Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1992-05-05 to 1992-06-05
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1992
Report Date:
1992

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
adopted 12th May 1981
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Version / remarks:
19.9.1984
Deviations:
no
GLP compliance:
yes
Type of study:
guinea pig maximisation test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
other: liquid

In vivo test system

Test animals

Species:
guinea pig
Strain:
other: Bor:DHPW
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Versuchstierzucht Winkelmann, Borchen/Kreis Paderborn
- Other: Animals of this strain were used at Bayer AG for toxicological studies for years. The health of the animals were checked regularly and randomly regarding the most important specific infectious agents. Results of these investigations are archived at Bayer AG.
The sensitivity of the guinea pig strain was checked regularly. The correspondent documents were archieved at Bayer AG.
- Strain: Bor: DHPW (SPF)
- Number of animals: 54
- Age at study initiation: 5-8 weeks
- Weight at study initiation: 351 g (average), 301-397 g
- Housing: Makrolon cages type IV, 5 animals per cage. Bedding: wood granulate.
- Health: Only healthy animals were used in the study. Animals were not treated with anti-infective.
- Diet: "Altromin 3020 - Haltungsdiät für Meerschweinchen". Both food was offered ad libitum.
- Water: drinking water (ad libitum), in each cage two 750 mL flasks made of poly cabonate were installed with drinking water.
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature: 21 +/- 1.5 °C
- Humidity: 40-70 %
- Air changes: ca. 12-15 chenges per hours
- Photoperiod: 12 hours (6:00 am -18:00 pm) artificial illumination

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
other: intradermal, epicutaneous
Vehicle:
propylene glycol
Concentration / amount:
Main test:
- intradermal induction: 5 %
- epicutaneous induction: 100 %
- 1. challenge: 100 % (epicutaneous)
- 2. challenge: 50 % and 5 % (epicutaneous)
Challengeopen allclose all
Route:
other: epicutaneous
Vehicle:
propylene glycol
Concentration / amount:
Main test:
- intradermal induction: 5 %
- epicutaneous induction: 100 %
- 1. challenge: 100 % (epicutaneous)
- 2. challenge: 50 % and 5 % (epicutaneous)
No. of animals per dose:
Main test:
3 animal groups: 20 animals for the test substance, and two control groups with each 10 animals. The second control group was prepared for a further second challenge, if needed.
Details on study design:
RANGE FINDING TESTS:
- dose finding for intradermal induction:
A volume of 0.1 mL of the following test substance concentration were injected intradermal in an animal: 0%, 1 %, 2.5%, 5%. There areas of the injections were assessed after 24 and 48 h: after 24 and 48 h: 0-5% white area with red border, diameter ca. 0.8 cm
- dose finding for topic induction:
Using 4 animals 5 concentrations (6%, 12.5%, 25%, 50%, 100%) were checked. Each animal got 4 patches each impreganated with 0.5 mL test substance formulation (occlusive conditions) for 24 h. After exposure test substance residues were washed with sterile physiologic salt solution. The skaround the treated areas was clipped. The skin reactions (skin reddening and formation of edemas) after 48 and 72 h after application were assessed.
No signs of skin reddening or formation of edemas were observed for all test substance concentrations.
- dose finding for challenge:
Using 5 animals 4 concentrations (12%, 25%, 50%, 100%) were checked. Each animal got 4 patches each impreganated with 0.5 mL test substance formulation (occlusive conditions) for 24 h. After exposure test substance residues were washed with sterile physiologic salt solution. The skaround the treated areas was clipped. The skin reactions (skin reddening and formation of edemas) after 48 and 72 h after application were assessed.
No signs of skin reddening or formation of edemas were observed for all test substance concentrations.

MAIN STUDY
INDUCTION EXPOSURE- Intradermal induction:
One day before application back and flank of the animals were clipped. Behind the neck on the left and the right side from the backbone each three injections which were positioned in a line, were conducted. The distance between the injections was 1-2 cm.
A volume of 0.1 mL was applied per injection. The animals of the different groups were treated as follows:
- test substance groups:
1. Injections (cranial): Complete adjuvant according to Freund (Difco Lab.) 1:1 diluted with physiologic salt solution (sterile)
2. Injections (meidal): test substance 5% in propylene glycole formulated
3. Injections (caudal): test substance 5% in propylene glycole and complete adjuvant according to Freund in equal parts formulated
- control groups:
The animals of the control groups were trated as the animals of the test substance group, but the formulations for the injections 2 and 3 did not contain any test substance , only polypropylene glycole.

INDUCTION EXPOSURE - epicutaneous induction:
One week after the intadermal induction a topic induction was performed. A day before the induction the animals were clipped and were treated with an 10% formulation of sodium lauryl sulafte in paraffin. Between and in the areas where the injections were done hypo allergenic patches (2x4 cm) were applied and were covered with aluminium foil and additionally were fixed on the skin with Fermo-flextape (Transatalantic GmbH, Schwarzenbach).
The patches were treated as follows:
- test substance groups: 0.5 mL test substance (100 %)
- control groups: 0.5 mL propylene glycole
After exposure for 48 hours residues of test item were washed with physiologic salt solution.

