Registration Dossier

Toxicological information

Skin sensitisation

Currently viewing:

Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to
Guideline:
other: JMAFF 12-Nousan-8147 (2000)
Deviations:
no
GLP compliance:
yes
Type of study:
other: Magnusson-Kligman Maximization Method
Justification for non-LLNA method:
An existing study existed (guinea pig Magnusson-Kligman maximization method) which sufficiently addressed the endpoint.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Purity: 92.1%

In vivo test system

Test animals

Species:
guinea pig
Strain:
Hartley
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: 7 weeks
- Weight at study initiation: 474-639 g
- Housing: The animals were gang housed in plastic solid bottom polycarbonate cages or stainless steel solid bottom cages
- Diet: Teklad Global Guinea Pig Diet® #2040, or equivalent. A designated amount of the diet was available to each guinea pig (approximately 20 grams/day).
- Water: Filtered tap water was supplied ad libitum
- Acclimation period: 5-28 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-23°C
- Humidity (%): 41-59%
- Air changes (per hr): 13
- Photoperiod: 12 hour light/dark cycle

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal and epicutaneous
Vehicle:
CMC (carboxymethyl cellulose)
Concentration / amount:
Induction intradermal injections: 1% w/w mixture of test substance in a 2% w/v solution of CMC in distilled water
Induction epicutaneous: 0.5 g of a 60% w/w mixture of the test substance in a 2% w/v solution of CMC in distilled water
Challenge epicutaneous: 0.5 mL of a 30% w/w mixture of the test substance in a 2% w/v solution of CMC in distilled water or 0.5 mL of a 10% w/w mixture in a 2% w/v solution of CMC in distilled water (33% dilution of the HNIC)
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
CMC (carboxymethyl cellulose)
Concentration / amount:
Induction intradermal injections: 1% w/w mixture of test substance in a 2% w/v solution of CMC in distilled water
Induction epicutaneous: 0.5 g of a 60% w/w mixture of the test substance in a 2% w/v solution of CMC in distilled water
Challenge epicutaneous: 0.5 mL of a 30% w/w mixture of the test substance in a 2% w/v solution of CMC in distilled water or 0.5 mL of a 10% w/w mixture in a 2% w/v solution of CMC in distilled water (33% dilution of the HNIC)
No. of animals per dose:
Test: 20
Control: 10

Details on study design:
RANGE FINDING TESTS: Prior to the challenge phase, a group of animals was used to determine the highest non-irritating concentration. The fur was removed by clipping the flanks of each guinea pig. The test substance was mixed with a 2% w/v solution of CMC in distilled water to yield w/w concentrations of 60%, 45%, 30% and 15%. Each concentration (0.5 g or mL) was applied to an occlusive 25 mm Hill Top Chamber and applied to the appropriate test site. After 24 hours sites were washed then read at 24 and 48 hours.
Based on these findings, the concentration which produced faint to moderate irritation (1-2) selected for the intradermal induction was a 1% w/w mixture in a 2% w/v solution of CMC in distilled water. The 3% w/w mixture also produced faint to moderate irritation (1-2). However, this concentration was very difficult to dose due to viscosity and was not selected for the intradermal induction based on this finding. That which produced very faint to faint irritation (0.5-1) selected for the topical induction was a 60% w/w mixture in a 2% w/v solution of CMC in distilled water. The HNIC (the highest concentration that produced responses in four guinea pigs no more severe than two scores of 0.5 and two scores of zero) selected for the challenge phase was a 30% w/w mixture in a 2% w/v solution of CMC in distilled water.


MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures:
Intradermal Phase: two intradermal injections of each preparation, each at a separate site (0.1 ml each) 1% w/w mixture of test substance in a 2% w/v solution of CMC in distilled water or 1% w/w mixture of test substance in an emulsion of Freund’s Adjuvant Complete (50% v/v in distilled water), treated once
Topical Induction Phase: one application, each at the injection site, 0.5 g of a 60% w/w mixture of the test substance in a 2% w/v solution of CMC in distilled water
-Controls: Emulsion of Freund’s Adjuvant Complete (50% v/v in distilled water), 100% of a 2% w/v solution of CMC in distilled water, 50% w/w mixture of a 2% w/v solution of CMC in distilled water in an emulsion of Freund’s Adjuvant Complete (50% v/v in distilled water)
- Exposure period:
Intradermal Phase: 7 days
Topical Induction Phase: 48 hours
- Site: suprascapular region or flanks
- Frequency of applications: once

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: After the 24 hour exposure period, the chambers were removed and all test sites were gently cleansed of any residual test substance. Approximately 24 and 48 hours after patch removal; these sites were evaluated for a sensitization response (erythema)
- Exposure period: 24 hours
- Site: right and left flank
- Concentrations: Three occluded topical applications were then placed on all test and test vehicle control group animals using 25 mm Hill Top Chambers. A quantity equal to 0.5 mL of a 2% w/v solution of CMC in distilled water was applied to one chamber and positioned on the right middle flank. The remaining two chambers containing 0.5 mL of a 30% w/w mixture of the test substance in a 2% w/v solution of CMC in distilled water (HNIC) and 0.5 ml of a 10% w/w mixture in a 2% w/v solution of CMC in distilled water (33% dilution of the HNIC) were positioned on the left front and rear flank, respectively.
- Evaluation (hr after challenge): 24 and 48 hours

