March 13th, 2017
EPA-HSRB-17-1
Robert J. Kavlock, Ph.D.
Acting EPA Science Advisor
Office of the Science Advisor
1200 Pennsylvania Avenue, NW
Washington, DC 20460
Subject: January 25-26, 2017 EPA Human Studies Review Board Meeting Report
Dear Dr. Kavlock,
The United States Environmental Protection Agency (l-IW or Agency) requested that the
Human Studies Review Board CTTSRB) pio\ ide scientific and ethics reviews of five items:
•	A published study. Cimclidinc-CarbarYl Interaction in Humans: Evidence for an
Active Metabolite of Carbarvl. authored by I). Gail May, Rebecca J. Naukam, J.
Neddy kamham. and Robert A. Branch. Journal of Pharmacology Exposure
Therapy (1992) 262(3). 1057-1061;
•	An unpublished study A randomized double blind study with malathion to determine
the residues of malathion dicarboxylic acid (DCA), malathion monocarboxylic acid
(MCA), dimethyl phosphate (DMP), dimethyl thiophosphate (DMTP), and dimethyl
dithiophosphate (DMDTP) in human urine;
•	A published study, Methylisothiazolinone contact allergy and dose-response
relationships, authored by Michael D. Lundov, Claus Zachariae, and Jeanne D.
Johansen. Contact Dermatitis (2011) 64, 330-336.
•	A published study, Methylisothiazolinone in rinse-off products causes allergic contact
dermatitis: a repeated open-application study, authored by K. Yazar, M. D.
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Lundov, A. Faurschou, M. Matura, A. Boman, J. D. Johansen, and C. Liden. British
Journal of Dermatology (2015) 173,115-122.
• A published study, An evaluation of dose/unit area and time as key factors
influencing the elicitation capacity of
methylchloroisothiazolinone/methylisothiazolinone (MCI/MI) in MCI/MI-allergic
patients, authored by Claus Zachariae, Anne Lerbaek, Pauline M. McNamee, John
E. Gray, Mike Wooder, and Torkil Menne. Contact Dermatitis (2006) 55,160-166.
The charge questions posed to the Board included assessing scientific and ethical aspects of these
studies viewed individually, as well as the proposed combined use of the three studies related to
methylisothiazolinone for a weight of evidence approach to risk assessment.
The Board's responses to the charge questions and detailed rationale and recommendations are
provided in the enclosed final meelinu report
Signed,
Liza Dawson. Phi)
Chair
EPA Human Studies Re\ie\\ Board
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INTRODUCTION
On January 25-26, 2017, the United States Environmental Protection Agency's (EPA or Agency)
Human Studies Review Board (HSRB or Board) met to address the scientific and ethical charge
questions related to five items:
•	A published study, Cimetidine-Carbaryl Interaction in Humans: Evidence for an
Active Metabolite of Carbaryl, authored by 1). (.nil May, Rebecca J. Naukam, J.
Reddy Kambam, and Robert A. Branch. Journal of Pharmacology Exposure
Therapy (1992) 262(3), 1057-1061;
•	An unpublished study: A randomized double blind study willi malathion to determine
the residues of malathion dicarboxylic acid (DCA), malathion monocarboxylic acid
(MCA), dimethyl phosphate (DMP), dimethyl lliiophosphate (DMTP). and dimethyl
dithiophosphate (DMDTP) in human urine:
•	A published study, Methylisolhiazolinone contact allergy and dose-response
relationships, authored by Michael I). I. undo v. (Inns Zachariae, and Jeanne D.
Johansen. Contact Dermatitis (2011) 64. 330-336.
•	A published study. Mclhylisolhiazolinonc in rinse-off products causes allergic contact
dermatitis: a repealed open-application study, authored by K. Yazar, M. D.
l.undoY. A. l-'aiirschou. M. Matura. A. IJomnn, J. D. Johansen, and C. Liden. British
Journal of Dermatology (2015) 173,115-122.
•	A pnhlishcd study. An evaluation of dose/unit area and time as key factors
influencing the elicitation capacity of
methylchloroisothiazolinone/methylisothiazolinone (MCI/MI) in MCI/MI-allergic
patients, authored by Claus Zachariae, Anne Lerbaek, Pauline M. McNamee, John
E. Gray, Mike Wooder, and Torkil Menne. Contact Dermatitis (2006) 55,160-166.
In addition, EPA requested that the HSRB respond to an additional charge question related to the
use of the three MI studies in a weight of evidence approach to establishing a point of departure
for risk assessment.
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REVIEW PROCESS
The Board conducted a public meeting on January, 25-26, 2017. Advance notice of the meeting
was published in the Federal Register as "Human Studies Review Board; Notification of a
Public Meetings" (EPA, December 19, 2016, 95982, Vol. 81, No.250).
Following welcoming remarks from Agency officials, the Board began its consideration of the
materials presented for review. On day one of the meeting. N\\ staff began by presenting an
overview of physiologically based pharmacokinetic (PlJI'k) modeling and its intended uses by
the Agency prior to the review of specific studies ofcimelidine caiharvl and malathion. On day
two of the meeting, Agency staff presented an o\ er\ iew of repealed open application test
(ROAT) studies prior to the review of the dermal exposure studies related to
methylisothiazolinone. These informational sessions provided ihe Board with context for the
scientific and ethical review of each study and help Board members understand how the data
would contribute to a larger process of risk assessment In the Agency.
On each day, the Board then heard presentations from I -PA for each agenda item in sequence,
consisting of the Agency's re\ iew of scientiiic and ethical aspects of the two studies, followed
by discussion from members of the I ISRIi who re\ iewed each of the studies for scientific and
ethical soundness This I 'inal Report of the meeting describes the HSRB's discussion,
recommendations, rationale and consensus in response to each charge question for each of these
items.
For each agenda item. Agency staff first presented their review of the science and the Board
asked the Agency presenters clari lying questions. The staff then described their review of the
ethical aspects and the Board asked clarifying questions about those. The HSRB solicited public
comments and next asked Agency staff to read the Charge Questions under consideration. The
Board discussed the science questions first and then the ethics question. The Chair then called for
a vote to confirm concurrence on a summary statement in response to each charge question.
For their evaluation and discussion, the Board considered materials presented at the meeting, oral
comments, related materials and documents provided by the study sponsors, the Agency's
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science and ethics reviews of the studies, oral responses from a study sponsor and protocol team,
and public comments made at the meeting.
HSRB review of a published article: Cimetidine-Carbaryl Interaction in Humans:
Evidence for an Active Metabolite of Carbaryl, authored by D. Gail May, Rebecca J.
Naukam, J. Reddy Kambam, and Robert A. Branch. Journal of Pharmacology Exposure
Therapy (1992) 262(3), 1057-1061
CHARGE TO THE BOARD AND BOARD RESPONSE
Charge to the Board: Is the research described in the published article "Cimetidine-Carbaryl
Interaction in Humans: Evidence for an Active Metabolite of Carbaryl" scientifically sound,
providing reliable data?
Board Response:
The Board concludes thai the research described in the published article "Cimetidine-Carbaryl
Interaction in Humans l-\ idence lor an Acli\ e Metabolite of Carbaryl" is scientifically sound
and pro\ ides reliable data that may or may not be uenerali/able to larger human populations.
