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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to
Guideline:
other: JMAFF 12-Nousan-8147 (2000)
Deviations:
no
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
LLNA study was not performed since in vivo guinea pig data were already available for the test substance.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Details on test material:
Purity: 99.4%
Impurities: Not reported

In vivo test system

Test animals

Species:
guinea pig
Strain:
other: Hartley albino
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Elm Hill Breeding Labs, Chelmsford, MA
- Age at study initiation: Preliminary Irritation Group: Young adult (5 weeks), Test and Test Vehicle Control Groups: Young adult (4 weeks)
- Weight at study initiation: 373-439 grams
- Housing: The animals were gang housed in plastic solid bottom polycarbonate cages or stainless steel solid bottom cages
- Diet: Approximately 20 grams per day
- Water: ad libitum
- Acclimation period: 14 or 19 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19-23°C
- Humidity: 53-65%
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal
Vehicle:
other: 2% w/v solution of CMC in distilled water
Concentration / amount:
The test animals received six intradermal injections (0.1 mL each) in the shaved suprascapular region as follows:
Emulsion of Freund’s Adjuvant Complete (50% v/v in distilled water), 1% w/w mixture of test substance in a 2% w/v solution of CMC in distilled water, 1% w/w mixture of test substance in an emulsion of Freund’s Adjuvant Complete (50% v/v in distilled water)
Day(s)/duration:
24 and 48 hours
Route:
other: Topical
Vehicle:
other: 2% w/v solution of CMC in distilled water
Concentration / amount:
0.5 grams of 65% w/w mixture of the test substance in a 2% w/v solution of CMC in distilled water
Day(s)/duration:
48 hours
Challenge
Route:
other: Topical
Vehicle:
other: 2% w/v solution of CMC in distilled water
Concentration / amount:
0.5 grams of a 65% w/w mixture of the test substance in a 2% w/v solution of CMC in distilled water (HNIC), 0.5 mL of a 22% w/w mixture in a 2% w/v solution of CMC in distilled water and 100% of a 2% w/v solution of CMC in distilled water
Day(s)/duration:
24 hours
No. of animals per dose:
Preliminary Irritation Testing: 12
Test Group: 20
Test Vehicle Control Group: 10
Details on study design:
RANGE FINDING TESTS:
Preliminary irritation testing
Preliminary Intradermal Injection: Prior to the induction phase, a group of four animals was used to determine the concentration of the test substance which produced faint to moderate irritation via intradermal injection. The fur was removed by clipping the suprascapular region of each guinea pig. This area was divided into six test sites (three sites on each side of the midline) on each animal. Each guinea pig received six intradermal injections (0.1 mL each); three concentrations (1, 3, and 5%) of the test substance in a 2% w/v solution of CMC in distilled water and the same concentrations in an emulsion of Freund’s Adjuvant Complete. All preparations in Freund’s Adjuvant Complete were thoroughly mixed with a tissue homogenizer prior to application. Approximately 24 and 48 hours after the injections, each site was evaluated for local reactions (erythema).
Preliminary Topical: Prior to the topical induction, a group of four animals was used to determine the irritation potential of the test substance to be used during the topical induction. The previously clipped flank area of each guinea pig was divided into two sites (one site on each side of the midline). The test substance was applied mixed with a 2% w/v solution of CMC in distilled water to yield w/w concentrations of 65%1 and 49%. Each concentration (0.5 g or mL) was applied to a 2 cm x 4 cm, 2-ply gauze patch and placed on one of the two test sites. The patch was covered with plastic wrap and secured in place with non-allergenic Durapore™ adhesive tape to avoid dislocation of the patch and to minimize loss of the test substance. After a 48 hour exposure period, the patches were removed and the test sites were gently cleansed of any residual test substance. One hour after patch removal readings were made of local reactions (erythema) according to the scoring system.
Highest Non-irritating Concentration (HNIC): Prior to the challenge phase, a group of four 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 65%, 49%, 33% and 16%. Each concentration (0.5 g or mL) was applied to an occlusive 25 mm Hill Top Chamber and applied to the appropriate test site. The sites were wrapped with non-allergenic Durapore™ adhesive tape. After 24 hours of exposure, the chambers were removed and the test sites were gently cleansed of any residual test substance. Approximately 24 and 48 hours after patch removal, each site was evaluated for local reactions (erythema) according to the scoring system.
Based on these findings, the concentration which produced faint to moderate irritation (1-2) selected for the intradermal induction was a 1%1 w/w mixture in a 2% w/v solution of CMC in distilled water. That which produced no irritation selected for the topical induction was a 65% w/w mixture in a 2% w/v solution of CMC in distilled water. Due to the lack of irritation produced during preliminary topical induction testing, a pretreatment of sodium lauryl sulfate was applied to all animals prior to the topical induction test substance application. 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 65% w/w mixture in a 2% w/v solution of CMC in distilled water.
MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 6
- Exposure period: 24 and 48 hours for Intradermal induction and 48 hours for topical induction
- Test groups: 1
- Control group: 1
- Site: Suprascapular region
- Concentrations: Induction phase: Emulsion of Freund’s Adjuvant Complete (50% v/v in distilled water), 1% w/w mixture of test substance in a 2% w/v solution of CMC in distilled water, 1% w/w mixture of test substance in an emulsion of Freund’s Adjuvant Complete (50% v/v in distilled water)
-Frequency of application: once in intradermal and once in topical
-Duration: 7 days

