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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1990
Report date:
1990

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
other: Mouse Ear Swelling Test (MEST)
Principles of method if other than guideline:
The objective of this study was to determine the sensitizing potential of isobutyraldehyde when applied dermally to female B6C3F1 mice. Thereby, measurement of the contact hypersensitivity was accomplished by the radioisotopic assay and the mouse ear swelling test (MEST) .
GLP compliance:
yes
Type of study:
mouse ear swelling test
Justification for non-LLNA method:
Reliable data was available. Study predates the adoption of the LLNA as OECD guideline.

Test material

Constituent 1
Chemical structure
Reference substance name:
Isobutyraldehyde
EC Number:
201-149-6
EC Name:
Isobutyraldehyde
Cas Number:
78-84-2
Molecular formula:
C4H8O
IUPAC Name:
2-methylpropanal
Details on test material:
- Name of test material (as cited in study report): Isobutyraldehyde (IBA)
- Storage condition of test material: - 20 °C

In vivo test system

Test animals

Species:
mouse
Strain:
B6C3F1
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Frederick Cancer Research Center or Taconic Farms
- Age at study initiation: 8 weeks of age
- Weight at study initiation: 19.9 g and 24.9 g
- Housing: 4 animals per cage
- Diet (e.g. ad libitum): Ziegler Rat and Mouse Ration (NIH 031), ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: at least one week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-24 °C
- Humidity (%): 40-68%
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 h / 12 h

Study design: in vivo (non-LLNA)

Induction
Route:
epicutaneous, open
Vehicle:
other: 4 parts acetone to one part olive oil (4:1)
Concentration / amount:
3, 10, 30 %
Challenge
No.:
#1
Route:
epicutaneous, open
Vehicle:
other: 4 parts acetone to one part olive oil (4:1)
Concentration / amount:
30%
No. of animals per dose:
8
Details on study design:
Mice received 20 µL by direct dermal application for 5 consecutive days to a prepared site. DNFB (1-fluoro-2,4-dinitrobenzene) (98%) was used as a positive control at a concentration of 0.5%. Measurement of the contact hypersensitivity was accomplished by the radioisotopic assay and the mouse ear swelling test (MEST).
Half the groups received Freund's Complete Adjuvant as part of the site preparation.

Group induction challenge adjuvant
1 vehicle vehicle y
2 vehicle 30% Isobutyraldehyde y
3 3% Isobutyraldehyde 30% Isobutyraldehyde y
4 10% Isobutyraldehyde 30% Isobutyraldehyde y
5 30% Isobutyraldehyde 30% Isobutyraldehyde y
6 DNFB DNFB n
7 vehicle DNFB n
8 vehicle 30% Isobutyraldehyde n
9 10% Isobutyraldehyde 30% Isobutyraldehyde n
10 30% Isobutyraldehyde 30% Isobutyraldehyde n


RANGE FINDING TESTS (Primary irritation assay):
A primary irritancy study of the potential sensitizer was performed in order to establish the concentrations for induction (sensitization) and challenge. Isobutyraldehyde was tested in the concentrations of 30, 10, 3.0, 1.0, 0.3 and 0.1%. A 20 µl aliquot of each of the concentrations of isobutyraldehyde or vehicle was administered to the prepared dorsal surface (shaved and dermabraded) of each mouse (4 mice/concentration). This treatment was repeated for five days. Mice were then rested for seven days. During this time mice were visually examined for signs of irritation or toxicity. Following the rest period 20 µl of the appropriate isobutyraldehyde concentration or vehicle was administered to the dorsal surface of the left ear of each mouse. For all mice, the right ear was untreated. These groups were compared to another group of four mice which were shaved and dermabraded but received no other treatment. 30 minutes before administration of the test article or the vehicle, all mice were injected (i.v.) with 0.2 ml of 125 I-bovine serum albumin (1.0 ml, 1 mCi/ml). Four hours after the administration of the test article or vehicle, all mice were sacrificed and ear biopsies taken and placed in counting tubes. All samples were counted in a Gamma Counter. Results were expressed as an irritancy index (I.I.). Thereby, the mean background (Bg) was first subtracted from all values. The left (treated) to right (untreated) biopsy fragment ratio was calculated by dividing the counts per minute in the left biopsy by the counts per minute in the right biopsy, and then the mean ratio for the naive group was subtracted. The resultant number was the irritancy index. A mean I.I. ± S.E. was determined for each concentration of the test article. The minimal irritating concentration (M.I.C.) was defined as the lowest concentration producing an I.I. significantly different from vehicle control. The maximal non-irritating concentration (M.N.C.) was defined as the highest concentration producing an I.I . not significantly different than control. No concentration of isobutyraldehyde could be considered the M.I.C., since none of the I.I. calculated was significantly greater than the vehicle control.

