Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
May 15 - June 8, 2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study (OECD), GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report Date:
2000

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
July 17, 1992
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to
Guideline:
other: Japan; MAFF
Deviations:
no
GLP compliance:
yes
Remarks:
Department of Toxicology, BASF AG
Type of study:
guinea pig maximisation test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name as cited in report: Adipic acid, cyclic ester with diethyleneglycol
- Analytical purity: ca. 90%
- Physical state: Solid/white
- Storage: Refrigerated
- Lot/batch No.: B 74
- Stability: The test substance was stable in 0 .5% Tylose CB 30.000 in aqua bidest for a time period of 4 hours.

In vivo test system

Test animals

Species:
guinea pig
Strain:
other: Hsd Poc: DH (SPF)
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Winkelmann GmbH, Borchen, FRG
- Age at study initiation: young adults
- Weight range at study initiation: 327 - 360 g
- Housing: 5 per cage
- Diet: ad libitum, Kliba Labordiaet (Kaninchen-Meerschweinchen-Haltungsdiaet). The feed was assayed for chemical and microbiological contaminants.
- Water: ad libitum, tap water (drinking water is regularly assayed for chemical and micriobilogical contaminants)
- Acclimation period: 6 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 - 25
- Humidity (%): 30 - 70
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal and epicutaneous
Vehicle:
other: 1% Tylose CB 30.000 (cleaned sodium carboxymethylcellulose from Hoechst AG) in aqua bidest
Concentration / amount:
- Pretest intradermal induction: 5% in Freund's adjuvant/ 0.9 / aqueous NaCl (1:1) or in 1% Tylose CB 30.000 in aqua bidest.
- Pretest epicutaneous induction: 10% and 25% test substance in 1% Tylose CB 30.000 in aqua bidest.

MAIN TEST:
Intradermal induction: test substance 5% in 1% Tylose CB 30.000 in aqua bidest. or 5% in Freund's adjuvant /0.9% aqueous NaCI solution (1:1).
Epicutaneous induction: 25% test substance 1% Tylose CB 30.000 in aqua bidest.
Challenge: 25% test substance 1% Tylose CB 30.000 in aqua bidest.
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
other: 1% Tylose CB 30.000 (cleaned sodium carboxymethylcellulose from Hoechst AG) in aqua bidest
Concentration / amount:
- Pretest intradermal induction: 5% in Freund's adjuvant/ 0.9 / aqueous NaCl (1:1) or in 1% Tylose CB 30.000 in aqua bidest.
- Pretest epicutaneous induction: 10% and 25% test substance in 1% Tylose CB 30.000 in aqua bidest.

MAIN TEST:
Intradermal induction: test substance 5% in 1% Tylose CB 30.000 in aqua bidest. or 5% in Freund's adjuvant /0.9% aqueous NaCI solution (1:1).
Epicutaneous induction: 25% test substance 1% Tylose CB 30.000 in aqua bidest.
Challenge: 25% test substance 1% Tylose CB 30.000 in aqua bidest.
No. of animals per dose:
PRETEST:
Intradermal pretest: 2 animals
Epicutaneous pretest: 5 animals

MAIN TEST.
Control group 1: 5
Control group 2: 5
Test group: 10
Details on study design:
PRETEST: INTRADERMAL INDUCTION
The pretest was used to determine the concentrations for the test substance suitable for use in the main experiment. 6 intradermal injections (per animal) in groups of two per animal were applied to the clipped shoulder of each animal (2 animals/dose). 24 h after injection, a reading of dermal response was performed.

- front row: 2 injections each of 0.1 ml Freund's adjuvant without test substance emulsified with 0.9% aqueous NaCI-solution in a ratio of 1 : 1.
- middle row: 2 injections each of 0.1 ml of a test substance formulation in an appropriate vehicle at the selected concentration.
- back row. 2 injections each of 0.1 ml Freund's adjuvant/ 0.9% aqueous NaCI-solution (1 : 1) with test substance at the selected concentration.

PRETEST: EPICUTANEOUS INDUCTION.
For detecting a possible influence on irritating effects of previous intradermal treatment with Freund's adjuvant, animals pretreated with Freunds's adjuvant 4 weeks prior to substance application were employed. The epicutaneous pretest was performed by applying 2 x 2 cm gauze patches (6 layers surgical gauze Ph. Eur. from Lohmann GmbH & Co. KG) containing 0.5 ml of the test substance formulation to the skin of the flanks under an occlusive dressing. The test substance was applied 2 times for 24 hours within a period of 96 hours in order to detect non-specific phenomena that are not caused by a sensitization reaction but could possibly be attributed to a shift in the irritation threshold. Readings were performed 24 h and 48 h after the beginning of application.

MAIN STUDY: INDUCTION EXPOSURE
6 intradermal injections in groups of two per animal were given on the clipped shoulders of the test animals. Skin responses were checked 24 h after the application of the intradermal injections. Injection of the test group was identical to the intradermal pretest (see above).
The animals of the control groups were used to rule out a substance induced primary skin irritation. A second control group (control group 2) was added for potential second challenge in case of borderline results at the first challenge. Control group 1 cannot be used in this case due to a possible sensitization by the single application of the test substance at the first challenge .

