Abstract
Capsule Summary
The Research Institute for Fragrance Materials, Inc. (RIFM) conducted a pilot study in order to more closely mimic the patch testing conducted by dermatologists assessing allergic contact dermatitis.
During this pilot, no significant differences in sensitization were noted between the two challenge protocols.
Adopting this new protocol for RIFM-conducted CNIHs does not invalidate previously published studies conducted according to the 2008 RIFM standard protocol.
INTRODUCTION
The Research Institute for Fragrance Materials, Inc. (RIFM) outlined a standard protocol for human-repeated insult patch tests (HRIPTs). 1 This protocol is appropriate for cosmetic and other nonprescription, topically applied test materials. The HRIPT, as conducted by RIFM, is never used to determine hazard but to confirm that a test material will not induce dermal sensitization in humans at a predetermined no observed effect level (NOEL). The studies conducted by RIFM are always in compliance with Good Clinical Practice guidelines and approved by the Institutional Review Board (IRB) for each testing laboratory. The methods and tools for determining a NOEL and defining the no expected sensitization induction level (NESIL) have previously been described. 2 Defining the NESIL is an essential step of dermal quantitative risk assessment, as outlined by Api et al.3,4
RIFM evaluated 30 years of HRIPT data that conformed to the RIFM standard protocol. 5 The studies selected each have approximately 100 participants completing the study, use a standard vehicle mixture of ethanol (EtOH) and diethyl phthalate (DEP), include a negative control, and use an occlusive patch (typically a 25-mm Hilltop Chamber). Data from 154 HRIPTs using 16,512 volunteers recorded only 20 instances of de novo skin sensitization induction (0.12%). Moreover, in the last 11 years, only 3 (0.03%) of 9854 subjects became sensitized. This analysis confirmed that the risk of inducing skin sensitization in the HRIPT using the RIFM standard protocol is very low. RIFM adopted the name “confirmation of no induction in humans” (CNIHs) to distinguish the confirmatory nature of RIFM-conducted HRIPTs from the exploratory nature of HRIPTs generally. 5 CNIH testing remains necessary to minimize the risk of inducing skin sensitization in a larger human population since no appropriate method exists to replace confirmatory human testing accurately.
At the recommendation of the Expert Panel for Fragrance Safety, 1 RIFM considered adopting a 48-hour challenge for future CNIH studies. This longer challenge phase would ensure that future studies would more closely mimic the patch testing conducted by dermatologists assessing allergic contact dermatitis. 6 A pilot study was undertaken to confirm that adopting a new protocol would not invalidate the 30+ years of human patch testing data collected by RIFM.
MATERIALS AND METHODS
Test Subjects
A sufficient number of subjects (approximately 120) were empaneled so that at least 100 fully completed the study. The subjects were informed of the nature of the test, including possible adverse reactions, and written, informed consent was obtained. Before starting the study, each subject completed a medical history form. Subjects were excluded if they exhibited any physical or dermatological condition that would preclude applying the test materials. The inclusion and exclusion of criteria are shown in Table 1.
Inclusion and Exclusion Criteria for Confirmation of No Induction in Human Study Subjects
Test Materials and Patch Conditions
A 0.3-mL aliquot of each material was evaluated under occlusive patch conditions. The materials were applied to a 25-mm Hill Top Chamber System® where it volatilized for at least 15 minutes but no longer than 40 minutes. The patches were applied to the infrascapular area of the back, either to the right or left of the midline, and were secured with hypoallergenic tape (Micropore) as needed. Saline served as a negative control and a vehicle control contained the solvent of a 1:3 mixture of EtOH and DEP.
Irritation Pretest
An irritation pretest was typically conducted 1–4 weeks before the start of the induction phase. Using a precise, repeating syringe, 0.3 mL of each test material, vehicle control, or saline was applied to each designated patch. The patches were applied to the backs of approximately 5 subjects. Each subject was instructed that the patches were to remain in place and kept dry for approximately 48 hours. After 48 hours, the patches were removed, the test sites were read, and the scores were recorded. Approximately 72 hours, 96 hours, and 168 hours after patching, the subjects returned to the testing facility. A technician reassessed the test sites and recorded the scores according to the scoring scale shown in Table 2.
