

           

LOQ : limit of quantification, estimated to be twice the background; 

n.a. : not applicable is this is a sample not a whole tissue

Animal numbers evaluable:a N=4 each, bN=3 each

c tissue was lost due to an instrumental malfunction during sample processing

d Variations in tissue concentrations in some animals were discussed to be related to leakage of HQ from the exposure site into the adjacent area resulting in contamination of tissues during the sampling procedure.

Applicant's summary and conclusion

Conclusions:
The low percent of applied dose recovered from urine and faeces (9 to 16%), and the low amount observed in the blood, in spite of the dermal irritation produced by HQ, indicate that HQ is not readily absorbed percutaneously. The systemic absorption was estimated to account to 11% based on a comparison of urinary excretion after oral and dermal absorption.
Executive summary:

Groups of male and female F344 rats were exposed to14C-labelled HQ (5.4% aqueous solution) at doses of 25 or 150 mg/kg bw applied to a skin area of ca. 2 cm2 to study dermal absorption. The test protocol was comparable to OECD Guideline 427. Columnators were used to contain the test solution (volumes of 60 or 500 µL) at the application site during the 24-h exposure period. Occlusive bandages were used to prevent ingestion of test substance. However, due to technical difficulties with the fixation of the columnators at the test site, leakage of HQ from the exposure site into the adjacent area occurred in some animals. This may have resulted in direct contamination of urine and faeces in the metabolism chambers and of chamber washes: On the other side unintended oral uptake of HQ may have occurred affecting recovery of14C in tissues, urine and faeces. Total recovery of14C was calculated based on recoveries in unabsorbed test solution, skin washes and occlusive bandages after the 24-h exposure period, on cumulative recoveries in urine, faeces and chamber rinses measured repeatedly for 7 days, and on recoveries in tissues, carcass and in the exposed skin site at 168 h after application of the test substance. Blood levels were determined for up to 96 h p.a.. Urine samples were hydrolized with glucuronidase, sulfatase, or hydrochloric acid before identification of excretion products by HPLC-MS.

Most of the applied14C-HQ (61-71%) was recovered from the skin surface at 24 h. Cumulative recoveries of14C by 168 h after application amounted to 7.8 to 12.8% of the material in urine, 3.8 to 8.9% in the chamber rinsings, and 1.7 to 3.7% in faeces. From 0.14 to 2.2% was found in the excised skin at the exposure site, and 2.6 to 12.9% was associated with the tissues and the carcass. Blood levels were found to be below or near the limit of quantification (ca. 0.01% of applied dose or 0.06 µg equivalents HQ/g blood) at all timepoints in male and female rats except for 25 mg/kg bw female rats at the 0.5 hr timepoint (0.47 µg equivalents HQ/g blood) and 150 mg/kg bw female rats at the 1 hr timepoint (1.13 µg equivalents HQ/g blood). Total recoveries were in the range of 90.1 to 94.6% being within the limits of OECD Guideline 427. In urine,4-8 % of the dose accounted to HQ glucuronide, < 2% to HQ sulfate, and 0.4-1.5% to unchanged HQ, mercapturic acid conjugate and benzoquinone were occasionally detected. 

The low percent of applied dose recovered from urine and faeces (9 to 16%), and the low amount observed in the blood, in spite of the dermal irritation produced by HQ, indicate that HQ is not readily absorbed percutaneously. The systemic absorption was estimated to account to 11% based on a comparison of urinary excretion after oral and dermal absorption (data from the same study). This value may represent a slight overestimation of the actual dermal absorption as contamination of urine may have occurred due to leakage of test substance from the dermal application device.