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Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 05 JUN 2001 to 06 JUL 2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study (OECD Guideline 406; EU Method B.6; OPPTS 870.2600), in compliance with GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2001
Report date:
2001

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
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
guinea pig maximisation test

Test material

Constituent 1
Reference substance name:
Reference substance 002
Cas Number:
99402-80-9
Test material form:
solid: nanoform, no surface treatment
Details on test material:
Name of test material (as cited in study report): Part of several studies carried out by Aventis (see study record for report name); Permanent-Rubin F6B;
Analytical purity: not specified

In vivo test system

Test animals

Species:
guinea pig
Strain:
other: HsdPoc:DH (Harlan)
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain specifics: HsdPoc (harlan)
- Source: Harlan Winkelmann GmbH, Borchen, Germany
- Weight at study initiation: mean 366 g (range: 345 to 414 g)
- Housing: in groups of five animals in Makrolon type 4 cages in fully air-conditioned rooms
- Diet: ssniff Ms-H (V2233), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 5 days
- Randomisation: randomisation scheme 2001.0316

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 +/- 3
- Humidity (%): 50 +/- 20
- Air changes (per hr): fully air-conditioned
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal and epicutaneous
Vehicle:
other: Freund's Adjuvant, deionized water, sesame oil
Concentration / amount:
Intradermal induction on day 1:
2 x 0.1 ml 50 % Freud´s Adjuvants alone , injection site 1
2 x 0.1 ml 5 % test item in sesame oil, injection site 2
2 x 0.1 ml 5 % test item in 50 % Freud´s Adjuvants, injection site 3

Dermal induction on day 8:
25 % test item in sesame oil (0.5 g)

Dermal challenge on day 22:
25 % test item in sesame oil (0.5 g)
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
other: Freund's Adjuvant, deionized water, sesame oil
Concentration / amount:
Intradermal induction on day 1:
2 x 0.1 ml 50 % Freud´s Adjuvants alone , injection site 1
2 x 0.1 ml 5 % test item in sesame oil, injection site 2
2 x 0.1 ml 5 % test item in 50 % Freud´s Adjuvants, injection site 3

Dermal induction on day 8:
25 % test item in sesame oil (0.5 g)

Dermal challenge on day 22:
25 % test item in sesame oil (0.5 g)
No. of animals per dose:
10 animals in the treatment group
5 animals in the control group
(in case of a questionable result additional animals will be tested to give a total of 20 test and 10 control animals)

3 animals in the group for determination of a primary non-irritant concentration
2 animals in the group for determination of tolerance of intradermal injections
Details on study design:
MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 2 intradermal injections and 1 dermal application
- Exposure period: intradermal injections on day 1 and dermal application on day 8 (occlusive, bandage removed after 48 h)
- Test groups: 3 sites (50% Freund's Adjuvant only, test substance in sesame oil and test substance in 50% Freund's Adjuvant) per injection, test substance in sesame oil for dermal application
- Control group: 3 sites (50% Freund's Adjuvant, sesame oil and equal volume of sesame oil and 50% Freund's Adjuvant) per injection, sesame oil for dermal application
- Site: dorsal area measuring 2 x 4 cm in the vicinity of the shoulders for intradermal injections, dermal application covered the same area where the intradermal injections had been placed
- Frequency of applications and duration: see above
- Concentrations: see above

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: 22
- Exposure period: occlusive for 24 h (removal of bandage on day 23)
- Test groups: test substance
- Control group: test substance
- Site: left flank
- Concentrations: see above
- Evaluation (hr after challenge): 48 h and 72 h
Challenge controls:
5 animals treated with the test substance (25 % in sesame oil, 0.5 g applied)
Positive control substance(s):
yes
Remarks:
alpha-hexylcinnamaldehyde

Results and discussion

Positive control results:
Positive control assay (report date: 2001.04-17):
After the challenge treatment (alpha hexylcinnamaldehyde) all animals (10) showed a positive skin reaction during the observation period.

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
48
Group:
test chemical
Dose level:
25 %
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no clinical signs of intoxication
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 48.0. Group: test group. Dose level: 25 %. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: no clinical signs of intoxication.
Reading:
2nd reading
Hours after challenge:
72
Group:
test chemical
Dose level:
25 %
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no clinical signs of intoxication
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 72.0. Group: test group. Dose level: 25 %. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: no clinical signs of intoxication.
Reading:
1st reading
Hours after challenge:
48
Group:
negative control
Dose level:
25 %
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 25 %. No with. + reactions: 0.0. Total no. in groups: 5.0.
Reading:
2nd reading
Hours after challenge:
72
Group:
negative control
Dose level:
25 %
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 72.0. Group: negative control. Dose level: 25 %. No with. + reactions: 0.0. Total no. in groups: 5.0.

Any other information on results incl. tables

Intradermal induction treatment: No effects on body weight gains or clinical signs were observed. The intradermal injection with Freund's adjuvant (with and without test substance) caused well-defined up to severe edema as well as indurations and encrustations. The application sites treated with Freund's adjuvant without the test substance also showed well-defined up to severe erythema. Administration sites treated with test item in sesame oil showed slight up to well-defined edema. The injection sites treated with the test substance were discolored red, therefore, erythema formation was not assessable. Intradermal injections of the vehicle alone exhibited no signs of iritation. Due to these strong irritation reactions of the skin, treatment with 10% sodium dodecylsulfate was not done on day 7.

Dermal Induction treatment

After the removal of the patches at day 10, well-defined erythema and edema, indurated and encrusted skin as well as necrosis were observed at the sites previously treated with Freund's Adjuvant (without test item). The administration sites treated with Freud´s Adjuvant (with test item) showed well-defined edema, indurated and encrusted skin as well as necrosis. Additionally, injection sites were red discolored. The administration sites treated with the test item showed well-defined edema. Additionally, injection sites were red discolored. The administration sites treated with the vehicle alone ahowed no signs of irritation. Due to the red discoloration of the injection sites the animals could not be assessed for erythema

Skin readings after challenge treatment (day 22)

Control animals No. 1 2 3 4 5

Treatment animals No. 6 7 8 9 10 11 12 13 14 15

1. reading (48 hours)

Control animals

Erythema 0 0 0 0 0  

Edema 0 0 0 0 0

Treatment animals

Erythema 0 0 0 0 0 0 0 0 0 0

Edema 0 0 0 0 0 0 0 0 0 0

2. reading (72 hours)

Control animals

Erythema 0 0 0 0 0  

Edema 0 0 0 0 0

Treatment animals

Erythema 0 0 0 0 0 0 0 0 0 0

Edema 0 0 0 0 0 0 0 0 0 0

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
In a guinea pig maximisation test, the test item caused no skin reaction in the ten animals of the treatment group or in the five control animals. Based on the results of this study the test item showed no evidence for sensitising properties.
Executive summary:

Testing for sensitizing properties of the test item was performed in female Guinea pigs according to the method of MAGNUSSON & KLIGMAN (OECD TG 406). Intradermal induction was performed using 5 % test item in sesame oil. Dermal induction and challenge was carried out with 25 % test item in sesame oil. The challenge treatment caused no skin reaction in the ten animals of the treatment group or in the five control animals. Based on the results of this study the test item showed no evidence for sensitizing properties.