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Toxicological information

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 December 11, 2008 to April 08, 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes
Type of study:
Buehler test
Justification for non-LLNA method:
The test substance is surfactants, which are known to give false-positive results in LLNA method.

Test material

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

In vivo test system

Test animals

Species:
guinea pig
Strain:
Hartley
Sex:
male/female
Details on test animals and environmental conditions:
The following information is for all animals used in this study, including those from the first experiment:
Number of Animals: 78
Number of Groups: 3
Preliminary Irritation Group: 18
Test Group: 20 per test
Naive Control Group: 10 per test
Sex: Male and Female. The females assigned to test were nulliparous and nonpregnant.
Species/Strain: Guinea pigs/Hartley albino
Age/Body weight: Preliminary Irritation Group: Young adult
Test and Naive Control Groups: Young adult/females 317-429 grams at experimental start
Source: Received from Elm Hill Breeding Labs, Chelmsford, MA on November 12, 2008, January 14, February 18 and March 11 and 18, 2009 (Preliminary Irritation Group) and December 30, 2008 and March 4, 2009 (Test and Naive Control Groups)

Housing: The animals were group housed in suspended stainless steel caging with mesh floors or plastic perforated bottom caging which conform to the size recommendations in the most recent Guide for the Care and Use of Laboratory Animals DHEW (NIH). Litter paper was placed beneath the cage and was changed at least three times per week.
Animal Room Temperature and Relative Humidity Ranges: 19-22 ºC and 40-60%, respectively
Photoperiod: 12-hour light/dark cycle
Acclimation Period: 5-22 days
Food: Pelleted Purina Guinea Pig Chow #5025
Water: Filtered tap water was supplied ad libitum by an automatic water dispensing system.
Contaminants: There were no known contaminants reasonably expected to be found in the food or water at levels which would have interfered with the results of this study.
Analyses of the food and water are conducted regularly and the records are kept in the study fecility
Identification of Animals: Each guinea pig was marked with a color code and given a sequential animal number assigned to study 26615, which constituted unique identification.

Study design: in vivo (non-LLNA)

Induction
Route:
epicutaneous, occlusive
Vehicle:
other: mineral oil
Concentration / amount:
undiluted test substance or a 50-75% w/w mixture (6-hour exposure)
Day(s)/duration:
Three times each week for three weeks
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Challenge
No.:
#20
Route:
epicutaneous, occlusive
Vehicle:
other: mineral oil
Concentration / amount:
four-tenths of a milliliter of a 0.8% w/w mixture of the test substance in mineral oil (HNIC)
Day(s)/duration:
24 and 48 h after the challenge application
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
Test Group: 20 per test
Naive Control Group: 10 per test
Details on study design:
Preliminary Irritation Testing
A group of animals was used to determine the highest non-irritating concentration (HNIC) of the test substance prior to the challenge dose. The fur was removed by clipping the dorsal area and flanks of each guinea pig. This area was divided into four test sites (two sites on each side of the midline) on each animal. The test substance was applied neat (100%) and also diluted in mineral oil to yield w/w concentrations of 75%, 50%, 25%, 12%, 6%, 3%, 1%, 0.8%, 0.6%, 0.4% and 0.2%. Each concentration was applied (0.4 mL) to a test site using an occlusive 25 mm Hill Top Chamber. The sites were wrapped with non-allergenic Durapore adhesive tape. After 6 hours of exposure, the chambers were removed and the test sites were gently cleansed of any residual test substance. Approximately 24 hours after application, each site was evaluated for local reactions (erythema) . From these results, the HNIC (the highest concentration that produced responses in 4 guinea pigs no more severe than two scores of 0.5 and two scores of zero) was established and used for challenge. The HNIC selected for the challenge phase was a 0.8% w/w mixture in mineral oil.

Preparation and Selection of Animals
On the day before initiation, the fur of a group of animals was removed by clipping the dorsal area and flanks. After clipping and prior to initiation, the animals were weighed and the skin was checked for any abnormalities. Only healthy, naive animals (not previously tested) without pre-existing skin irritation were selected for test. Animals were re-clipped prior to each dose.

