Dibutyl fumarate

Endpoint summary

Description of key information

In the Guinea Pig Maximisation Test, with an intradermal induction of 10%, all test substance group animals (100 %) had positive skin reactions at the test substance treated sites. 1/5 of the control group animals (20 %) also showed a well defined erythema. The control sites of all animals of both groups were normal at each reading time.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 September 2002 to 22 January 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Fully GLP- and guideline compliant study

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Deviations:

no

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

A LLNA study does not need to be conducted because adequate data from an in vivo skin sensitisation study are available.

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS- Source: Harlan Winkelmann, Gartenstrasse 27, D-33178 Borchen.- Age at study initiation: 5 - 7 weeks at the first application- Weight at first application: 290 g - 330 g- Housing: Until Day 0 single caging in Makrolon cages type III (23 cm x 39 cm x 18 cm) with wire mesh lids. From Day 0 group caging in plastic containers (46 cm x 105 cm x 36 cm), partly shaded, 6 (control group) or 11 (test substance group) animals per container.- Diet (ad libitum): Altromin Maintenance Diet No. 3122 offered in stainless steel containers. Analysis of the feed for ingredients and contaminants are performed randomly by Altromin GmbH, D-32791 Lage.- Water (ad libitum): Tap water offered in Makrolon bottles with stainless steel canules ad libitum. - Acclimation period: 5 days.ENVIRONMENTAL CONDITIONS- Temperature: Average of 21.9 °C- Humidity: Average of 49.5 %.- Air changes (per hr): 12- Photoperiod: 12 hrs dark, 12 hrs lightIN-LIFE DATES: From: 7 October 2002 To: 13 December 2002

Route:

intradermal and epicutaneous

Vehicle:

other: corn oil for intradermal and acetone for the epicutaneous induction and challenge exposure

Concentration / amount:

10 % (v/v) in corn oil for the intradermal induction25 % (v/v) in acetone for the epicutaneous induction and10 % (v/v) in acetone for the challenge exposure

Route:

epicutaneous, occlusive

Vehicle:

other: corn oil for intradermal and acetone for the epicutaneous induction and challenge exposure

Concentration / amount:

10 % (v/v) in corn oil for the intradermal induction25 % (v/v) in acetone for the epicutaneous induction and10 % (v/v) in acetone for the challenge exposure

No. of animals per dose:

10 animals were used for as test substance group and 5 animals as negative control group. Spare animals: One additional animal per group was kept and administered under the same conditions as the other animals of the respective group. Findings on the spare animals were only to be incorporated into this report if other animals of the test substance group or of the control group would have died spontaneously. Otherwise, the skin reactions of these animals were not used for the interpretation of the results.

Details on study design:

RANGE FINDING TESTS:To obtain the appropriate concentrations of the test substance for the definitive study, a preliminary test was carried out with 3 female guinea pigs. 4 different concentrations of the test substance and FCA were administered intradermally and 7 days later 4 concentrations of the test substance were administered epicutaneously. The modes of application were the same as in the definitive study. The duration of the epicutaneous exposure was 24 hours.The test substance was dissolved in corn oil (v/v) for the intradermal injections and in acetone (v/v) for the epicutaneous administration. For results see "Any other information on results incl. tables".MAIN STUDYA. INDUCTION EXPOSURE- Day of exposures: Intradermally on Day 0; epicutaneously on Day 7.- Method, intradermally: Hair was clipped. The application site for all injections was an area of about 2 cm x 4 cm in the interscapular region. In each of the injection rows listed in "Any other information on methods and materials incl. tables" two intradermal injections were made side by side. A volume of 0.1 ml was applied for each injection site.- Method, epicutaneously: Test patches (Pur Zellin-Tupfer, obtained by Fa. Hartmann , A-2355 Wiener Neudorf), about 2 cm x 4 cm, soaked with the test substance (test substance group) and with acetone (control group), respectively, were applied to the area of the intradermal injections. They were fixed with a strip of non-irritating tape ("Blenderm*" surgical tape, hypoallergenic, 3M, made in USA, Medical Products Division, St. Paul, MN 551444). The area of administration was then covered occlusively with aluminium foil and finally fixed with "Fixomull* stretch" (self adhesive non woven fabric, hypoallergenic, made by Beiersdorf AG, D-20245 Hamburg). The exposure time was 48 hours.0.5 mL of test substance formulation or 0.5 mL acetone were applied to each animal.- Concentrations: 10 % (v/v) in corn oil for the intradermal induction, 25 % (v/v) in acetone for the epicutaneous induction.B. CHALLENGE EXPOSURE- No. of exposures: 1- Day(s) of challenge: Day 21- Exposure period: 24 hours- Site: left flanks: test substance, right flanks: vehicle- Concentrations: 10 % in acetone- Evaluation: 24 h after the end of the exposure period (Day 23) and a second skin examination further 24 h later (Day 24).0.5 mL of test substance formulation and 0.5 mL of acetone were applied to each animal.

Challenge controls:

A negative control group (5 animals) was tested in parallel.