HSRI? Detailed Recommendations and Rationale:
This study, performed in the curly I990's, involved in vitro tests of carbaryl, alone or together
with the histamine-2 (112) receptor antagonist, cimetidine, using erythrocyte acetylcholinesterase
(Acse) activity as a pharmacodynamics endpoint of carbaryl-related bio-activity. In vivo study
in this work, in four male subjects, followed plasma carbaryl and erythrocyte Acse activity over
time following a single, oral dose. Subsequently the impact of pretreatment with a known
cytochrome P450 inhibitor on pharmacokinetics and pharmacodynamics in those same study
participants was assessed by pretreating them with 600 mg of cimetidine (divided into three, 8-
hour doses) daily for three days, followed by a single carbaryl oral dosing. The investigators
found that when cimetidine was pre-administered, that carbaryl peak plasma concentration was
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higher, although not statistically significant; oral clearance was lower and statistically significant
(p<0.05); and plasma half-life was longer, but not statistically significant. These findings
suggested that metabolism of carbaryl was inhibited by cimetidine. Unexpectedly, the in vivo
studies suggested that, in spite of the increased body exposure to carbaryl caused by cimetidine
pretreatment, the inhibition of Acse by carbaryl was reduced in the pretreated individuals.
Authors concluded with a tenable hypothesis that carbaryl is metabolized (by a cimetidine-
inhibitable enzyme) to a more pharmacodynamically active metabolite.
The HSRB commented that one of the staten»-:>!s maiie in sh«-' '.!\\ summary of the paper
appeared to be incorrect, namely, "The results of the in vitro assays confirmed that, at a
concentration of 10 or 100 pg/mL, cimetidine did not impact the RBC AChE inhibition caused
by increasing concentrations of carba. v 1." In fact, at 1 OOug/ml both cimetidine and carbaryl
inhibit erythrocyte Acse, so the tota' ohser\ u.1 inhibition is imix-.ted. Furthermore, EPA's
statement implies that the combined Hlou is :=cklili\ hut llv: u=ii;==ss at least raise the possibility
that the effect may not be additive ot 1 00ml; mi dii^iidhie Th< 'iraphs appear not to be
completely additive, and in ;=n\ case n»e are not conducive on this point. The board also
pointed out an inconsistency 1:» n»'- paper. 1:» n»ai fin* n;:per reports 4 human subjects, but reports
n=6 ir.i ;;I \ SllKh' S "his tiiSCIVpiUK'V IS <\p!:;;M!'d.
The Hoard opines thai the fundamental study design seems sound, and the results suggest that
there is a real possibility of a drug-drug interaction between carbaryl and cimetidine. The second
element of the charge is related to provision of reliable data. The Board expressed confidence in
the analytical/experimental reliability of the results. With regard to the human population-level
reliability and generalizabilily of the results, the small number of subjects, the unclear process
for selecting study participants, and the fact that the putative underlying processes uncovered in
this study (activity of drug metabolizing enzymes) is subject to substantial individual variability
in humans, all taken together reduce the Board's confidence in the population level reliability of
the data. Nevertheless, the study is of acceptable scientific merit for EPA scientists to use the
data.
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Comments on statistical aspects of the study
Cimetidine-carbaryl interaction in humans is investigated in study with four non-smoking, drug-
free normal males. In vitro assays, cimetidine did not impact the outcome, whereas with vivo
exposure, pre-treatment of cimetidine had an effect. The results support the hypothesis that the
main site of metabolism for carbaryl is the liver by drug-metabolizing enzymes that can be
inhibited by cimetidine.
The sample size of n=4 subjects seems small and it is unclear how the sample size was
determined. However, this may be a pilot study, as the purpose appears to be to investigate the
pathway for these four individuals in depth and there is not as much concern about the
population-level inference.
Statistical methods used for the data analysis include regression analysis, A\()Y.\, and repeated
measures analysis. However, details are largely missing about the software package and
procedures used. It appears that data and the code for analyzing the data are not available,
making it a challenge to e\ aluate the quality of the data analysis.
Ethics
Charge to the Board:
Does a\ ailahle information support a determination that the study was conducted in substantial
compliance with subparts k and I. of 4<) (TR part 26?
Board Response
While the research was conducted before implementation of 40 CFR, the information provided
supports a determination that the studies were conducted in substantial compliance with subparts
K and L of 40 CFR Part 26.
HSRB Detailed Recommendations and Rationale:
40 CFR 26 subpart K requires that studies initiated on or after April 7, 2006 involving intentional
exposure of human subjects to a pesticide be reviewed and approved by an institutional review
board (IRB) that meets the membership and review criteria listed in that subpart. The Vanderbilt
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Institutional Committee for the Protection of Human Subject reviewed and approved of this
study. While we do not know if this Committee meets the criteria stated in 40 CFR 26 subpart K,
there was independent review at a reputable research university, the HSRB believes this 1991
study complied with spirit of independent review.
40 CFR 26 subpart K mandates studies minimize risk to subjects, equitably select subjects, seek
and appropriately document informed consent, make ac1c(.|iiate pro\ isions to ensure safety of
subjects, and protect the privacy of subjects and confidentiality of data.
Minimize Risk: There is not a great deal of information pro\ ided ah out steps the investigators took
to minimize participant risk. The dosages used in the study were below the level to cause symptoms
and were "not expected to cause any additional short term adverse e\ enls
Equitable Selection: The investigator did not pro\ ide information about equitahility of selecting
the 4 male participants in this study It appears to he a coin cnience sample of individuals
affiliated with the University of Pittsburgh Center of Clinical Pharmacology, probably including
the principal investigator. The Board did discuss the inclusion of only men in this study and
noted the time period of the research (early llwn"S). prior to the widespread recognition of the
importance of including women as well as men in biomedical research in order to obtain reliable
information about the effects of treatments or exposures in females. The Board agreed that,
given the time period of the study, inclusion of only male participants was consistent with typical
research practice, particularly for non-henelicial exposure studies.
Informed Consent As the research w as done 26 years ago, neither the investigators nor the
independent re\ iew hoard could not pro\ ide informed consent documentation, but the
investigator told N\\ that participants 1) were told of the science and risks of the study, 2) were
sufficiently educated to understand the risks, and 3) signed informed consent documentation. The
investigator does not pro\ ide information about provisions to ensure safety, protect privacy or
confidentiality. While not ideal, the board does not feel the lack of this information violates the
intention of 40 CFR 26 subpart K.
40 CFR 26 subpart L prohibits the EPA from relying on third-party research involving
intentional exposure to a pesticide of human subjects who are children or pregnant or nursing
women. The 4 participants were male, so this does not violate 40 CFR 26 subpart L.
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HSRB review of an unpublished study: A randomized double blind study with malathion to
determine the residues of malathion dicarboxylic acid (DCA), malathion monocarboxylic
acid (MCA), dimethyl phosphate (DMP), dimethyl thiophosphate (DMTP), and dimethyl
dithiophosphate (DMDTP) in human urine.
CHARGE TO THE BOARD AND BOARD RESPONSE
Charge to the Board: Did the research on plasma le\ els of malalhion and malaoxon, and
urinary metabolites of malathion, as described in the study reports "A randomized double blind
ascending single oral dose study with malathion lo determine the No Tflcct I .e\ el on plasma and
RBC cholinesterase activity" and "Determination of residues of malathion dicarboxylic acid
(DCA), malathion monocarboxylic acid (MCA), dimethyl phosphate (DMP), dimethyl
thiophosphate (DMTP), and dimethyl dilhiophosphate (DMDTP) in human urine," generate
scientifically sound, reliable data '
Board Response:
TheBiuisci cond titles Hum iho resenivh on plnsnui i"\els of malathion and malaoxon, and urinary
metabolites of nuikilhion. as ("S'.-nbet1 liic study repuiis "A randomized double blind
ascending single oral dose study with malathion to determine the No Effect Level on plasma and
RBC cholinesterase activity" and "Determination of residues of malathion dicarboxylic acid
(DCA), malathion mo;H;v;.;: ho-;yiie acid (MCA), dimethyl phosphate (DMP), dimethyl
thiophosphate ( >V .'M. nnt1 dimethyl dithiophosphate (DMDTP) in human urine," generated
scientifically sound, reliable duui liiat may or may not be generalizable to larger human
populations.