B. CHALLENGE EXPOSURE
Epicutaneous challenge: Challenge was performed 3-4 weeks after the intradermal induction.
Animals that were treated as control anilamls were used as for a dose range finding test for the test substance concentration in the first challenge.
One day before the challenge the animals were clipped on the back and flank. The animals of the test substance group and of the first control group were treated in the course of the first challenge with 100% test substance which was applied on hypoallergenic patch. The patch was applied on the right flank for 24 h using a Fermoflex-tape. On the right flank an untreated patch was fixed (control).
In the second challenge the animals of the test substance group and the second control group were treated with 50% and 5% test substance formulation, respectively, which was applied on a hypo allergenic patch. This patch was fixed with a Fermoflex tape on the left flank for 24 h. The patches with both test substance concentrations were fixed on the animals alternately cranial or caudal. On the right flank two patches which were impreganated with propylene glycol were applied (control). A volume of 0.5 mL was applied. After exposure residues of test item were washed with sterile physiologic salt solution and the skin around the application fields was clipped.

Challenge controls:
Yes, concurrent vehicle during induction exposure.
Positive control substance(s):
no

Results and discussion

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
48
Group:
test group
Dose level:
100%
No. with + reactions:
8
Total no. in group:
19
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 48.0. Group: test group. Dose level: 100%. No with. + reactions: 8.0. Total no. in groups: 19.0.
Reading:
1st reading
Hours after challenge:
72
Group:
test group
Dose level:
100%
No. with + reactions:
9
Total no. in group:
19
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 72.0. Group: test group. Dose level: 100%. No with. + reactions: 9.0. Total no. in groups: 19.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
50%
No. with + reactions:
12
Total no. in group:
19
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 50%. No with. + reactions: 12.0. Total no. in groups: 19.0.
Reading:
2nd reading
Hours after challenge:
72
Group:
test group
Dose level:
50%
No. with + reactions:
9
Total no. in group:
19
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 72.0. Group: test group. Dose level: 50%. No with. + reactions: 9.0. Total no. in groups: 19.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
5%
No. with + reactions:
2
Total no. in group:
19
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 5%. No with. + reactions: 2.0. Total no. in groups: 19.0.
Reading:
2nd reading
Hours after challenge:
72
Group:
test group
Dose level:
5%
No. with + reactions:
1
Total no. in group:
19
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 72.0. Group: test group. Dose level: 5%. No with. + reactions: 1.0. Total no. in groups: 19.0.
Reading:
1st reading
Hours after challenge:
48
Group:
negative control
Dose level:
100%
No. with + reactions:
0
Total no. in group:
19
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 100%. No with. + reactions: 0.0. Total no. in groups: 19.0.
Reading:
1st reading
Hours after challenge:
72
Group:
negative control
Dose level:
100%
No. with + reactions:
0
Total no. in group:
19
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 72.0. Group: negative control. Dose level: 100%. No with. + reactions: 0.0. Total no. in groups: 19.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
50%
No. with + reactions:
0
Total no. in group:
19
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 50%. No with. + reactions: 0.0. Total no. in groups: 19.0.
Reading:
2nd reading
Hours after challenge:
72
Group:
negative control
Dose level:
50%
No. with + reactions:
0
Total no. in group:
19
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 72.0. Group: negative control. Dose level: 50%. No with. + reactions: 0.0. Total no. in groups: 19.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
5%
No. with + reactions:
0
Total no. in group:
19
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 5%. No with. + reactions: 0.0. Total no. in groups: 19.0.
Reading:
2nd reading
Hours after challenge:
72
Group:
negative control
Dose level:
5%
No. with + reactions:
0
Total no. in group:
19
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 72.0. Group: negative control. Dose level: 5%. No with. + reactions: 0.0. Total no. in groups: 19.0.

Any other information on results incl. tables

General investigations:

All animals did not show any symptoms. On day 5 one animal of the group was dead. The following autopsy findings were recorded: coagulated blood in chest and abdomen, liver faded.

After removal of the patches of the second induction (day 9) some animals (animal no. 7, 15, 25, 28, 32) showed wounds in places. On day 10 some animals (animal no. 7, 15, 22, 24, 25, 28, 32, 33, 40) had a crusted skin at the treated areas. These crusts were healed on day 13 (animal no. 22, 24, 28) , on day 14 (animal no. 15) , on day 15 (animal no. 7), on day 16 (animal no. 32, 33, 40), and on day 17 (animal 25).

On day 24 the body weights of the animals of the test substance group and of the first control group were slightly reduced compared to the body weights of the second control group. On day 31 the body weights of the animals of the second control group and of the test item group were reduced and slightly increaced, respectively, compared to the body weights recorded on day 24.

Applicant's summary and conclusion

Interpretation of results:
sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Under the conditions investigated the test substance showed a sensitizing potential to skin.
Executive summary:

The test substance was tested in a Guinea Pig Maximization Test according to EU method B.6 and OECD guideline no. 406. The test substance concentrations selected for the main study were based on the results of preliminary skin irritation tests.

In the main study, 20 animals were intradermally induced with a test substance concentration of 5% in propylene glycol. One week later the test substance in its pure form (100%) was applied epicutaneously on rabbit’s skin. After 48 hours, the test substance was removed with physiological saline. Ten control animals were similarly treated, but with vehicle only. Three to four weeks after the epicutaneous application all animals were challenged with the test substance in its pure form (100%). The second challenge was performed with 50% and 5% of the test substance. Vehicle was used as control.

After the first challenge 63 % of the treated animals showed signs of skin reactions (redness). The skin of the control animals was unaffected. After the second challenge skin reactions were noted in 63 % and 50 % of the animals treated with 50% and 5 %, respectively. No skin irritation was observed in control animals.

Body weight was reduced in animals of the treatment group and of control animals after challenge with the test substance compared to control group indicating a slightly systemic effect.

Under the conditions of the experiment, the test substance is considered to have a skin sensitising potential.