Registration Dossier

Administrative data

Description of key information

A maximisation assay in guinea pigs was performed to evaluate the skin sensitizing potential of the test material (GLP, similar to OECD guideline 406). The test material did not provoke an allergic reaction or skin irritation after repeated intradermal and topical application. The substance is therefore not regarded as a skin sensitizer.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1998-03-30 - 1998-05-22
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
adopted on 17th July 1992
Deviations:
yes
Remarks:
temperature and relative humidity of the animal room exceeded the recommended range
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
Valid in vivo data with guinea pigs are available, therefore no further studies are required.
Species:
guinea pig
Strain:
other: Hartley-derived albino guinea pigs
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Hilltop Lab Animals, Inc., Scottdale Pennsylvania
- Age at study initiation: young adult Hartley-derived albino guinea pigs
- Weight at study initiation: 407 – 474 g (males), 382 – 461 g (females)
- Housing: housed individually in suspended stainless steel cage
- Diet: PMI Certified Guinea Pig Chow (Purina Mills, Inc.), ad libitum
- Water: Municipal tap water treated by reverse osmosis, ad libitum
- Acclimation period: five days
Other:
- Only healthy animals were chosen for study use
- Females were nulliparous and nonpregnant

ENVIRONMENTAL CONDITIONS
- Temperature: 61 – 74 °F [16 – ca. 23 °C] (recorded daily)
- Humidity: 31 -75 % (recorded daily)
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: 1998-04-01 To: 1998-05-02
Route:
intradermal and epicutaneous
Vehicle:
propylene glycol
Concentration / amount:
3 %
Adequacy of induction:
highest technically applicable concentration used
Route:
epicutaneous, occlusive
Vehicle:
propylene glycol
Concentration / amount:
50 %
Day(s)/duration:
48 h
Adequacy of induction:
non-irritant substance, but skin pre-treated with 10% SDS
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
propylene glycol
Concentration / amount:
50 %
Day(s)/duration:
24 h
Adequacy of challenge:
other: non irritating concentration allowing for the highest amount of test material to be placed in the chamber
No. of animals per dose:
Experimental group: 20 animals
Control group: 10 animals
Details on study design:
RANGE FINDING TESTS:

Topical Range-Finding Study
On the day prior to dose administration, four topical range-finding guinea pigs were weighed and the hair removed from the right and left sides of the animals with a small animal clipper. Care was taken to avoid abrading the skin during clipping procedures. On the following day, four concentrations of the test article were prepared and each concentration was applied to the clipped area of each topical range-finding animal using a 25 mm Hilltop Chamber. Concentrations used were 10%, 25%, 50% and 100%. Following chamber application, the trunk of the animal was wrapped with elastic wrap which was secured with adhesive tape to prevent removal ofthe chambers and the animal was returned to its cage. Approximately 24 hours after chamber application, the elastic wrap, tape and chambers were removed. The test sites were then wiped with gauze moistened in deionized water, followed by dry gauze, to remove test article residue and the animals returned to their cages. The test sites of the topical range-finding animals were graded for irritation at approximately 24 and 48 hours following chamber removal.
Although the scores were similar at 50% and 100% there was actually a greater amount of test article that could be put in the Hilltop chamber at the 50% w/v concentration (0.3 mL) than at the 100% concentration (0.10 g) due to the great volume (low density) that the powdered test article occupied, therefore, the results of the range-finding study indicated that a test article concentration of 50% w/v in propylene glycol was considered appropriate for topical induction.

Intradermal Range-Finding Study
On the day prior to dose administration, four intradermal range-finding guinea pigs were weighed and the hair removed from the right and left sides of the animals with a small animal clipper. Care was taken to avoid abrading the skin during clipping procedures. On the following day, four concentrations of the test article were prepared and each concentration was intradermally injected into each intradermal range-finding animal using a syringe attached to a hypodemnic needle. Tested concentratios were 01%, 1.0%, 3.,% and 5.0%. The test sites of the intrademnal range-finding animals were graded for irritation at approximately 24 and 48 hours following intradermal injections.
Due to difficulty dosing the 5% w/v concentration in propylene glycol, a test article (ground/sieved) concentration of 3% w/v in propylene glycol was considered appropriate for intrademnal induction.


MAIN STUDY

A. INDUCTION EXPOSURE

- No. of exposures: 2 (a single intradermal exposure on day 1 (6 injections/animal) and an epicutaneous exposure starting on day 7 (closed patch))
- Exposure period: single application (intradermal exposure), 48 hours (epicutaneous exposure)
- Site: scapular region (intradermal exposure), test item placed over injection sites (epicutaneous exposure)
- Frequency of applications: 1 intradermal application, 1 epicutaneous application
- Concentrations: 3 % (intradermal exposure), 50 % (epicutaneous exposure)

- Test groups:
Intradermal injections:
On the day prior to induction (day -1), all test and control animals were weighed and an area of the dorsal skin from the scapular region (approximately 4 x 6 cm) was clipped free of hair. Three pairs of intradermal injections (0.1 mL/site) were made at the border of a 2 x 4 cm area in the clipped region as follows:
A) Injection Pair A: 0.1 mL of FCA emulsion
B) Injection Pair B: 0.1 mL of 3% (w/v) test item in propylene glycol
C) Injection Pair C: 0.1 mL of 3% (w/v) test item/FCA emulsion

On the day prior to topical induction (day 6), the guinea pigs had the hair removed from the scapular area with a small animal clipper. Care was taken to avoid abrading the skin during the clipping procedures. Following clipping, 0.5 mL of 10% (w/w) sodium lauryl sulfate in petrolatum was spread over the intradermal injection sites of all study animals.