Positive control substance(s):

yes

Remarks:

HEXYL CINNAMIC ALDEHYDE (HCA), controls tests performed periodically, data attached to the report

Statistics:

- Randomisation: The individual animals were allocated to their groups by random numbers. The order of animals for the evaluation of the skin of test and control animals was randomised. A "blind" evaluation was performed.The t-test was used to evaluate differences of the mean body weights between the test substance group and the control group on Days 0 and 24 (p = 0.05).

Positive control results:

Positive control group:Vehicle site: no positive skin reaction in any animal at any reading time.Substance site: very slight to severe erythema and/or oedema in 7/10 animals 24 and/or 48 hours after the challenge exposure. 7/10 animals had a "positive skin reaction".

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

Test substance 10 % in acetone (v/v)

No. with + reactions:

1

Total no. in group:

5

Clinical observations:

none

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: Test substance 10 % in acetone (v/v). No with. + reactions: 1.0. Total no. in groups: 5.0. Clinical observations: none.

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

Test substance 10 % in acetone (v/v)

No. with + reactions:

10

Total no. in group:

10

Clinical observations:

none

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: Test substance 10 % in acetone (v/v). No with. + reactions: 10.0. Total no. in groups: 10.0. Clinical observations: none.

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

Test substance 10 % in acetone (v/v)

No. with + reactions:

1

Total no. in group:

5

Clinical observations:

none

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: Test substance 10 % in acetone (v/v). No with. + reactions: 1.0. Total no. in groups: 5.0. Clinical observations: none.

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

Test substance 10 % in acetone (v/v)

No. with + reactions:

10

Total no. in group:

10

Clinical observations:

none

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: Test substance 10 % in acetone (v/v). No with. + reactions: 10.0. Total no. in groups: 10.0. Clinical observations: none.

Results (scores) of the preliminary tests:

Intradermal exposure:

test substance	Scores for animal Nos.^a)
concentration (v/v)	10	11	12
25.0 %	3/3	3/3	3/3
10.0 %	2/2	2/2	2/2
1.0 %	0/0	0/0	1/0
0.1 %	0/0	0/0	0/0

^a)first numbers / second numbers: scores 24/48 hours after intradermal injection.

Epicutaneous exposure:

test substance	Scores for animal Nos.^a)
concentration (v/v)	10	11	12
100.0 %	3/3	3/3	3/3
50.0 %	3/3	2/2	3/3
25.0 %	2/2	0/1	2/2
10.0 %	0/0	0/0	0/0

^a)first numbers / second numbers: scores 24/48 hours after the end of the epicutaneous exposure.

Interpretation of results:

sensitising

Remarks:

Migrated information

Conclusions:

In the Guinea Pig Maximisation Test, with an intradermal induction of 10%, all test substance group animals (100 %) had positive skin reactions at the test substance treated sites. 1/5 of the control group animals (20 %) also showed a well defined erythema. The control sites of all animals of both groups were normal at each reading time.

Executive summary:

Method

10 female guinea pigs were used as a test substance group and another 5 females were used as a negative control group. No repetition with another 10 + 5 animals was performed due to positive results (see below). There were two induction exposures (intradermally and epicutaneously) and one epicutaneous challenge exposure.
Concentrations of "DIBUTYLFUMARATE" were:

10 % (v/v) incorn oil for the intradermal induction

    25 % (v/v) in acetone for the epicutaneous induction and

10 % (v/v) in acetone for the challenge exposure.

Investigations performed were in conformance with the Directive 96/54/EC, B.6. and with the OECD-Guideline 406.
Application of Freund's complete adjuvant was included in the intradermal exposure of both groups to enhance a possible sensitisation.
For the epicutaneous exposures occlusive dressings were used.

Results

General

All animals survived till the end of the study.
Intradermal injections of Freund's adjuvant caused severe local reactions in all animals, a known effect of the adjuvant. Sensitisation excluded, no other adverse effects were noted.

Skin reactions after the challenge exposure

The results of these skin examinations were decisive for the grading of the potential of sensitisation.

The control sites of all animals of both groups were normal at each reading time.

24 and/or 48 hours after the end of the challenge exposure, all test substance group animals (100 %) had positive skin reactions at the test substance treated sites. 1/5 of the control group animals (20 %) also showed a well defined erythema.

The potential of the test substance to cause skin sensitisation was established by subtracting the percentage of animals exhibiting an inflammatory reaction in the control group from the percentage of positively reacting test substance group animals. As the rate of animals with positive skin reactions was 20 % in the control group and 100 % in the test substance group, 80 % (100 % - 20 %) of the test substance group animals were regarded as sensitised.

According to the results of this study and to the Directive 2001/59/ECfor classification, the test substance "DIBUTYLFUMARATE" needs to be labelled with "R43 May cause sensitisation by skin contact".

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

Migrated from Short description of key information:
Skin sensitisation, Guinea Pig Maximisation Test, OECD406, EU B. 6: sensitising.

Justification for classification or non-classification

According to positive results of a guinea pig maximisation test the test substance has sensitising potential and has to be classified as "H317 May cause sensitisation by skin contact".