Induction Phase
Three times each week for three weeks, four-tenths of a milliliter of the undiluted test substance or a 50-75% w/w mixture in mineral oil was applied to the left side of each test animal using an occlusive 25 mm Hill Top Chamber. The chambers were secured in place and wrapped with non-allergenic Durapore adhesive tape to avoid dislocation of the chambers and to minimize loss of the test substance. After the 6-hour exposure period, the chambers were removed and the test sites were gently cleansed of any residual test substance. For the first and second inductions the test substance was applied undiluted (100%). Due to severe irritation noted following the second application, the concentration of the test substance was reduced to a 75% w/w mixture in mineral oil for the third induction. Similar irritation persisted after this application; therefore the concentration was lowered to 50% in mineral oil for the fourth through ninth applications. Approximately 24 and 48 hours after each induction application, readings were made of local reactions (erythema).
Challenge controls:
Challenge Phase
Twenty-eight days after the first induction dose, four-tenths of a milliliter of a 0.8% w/w mixture of the test substance in mineral oil (HNIC) was applied to a naive site on the right side of each animal as a challenge dose, using the procedures described above. These sites were evaluated for a sensitization response (erythema) approximately 24 and 48 hours after the challenge application. In addition to the test animals, 10 guinea pigs from the same shipment were maintained under identical environmental conditions and were treated with the HNIC of the test substance at challenge only. These animals constituted the "naive control" group.
Positive control substance(s):
yes

Results and discussion

Positive control results:
Induction Phase - Historical Positive Control Animals (100% HCA): Very faint erythema (0.5) was noted for six of ten positive control sites at various intervals during the induction phase.
Challenge Phase - Historical Positive Control Animals (100% HCA): Three of ten positive control animals exhibited signs of a sensitization response (faint erythema) 24 hours after challenge. Similar indications persisted at one of these sites through 48 hours. Very faint erythema (0.5) was noted for most other sites following challenge.

In vivo (non-LLNA)

Resultsopen allclose all
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
four-tenths of a milliliter of a 0.8% w/w mixture of the test substance in vehicle
No. with + reactions:
2
Total no. in group:
20
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
100% HCA
No. with + reactions:
7
Total no. in group:
10
Remarks on result:
positive indication of skin sensitisation

Any other information on results incl. tables

Induction Phase

Test Animals (undiluted or as a 75% or 50% w/w mixture of the test substance in mineral oil): Very faint to severe erythema (0.5-3) was noted for all test sites throughout the induction phase. An area of corrosion was noted for most sites following the second and third applications. Due to irritation present at the dose sites after the second through eighth inductions, the dose site for all animals was relocated to an adjacent site for each subsequent application. In addition, due to the severity of irritation noted after the second and third induction applications, the concentration was lowered to 75% or 50% in mineral oil for the third and fourth through ninth inductions, respectively.

Challenge Phase

Test Animals (0.8% w/w mixture of the test substance in mineral oil): Very faint erythema (0.5) was noted for nine of twenty test sites 24 hours after challenge. Irritation persisted at two of these sites through 48 hours. Naive Control Animals (0.8% w/w mixture of the test substance in mineral oil): Very faint erythema (0.5) was noted for one of ten naive control sites 24 hours after challenge. Irritation cleared from the affected site by 48 hours.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Under the study conditions, the test substance was not considered to be a contact sensitizer.
Executive summary:

A study was conducted to determine the potential for the test substance to produce sensitization after repeated topical applications to guinea pigs according to OECD Guideline 406, EU Method B.6 and EPA OPPTS Method 870.2600, in compliance with GLP. The study was conducted twice. The results of the first study were disregarded because  the induction scores did not support the HNIC scores, which questioned the concentration used for the challenge phase. In the second test, the undiluted test substance or a 50-75% w/w mixture in mineral oil of the test substance was topically applied to 20 healthy test guinea pigs, 3 times each week for a 3 week induction period. Twenty-eight days after the first induction dose, a challenge dose of the test substance at its highest non-irritating concentration (HNIC, determined in the preliminary irritation screen to be a 0.8% w/w mixture in mineral oil) was applied to a naive site on each guinea pig. A naive control group (10 animals) was maintained under the same environmental conditions and treated with the test substance at challenge only. Approximately 24 and 48 h after each induction and challenge dose, the animals were scored for erythema. The positive response observed in the historical positive control validation study with alpha-Hexylcinnamaldehyde Technical (HCA) validated the test system used in this study. Under the study conditions, the test substance was not considered to be a contact sensitizer (Oley, 2009).