Epicutaneous applications:
On study day 7, any residual sodium lauryl sulfate preparation was removed with dry gauze and the scapular area was again treated. A Webril patch (2 x 4 cm) was applied with 0.8 mL of a 50 % (w/w) concentration of the test item in propylene glycol and placed between the injection sites of the test animals. The trunk of each animal was wrapped with elastic wrap which was secured with adhesive tape to prevent removal of the patch and the animal returned to its cage. Approximately 48 hours after dosing, the elastic wrap, tape and patch were removed. The test sites were wiped with gauze moistened in deionized water or propylene glycol, followed by dry gauze, to remove test article residue and the animals were returned to their cages.

- Control group:
The guinea pigs of the control group were treated in a similar method as described above by the intradermal and epicutaneous inductions with omission of the test item (challenge control and rechallenge control: 0.8 mL pure propylene glycol). Reaction sites were assessed and summarised similarly.


B. CHALLENGE EXPOSURE

- No. of exposures: 1 epicutaneous exposure
- Day of challenge: 21
- Exposure period: 24 hours
- Concentration: 50 %

- All animals:
On the day prior to challenge dose administration, the hair was removed from the right side of the test and challenge control animals with a small animal clipper. Care was taken to avoid abrading the skin during the clipping procedures.

On the following day (day 21), the appropriate concentration of the test item was prepared and applied. The experimental and control guinea pigs were challenged two weeks after the epicutaneous induction application. A volume of 0.3 mL of the following test item concentrations were applied using 25 mm Hilltop Chambers:

a = 50 % (w/v) in propylene glycol (Test)
b = 50 % (w/v) in propylene glycol (Challenge Control)

The trunk of each animal was wrapped with elastic wrap which was secured with adhesive tape to prevent removal of the chamber and the animal returned to its cage. Approximately 24 hours after dosing, the elastic wrap, tape and chamber were removed. The test sites were wiped with gauze moistened in deionized water, followed by dry gauze, to remove test article residue and the animals were returned to their cages.

- Evaluation: Dermal observations
The test sites at challenge were graded for dermal irritation at approximately 24 and 48 hours following chamber removal using the Dermal Grading System.

- Other: Clinical observations
Any unusual observations and mortality were recorded. The animals were observed for general health/mortality twice daily, once in the morning and once in the afternoon. All sensitization study animals were sacrificed by carbon dioxide inhalation following each animal's final scoring interval. Gross necropsy examinations were not required for these animals.
Positive control substance(s):
yes
Remarks:
Alpha-Hexylcinnamaldehyde
Positive control results:
Using Alpha-Hexylcinnamaldehyde as positive control, a study was performed during the past six months which provided historical control data for contact sensitization to this agent utilizing the test system described herein (Maximization Design). Following topical induction and intradermal induction at 5.0% (w/v) Alpha-Hexylcinnamaldehyde in propylene glycol and challenge at levels of 1.0% and 0.5% (w/v) Alpha-Hexylcinnamaldehyde in propylene glycol, a contact sensitization response was observed, thereby demonstrating the susceptibility of the test system to this sensitizing agent.
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
50 %
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
Animals appeared in good health
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
50 %
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
Animals appeared in good health
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
50 %
No. with + reactions:
4
Total no. in group:
20
Clinical observations:
Animals appeared in good health
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
50 %
No. with + reactions:
2
Total no. in group:
20
Clinical observations:
Animals appeared in good health

Following challenge with 50% (w/v) test item in propylene glycol, dermal scores of 1 were noted in 4/20 test animals at the 24-hour and in 2/20 test animals at the 48-hour scoring interval. Dermal reactions in the remaining test and all challenge control animals were limited to scores of 0. Group mean dermal scores were noted to be similar to the test animals as compared with the challenge control animals.

Interpretation of results:
GHS criteria not met
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

Procedure and observations

The dermal sensitization potential of the test item was evaluated in Hartley-derived albino guinea pigs. Ten male and ten female guinea pigs received intradermal injections of 3.0% material in propylene glycol along with injections of FCA and test substance in FCA. One week later, the test animals received a topical application of 50% test material in propylene glycol. Challenge and rechallenge control animals received similar intradermal and topical treatments except propylene glycol was used in place of the test article. Following a two-week rest period, a challenge was performed whereby the twenty test and ten previously untreated (naive) challenge control guinea pigs were topically treated with a 50% solution. Following challenge, dermal scores of 1 were noted in 4/20 test animals at the 24-hour and in 2/20 test animals at the 48-hour scoring interval. Dermal irritation was not observed. Based on the results of this study, the test substance is not considered to be a contact sensitizer in guinea pigs.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substance is not considered to be classified for skin sensitization under Regulation (EC) No. 1272/2008.