MAIN STUDY
No concentration of isobutyraldehyde could be considered the M.I.C., since none of the I.I. calculated was significantly greater than the vehicle control. Visual evaluation also indicated that no concentration tested was significantly irritating. Therefore, 30%, 10% and 3% isobutyraldehyde for treatment and 30% isobutyraldehyde were used for challenge in the contact hypersensitivity study.

Mice were sensitized on dorsal sites prepared by shaving and dermabrading and in appropriate mice i.d. administration of Freund's Complete Adjuvant (an emulsion of 50% adjuvant and 50% sterile water). All other reagents were administered by delivery of 20 µl directly to the prepared site with a Finn pipette. Induction treatment was repeated on 5 consecutive days followed by a on week rest period, but dermabrasion was only performed on every other day.
Challenge occurred at the dorsal side and ventral sides of the left ear; the right ear remained untreated. 24 hours before the challenge, 125 I-IUDR (0.2 ml, 10 uCi/ml) were injected i.v. into the tail vein of all mice. On the first and second day following challenge mice were examined for a contact hypersensitivity response by the mouse ear swelling test (MEST) based on the method of Gad et al . (Toxicol. and Appl. Pharmacol., 84:93, 1986). Thereby, the thickness of the challenged left ear and untreated right ear of each mouse was measured. The mean ear thickness of the treated mice was compared to the mean ear thickness of the control mice and the percent ear swelling for each mouse was calculated. Following the MEST on day 2 after challenge the mice were sacrificed by CO2 exposure and cervical dislocation, and the challenged and untreated ears were biopsied and counted in a gamma counter. Hypersensitivity indexes (H.I.) were calculated as described above.
Challenge controls:
yes
Positive control substance(s):
yes
Remarks:
DNFB (1-fluoro-2,4-dinitrobenzene) (98%) was used as a positive control (0.5%)

Results and discussion

Positive control results:
HI for DNFB was 0.8 compared to 0.2 in control mice (p < 0.01%)

In vivo (non-LLNA)

Results
Key result
Reading:
other: Ear Swelling and cell count
Hours after challenge:
48
Group:
test chemical
Dose level:
all
No. with + reactions:
0
Total no. in group:
8
Remarks on result:
no indication of skin sensitisation

Any other information on results incl. tables

Mouse ear swelling test (MEST)

No statistically significant dose-dependent contact hypersensitivity response to isobutyraldehyde was demonstrated in mice when the site of sensitization was prepared using shaving and dermabrasion with or without adjuvant.

PRIMARY IRRITANCY RESPONSE

The primary irritancy response, using the extravasation of 125 -I-bovine serum albumin into the exposed area, demonstrated that concentrations as high as 30% were not significantly irritating.

CONTACT HYPERSENSITIVITY STUDY

No hypersensitivity response was elicited in mice using sensitizing concentrations of up to and including 30% and challenging concentrations of 30%. No response was significantly different from vehicle control and there was no indication of a dose response.

The positive control DNFB induced a positive response. The H.I. for DNFB was 0.8 as compared to the control of 0.2. The % Ear Swelling for DNFB was 183.5 as compared to the control of 127.6 on the first day after challenge, and 163.8 as compared to 119.5 for the control on the second day after challenge.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Conclusions:
No statistically significant dose-dependent contact hypersensitivity response to isobutyraldehyde was demonstrated in mice when the site of sensitization was prepared using shaving and dermabrasion with or without adjuvant.