Diethyl maleate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 March to 12 May 1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1999

Report date:

1999

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Deviations:

no

Test guideline 2

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:

The testing using the guinea pig maximisation test protocol was completed on 12 May 1999. The LLNA OECD Guideline 429 was not formally adopted by the OECD until 22 July 2010.

Test material

Test material information

Constituent 1

Reference substance name:

Diethyl maleate

EC Number:

205-451-9

EC Name:

Diethyl maleate

Cas Number:

141-05-9

Molecular formula:

C8H12O4

IUPAC Name:

diethyl (Z)-but-2-enedioate

Details on test material:

- Name of test material (as cited in study report): Diethylmaleinate
- CAS No.: 141-05-9
- Trade name: DEM
- Physical state: Colourless liquid
- Purity: 99 %
- Lot/batch No.: EMAM 201
- Expiration date of the lot/batch: June 1999
- Storage condition of test material: In the refrigerator, in the dark

In vivo test system

Test animals

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: Approx. 6 weeks at the first application
- Weight at first application: 281 g - 369 g
- Housing: Single caging in Makrolon cages type III (23 cm x 39 cm x 18 cm) with wire mesh lids
- Diet (ad libitum): Altromin Standard Diet No. 3022. Analysis of the feed for ingredients and contaminants are performed randomly by Altromin GmbH, D-32791 Lage.
- Water (ad libitum): Tap water, acidified with HCl to pH = 3, offered in Makrolon bottles with stainless steel canules ad libitum.
- Acclimation period: Approx. 2 weeks.


ENVIRONMENTAL CONDITIONS
- Temperature: Approx. 22 °C
- Humidity: Approx. 60 %.
- Air changes (per hr): 12
- Photoperiod: 12 hrs dark, 12 hrs light

IN-LIFE DATES: From: 24 March 1999 To: 22 April 1999

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

intradermal and epicutaneous

Vehicle:

other: corn oil for intradermal and acetone for challenge exposure

Induction 2

Concentration / amount:

5 % (v/v) in corn oil for the intradermal induction
100 % (undiluted) for the epicutaneous induction and
25 % (v/v) in acetone for the challenge exposure

Challenge

Challenge 1

Route:

epicutaneous, occlusive

Vehicle:

other: corn oil for intradermal and acetone for challenge exposure

Challenge 2

Concentration / amount:

5 % (v/v) in corn oil for the intradermal induction
100 % (undiluted) for the epicutaneous induction and
25 % (v/v) in acetone for the challenge exposure

No. of animals per dose:

20 animals were used for as test substance group and 10 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 for the intradermal injections and in acetone for the epicutaneous administration.

For results see "Any other information on results incl. tables".

MAIN STUDY
A. INDUCTION EXPOSURE
- Day of exposures: Intradermally on Day 1; 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 deionised water (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.

- Concentrations: 5 % (v/v) in corn oil for the intradermal induction, 100 % (undiluted) 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: 25 % 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).

Challenge controls:

A negative control group (10 animals) was tested in parallel.

Positive control substance(s):

yes

Remarks:

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

Results and discussion

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".

In vivo (non-LLNA)

Results

Results 1

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

Test substance 25 % in acetone (v/v)

No. with + reactions:

1

Total no. in group:

10

Clinical observations:

none

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: Test substance 25 % in acetone (v/v). No with. + reactions: 1.0. Total no. in groups: 10.0. Clinical observations: none.

Results 2

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

Test substance 25 % in acetone (v/v)

No. with + reactions:

19

Total no. in group:

20

Clinical observations:

none

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: Test substance 25 % in acetone (v/v). No with. + reactions: 19.0. Total no. in groups: 20.0. Clinical observations: none.

Results 3

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

Test substance 25 % in acetone (v/v)

No. with + reactions:

1

Total no. in group:

10

Clinical observations:

none

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: Test substance 25 % in acetone (v/v). No with. + reactions: 1.0. Total no. in groups: 10.0. Clinical observations: none.

Results 4

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

Test substance 25 % in acetone (v/v)

No. with + reactions:

19

Total no. in group:

20

Clinical observations:

none

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: Test substance 25 % in acetone (v/v). No with. + reactions: 19.0. Total no. in groups: 20.0. Clinical observations: none.

Results 5

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Dose level:

Test substance 10% in acetone (v/v)

No. with + reactions:

7

Total no. in group:

10

Clinical observations:

none

Remarks on result:

positive indication of skin sensitisation

Results 6

Reading:

2nd reading

Hours after challenge:

48

Group:

positive control

Dose level:

Test substance 10 %in acetone (v/v)

No. with + reactions:

7

Total no. in group:

10

Clinical observations:

none

Remarks on result:

positive indication of skin sensitisation

Any other information on results incl. tables

Results (scores) of the preliminary test:

Intradermal exposure:

test substance	scores for animal Nos. *
concentration (w/v)	16	17	18
20.0 %	3/3#	3/3#	3/3#
5.0 %	1/0	3/3#	0/0
1.0 %	0/0	0/0	0/0
0.1 %	0/0	0/0	0/0

*   first numbers / second numbers: scores 24/48 hours after intradermal injection

#   eschars

Epicutaneous exposure:

test substance	scores for animal Nos. *
concentration (w/w)	16	17	18
100 %	2/0	3/1	2/2
50 %	1/0	2/0	1/2
10 %	0/0	0/0	0/0
1 %	0/0	0/0	0/0

* first numbers / second numbers: scores 24/48 hours after the end of the epicutaneous exposure

Results of the main study:

All animals survived till the end of the study.

There were no significant differences in the mean body weights between teh test substance group and the control group on Day 0. A significantly lower mean body weight of the thest substance group animals on Day 24, compared to the controls, was possibly caused by a systemic effect of the test substance.

No abnormal behaviour or clinical signs were detected during the experiment in the animals.

The net rate of animals with positive skin reactions was 85 %.

Applicant's summary and conclusion

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

One out of 10 control animals and 19 out of 20 test animals had a positive skin reaction after the challenge exposure. Diethylmaleinate is therefore considered to be a skin sensitiser.

Executive summary:

Method The "maximisation test" of B. Magnusson and A.M. Kligman was performed to reveal a possible sensitising potential of"DIETHYLMALEINATE". Twenty female guinea pigs were used as a test substance group and another 10 females were used as a negative control group. There were two induction exposures (intradermally and epicutaneously) and one epicutaneous challenge exposure. Test substance concentrations were 5 % (v/v) in corn oil for the intradermal induction, 100 % (undiluted) for the epicutaneous induction and 25 % (v/v) in acetone for the challenge exposure. Investigations performed were in conformance with the OECD-Guideline 406 and with the Directive 96/54/EC, B.6. Application of Freund's complete adjuvant was included in the intradermal exposure of both groups to enhance a possible sensitisation. Occlusive dressings were used for the epicutaneous exposures. Results All animals survived till the end of the study. Sensitisation excluded, no other adverse effects were noted. Skin reactions after the challenge exposure: The control sites of all animals of both groups were normal at each reading time. In the test substance group, positive skin reactions (well defined to severe erythema and/or oedema) were noted in 19/20 animals (95 % of the animals) at the test substance treated areas 24 and/or 48 hours after the end of the challenge exposure. As 1/10 animals of the control group (10 % of the animals) had severe erythema and/or oedema at the test substance treated site, the net rate of animals in the test substance group regarded as sensitised is therefore 85 % (95 % - 10 %).Diethylmaleinate is therefore considered to be a skin sensitiser.