HSRB Detailed Recommendations and Rationale:
The unpublished study report describes two studies. The first was a study in which 48 human
participants were administered placebo or malathion at one of 5 different doses ranging from 0.5
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mg/kg to 15 mg/kg. The study was entitled "A randomised double blind ascending single oral
dose study with malathion to determine the no effect level on plasma and RBC cholinesterase
activity" and it was conducted by Inveresk Research, Elphinstone Research Centre, Tranent, EH
32 2NE, Scotland.1 In this study, the pharmacodynamic endpoints of plasma and erythrocyte
acetylcholinesterase (Acse) inhibition were assessed. The second study was entitled
"Determination of residues of malathion dicarboxylic acid (DCA), malathion monocarboxylic
acid (MCA), dimethyl phosphate (DMP), dimethyl thiop;><-sphai
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subject level. That is, it is unclear why were the data analyzed separately for male and female
subjects. The model diagnostics are also unclear. More details and justifications for a particular
choice (e.g. linear trend detection versus Bonferroni correction for multiple comparison) would
be helpful to have. In addition, it is not clear how much the analysis can leverage the robustness
of these methods against non-normality. Based on the description about the residuals, it seems
that there is some heavy-tail (i.e., non-normal) distribution in the error term. It may be interesting
to examine the robustness issue further.
Ethics
Charge to the Board:
Does available information support a determination that the study was conducted in substantial
compliance with subparts K and L of 40 CFR pai l 2(->?
Board Response
While the research was conducted before implementation of 4<) (T R, the information provided
supports a determination thai the studies were conducted in substantial compliance with subparts
K and L of 40 CFR Part 2o
HSRB Detailed Recommendations and Rationale:
40 CI R 2(-> subpart k ivcjuiivs that studies initiated on or after April 7, 2006 involving intentional
exposure of human subjects to a pesticide be re\ iewed and approved by an institutional review
board (IRBj that meets the membership and review criteria listed in that subpart. The Inveresk
Research Independent l-thics Review Committee reviewed and approved of this study. Based on
information provided, the Committee meets the criteria stated in 40 CFR 26 subpart K. The study
was conducted in accordance with the guidelines set out in Declaration of Helsinki, 1964 and its
1975, 1989, 1996 amendments. The HSRB believes this study complied with spirit of
independent review. Amendments not reviewed by the Review Commitment did not affect
patient safety.
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40 CFR 26 subpart K mandates studies minimize risk to subjects, equitably select subjects, seek
and appropriately document informed consent, make adequate provisions to ensure safety of
subjects, and protect the privacy of subjects and confidentiality of data.
Minimize Risk: The study listed above minimized risk to subjects by recruiting participants who
were healthy (normal physical exam, laboratory data, and ECG), choosing a dose low not
expected to cause adverse effects, and not escalating doses if there were any significant
inhibition of cholinesterase. The study also contacted the participants' primary physician to
determine if there was a concern with that individual participating in this study.
Equitable Selection: There are no specific information regarding the population from which
participants were recruited, except that they were from the surrounding area and a generic
advertisement was used. The use of randomization to different dosage or placebo allowed for
equitable exposure to risk.
Informed Consent: The study physician explained the stuck and participants were allowed to ask
questions. Written information regarding nialathion Additionally, this document contained most
of the elements contained in a standard informed consent document (procedures, risks and
benefits, withdrawing from the study, confidentiality, and physician contact information).
Safety: A physician pro\ ided o\ ersight for the entire study Participants resided in the study
clinic for 2 days and were monitored at outpatient \ isits 3, 5, and 13 days after dosing.
Privacy and Confidentiality luich participant was assigned a random individual identification
number which protected pri\acy and confidentiality
40 CI R 2(-> subpart I. prohibits the l-IW from relying on third-party research involving
intentional exposure to a pesticide of human subjects who are children or pregnant or nursing
women Materials submitted to l-l'A and HSRB indicate that no children or pregnant or nursing
women enrolled in this stuck Pregnancy testing occurred up to 21 days prior and again the day
prior to the study. As a result, the study was conducted in substantial compliance with 40 CFR
part 26, subpart L.
HSRB review of a published study Methylisothiazolinone contact allergy and dose-response
relationships, authored by Michael D. Lundov, Claus Zachariae, and Jeanne D. Johansen.
Contact Dermatitis (2011) 64, 330-336.
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CHARGE TO THE BOARD AND BOARD RESPONSE
Charge to the Board: Is the research described in the published article "Methylisothiazolinone
contact allergy and dose-response relationships" scientifically sound, providing reliable data?
Board response:
The board after discussion agreed that the research in the published article
"Methylisothiazolinone contact allergy and dose-response relationships" is scientifically
sound, providing reliable data. In the use of this data for risk assessment to establish the
elicitation threshold for methylisothiazoline, the board wishes lo highlight some relevant
exposure conditions or statistical concerns thai should lx- considered when using this data.
HSRB detailed recommendations and rationale:
In this study authors wanted to 1) in\ estigalc the eliciting doses of MI in a patch test and in a
ROAT for already sensitized subjects. 2) in\ estigate w hether pheno-oxyethanol (PE) influenced
reactivity to MI, and 3) test a model lor an exposure coin ersion between ROAT and Patch test.
The model will not be discussed here Tor this study I I lest subjects (2 women and 9 men,
average age of 49.7 years) with pre\ ions positive reactions to MCI/MI or MI were included.
There were 14 controls ((•> women and N men. average age 27.5) recruited into study. Subjects
and controls were patch-tested first with 12 decreasing doses of MI (60 down to 0.0105 |ig/cm2)
in 10".. ethanol and W'n aqua, and then again with those same doses of MI in same mixture but
with i) 4".. pheno-oxyethanol (l) 2o ug cm2). Blanks consisted of just the 0.4% pheno-oxyethanol
in aqua and ethanol Readings occurred on days 2, 3, 4 and 7 following occlusion for 2 days on
the subject's backs Placement of doses was double blinded. For the ROAT arm of study,
patients applied 20 ul of Iooppm, 50 ppm and 5 ppm of MI mixture containing pheno-
oxyethanol (using a fixed micropipette) twice a day on 3 x 3 cm on the volar part of forearm
resulting in concentrations of 0.42, 0.21 and 0.021 |ig/cm2/day (this amount is double the per
application dose because the study involved two applications a day with no rinse off). Response
were determined on day 2, 3, 4, 7, 14, and 21. In this study, an 8-point response scale from 0 (no
reaction) to 8 (intensive erythema, infiltration and papules) was used for patch and ROAT.
Responses greater than 5 on the response scale triggering stopping exposures in the ROAT arm.
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Results: The study reported no statistical difference in reactions for test subjects in patch test
with or without phenoxyethanol, or across threshold doses. Responses were seen at
concentrations starting at 1.47 |ig/cm2 (55%) in the PATCH, while there was no response to 60
|ig/cm2for controls in PATCH. In the ROAT study, for test subjects, 7/11 (64%) reacted to the
0.21 |ig/cm2/day, and 2/11 (18%) to the 0.021 |ig/cm2/day (this dose is twice the per application
dose due to twice daily exposure). Controls had no reaction in the ROAT study. Doses are
described as low 1.47 |ig/cm2 for MI in PATCH test (55%. which would mean a concentration in
cream of 49 ppm). The percentage of individuals who responded lo 0.021 |ig/cm2/day in the
ROAT was 18%.