B. CHALLENGE EXPOSURE
- No. of exposures: 6
- Day of challenge: 21
- Exposure period: 24 hours
- Test groups: 1
- Control group: 1
- Site: Right and left flank of each animal
- Concentrations: 0.5 grams of a 65% w/w mixture of the test substance in a 2% w/v solution of CMC in distilled water (HNIC), 0.5 mL of a 22% w/w mixture in a 2% w/v solution of CMC in distilled water and 0.5 mL of a 2% w/v solution of CMC in distilled water
- Evaluation (hr after challenge): 24 and 48 hours
Challenge controls:
2% w/v solution of CMC in distilled water
Positive control substance(s):
yes
Remarks:
Alpha-Hexylcinnamaldehyde

Results and discussion

In vivo (non-LLNA)

Resultsopen allclose all
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
65% w/w in a 2% w/v solution of CMC in distilled water
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
Very faint erythema (0.5) was noted for four of twenty test sites 24 hours after challenge.
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
65% w/w in a 2% w/v solution of CMC in distilled water
No. with + reactions:
0
Total no. in group:
20
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
22% w/w in a 2% w/v solution of CMC in distilled water
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
Very faint erythema (0.5) was noted for three of twenty test sites 24 hours after challenge.
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
22% w/w in a 2% w/v solution of CMC in distilled water
No. with + reactions:
0
Total no. in group:
20
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
100% of a 2% w/v solution of CMC in distilled water
No. with + reactions:
0
Total no. in group:
20
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
100% of a 2% w/v solution of CMC in distilled water
No. with + reactions:
0
Total no. in group:
20
Reading:
1st reading
Group:
positive control
Remarks on result:
other: Historical positive control data were used. Positive indication of sensitization response was observed.
Reading:
1st reading
Group:
negative control
Dose level:
control group for 65 % w/w
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Group:
negative control
Dose level:
control group for 22% w/w
Remarks on result:
no indication of skin sensitisation

Any other information on results incl. tables

Table 1: Incidence of the sensitization response noted after challenge

Dose

Incidence with Skin Reactions1

Test animals

Test Vehicle Control/ Naïve Animals

Hours

24

48

24

48

65% w/w in a

2% w/v solution of CMC in distilled water

0/20

0/20

0/10

0/10

22% w/w in a

2% w/v solution of CMC in distilled water

0/20

0/20

0/10

0/10

100% of a 2% w/v solution of CMC in distilled water

0/20

0/20

0/10

0/10

1 Animals with scores greater than 0.5

Table 2: Severity of the sensitization response noted after challenge

Dose

Severity2

Test animals

Test Vehicle Control/ Naïve Animals

Hours

24

48

24

48

65% w/w in a

2% w/v solution of CMC in distilled water

0.10

0.00

0.00

0.00

22% w/w in a

2% w/v solution of CMC in distilled water

0.08

0.00

0.10

0.00

100% of a 2% w/v solution of CMC in distilled water

0.00

0.00

0.00

0.00

2 The test substance, as received, was a solid. To enhance skin contact, the test substance was moistened with a 2% w/v solution of CMC in distilled water prior to application.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Non sensitizer
Executive summary:

The study was conducted according to guidelines, U.S. EPA OPPTS 870.2600 and OECD Guideline 406. A Magnusson-Kligman maximization test was conducted with guinea pigs to determine the potential for 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. Preliminary irritation testing was performed on 12 animals to determine appropriate concentrations of the test substance that could be used for both intradermal and topical induction as well as topical challenge.

An emulsion of 50% v/v Freund’s Adjuvant Complete in distilled water was used during the intradermal injection screening and the injection induction phases. This emulsion was thoroughly mixed using a stir plate and is referred to throughout the report as an emulsion of Freund’s Adjuvant Complete.

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 carboxymethylcellulose (CMC) 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. A sham control group (ten animals) was maintained under the same environmental conditions and received injections of a 2% w/v solution of CMC in distilled water, a 2% w/v solution of CMC in distilled water (50% w/w) combined with an emulsion of Freund’s Adjuvant Complete, as well as an emulsion of Freund’s Adjuvant Complete alone. Approximately 24 and 48 hours after the injections, all sites were evaluated for an irritation response (erythema).

Approximately one week later, the second phase of induction was conducted. Due to a lack of irritation produced during preliminary testing, all animals received a pretreatment of sodium lauryl sulfate prior to test substance application. A 65% 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 then applied topically for a period of 48 hours to the area encompassing the intradermal injection sites. Approximately one hour after the topical induction patches were removed, all animals were scored for erythema.

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 65% 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 (22% 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 at challenge. Approximately 24 and 48 hours after challenge patch removal, all animals were scored for a sensitization response (erythema).

The results showed no incidence of skin reactions at 24 and 48 hours after challenge doses of 65% w/w in a 2% w/v solution of CMC in distilled water, 22% w/w in a 2% w/v solution of CMC in distilled water and 100% of a 2% w/v solution of CMC in distilled water. Based on these results, test substance is considered not to be a contact skin sensitizer.