Diethyl acetylsuccinate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 February - 4 May 1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study according to GLP

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:

test was performed in 1999.

Test material

Test material information

Constituent 1

Reference substance name:

Diethyl acetylsuccinate

EC Number:

214-223-8

EC Name:

Diethyl acetylsuccinate

Cas Number:

1115-30-6

Molecular formula:

C10H16O5

IUPAC Name:

1,4-diethyl 2-acetylbutanedioate

Details on test material:

- Name of test material (as cited in study report): "Diethylacetylsuccinate"- Physical state: Bright yellow liquid.- Lot/batch No.: SLBE 101- Solubility i nwater: ca. 15g/L- Melting point: -8 °C- Boiling point: 180 °C/67 mbar- Density: 1.081- Expiration date of the lot/batch: December 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: 348 g - 459 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

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

intradermal and epicutaneous

Vehicle:

other: corn oil/acetone (9+1, v/v) for intradermal induction exposure

Induction 2

Concentration / amount:

5 % (v/v) in corn oil/acetone (9+1, v/v) for the intradermal induction100 % (undiluted) for the epicutaneous induction and100 % (undiluted) for the challenge exposure

Challenge

Challenge 1

Route:

epicutaneous, occlusive

Vehicle:

other: corn oil/acetone (9+1, v/v) for intradermal induction exposure

Challenge 2

Concentration / amount:

5 % (v/v) in corn oil/acetone (9+1, v/v) for the intradermal induction100 % (undiluted) for the epicutaneous induction and100 % (undiluted) for the challenge exposure

No. of animals per dose:

20 animals were used 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 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 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: deionised water- Concentrations: 100 % (undiluted)- 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

Study design: in vivo (LLNA)

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

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 weiths between the test substance group and dthe control group on Day 0 and 24 (P=0.05).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 25 of each step (P = 0.05).

Results and discussion

Positive control results:

See attachment.

In vivo (non-LLNA)

Results

Results 1

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

Test substance 100 % (undiluted)

No. with + reactions:

0

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 100 % (undiluted). No with. + reactions: 0.0. 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 100 % (undiluted)

No. with + reactions:

2

Total no. in group:

20

Clinical observations:

very slight erythema and severe erythema/oedema

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: Test substance 100 % (undiluted). No with. + reactions: 2.0. Total no. in groups: 20.0. Clinical observations: very slight erythema and severe erythema/oedema.

Results 3

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

Test substance 100 % (undiluted)

No. with + reactions:

0

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 100 % (undiluted). No with. + reactions: 0.0. 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 100 % (undiluted)

No. with + reactions:

1

Total no. in group:

20

Clinical observations:

severe erythema/oedema

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: Test substance 100 % (undiluted). No with. + reactions: 1.0. Total no. in groups: 20.0. Clinical observations: severe erythema/oedema.

Any other information on results incl. tables

Results of the preliminary test:

To obtain the appropriate concentrations of the test substance for the definitive study, a preliminary test was carried out with 3 female guinea pigs, pretreated with FCA. 4 different concentrations of the test substance 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.

Results (scores) of the preliminary test (for scale see Table 4):

Intradermal exposure:

test substance	Scores for animal Nos. *
concentration (w/v)	4	5	6
20.0 %	3/3	3/3	3/3
5.0 %	2/1	0/0	3/3
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

 

Epicutaneous exposure:

test substance	Scores for animal Nos. *
concentration (w/w)	4	5	6
100 %	0/0	0/0	0/0
50 %	0/0	0/0	0/0
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

For the main study the following concentrations of "DIETHYLACETYLSUCCINATE" were therefore selected:

5 % (v/v) in corn oil/acetone (9+1,v/v) for the intradermal induction, 100 % (undiluted) for the epicutaneous induction and for the challenge exposure.

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information

Conclusions:

10 % of the test substance group aminals were regarded as sensitised. According to the EC Guideline 93/21, the threshold limit for a classification of a test substance as a sensitiser is 30 % of positively reacting animals in the GPMT. As the sensitisation rate in this study was clearly below this threshold, the test substance needs not to be labelled with "R43 may cause sensitisation by skin contact".

Executive summary:

Method 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/acetone (9+1, v/v) for the intradermal induction    100 % (undiluted) for the epicutaneous induction and 100 % (undiluted) 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 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. In the control group, no positive skin reactions were noted in any animal. In the test substance group, very slight to severe erythema and/or oedema were noted in 2/20 animals at the test substance treated areas 24 hours and/or 48 hours after the end of the challenge exposure. Therefore 2/20 animals (10 % of the test substance group animals) were regarded as sensitised. According to the EC Guideline 93/21, the threshold limit for a classification of a test substance as a sensitiser is 30 % of positively reacting animals in the Guinea Pig Maximisation Test. As the sensitisation rate in this study was clearly below this threshold, the test substance "DIETHYLACETYLSUCCINATE" needs not to be labelled with "R43 May cause sensitisation by skin contact".