The HSRB expresses confidence that this stuck pro\ ides reliable and \ alicl data. The Board
notes that sufficient information is provided In the authors that the stuck has the potential to be
repeated and validated. The study objectives are stated: an acceptable control group is used; and
the investigators use measures to reduce bias in the stuck In conducting a double blind study and
by using an acceptable reading scale lor responses The in\ estigators use methodologies to help
assure adherence to study protocols (e.g.. instructions to subjects, micropipette for application),
and response rates related to application rates concentrations are reported and related back to the
objectives. These study characteristics provide support for the Board's conclusion that the data
are scientifically sound
The Board further recommends that l-l'.V consider the following issues that may affect elicitation
thresholds in the stuck
1)	Five sets of ROAT bottles were weighed to determine application conformance by
subjects. howe\ er beyond stating adherence, no exact weights were reported and
compared between control and test subjects.
2)	Accumulated doses are mentioned in this study and compared to the PATCH results
(again PATCH studies are occluded). This is done to build their model comparison
between PATCH and ROAT. EPA may want to consider for risk assessment purposes
the relationship of ROAT studies looking at elicitation threshold and potentially to relate
back to induction threshold or even irritant thresholds that avoid induction and therefore
elicitation points in participants. These relationships are only mentioned as a potential
'model' to explore elicitation thresholds, where induction of participants can be avoided
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or where human subjects for studies who have been induced are hard to find. Authors
suggest repeated lower doses elicited quicker responses based on their comparison curves
between PATCH and ROAT using equivalent ROAT doses, and an algorithmic
relationship between the two is derived.
3)	Although it is established that pheno-oxyethanol is not a sensitizer based on lack of
response in subjects when pheno-oxyethanol was applied alone, this does not necessarily
preclude that it could act synergistically or antagonistically with MI. For the ROAT arm,
MI was not applied alone and therefore pheno-oxyethanol should not be entirely
dismissed as having any vehicle effects when comparing lo other studies.
4)	Mi-allergic subjects were previously tested at concentrations of up to 2000 ppm.
Confirmatory PATCH testing was also conducted at this level These are potentially high
induction threshold levels and the relationship between induction thresholds and
elicitations thresholds are not well understand, thus the PATCH testing itself could have
contributed to elicitation thresholds, although this is not known.
5)	The average days to respond in the ROAT are not specifically reported and might be of
interest. In addition, two of the I I subjects in the ROAT did not follow the application
scheme. It is not clear how this affected the statistical analysis
Comments on statistic:il aspects of (lie study
Standard logistic regression analysis was used to estimate the dose-response relationship in the
patch tests. The l-l)5<) and l>5"o CI were estimated from the dose response curve. Comparison
between the patch test reactions with or without phenoxyethanol was performed with Wilcoxon's
ranked sums lest, and correlations between the individual threshold doses were investigated by
Spearman's ranked correlation Differences in reactions to the same doses in the patch test and
ROAT were investigated with VLcNemar's test.
Based on the information related to statistical analysis provided in the article, the statistical
validity of patch test results is questionable due to the following reasons: (1) because same
subjects were used for with and the without treatments in the patch tests, the observed responses
derived from treatments with and without phenoxyethanol were usually correlated, and this
correlation was ignored in the dose-response models; and (2) the use of the statistically
significant Spearman's ranked correlation to conclude that treatments with and without phenoxy
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ethanol produced similar rankings was inappropriate; (3) the Agency's statistical validation
confirmed the statistical results reported in the paper (ED50 and 95% CI were estimated from the
dose response curve) was non-reproducible.
In summary, although there were flaws in the statistical analysis, the data presented in this article
could be considered as scientifically sound and providing reliable data but limited to weight of
evidence (WOE) based applications when combined with other two reviewed papers.
Ethics
Charge to the Board: Does available information siipptx l a delermination that the study was
conducted in substantial compliance with subparts k and L of 40 (T R pai l 26?
Recommendation for the Board: The portions of the I-PA regulations regarding the conduct of
research with human subjects, 40 CI 'R pai l 2o subpart k and I.. do not apply because the
research was not conducted or supported In I-PA. nor was it conducted with the intention to
submit the results to F.PA. F.P A identified this study through a re\ ieu of the public literature; the
published article or any results of this research were not independently submitted to EPA.
The study was conducted in Denmark, therefore, the I S federal Insecticide, Fungicide and
Rodenticide Act (IIIRA) did not apply to the conduct of this research, but does for EPA use of
data generated
The available information supports a determination that the study is in substantial compliance
with subpart Q of 4<) CI 'R Part 2(-> (Ethical Standards for Assessing Whether to Rely on the
Results of Human Research in EPA Actions). Assuming scientific validity, the study may be
used in actions under FIFRA (7 U.S.C. 136 et seq.) and section 408 of the Federal Food, Drug
and Cosmetic Act (FFDCA).
Rationale: In accordance with 40 CFR 26.1703, the research did not involve intentional
exposure of any pregnant or nursing female subjects or any children. All of the subjects in this
study were adults. Pregnancy and nursing were exclusion criteria for both test and control
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subjects. Female subjects were not tested for pregnancy before participation; they were asked if
they were breastfeeding or could be pregnant. Given the minimal risk nature of the study (no
subject had any severe or widespread reaction) and that patch testing during pregnancy or
lactation is not known to be harmful, this seems adequate.
In accordance with 40 CFR 26.1704, the study was conducted under procedures at least as
protective as those in subparts K and L of 40 CFR Part 26. The protocol and the published article
state that the study was conducted according to the principles in the Declaration of Helsinki.
Documents provided to the EPA indicate that the study was re\ iewed and approved by the
Capital Region of Denmark (the ethics committee with jurisdiction) on May 17, 2010. The ethics
committee reviewed the study prior to initiation, amendments and reports with standards similar
to those required by EPA (Danish Act on Research l-thics Review of Health Research Projects
and the Ministerial Order No 806 of 12 July 2004 on Information and Consent)
The informed consent materials contained adequate information for the subjects (n=25) about the
risks, discomforts and benefits of participation, and of their right not to participate or to
withdraw (n=0). The risk-benefit ratio was determined to he acceptable and risks were
minimized appropriately and were justified by the potential societal benefits associated with
gathering data to determine the dose-response relationship in persons already sensitized to or
allergic to the test article
The selection of study participants appeared to be equitable and no children or pregnant women
were enrolled No conflicts of interest were declared. There is no evidence that the conduct of the
research was fundamentally unethical or deficient relative to ethical standards and minimized the
risk of harm to subjects
HSRB review of a published study, Methylisothiazolinone in rinse-off products causes
allergic contact dermatitis: a repeated open-application study, authored by K. Yazar, M. D.
Lundov, A. Faurschou, M. Matura, A. Boman, J. D. Johansen, and C. Liden. British
Journal of Dermatology (2015) 173,115-122.
17
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CHARGE TO THE BOARD AND BOARD RESPONSE
• Charge to the Board: Is the research described in the published article
"Methylisothiazolinone in rinse-off products causes allergic contact dermatitis: a
repeated open-application study" scientifically sound, providing reliable data?
The board after discussion agreed that the research in the published article
"Methylisothiazolinone in rinse-off products causes allergic contact dermatitis: a repeated
open-application study" is scientifically sound, pro\ idinu reliable data.
HSRB detailed recommendations and rationale
In the use of this data for risk assessment to establish the elicitation threshold for
methylisothiazoline, the board wishes to highlight some rele\ anl exposure conditions or
statistical concerns that should be considered when using these data.
