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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:
August-September 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 guideline
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Version / remarks:
Buehler method
Deviations:
yes
Remarks:
One minor deviation that did not have any negative impacts on the results were described in the full study report and given in more detail below.
GLP compliance:
yes
Remarks:
GLP compliance statement is included in attached full study report
Type of study:
Buehler test
Justification for non-LLNA method:
There was no skin irritation observed (see section 7.3.1) during the in vivo study with rabbits. Because the substance is typically 98% water, and no toxicity or skin/eye irritation has ever been observed, a skin sensitization reaction is not expected to occur. No skin sensitization is shown here using the Buehler method.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
Sample ID: CP-100
0.18% complex polymeric polyhydroxy acids (based on total organic carbon content of 0.1%)
Storage at room temperature

In vivo test system

Test animals

Species:
guinea pig
Strain:
Hartley
Sex:
male/female
Details on test animals and environmental conditions:
Experimental Animals
Source: Charles River Laboratories; Wilmington, MA
Quantity & Sex: 2/sex (range-finding); 15/sex (definitive); females nulliparous and non-pregnant
Quarantine Period: 5 days
Date Born / Date Received: 30 Jun & 04 Jul 2009 / 03 Jul 2009
Animal/Group Identification: Ear punch
Weights When Tested: Male: 342-399 g ; Female: 343-386 g

Animal Husbandry
Cage Type: suspended, wire bottom, stainless steel
House: 1-5 per cage (males separate from females)
Environmental Controls: set to maintain 20 °C (+/- 3 °C), 30-70% relative humidity, 12 hour light/dark cycle, 10-12 air changes/hour. Actual temp/humidity: 19-23 °C, 68-97% relative humidity
Food: PMI Feeds Inc. Guinea Pig Diet #5025, available ad libitum
Water: Municipal water supply analyzed by TCEQ Water Utilities Division; available ad libitum from water bowls
Animal husbandry and housing at STILLMEADOW, Inc. comply with standards outlined in the “Guide for the Care and Use of Laboratory Animals” (NRC Publ.). No contaminants were expected to have been present in the feed or water that would have interfered with or affected the results of the study.

Study design: in vivo (non-LLNA)

Induction
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
Based on irritation screening [where 100% (undiluted) and 75%, 50%, and 25% v/v dilutions in deionized water, with each animal receiving 0.4 mL of each concentration at different test sites], 0.4 mL of the undiluted test substance was administered.
Day(s)/duration:
Exposure period for each induction was 6 hours. Group II animals were treated once weekly for three weeks (with 0.4 mL of the undiluted test substance). Induction treatments were on Days 1, 8, and 15.
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Challenge
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
After a two-week rest period, all animals (Groups I and II) were each challenged at a virgin test site with an application of 0.4 mL of the undiluted test substance.
Day(s)/duration:
Exposure period for the challenge treatment was 6 hours. The challenge treatment was on Day 29.
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
20 animals per dose (10 per sex).
Details on study design:
Irritation Screening
Two male and two female albino guinea pigs were selected for irritation screening (Diagram 1) to determine both the maximum dose producing no more than moderate irritation, and the maximum non-irritating dose. Concentrations tested in the screening were 100% (undiluted), and 75%, 50%, and 25% v/v dilutions in deionized water, with each animal receiving 0.4 mL of each concentration at different test sites.

Preparation of Animals
Males and females were selected for each of two treatment groups. Group I animals (5/sex) served as a naive control group and Group II animals (10/sex) were designated the test group. On the day prior to each treatment, the animals were prepared by clipping the back of the trunk free of hair to expose a longitudinal area of at least 8 x 10 cm on each animal. Individual body weights were recorded on Days 0 and 31.

Test Substance Administration
Based on the results of the irritation screening, the dose administered was an application of 0.4 mL of the undiluted test substance. For each induction treatment, Group II animals were treated by introducing the test substance beneath a 4-ply, 2.5 x 2.5 cm surgical gauze patch. Each gauze patch was placed laterally from the midline of the back on the left front quadrant of the exposure area (Diagram 2) and secured with a strip of non-irritating adhesive tape. A strip of clear polyethylene film was placed over the patch and securely taped. At the end of the 6-hour exposure period, the wrappings and patches were removed, and the animals were returned to their cages. Group II animals were treated once weekly for three weeks with 0.4 mL of the undiluted test substance. Induction treatments were on Days 1, 8, and 15. The same treatment regimen and test site location was used for all three induction treatments. Group I animals remained untreated during the induction phase of the study.

Challenge Treatment
After a two-week rest period, all animals (Groups I and II) were each challenged at a virgin test site with an application of 0.4 mL of the undiluted test substance. The challenge treatment was on Day 29. The dose was applied in a manner identical to the induction treatments, except the test site was placed laterally on the right rear quadrant of the exposure area (Diagram 2).