Confirmation of No Induction in Human Study Scoring System
CNIH Protocol
Using a precise, repeating syringe, 0.3 mL of the test material, vehicle control, or saline was applied to each designated patch. The left side of the back was usually the test area for the induction phase. The subject’s skin was marked with a gentian violet surgical marker on the left side of the test site. The test site was recorded on the anatomical diagram of each subject’s individual data form. The induction phase consisted of a series of 9 applications of the study materials and subsequent evaluations of the application sites. Patches were applied on Mondays, Wednesdays, and Fridays for 3 consecutive weeks. The patches remained in place, and the subjects were instructed to keep the area dry for approximately 24 hours, at which time they were to remove the patches. Approximately, 24 hours after patch removal (48 hours after patch application), the subjects returned to the facility to have the sites evaluated and identical patches applied to the same sites. Patches applied on Friday were removed by the subjects at home approximately 24 hours after application and then evaluated on Monday. Reactions of erythema with edema (1E or 2E and greater) observed at the first or second evaluation were considered evidence of possible pre-sensitization, and application of the product would be discontinued on the affected subjects. Product application would continue on all other subjects. Subjects absent once during the induction phase would either receive a makeup (MU) patch during or at the end of the induction phase or be discontinued. The MU applications were graded 48 hours later at the MU visit or recorded as no ninth grading (N9R). Subjects who missed the ninth evaluation (N9R) but had received 9 patch applications were considered to have completed the induction phase. If an MU visit was required at the end of the induction phase, grading would be performed either 24 or 48 hours after patch application, then either immediately after removal of the patch or 24 or 48 hours later.
A rest period of approximately 2 weeks followed the last induction patching; no test material was applied during the rest Period. The preferred minimum rest interval was 9 days, and the preferred maximum was 22 days.
At the challenge phase, the original induction test site was observed, and each subject was queried as to whether any reaction was experienced during the rest Period. Materials were applied similarly to the induction phase. The opposite side of the back was usually the virgin test site for the challenge phase. The challenge patches were applied to the virgin site only. Two identical series of patches were applied to each subject. Each subject was again instructed to keep the patches on and dry. Each subject returned to the testing site approximately 24 hours later (Challenge Reading 1), at which time 1 series of the patches was removed, and the challenge site was scored and recorded by the technician. The original (induction) test site was also observed. Each subject returned to the testing site at approximately 48 hours after patching (Challenge Reading 2), at which time the other patch was removed, and the challenge site was scored, and results were recorded by the technician. Each subject returned to the testing site approximately 72 hours (Challenge Reading 3), approximately 96 hours (Challenge Reading 4), and approximately 168 − 192 hours (Challenge Reading 5) after patching for additional evaluation; reactions of both test sites were scored and recorded.
To be considered a completed case, a subject was required to have 9 applications of the study materials with no fewer than 8 subsequent readings during induction and a single application and 4 readings during the challenge. Only completed cases were used to assess sensitization.
RESULTS
In total, there were 21 CNIHs conducted using 19 fragrance ingredients. More information about these materials, including the dosage tested, is shown in Table 3. Reactions level 1E or level 2E and greater were observed in 3 out of the 21 panels. These reactions were attributable to the saline control (1 reaction) or the vehicle control (2 reactions), and none were due to the test materials alone. While there were reactions in 14% of the panels during the pilot, they (saline or vehicle control) only occurred in 0.13% (3 out of 2317) of the subjects who fully completed a study during the pilot. Additionally, these reactions were only observed in the 48-hour challenge patching as opposed to the 24-hour challenge patching. The specific reactions are described here:
Test Material Information and Dosage
RIFM, Research Institute for Fragrance Materials, Inc.
1,3,4,6,7,8-Hexahydro-4,6,6,7,8,8-hexamethylcyclopenta-γ-2-benzopyran (CAS No: 1222-05-5) was tested in panel #21 − 110. During the challenge phase, one subject exhibited a reaction at the site that was patched with the saline control for 48 hours. The reaction—erythema, covering the whole test area, with edema (1E)—was observed approximately 48 hours after patch removal (96 hours after patch application). When the subject returned approximately 168 hours after the patch application, the reaction had subsided to an unspecified reaction in which the subject displayed faint, minimal erythema with dryness (±DR).
Nerolidol (isomer unspecified; CAS No: 7212-44-4) was tested in Panel #21 − 116. When the challenge patches were applied for 48 hours, one subject exhibited a level 1E reaction with the test material as well as the vehicle control approximately 48 hours after patch removal (96 hours after patch application). When the subject returned approximately 168 hours after patching, the reactions had subsided to an unspecified reaction where the subject displayed erythema, covering the whole test area with dryness (1DR). Based on the guidance of the Expert Panel for Fragrance Safety, considering that an equivalent edematous reaction was observed in the same subject for both the test material and the vehicle, the observed edematous reaction was due to the vehicle (1:3 EtOH:DEP). Based on the pattern observed, the level 1 reactions to the test material, vehicle control, and saline control that were experienced as a result of the 48-hour challenge patching were likely false positive reactions due to irritancy.
Tetrahydro-4-methyl-2-phenyl-2H-pyran (CAS No: 94201-73-7) was tested in Panel #22 − 106. During the challenge in which the patches were applied for 48 hours, one subject exhibited intense erythema, covering the whole test area, with edema (2E) the test site for the vehicle control. This reaction was observed when the patch was removed (48 hours after patch application) and 24 hours later (72 hours after patch application). Forty-eight hours after patch removal (96 hours after patch application), the reaction subsided to erythema, covering the whole test site with edema (1E). When the subject returned approximately 168 hours after patch application, the reaction had subsided to an unspecified reaction in which the subject displayed erythema, covering the whole test area (1).