Injection pattern for control groups:
- front row: 2 injections each of 0.1 ml Freund's adjuvant without test substance emulsified with 0.9% aqueous NaCI-solution in a ratio of 1 : 1.
- middle row: 2 injections each of 0.1 ml of the undiluted vehicle
- back row. 2 injections each of 0.1 ml of a 50% formulation of the vehicle without test substance emulsified with Freund's adjuvant/ 0.9% aqueous NaCI-solution (1 : 1 )

One week after the intradermal injections, the epicutaneous induction was carried out by applying 2 x 4 cm gauze patches (6 layers surgical gauze Ph. Eur. from Lohmann GmbH & Co. KG) containing 1 ml of the test substance formulation to the skin of the shoulder (same area as in the case of the previous intradermal application) of each animal under occlusive dressing. The patches were removed after 48 h, the skins were washed with water and the skin was assessed.

B. CHALLENGE EXPOSURE
The challenge exposure was performed 14 days after the epicutaneous induction. 2 x 2 cm gauze patches (6 layers surgical gauze Ph. Eur. from Lohmann GmbH & Co . KG) containing 0.5 ml of the test substance formulation were applied to the skin of the intact flank of each animal under an occlusive dressing. The test group and control group 1 were treated with the test substance formulation. Additionally, 1% Tylose CB 30 .000 in aqua bidest. was applied as a vehicle control. Control group 2 only were treated with only the vehicle. The exposure lasted 24 hours. The skins were washed with water upon removal of the patch and checked for dermal response 24 and 48 h after removal of the patch.

OTHER:
Weight check of the individual animals was performed at the beginning of the study (day 0) and at the end of the study (last day of observation). Clipping of the test animals was carried out at least 2 hours before each test substance application at the appropriate application sites. A check for dead or moribund animals was made twice each workday and once on Saturdays, Sundays and on public holidays.

The number of animals with skin findings at 24 and/or 48 hours after the removal of the patch is taken into account for the determination of the sensitization rate. The evaluatio "sensitizing" results if 30% of the test animals exhibit skin reactions in the adjuvant test.
Positive control substance(s):
no
Remarks:
- However, separate studies performed in the lab. including the positive control substance, alpha-hexylcinnamaldehyde, indicated that the test system was able to detect sensitizing compounds under the laboratory conditions chosen.

Results and discussion

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
other: Control group 1
Dose level:
25% Test substance in 1% Tylose CB 30.000 in aqua bidest.
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: other: Control group 1. Dose level: 25% Test substance in 1% Tylose CB 30.000 in aqua bidest.. No with. + reactions: 0.0. Total no. in groups: 5.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
other: Control group 1
Dose level:
25% Test substance in 1% Tylose CB 30.000 in aqua bidest.
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: other: Control group 1. Dose level: 25% Test substance in 1% Tylose CB 30.000 in aqua bidest.. No with. + reactions: 0.0. Total no. in groups: 5.0.
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
25% Test substance in 1% Tylose CB 30.000 in aqua bidest.
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 25% Test substance in 1% Tylose CB 30.000 in aqua bidest.. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
25% Test substance in 1% Tylose CB 30.000 in aqua bidest.
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 25% Test substance in 1% Tylose CB 30.000 in aqua bidest.. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
1st reading
Hours after challenge:
24
Group:
other: Control group 1
Dose level:
1% Tylose CB 30.000 in aqua bidest.
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: other: Control group 1. Dose level: 1% Tylose CB 30.000 in aqua bidest.. No with. + reactions: 0.0. Total no. in groups: 5.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
other: Control group 1
Dose level:
1% Tylose CB 30.000 in aqua bidest.
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: other: Control group 1. Dose level: 1% Tylose CB 30.000 in aqua bidest.. No with. + reactions: 0.0. Total no. in groups: 5.0.
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
1% Tylose CB 30 .000 in aqua bidest.
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 1% Tylose CB 30 .000 in aqua bidest. . No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
1% Tylose CB 30.000 in aqua bidest.
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 1% Tylose CB 30.000 in aqua bidest.. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
1st reading
Hours after challenge:
24
Group:
other: Control group 2
Dose level:
1% Tylose CB 30 .000 in aqua bidest.
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: other: Control group 2. Dose level: 1% Tylose CB 30 .000 in aqua bidest. . No with. + reactions: 0.0. Total no. in groups: 5.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
other: Control group 2
Dose level:
1% Tylose CB 30 .000 in aqua bidest.
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: other: Control group 2. Dose level: 1% Tylose CB 30 .000 in aqua bidest. . No with. + reactions: 0.0. Total no. in groups: 5.0.

Any other information on results incl. tables

Comments to Maximization test in Guinea pig

Control group 2 had been intended for a potential 2nd challenge. This group was not treated with the test substance, since a 2nd challenge was not necessary on the basis of the unambiguous results of the challenge.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information