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Toxicological information

Dermal absorption

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Administrative data

Endpoint:
dermal absorption in vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: study meets basic scientific principles with the following restrictions: documentation limited; low animal numbers

Data source

Reference
Reference Type:
publication
Title:
The influence of limonene on induced delayed hypersensitivity to citral in Guinea Pigs. II. Label distribution in the skin of 14C-labelled citral
Author:
Barbier P, Benezra C
Year:
1983
Bibliographic source:
Acta Dermatovener (Stockholm) 63, 93-96

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
Distribution of 14C in different layers of skin and in urine and faeces investigated by liquid scintillation counting
GLP compliance:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): citral and [1,2-14C]-citral
- Analytical purity: not specified
- Specific activity (if radiolabelling): 83 µCi/mg
- Locations of the label (if radiolabelling): C1 and C2
Radiolabelling:
yes
Remarks:
[1,2-14C]-citral

Test animals

Species:
guinea pig
Strain:
Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Versault, Luisetaines, France
- Weight at study initiation: 300-350 g

PRETREATMENT: as the study was designed to investigate the role of dermal absorption in elicitation of hypersensitivity, groups of guinea pigs were used that had been subjected to different induction procedures by applying the Freund Complete Adjuvant (FCA)Test:
Group A - citral treatment: 0.5 mL citral was dissolved in 4.75 mL FCA and then emulsioned with 4.75 mL saline, using a syringe. Each animal received intradermally 5 injections of 0.1 mL each, on alternate days.
Group B - FCA-treated control: 4.75 mL FCA was emulsioned with 4.75 mL saline, using a syringe. Each animal received intradermally 5 injections of 0.1 mL each, on alternate days.
Group C - untreated control: no pretreatment procedure
All groups: This induction procedure was followed by a 2-week rest, before elicitation (see below) was performed.

Administration / exposure

Type of coverage:
open
Vehicle:
ethanol
Duration of exposure:
single
Doses:
- Nominal doses: 1.88 mg/animal, ca. 63 µg/cm2 skin area
- Dose volume: 188 µl of a 0.5% solution per area, 2 areas in total
- Rationale for dose selection: no data
No. of animals per group:
1
Details on study design:
VEHICLE
- Justification for use and choice of vehicle (if other than water): ethanol due to solubility
- Amount(s) applied (volume or weight with unit): 2 x 188 µL per animal
- Concentration (if solution): 0.5%

TEST SITE
- Preparation of test site: shaving
- Area of exposure: circular 15 cm2 area on each flank

SITE PROTECTION / USE OF RESTRAINERS FOR PREVENTING INGESTION: no

SAMPLE COLLECTION
- Collection of urine and faeces: during 16 hr via a metabolism cage
- Terminal procedure: 16 hr p.a.
- Analysis of organs: skin

SAMPLE PREPARATION
- Storage procedure:
- Preparation details:
Step 1: Unresorbed citral: Skin was collected and wiped with a cotton swab containing methanol to remove unresorbed citral. Citral was extracted with toluene.
Step 2: Total skin: The skin was stretched on a board in such a way as to cover its initial area. The 15 cm2 marked area was punched with drills. Radioactivity was recovered from the punched skin areas and the used drills by extraction with soluene (60 °C for 4 hrs).
Step 3: Preparation of stratum corneum: The skin was stripped with a cellulose tape (15 strips) until the wet layer appeared. The tapes were extracted with toluene and radioactivity was measured.
Step 4: Stripped skin: radioactivity was extracted from the skin depleted from the stratum corneum in step 3
Step 5: Soluble skin before dialysis: remaining skin was cut into small pieces (area < 1 cm2) dipped in liquid nitrogen and blended. The preparation was stirred with phosphate buffer in cold room (5 °C) for 40 hrs. Then the buffer was centrifuged and filtered and radioactivity in the buffer was measured.
Step 6: Insoluble skin protein extracts: insoluble portion from step 5 homogenized with a spatula, aliquots were dissolved in soluene and heated for 4 hrs at 60 °C.
Step 7: Dialysate: the filtered buffer from step 5 was placed in dialysis bags (10,000 daltons exclusion) and dialyzed against deionized water. 14C in the dialysate was counted.
Step 8: Precipitate formed during dialysis in the dialysate, probably corresponding to some protein denaturation
Step 9: Unfiltered dialysate: the content of the dialysis bag from step 7 was counted.
Urine: direct measurement by dissolution in aquasol.
Faeces: After grounding dissolution in soluene and left at 60 °C for a few hrs. Aliquots of solution dissolved in mixture of aquasol/soluene.

ANALYSIS
- Method type(s) for identification: Liquid scintillation counting

Results and discussion

Signs and symptoms of toxicity:
not examined
Dermal irritation:
not examined
Absorption in different matrices:
See table
Total recovery:
Total recovery from urine and feces, total skin and unresorbed citral at the skin surface: 42.1-47.7%

Any other information on results incl. tables

The total recovery of radioactivity from the excreta urine and feces, from total skin and from unresorbed citral at the skin surface was 42.1% in a guinea pig without pre-treatment (C) and 47.7% in a guinea pig that had been subjected to an induction treatment with citral (A). Amounts evaporated from the site of application, excreted via exhalation of 14CO2, or deposited in body tissues were not recorded in this study. The amounts absorbed into the skin within 16 hrs p.a. were 23.9% (C) and 27.5% (A). The amount of 14C in the stratum corneum was comparable in all groups (10.0-12.2 %). There was a greater variation in the penetration to deeper skin layers between the single animals (6.4-10.5%). However, the 14C-recovery after stripping the stratum corneum from the deeper skin layers was incomplete compared to 14C-acitivity in total skin. Additionally, the values were recorded from single animals only, so that the values may represent individual variation.

Table: Label distribution (% of initial deposit) of14C-citral in sensitized guinea pigs and controls

Test group (1 animal/group)

A

B

C

Sensitization: induction treatment

citral

FCA-treated control

Untreated control

Elicitation

0.5% citral

0.5% citral

0.5% citral

Excreta

Feces

0.3

0.1

0.4

Urine

11.6

14.1

11.7

Skin samples

Skin sample preparation step

Unresorbed citral (cotton swab)

1

8.3

7.1

6.1

Total skin

2

27.5

17.1

23.9

Stratum corneum

3

12.2

10.0

10.8

Stripped skin

4

10.5

7.2

6.4

Soluble skin before dialysis

5

5.1

3.3

3.5

Insoluble skin

6

5.3

3.2

2.5

Dialysate

7

4.6

3.0

2.4

Precipitate

8

0.1

0.2

0.4

Unfiltered dialysate

9

0.6

0.8

0.6

Ratio soluble to insoluble proteins

10.2

20

19.3

Total 14C recovered

47.7

38.4

42.1

FCA: Freund's complete adjuvant

Ratio soluble to insoluble proteins =100x [Step 9]/[Step9] + [Step6]

Applicant's summary and conclusion