Cumulative results for the pilot study are summarized in Table 4 for the test materials, Table 5 for the vehicle (1:3 EtOH:DEP) control, and Table 6 for the saline control. Individual tables for each study and material are available as Supplemental Tables. In addition to the reactions described above, more unspecified reactions were observed with the 48-hour challenge patching. Unspecified reactions were especially higher with the saline control.
Cumulative Summary Tables for Test Materials
Cumulative Summary Tables for Vehicle (1:3 EtOH:DEP) Control
DEP, diethyl phthalate.
Cumulative Summary Tables for Saline Control
DISCUSSION
RIFM has a long history of conducting CNIH tests as part of its safety assessment program for dermal sensitization evaluations on fragrance ingredients. 5 These studies are essential for confirming a NESIL to perform dermal quantitative risk assessment on fragrance ingredients with the potential to induce skin sensitization. As suggested in the name, the CNIH test is conducted to confirm a NOEL, as it is the most reliable test method by which confirmatory human data can be generated. Historically, RIFM has performed these tests according to the published standard protocol. 1 Volunteers are patch tested with a fragrance ingredient under exaggerated conditions at a predetermined dose that is not expected to induce skin sensitization. The protocol mimics the 2 phases of the skin sensitization adverse outcome pathway—the induction phase and the elicitation phase (typically referred to as the challenge phase).
The present study was conducted upon recommendation by the Expert Panel for Fragrance Safety. All the key steps of the RIFM standard protocol remained unchanged, except for the introduction of a 48-hour patch, in addition to the 24-hour patch, during the challenge phase. An irritation pretest screening and an additional reading 168 hours post-patching were also added. The goal of this pilot study was to test the outcome of adopting a 48-hour challenge in place of the 24-hour challenge in the RIFM standard protocol. The 48-hour challenge patch would enhance the exaggerated exposure to the test material (thus increasing the sensitivity of the test) and align with how current clinical patch testing is conducted.6–8
Even with the exaggerated 48-hour challenge patching, during this pilot, the chosen NOEL was confirmed through CNIH testing. RIFM, at the recommendation of the Expert Panel for Fragrance Safety, has now adopted the 48-hour patching for the challenge phase of CNIH testing. To address the increase in unspecified reactions of faint, minimal erythema (±) observed when using saline as the negative control, the Expert Panel for Fragrance Safety recommended switching to distilled water as a negative control instead of saline.
It is important to note that the 48-hour challenge patching used in CNIH testing may increase the likelihood of irritation, as suggested by the reactions observed with the saline negative control. This heightened response could also lead to materials with irritation potential being mistakenly identified as sensitizers under these exaggerated conditions during CNIH testing. RIFM uses CNIH testing specifically to confirm a NOEL, which is a distinct purpose from that of diagnostic patch testing. Anyone choosing to adopt the protocol outlined in this paper should be aware that it may overstate a material’s irritation potential compared to typical use conditions.
Following the Politano and Api (2008) protocol, the material was allowed to volatilize prior to patch application for at least 15 minutes but no longer than 40 minutes. The Expert Panel for Fragrance Safety recommended that volatilization should no longer be utilized due to concerns about evaporation leading to a reduced tested dose. 9 Therefore, at the conclusion of this pilot study, the new RIFM CNIH protocol would also remove the 15-minute requirement for volatilization and instead apply patches immediately after preparation and discard them if they are not applied within 5 minutes.
The 3 positive reactions observed in this pilot study were attributed to either the saline control or the vehicle control, and the subjects did not return to the testing facility for a rechallenge, as outlined in the published standard protocol. The Expert Panel has advised that, in cases where reactivity suggestive of sensitization is observed, a rechallenge conducted only on the subjects showing reactions, and not the whole panel, would not be sufficient to confirm lack of skin sensitization due to the test material. In these cases, the Expert Panel recommends that all subjects be rechallenged.
Another protocol modification in this study, and adopted in the new RIFM CNIH protocol, is that scoring of the test site did not end at the 72-hour reading but continued with the addition of 96-hour and 168- to 192-hour readings. The latter reading/scoring occurred approximately 1 week after the application of the challenge patch, which is not only in line with current clinical patch testing protocols but allows for sufficient time to make a conclusive analysis of the patched site.
CONCLUSIONS
In brief, the new RIFM CNIH protocol includes the following changes: (1) removing the 15-minute volatilization requirement to apply patches immediately and discarding them after 5 minutes instead of 40 minutes; (2) replacing saline with distilled water for the negative control; (3) adding the irritation pretest screening; (4) adding an additional reading at 168 hours post-patching; and (5) changing the challenge patching duration from 24 to 48 hours.
Although patching for 48 hours is more sensitive than patching for 24 hours, during this pilot, there was no significant increase in sensitization when comparing the 24-hour challenge patching to the 48-hour challenge patching. Therefore, adopting this new 48-hour challenge patching for RIFM-conducted CNIHs does not invalidate previously published studies conducted according to the 2008 RIFM standard protocol, which utilized a 24-hour patching during the challenge phase.