Observations and Scoring Method
Observations for skin reactions at each test site were made approximately 24 hours after each treatment. In addition, observations for skin reactions were made approximately 48 hours after the first induction treatment and 48 hours after the challenge treatment. The scoring scale used for grading skin reactions is presented as follows:
Erythema:
• No reaction: 0
• Very faint, usually nonconfluent: 0.5
• Faint, usually confluent: 1
• Moderate: 2
• Strong, with or without edema: 3
An average score for each time period was obtained by adding all of the scores for each time period and dividing by the number of test sites scored for that time period. The test substance is considered a sensitizer if the mean irritation scores, the total number of animals with scores, and/or the total number of scores for the virgin test site in the test group after the challenge treatment are appreciably greater than those for the naive challenge group.

Positive Control Material:
Label: Alpha-hexylcinnamaldehyde, tech., 85%, Batch # MKAA2596, Stabilized with 0.3 ppm BHA
Manufacturer: Aldrich
Physical Description: Clear yellow liquid
Storage: Room temperature
Purity, Composition, & Stability: Certificate of Analysis available from manufacturer
Concentrations Administered: Induction: 0.4 mL as received; Challenge: 0.4 mL as received

Positive Control Testing
The sensitivity of guinea pigs to a positive control material is confirmed in this laboratory periodically. The positive control animals used to conduct this study were supplied by Charles River Laboratories, and were tested according to the Buehler Method.

STILLMEADOW, Inc. Study No. 12972-09
In-life started: 6 May 09
In-life completed: 5 Jun 09

Positive Control Results
Data from this study are presented in Appendix A. A mean score of 0.6 for the test group after challenge treatment, when compared with naive control group mean score of 0.0, confirmed the sensitivity of guinea pigs to the positive control material.
Challenge controls:
The naïve control group (Group I) consisted of 10 animals (5 per sex). Group I animals remained untreated during the induction phase of the study. Group I animals were treated during the challenge phase, along with the Group II animals.
Positive control substance(s):
yes
Remarks:
hexyl cinnamic aldehyde (CAS# 101-86-0)

Results and discussion

Positive control results:
Data from this study are presented in Appendix A in the attached full study report. A mean score of 0.6 for the test group after challenge treatment, when compared with naïve control group mean score of 0.0, confirmed the sensitivity of guinea pigs to the positive control material.

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
0.4 mL of 85% alpha-hexylcinnamaldehyde
No. with + reactions:
9
Total no. in group:
10
Clinical observations:
One animal had no reaction (score of 0). Seven animals had very faint, usually nonconfluent reactions (score of 0.5). Two animals had faint, usually confluent reactions (score of 1).
Remarks on result:
positive indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
0.4 mL of 85% alpha-hexylcinnamaldehyde
No. with + reactions:
9
Total no. in group:
10
Clinical observations:
One animal had no reaction (score of 0). Seven animals had very faint, usually nonconfluent reactions (score of 0.5). Two animals had faint, usually confluent reactions (score of 1).
Remarks on result:
positive indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
0.4 mL of undiluted substance
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
No erythema reactions were observed for any animals in Group I (naïve control group) during the challenge phase
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
0.4 mL of undiluted substance
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
No erythema reactions were observed for any animals in Group I (naïve control group) during the challenge phase
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
0.4 mL of undiluted substance
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
No erythema reactions were observed for any animals in Group II (test group) during the challenge phase
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
0.4 mL of undiluted substance
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
No erythema reactions were observed for any animals in Group II (test group) during the challenge phase
Remarks on result:
no indication of skin sensitisation
Reading:
other: Induction Treatments, Group II
Hours after challenge:
48
Group:
test group
Dose level:
0.4 mL of undiluted substance
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
No erythema reactions were observed for any animals in Group II (test group) during the induction phase
Remarks on result:
no indication of skin sensitisation
Reading:
other: Induction Treatments, Group II
Hours after challenge:
24
Group:
test group
Dose level:
0.4 mL of undiluted substance
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
No erythema reactions were observed for any animals in Group II (test group) during the induction phase
Remarks on result:
no indication of skin sensitisation

Any other information on results incl. tables

Body weights are given in Table 3 of the attached full study report.


 


Protocol Deviations:


1) Relative humidity was outside protocol range but did not affect study outcome


 

Applicant's summary and conclusion

Interpretation of results:
other: does not elicit a skin sensitizing reaction
Conclusions:
The test substance, Carbon Power, produced no irritation in the test animals (Group II) or in the naïve control animals (Group I) after the challenge treatment, and therefore did not elicit a sensitizing reaction in guinea pigs.