The objective of this study was to determine if currently allowed concentrations of MI in rinse-
off products can cause allergic dermatitis In this study consisting of 19 MI allergic subjects
(mean age 40 years) and N control non-MI allergic subjects (mean age 37 years) participated. In
one arm of the study ^ Ml allergic subjects and all controls applied a 100 ppm MI soap five
times a day to a 5 cm \ I n cm area of the \ entral side of the forearm, resulting in a concentration
of 0.48 ug cm: per application and a daily concentration of 2.4 |ig/cm2. In the other arm of the
study I ii MI allergic subjects and all controls applied a 50 ppm MI soap five times a day to a
5cm x 11) cm area to the \ entral side of the forearm resulting in a concentration of 0.24 |ig/cm2
per application and a daily concentration of 1.2 |ig/cm2. Controls used soaps with no MI.
Confirmatory patch test lor Ml subjects was 6 solutions of MI soap from 60 down to 0.48
|ig/cm2, and vehicle control of aqua and 16% petrolatum. Control subjects only received the 60
|ig/cm2 concentration soap. Patch tests were applied to back, occluded for 2 days, with readings
on 4 day only. Readings for the ROAT were conducted once a week unless a reaction was
experienced. Trained nurses and researchers conducted the reading for the PATCH, and
researchers conducted readings for the ROAT. The study used an 8-point response scale from 0,
(no reaction) to 8 (intensive erythema, infiltration and papules) for patch and ROAT. The
investigators also used the Fisher scale ( >25% area of application). Threshold concentration for
18
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a positive reaction in this study was a visible reaction of 1 on the scale on day 4 (if there was a
continuous reaction). Subjects received instruction manuals and an instruction video on the
application methods. The area of skin is moistened, soap applied and spread evenly and allowed
to dry for 25s (some absorption into the skin is expected), area is rinsed pat dried, and then a
moisturizer (supplied by team) is applied once daily to area. Study participants were instructed
not to use any of their own products during the study.
Results: 10/10 (100%) of participants who used the 1( »< >ppni (<) 4S ug/cm2/application)
developed a reaction to MI soaps within 4-11 days (a\ crime 7 3 days); while 7/9 (78%)
developed a reaction to the 50 ppm soaps within 5-21 days (average X days). Controls had no
reaction to MI soaps and neither subjects nor controls had reaction lo the Mi-free soap. There
was statistical significance for the 100 ppm and 5<) ppm soap compared lo controls (Fisher test).
In the PATCH test, some MI -allergic subjects (3 I ^) reacted lo doses as low ns <) 48 |ig/cm2. All
Mi-allergic subjects reacted to the 15 uu cm2 level.
The HSRB expresses confidence that this study pro\ ides reliuhlc and valid data. The Board
notes that sufficienl information is provided In the authors tliiit the study has the potential to be
repeated and validated. The study ohjccth es are stated, an acceptable control group is used; and
the investigators used measures to reduce bias in the study by conducting a double blind study
and by using an acceptable reading scale lor responses The investigators use methodologies to
help assure adherence to study protocols (eg. instructions to subjects, soap bottles with pump
rates), and response rates related to application rates/concentrations are reported and related back
to the objecti\ es These study characteristics provide support for the Board's conclusion that the
data are scientifically sound.
The Board further recommends that EPA consider the following issues that may affect elicitation
thresholds in the study:
1) There is a need to clarify locations (anatomical) of application across studies.
Anatomical areas will differ in their sensitivity due to Stratum Corneum/Viable epidermis
thickness and other structural differences resulting in varying local reactions. If cosmetics
using MI are applied to various parts of the body, the most sensitive areas should be
tested/considered in a risk assessment.
19
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2)	It is not clear why readings occurred on day 4 as opposed to day 2 and day 4 for the
PATCH test.
3)	Detailed information is provided in this study on where the MI and liquid soap
preparation was obtained and chemical content of all applied test and control products.
This will allow further analysis of the potential role of vehicle effects on MI elicitation
thresholds.
4)	Because this is a rinse-off study, the application, daily dose or accumulated may not be
reflective of actual dose that remains on the skin, in the skin or absorbed into the skin
(total dose compared to the ROAT). The actual I ,().\l-1. in this study may in fact be
lower due to removal of the product following rinse acti\ilics. and pat and dry activities.
5)	The control soap in the MI studies was preserved with methyl and ethyl paraben.
Although there was no reaction to control soap in the ROAT, il might he i mportant to
note the presence of these preser\ ati\ es
6)	MI for rinse off products can be found in liquid soaps and shampoos. Exposure rates will
depend on customary uses of the products This study tested liquid soap at 5 times a day
and makes a statement that this may underestimate usage rate There is no study
reference lor usage rates (\olumes applied and areas of application), although a
conservati\ e approach (testing at higher doses and for higher usage rates) is appropriate
in order to establish a safety factor In the risk assessment, some questions arise in
comparing Ml studies with \arious application rates and therefore applied dose, or
loading rates.
7)	Vehicles effects for those w ho applied the optional provided lotion are not discussed.
Comments on sl:ilislic:il sispeels of the study
The study goal was to determine if MI concentrations of 100 ppm and 50 ppm have the potential
to elicit an allergic response in previously sensitized individuals. The treatment group consisted
of 19 subjects with dermatitis and contact allergy to MI. Assignment to treatment group was
determined by patch testing at 2000 ppm prior to start of the study. Ten subjects were assigned
to 100 ppm dosages, and 9 subjects to 50 ppm. There was no indication of how the assignments
were determined. The control group consisted of 19 subjects without contact allergy to MI as
determined by no allergic reaction to patch testing prior to the start of the study. The
20
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investigators used Fisher's Exact Test to compare differences in the proportion of allergic
responses for the two groups. This test would have been appropriate if underlying assumptions
were met, however there was a problem related to study design, because there was no 50ppm
control group. For MI of 100 ppm there was significant difference (p = 5.0 x 10"8) based on 10
allergic and 10 controls. The authors also claim there was a significant difference for an MI of
50 ppm (p = 3.0 x 10"5), but the control subjects were nol tested at 50 ppm. The apparent
assumption of the investigators is that the fact that suh jecls had no reaction at 100 ppm of MI for
a control subject implies they would have no reaction at 50 ppm of MI This might be a
reasonable guess, but for statistical purposes, the design of the study should have included a 50
ppm control group.
The investigators used McNemar's lest lor equality of proportions lor comparison of pre-study
patch test and ROAT on Ml allergic subjects. For 10 allergic subjects at 100 ppm of MI there
was a significant difference (p = 0.00195). None reacted to the patch test but all 10 reacted to the
ROAT testing. The problem with the use of this test. ho\\e\ er. is that the p-value is for two
sided alternative (difference in either direction) Init the authors" conclusion is that ROAT
reactivity is higher (one sided conclusion) I or all I ^ allergic subjects,3 there was a significant
difference (p <> <)<)<).122) between the reaction to the patch test at 100 pm and the combined 100
ppm and 5<) ppm reactions to the ROAT testing
Finally, the in\ estigators used Kendall's tau-B for an association for the allergic subjects
between a positi\ e MI patch test threshold and the threshold for ROAT. Two subjects with no
ROAT reaction were not included Kendall's tau-B is the difference between the number of
concordant and discordant pairs of observations, which is not quite the same as the usual
correlation coefficient, although the authors describe this as a test for correlation. The results
show that Kendall's tau-B = 0.381 and p= 0.062; the p value from SAS is 0.0569 which indicates
a computational difference, but does not change the overall finding that the test was not quite
statistically significant.
3 The Board notes a typographical error in the paper: the n = 49 for ROAT at 50 ppm should be n =
19.
21
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In summary, the chosen statistical analyses are appropriate but some of the conclusions have
what might be considered as minor flaws because the underlying assumptions are not met.
Specifically, the lack of a 50 ppm control group, the one-sided conclusion from a two sided test,
and the [problem with the Kendall's tau-B test] somewhat weaken the statistical analysis.
Ethics
Charge to the Board:
Does the available information support a determination that the studies were conducted in
substantial compliance with subparts K and L of 4<) CFR Part 20?
