Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21 February to 20 August 2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Fully GLP- and guideline compliant study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report date:
2002

Materials and methods

Test guidelineopen allclose all
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

Test material

Constituent 1
Chemical structure
Reference substance name:
Diisopropyl succinate
EC Number:
213-110-0
EC Name:
Diisopropyl succinate
Cas Number:
924-88-9
Molecular formula:
C10H18O4
IUPAC Name:
1,4-bis(propan-2-yl) butanedioate
Details on test material:
- Name of test material (as cited in study report): Diisopropyl succinate
- CAS No: 924-88-9
- Trade name: DIPS
- Hill formula: C10H18O4
- Physical state: Clear, colourless liquid
- Lot/batch No.: EPLS 301
- Expiration date of the lot/batch: 31. December 2002
- Stability under test conditions: Until December 2002
- Storage condition of test material: In the refrigerator, in the dark, may be used under light.

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. 5 - 7 weeks at the first application
- Weight at first application: 294 g - 385 g
- Housing: 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 (e.g. ad libitum): Altromin Maintenance Diet No. 3122, rich in crude fiber. Analysis of the feed for ingredients and contaminants are performed andomly by Altromin GmbH, D-32791 Lage.
- Water (e.g. ad libitum): Tap water offered in Makrolon bottles with stainless steel canules ad libitum.
- Acclimation period: 4 days.


ENVIRONMENTAL CONDITIONS
- Temperature: Step 1: Mean of 22.1 °C, Step 2: Mean of 21.9 °C
- Humidity: Step 1: Mean of 50.8 %, Step 2: Mean of 61.8 %.
- Air changes (per hr): 12
- Photoperiod: 12 hrs dark, 12 hrs light

IN-LIFE DATES: From: 25 February 2002 To: 21 June 2002

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal and epicutaneous
Vehicle:
corn oil
Remarks:
corn oil for intradermal exposure
Concentration / amount:
25 % (v/v) in corn oil for the intradermal induction
100 % (undiluted) for the epicutaneous induction and
100 % (undiluted) for the challenge exposure
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
corn oil
Remarks:
corn oil for intradermal exposure
Concentration / amount:
25 % (v/v) in corn oil for the intradermal induction
100 % (undiluted) for the epicutaneous induction and
100 % (undiluted) for the challenge exposure
No. of animals per dose:
The study was performed in two steps. For each step 10 animals were used as test substance group and another 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 for the intradermal injections and in acetone for the epicutaneous administration.

There were no differences in the skin reactions after the intradermal exposure, regardless how high or low the test substance concentrations were. To assure a maximum induction of a possible allergenic reaction, the highest tested concentration of 25 % (v/v) in corn oil was selected for the intradermal induction exposure for the main study.
100 % (undiluted) was selected for the epicutaneous induction and for the challenge exposure.
As the highest test substance concentration of 100 % did not cause markable skin irritations in the preliminary experiment it was also decided to pretreat all animals of both groups with a formulation of n-dodecylsulfate, sodium salt, in white petrolatum, one day before the epicutaneous induction exposure.


MAIN STUDY
The study was performed in two consecutive steps: In the first step 5 control and 10 test substance animals were used and as after the challenge exposure negative results were obtained, additional 5 control animals and 10 test substance animals were exposed.
A. INDUCTION EXPOSURE

Intradermal induction exposure:
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 below two intradermal injections were made side by side.
A volume of 0.1 mL was applied for each injection site.
For further details see "Any other information about materials and methods incl. tables".

Epicutaneous induction exposure:
The highest test substance concentration of 100 % (undiluted) did not cause markable skin irritations in the preliminary test in the animals. According to B. Magnusson and A.M. Kligman, all animals of both groups were therefore pretreated with a formulation of
n-dodecylsulfate, sodium salt (E. Merck, D-64271 Darmstadt, Art. No. 13760), 10 % (w/w) in white petrolatum, one day before the epicutaneous induction exposure. About 0.5 g per animal (step1 and step 2) was gently applied with a plastic spatula onto the appropriate area to give a slight local hyperaemia. The area was left uncovered.
The test substance was applied undiluted.
After clipping the hair test patches (Pur Zellin-Tupfer, obtained by Fa. Hartmann , A-2355 Wiener Neudorf), about 2 cm x 4 cm, were soaked with the test substance (test substance group) and with deionised water (control group), respectively, and were applied to the area of the intradermal injection. 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 the test substance or 0.5 mL deionised water were applied to each animal.

B. CHALLENGE EXPOSURE
The test substance was applied undiluted.
After clipping the hair test patches (Pur Zellin-Tupfer, obtained by Fa. Hartmann , A-2355 Wiener Neudorf), about 2 cm x 2 cm, were soaked with the test substance, and were applied to the left flanks of all animals of both groups. Test patches of the same size, soaked with deionised water, were applied to the right flanks of all animals. Mode of fixation was the same as for the epicutaneous induction exposure.
The exposure time was 24 hours.
0.5 mL of the test substance and 0.5 mL of deionised water were applied to each animal.

- Evaluation: 24 h after the end of the exposure period (Day 23) and a second skin examination further 24 h later (Day 24).
A skin reaction after the challenge exposure was regarded as positive when the site, where test substance formulation was applied, was more irritated than the area of the site, where the vehicle was applied. The rate of these positively reacting animals in the test substance group minus the rate of positively reacting animals in the negative control group gave the net percentage of sensitised animals.
Challenge controls:
For each of the two steps 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

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.
A skin reaction after the challenge exposure was regarded as positive when the site, where test substance formulation was applied, was more irritated than the area of the site, where the vehicle was applied. The rate of these positively reacting animals in the test substance group minus the rate of positively reacting animals in the negative control group gave the net percentage of sensitised animals.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:
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)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
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: 100 % undiluted. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: none.
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
100 % undiluted
No. with + reactions:
1
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: 100 % undiluted. No with. + reactions: 1.0. Total no. in groups: 20.0. Clinical observations: none.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
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: 100% undiluted. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: none.
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
100 % undiluted
No. with + reactions:
0
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: 100 % undiluted. No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: none.

Any other information on results incl. tables

All animals survived till the end of the study.

Some slight, but statistically significant differences in the mean body weights between the control and test substance group animals were considered of no biological relevance.

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.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information
Conclusions:
No control animal and 1 out of 20 test animals had a positive skin reaction after the challenge exposure. Diisopropyl succinate is therefore considered not 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"Diisopropyl succinate".

The study was performed in two consecutive steps: In each step 10 female guinea pigs were used as a test substance group and another 5 females were used as a negative control group.
There were two induction exposures (intradermally and epicutaneously) and one challenge exposure.

Test substance concentrations were:

25 % (v/v) incorn oilfor the intradermal induction

100 % (undiluted) for the epicutaneous induction and

100 % (undiluted) 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. Before the epicutaneous induction exposure, the animals were pretreated with n-dodecylsulfate, sodium salt (10 % w/w, in white petrolatum) to give a slight local hyperaemia. For the epicutaneous exposures occlusive dressings were used.

 

Results

General

The results of both steps were combined for the final conclusion.

All animals survived till the end of the study.

Some slight, but statistically significant differences in the mean body weights between the control and test substance group animals were considered of no biological relevance.


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 control sites of all animals of both groups were normal at each reading time. In step 1, 1/10 of the test substance group animals had a well defined erythema at the test substance treated site 24 hours after the end of the challenge exposure. No adverse skin reactions were observed in the control animals.
There were no adverse skin reactions in any animal of any group in step 2.

Combined result

1/20 animals of the test substance group (5 %) was regarded as sensitised.