Results and discussion

In vivo (non-LLNA)

Resultsopen allclose all
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
all
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
8 Test group animals at the 30% dose had a reading of 0.5; 3 Test group animals at the 10% dose had a reading of 0.5. A reading of 0.5 - very faint erythema is not considered a positive reaction by the scoring system.
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
all
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
3 Test group animals at the 30% dose had a reading of 0.5; 1 Test group animal at the 10% dose had a reading of 0.5. A reading of 0.5 - very faint erythema is not considered a positive reaction by the scoring system.
Reading:
1st reading
Hours after challenge:
24
Group:
other: Test Vehicle Control/ Naive Animals
Dose level:
all
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
2 Test Vehicle Control/Naive Animals at the 30% dose had a reading of 0.5. A reading of 0.5 - very faint erythema is not considered a positive reaction by the scoring system.
Reading:
2nd reading
Hours after challenge:
48
Group:
other: Test Vehicle Control/ Naive Animals
Dose level:
all
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
1 Test Vehicle Control/Naive Animal at the 30% dose had a reading of 0.5. A reading of 0.5 - very faint erythema is not considered a positive reaction by the scoring system.

Any other information on results incl. tables

All test and vehicle control animals survived and appeared active and healthy during the study. Although one test and one vehicle control animal lost weight over the course of the study, there was no statistical significant difference in the overall body weight gain of the test substance animals as compared to the test substance vehicle control animals.

At the 48 hour Challenge reading the irritation score for the Test Group Animals and Test Vehicle Control/Naive Animals was 0 for 25 of 30 animals. For the remaining animals the following was observed:

3 Test group animals (of 20) at the 30% dose had a reading of 0.5.

1 Test group animal (of 20) at the 10% dose had a reading of 0.5.

1 Test Vehicle Control/Naive Animal (of 10) at the 30% dose had a reading of 0.5.

A reading of 0.5 - very faint erythema is not considered a positive reaction by the scoring system.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Based on these findings and on the evaluation system used, the test substance is not considered to be a contact skin sensitizer.
Executive summary:

A Magnusson-Kligman maximization test was conducted with guinea pigs to determine the potential for the test substance to invoke dermal skin sensitization reactions. The study was conducted using four stages; preliminary irritation screens, a two-stage induction phase, and a challenge phase. The first induction phase involved six intradermal injections into the suprascapular area of each of 20 guinea pigs. These doses were comprised of pairs of injections of the test substance in a 2% w/v solution of CMC (carboxymethylcellulose) in distilled water (1% w/w), the test substance (1% w/w) combined with an emulsion of Freund’s Adjuvant Complete, as well as an emulsion of Freund’s Adjuvant Complete alone. Approximately one week later, the second phase of induction was conducted. A 60% w/w mixture of the test substance in a 2% w/v solution of CMC in distilled water (test group) or a 2% w/v solution of CMC in distilled water (test vehicle control group) was applied topically for a period of 48 hours to the area encompassing the intradermal injection sites.

Approximately two weeks later, a primary challenge consisting of three occluded applications was conducted on each animal. One Hill Top Chamber containing 0.5 mL of a 2% w/v solution of CMC in distilled water was applied to a naive site on the right middle flank of each animal. The remaining two Hill Top Chambers containing 0.5 mL of the HNIC (Highest Non-Irritating Concentration, determined in the preliminary irritation screen to be a 30% w/w mixture in a 2% w/v solution of CMC in distilled water) of the test substance and 0.5 ml of a 33% dilution of the HNIC (10% w/w mixture in a 2% w/v solution of CMC in distilled water) were positioned on two naive sites on the left front and rear flank, respectively, for approximately 24 hours. The test vehicle control group was also treated with the test substance and test vehicle (as described above) at challenge. Approximately 24 and 48 hours after challenge patch removal, all animals were scored for a sensitization response (erythema).

All test and vehicle control animals survived and appeared active and healthy during the study. Although one test and one vehicle control animal lost weight over the course of the study, there was no statistical significant difference in the overall body weight gain of the test substance animals as compared to the test substance vehicle control animals.

At the 48 hour Challenge reading the irritation score for the Test Group Animals and Test Vehicle Control/Naive Animals was 0 for 25 of 30 animals. For the remaining animals the following was observed:

3 Test group animals (of 20) at the 30% dose had a reading of 0.5.

1 Test group animal (of 20) at the 10% dose had a reading of 0.5.

1 Test Vehicle Control/Naive Animal (of 10) at the 30% dose had a reading of 0.5.

A reading of 0.5 - very faint erythema is not considered a positive reaction by the scoring system.

Based on these findings and on the evaluation system used, the test substance is not considered to be a contact skin sensitizer.