Board response:
HSRB detailed recommendations and rationale:
The portions of the EPA regulations regarding the conduct of research with human subjects, 40
CFR part 26 subpart k and I.. do not apply because the research was not conducted or supported
by EPA, nor was it conducted with the intention to submit the results to EPA. EPA identified this
study through a re\ iew of the public literature, the published article or any results of this research
were not independently submitted to l-l\\.
The study was conducted in Sweden and Denmark; therefore, the U.S. Federal Insecticide,
Fungicide and Rodenlicide Act (F1FR A) did not apply to the conduct of this research, but does
for EPA use of data to set a human dermal sensitization endpoint/point of departure in its risk
assessment for niethylisothia/olinone.
The available information supports a determination that the study is in substantial compliance
with subpart Q of 40 CFR Part 26 (Ethical Standards for Assessing Whether to Rely on the
Results of Human Research in EPA Actions). Assuming scientific validity, the study may be
used in actions under FIFRA (7 U.S.C. 136 et seq.) and section 408 of the Federal Food, Drug
and Cosmetic Act (FFDCA).
In accordance with 40 CFR 26.1703, the research did not involve intentional exposure of any
pregnant or nursing female subjects or any children. All of the subjects in this study were adults.
22
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Pregnancy and nursing were exclusion criteria for both test and control subjects. Female subjects
were not tested for pregnancy before participation; they were asked if they were breastfeeding or
could be pregnant. Given the minimal risk nature of the study (no subjects had any severe or
widespread reaction) and that patch testing during pregnancy or lactation is not known to be
harmful, this seems adequate.
In accordance with 40 CFR 26.1704, the study was conducted under procedures at least as
protective as those in subparts K and L of 40 CFR Part 20 The protocol and the published article
state that the study was conducted according to the principles in the Declaration of Helsinki.
Documents provided to the EPA indicate that the study was re\ iewed and approved by regional
ethics review boards in Stockholm, Sweden and Capital Region, Denmark (the ethics committees
with jurisdiction) in July 2013. The ethics committees reviewed the study prior to initiation with
standards similar to those required by EPA (Swedish Act on the Ethical Re\ iew of Research
Involving Humans; Danish Act on Research I vthics Re\ iew of I lealth Research Projects; and the
Ministerial Order No 806 on Information and Consent )
The informed consent materials contained adequate information lor the subjects (n=38) about the
risks, discomforts and benefits of participation, and of their l ight not to participate or to
withdraw (n=2). The risk-benefit ratio was determined to be acceptable and risks were
minimized appropriately and were justified In the potential societal benefits associated with
gathering data to determine if products preser\ ed with allowed concentrations of the test article
have the potential to elicit allergic contact dermatitis in previously sensitized individuals.
The selection of study participants appeared to be equitable and no children or pregnant women
were enrolled. T\o eonllicts of interest were declared. There is no evidence that the conduct of the
research was fundamentally unethical or deficient relative to ethical standards and minimized the
risk of harm to subjects.
HSRB review of a published study, An evaluation of dose/unit area and time as key factors
influencing the elicitation capacity of methylchloroisothiazolinone/methylisothiazolinone
(MCI/MI) in MCI/MI-allergic patients, authored by Claus Zachariae, Anne Lerbaek,
Pauline M. McNamee, John E. Gray, Mike Wooder, and Torkil Menne. Contact Dermatitis
(2006) 55,160-166.
23
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CHARGE TO THE BOARD AND BOARD RESPONSE
Charge to the Board: Is the research described in the published article "An evaluation of
dose/unit area and time as key factors influencing the elicitation capacity of
methylchloroisothiazolinone/methylisothiazolinone (MCI/MI) in MCI/MI-allergic patients"
scientifically sound, providing reliable data?
Board Response:
The board after discussion agreed that the research in the published article "An evaluation of
dose/unit area and time as key factors influencing the elicitation capacity of
methylchloriisothiazoline/ methylisothiazoline (MCI Ml) in MCI/MI allergic patients" is
scientifically sound, providing reliable data
In the use of this data for risk assessment to establish the elicitation threshold for
methylisothiazoline. the board wishes to highlight some rele\ant exposure conditions or
statistical concerns 1hat should be considered when using this data.
The authors evaluated the allergic response of MCI Ml patients to the application of a mixture of
MCI/MI where authors wanted to determine the elicitation response to a mixture of MCI/MI and
the influence of time and concentration (i.e., the exposure scenario) on elicitation thresholds.
This study enrolled 29 eczema patients aged under 18 who have a previous response to 100 ppm
MCI/MI in a patch test, where response was confirmed for 25 with a diagnostic patch test to 100
ppm MCI/MI. There were healthy 10 volunteers enrolled with no response to 100 ppm MCI/MI
in a patch test. The dose of MCI MI for Diagnostic Patch was 3 |ig/cm2 (determined from
applying 15ju.l of 100 ppm on a 0.5 cm2). A controlled patch using deionized water with 10%
ethanol was applied for two days with readings at days 2, 3, 4, and 7. The design of the double
blind placebo ROAT was for subjects to apply 2 drops (estimate volume of 0.05668|il) of a
MCI/MI mixture, twice a day for 4 weeks to 9 cm2 area of the volar part of forearm, and allow to
dry before putting on clothing. ROAT 1 was a total dose of 0.025 |ig/cm2 per day (4 drops over
the course of a day using a concentration of 2 ppm MCI/MI over a 9 cm2 area). ROAT 2 was
conducted 4 weeks after the end of ROAT 1, and also for 4 weeks. ROAT 2 was a total dose of
24
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0.094 |ig/cm2 per day (4 drops over the course of a day using a concentration of 7.5 ppm
MCI/MI over a 9 cm2 area). Response was graded as weak, moderate or strong for outcomes of
erythema, papules and vesicles. Test bottles were weighed before and after use, which helps to
confirm overall dose and daily dose over the 4 week period. Average daily dose across subjects
(those who had negative or positive responses and from ROAT 1 and ROAT 2) and controls was
comparable according to the authors, although no significance testing was reported on that
comparison.
Seven out of 25 (28%) subjects showed a positive response in ROAT 1 and the average number
of days to response was 16.5 (reported as non-significaul compared to vehicle control). Fourteen
out of 25 (56%) subjects showed a positive response in ROAT 2 and a\ erage days to response
was 12.1 days (reported as significant compared lo \ ehicle). The difference between ROAT 1
and ROAT 2 responses was statistically significant The majority of reactions were classified as
weak or moderate; the 10 non-allergic controls did not show reactions in the ROAT arms of the
study.
The HSRB expresses confidence that this study pro\ ides reliable and valid data. The Board
notes that sufficient information is pro\ ided by the authors that the study has the potential to be
repeated and validated The study objecti\ es are stated, an acceptable control group is used; and
the in\estimators used measures to reduce bias in the study by conducting a double blind study
and by using an acceptable reading scale lor responses The investigators use methodologies to
help assure adherence to study protocols (e g., instructions to subjects, bottles for application),
and response rates related to application rates/concentrations are reported and related back to the
objectives. These stuck characteristics provide support for the Board's conclusion that the data
are scientifically sound
The HSRB recommends that EPA consider the following scientific issues that may affect
elicitation responses.
1) Application to same area, following a rinse out period might be expected to result in
more rapid sensitivity for the ROAT 2 arm. Given the number of positive reactions
and the days to reaction in ROAT 1 versus ROAT 2, this outcome is in fact reported.
This may indicate a growing sensitivity to exposures of MCI/MI mixtures, for
25
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especially sensitive individuals. However, because a higher dose of MCI/MI was used
for ROAT 2, it is not possible to determine whether repeated application, or higher
dose, or both factors play a role. It unclear what the outcome would have been if the
same dose or even lower was used in ROAT 2 arm compared to ROAT 1. In this
study, 5 of the 7 who showed positive responses to ROAT 1, showed positive
responses also to ROAT 2, with reactions in first week. The HSRB suggests that
ROAT 2 may not offer any additional information on exposure conditions and
threshold responses, given the higher dose used
2)	The mixture of MCI/MI is not confirmed although authors mention a typical mixture
of 3:1. If this assumption is used to determine the concent rati on of MI, the ROAT 1
results is (0.025 |ig (MCI/MI)/cm: day 4) x 1 = 0.00625 uu (MTVcm2/ day and for
ROAT 2 = (0.094 |ig (MCI/MI)/cm: day 4) \ I n 0235 |ig (Ml) cm2/day.
Although authors state that they belie\ e that the elicitation threshold is in the
proximity of 0.025 |ig(MCI Ml) cm2 day (bused on response in ROAT 1 to daily
applications of this concentration), ihey found no significance for this level compared
to control In addition, this would indicate a dose of <> oii625 Hg (MI)/cm2/day of MI
alone (2S"() responded). assuming all the response is attributed to the MI in the
mixture I lo\\e\ er. it may not be possible to disaggregate the response to the
indi\idual components in the mixture In Lundov et al., 2011, page 334, MCI is
mentioned as a more potent sensitizer than MI. This is not a study to determine the
sensitix el\ of allergic patients to MI but to a mixture of MCI/MI. It is possible that
MCI can act to increase or decrease the activity of MI (i.e., synergistic or antagonistic
effects) The potential synergy or antagonism is not tested in this study but may be an
important consideration depending on whether a determination is to be made of MI in
specific mixtures, for specific application types. EPA may want to clarify if a
distinction between MCI and MI is necessary in this risk assessment determination.
3)	Authors have not confirmed what other ingredients are in the MCI/MI mixtures that
might act as sensitizers or influence elicitation responses. Ethanol is mentioned an
ingredient.
4)	This study, compared to those by Lundov et al., 2011 and Yazar et al., 2015 used
eczema patients as study subjects (although none with active eczema). This could
26
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result in higher response rates, or higher sensitivities for subjects, because these
subjects might be more sensitive to lower concentrations of MI or MCI/MI mixtures.
However the various skin conditions (and their population prevalence) that influence
elicitation thresholds is not well understood.
5)	Age or sex of participants is not reported to determine demographic representation.
Greater skin sensitivities might be expected for the very young and in the elderly.
Sensitivity might also be greater on areas like the face or scrotum. In this study the
forearm is used. In an elicitation response, greater sensitivity may be related to
thickness of the stratum corneum and access lo the \ iahle epidermis. Thickness of
layers of the skin vary over the body, and also In age and sex
6)	There is some suspicion that elicilalion response (i.e., threshold") may be related to
thresholds for induction. Full history of the parlici pants are not know n, and the study
does not report whether these participants were already induced/sensitized and at
what level, or if the conllrmalory patch at I'm ppm is their threshold for induction.
This is a lower level confirmatory patch than used in the other studies compared here.
7)	Response was based on a grading scale or weak, moderate or strong for outcomes of
erythema, papules and \esicles It is not clear is this is a slightly different reading
scale than that used in the two other studies under review, in which an 8-point reading
scale is mentioned
Comments on slsilislicsil sispecls of (lie study
The study goal was to determine the effect of concentration and time on elicitation capacity of
MCI/MI in sensitized subjects using ROAT testing. The treatment group consisted of 29 eczema
subjects; the number of males and females is not specified. Assignment to treatment group
required at least one prior positive reaction to patch test with MCI/MI concentration of 100 ppm.
The control group consisted of 10 healthy subjects with no allergic reaction to pre-study patch
test at 100 ppm. In the study, a pre-study patch test followed by two 4 week stages of ROAT
testing separated by a 4 week washout period. Stage 1 was ROAT with an MCI/MI
concentration of 2 ppm (0.025 (j,g/cm2); stage 2 was ROAT with an MCI/MI concentration of 7.5
ppm (0.094 (j,g/cm2). The investigators used Fisher's Exact Test which was appropriate for
27
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comparing the proportion of subjects in the allergic group having a positive reaction to the
control group proportion. A logistic regression was used for the allergic group subjects to
compare the proportions of positive reactions in the stage 1 ROAT and stage 2 ROAT. A subject
effect was included in the analysis. The problem with the logistic regression analysis is that
apparently subjects were not considered as random effects. Also, with a random subject effect,
significance of the results using SAS GLIMMIX depended on the method used to fit the logistic
model (p = 0.0407 with Residual pseudo-likelihood; p 4036 w iih Laplace). The investigators
also used Kaplan-Meier survival (no positive reaction) esti males o\ er time to a positive reaction
were calculated from the stage 1 and stage 2 data. The problem w ilh this analysis is that the
stage 1 and stage 2 are not independent because lliev come from the same set of subjects. In
summary, the chosen statistical analyses might be appropriate but there are issues with whether
or not the authors' underlying assumptions w ere reasonable
Ethics
Charge to the Board
Does available information support a determination that the study was conducted in substantial
compliance witil subparts k and I. of4<) (T'R part 2(->?
Board Response lo (lie Charge
HSRIS Recommendation (statement to \ote on)
The HSRB concludes that the a\ ailable information supports a determination that the Zachariae
et al. (2006) study was conducted in substantial compliance with subpart Q of 40 CFR part 26.
HSRB Detailed Recommendations and Rationale
The Agency's rules at 40 CFR part 26 subpart Q that are applicable to this review include the
following:
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§26.1703: Except as provided in §26.1706, EPA must not rely on data from any research subject
to this subpart involving intentional exposure of any human subject who is a pregnant woman
(and therefore her fetus), a nursing woman, or a child.
And,
§26.1704: EPA must not rely on data from any research subject to this section if there is clear
and convincing evidence that: (1) The conduct of the research was fundamentally unethical (e.g.,
the research was intended to seriously harm participants or failed to obtain informed consent); or
(2) The conduct of the research was deficient relative to the ethical standards prevailing at the
time the research was conducted in a way that placed participants at increased risk of harm
(based on knowledge available at the time the study was conducted) or impaired their informed
consent.
§26.1703:
The manuscript indicates (p. 161-162) that subjects were at least IS years of age and that
pregnant or lactating women were excluded from study participation I-PA follow-up with the
author notes that there was no pregnancy testing to confirm non-pregnant status.
§26.1704:
Ethics Committee Kc\ iew The manuscript (p. l(->2) indicates that a local ethics committee had
reviewed and approved the study and that it was conducted according to Good Clinical Practice
guidelines The author states that he no longer has copies of the ethics committee approval since
the study is more than a decade old. GCP standards (adopted June 1996) has clear standards for
ethic committee membership and review processes which are consistent with Agency rules at 40
CFR part 26, Subpart K (iCP also requires compliance with the ethical standards of the
Declaration of Helsinki, including risk minimization, informed consent of human subjects, and
protection of vulnerable populations.
Informed Consent: The manuscript also indicates that informed consent from all subjects was
obtained. Denmark's "Ministerial Order No 806 of July 2004 on Information and Consent at
Inclusion of Trial Subjects in Biomedical Research Projects" provides robust guidelines for
29
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informed consent standards in this trial. There is no evidence to suggest that this study was in
violation of these standards. As with ethics committee appi«.>\ a I records, the author did not have
a copy of the consent form available when requested by I -PA
Risk Minimization: Principal risk associated with this research in\ol\ es allergic reaction to the
MCI/MI material. This risk was minimized by pro\ iding subjects with instructions to visit the
institution's dermatology center in the e\ ent of an allergic reaction outside of the study's
scheduled monitoring visits. The author does not recall any ad\ erse events that required medical
treatment for subjects.
HSRB review of I lie KOAT studios considered together
Charge lo Hie Board:
When considered all together, do the three studies described in Lundov et al., Yazar et al., and
Zachariae et al . pro\ ide a scientific weight of evidence in support of establishing a point of
departure for the deternii nation of an elicitation threshold for methylisothiazolinone (as
identified by the Lundo\ et al . study) for use in risk assessments?4
4 For all three articles EPA has provided in English the following materials: Zachariae et al.,
2006: Data Evaluation Record by two EPA reviewers, Ethics review by an EPA ethics Review
officer, Ethics Questions completed by Dr. Zacharie, and the Ministerial Order 806 for
Biomedical Research from the Danish Ministry of Interior and Health; Lundov et al., 2011: Data
Evaluation Record by two EPA reviewers, Ethics Review by an EPA ethics review officer,
Ethics Questions completed by Dr. Johansen, Ethical Application to the Danish National
Bioethics Committee, the Ministerial Order 806 for Biomedical Research from the Danish
Ministry of Interior and Health, and Danish Act on Research Ethics Review; Yazar et al., 2011:
Data Evaluation Record by two EPA reviewers, Ethics Review by an EPA ethics review officer,
Ethics Questions completed by Dr. Yazar, Ethical Application to the Danish National Bioethics
Committee, Danish Approved Amendments, Swedish Ethical Application and Approval,
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HSRB Response:
The HSRB board agrees that when considered all together, the three studies described in Lundov
et al., Yazar et al., and Zachariae e tal., do provide a scientific weight of evidence in support of
establishing a point of departure for the determination of an elicitation threshold for
methylisothiazolinone (as potentially identified by the I .nndo\ el al., study) for use in risk
assessments.
HSRB detailed recommendations and rationale:
For all three studies EPA provides a summary of ilie study and findings and conclude that these
studies do provide a weight of evidence lo suppoi'i the dei i\ alion of an elicilalion point of
departure for MI. For the Lundov study. the I .owesl ()hsei \ ed Adverse Effect Level (LOAEL) is
mentioned as 0.0105 |ig/cm2 for Ml. where lor /achariae el al., O.025 |ig/cm2 for MI (mixture of
MCI/MI) is mentioned, and for Yazar el al . <> 24 tig cnr for MI (rinse-off product containing
MI) is mentioned. LIW has e\ alualed all studies with respect to "Guidance for Considering and
Using Open Literature Toxicity Studies to Support I luman Health Risk Assessment (USEPA,
2012)" and based on lo screening criteria determined that all three studies are appropriate for
quantitati\ e use in establishing a point of departure lor risk assessment. There was a finding for
Zachariae et al., 2006 that for criteria 13' Adequate data provided on the chemical tested.. .that
not enough information was pro\ ided I lowever, this lack of information does not prevent the
study from being found to be scientifically sound and providing reliable data. There is a
comparison of the /achariae et al . and the Yazar et al., studies to the Lundov et al., et al., where
Lundov et al., have established the (lowest) LOAEL of 0.0105 |ig/cm2. These are all considered
acceptable/non-guideline, none-registrant submitted studies.
EPA recognizes the small number of participants in each study, problems with demographic
representation or reporting, lack of knowledge on the history of MI exposure for participants, and
Swedish Approved Amendments to the Ministerial Order 806 for Biomedical Research from the
Danish Ministry of Interior and Health, and Danish Act on Research Ethics Review, Swedish
Ethical Review Act
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in some cases issues with adequate data for repeatable statistical analysis. In addition, EPA finds
that the studies do not provide enough information to determine or construct a dose-response
curve (Lundov et al with 3 applied concentration points is lacking in that regard). EPA also
recognizes the potential influence of vehicles on the elicitation threshold for MI, and for some
studies not enough information has been provided regarding the vehicles or their potential for
influencing response. However, the intent is to establish weight of evidence in deriving an
LOAEL for elicitation thresholds across the three studies Because immune responses in
individuals can vary so greatly, understanding or even repeating elicitation responses can be
challenging. Elicitation thresholds are a relatively new area research for preservatives, and the
influence of exposure conditions is also not well understood.
The HSRB recommends that the EPA take into account the following scienlilic issues when
considering risks assessment based on these data There is \ ai iability across the three studies in
types of applications and methods of applications. The ohjecli\ es of the studies also vary. It
recognized that sensitization to MI products might occur at lower product concentrations than
currently allowed, however the limit or elicitation point is difficult to narrow down from these
diverse studies, and lor a particular product Typical usage rates and application rates for the
general public or even for the high-risk indi\ idual (high usage rate) is not discussed, making it
more difficult to associate practical and likely dosage rate with a general and reasonable
elicitation threshold Care must he taken to compare appropriate doses/loadings across studies,
where product type and application method must also be considered. In a risk assessment,
appropriate comparable doses might best he represented by daily doses. For the Lundov study,
0.021 iig cm: day can be compared with 0.025 |ig/cm2/ day MCI/MI in the Zachariae study
(where MI is only 1 i of this), and for comparison with the 1.2 |ig/cm2/day (note rinse-off may
greatly lower this le\ el) in the Ya/.ar study. The statistical significance reported across these
doses, however, was not all strong, and the association of response to concentration in a product
may be variable. Another option is to compare accumulated applied dose to an elicitation
threshold across MI ROAT studies.
In establishing LOAEL and applying reasonable safety factors it is important to understand how
consumers use these products in terms of number of applications, volume per application, and
area of application. Determining an elicitation threshold and applied safety factors are dependent
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on understanding consumer behavior and the level of guidance or use recommendations that will
or can be provided. For manufacturers, a |ig/cm2/day must be translated to ppm in the product
(or particular products) with consideration for vehicle effects and usage patterns.
There may be a rationale to further study the concentration le\ els of MI or MCI/MI to be
allowed in a variety of consumer products, and to examine the extent to which individuals have
become sensitized (induced) to MI. Worst-case tests should he performed taking into account
sensitive areas, sensitive individuals, chemicals in the products that either promote absorption in
the skin or retention in the skin.
Another potential risk assessment approach is to look at minimum levels of MI (or other
products) needed for preservation If it can he established that preservation of a product can be
achieved at lower concentrations and with other established safer products, then this option needs
to be explored. For example, Lundo\ et al mention pre\ ions studies showing that 5 ppm MI with
0.4% pheno-oxyethanol was adequate to preser\ e standard cosmetic creams (where pheno-
oxyethanol rarely causes allergic response in subjects) I Iltimately lower and safer levels of MI
applied to protect MI allergic patients will need to satisfy those acceptable preservation levels.
In addition, in the risk assessment process, it would be of interest to relate elicitation thresholds
back to effective concentration inducing a stimulation index of 3 (EC3 values). This is a
measure used in the LI.N A (local lymph node assay ) to test whether a chemical is a sensitizer
and potentially how potent ~ Ml has an EC3 value of 0.4%6 (Yazar et al., 2015 pg. 116) and
considered a strong sensitizer
In conclusion, there may not be full confidence that these three studies overall definitely
establish the point of departure for an elicitation threshold in MI allergic patients, but that there
may be a point of departure for an elicitation threshold in MI allergic patients. The studies do
5 World Health Organization, 2008 "Skin Sensitization in Chemical Risk Assessment" Harmonization
Project Document 5, found at http://www.inchem.org/documents/harmproj/harmproj/harmproj5.pdf
7 Leiva-Salinas and Silvestre (2013) "Update on Allergic Contact Dermatitis due to
Methylchloroisothiazolinon/Methylisothiazolinon and Methylisothiazolinone" Actas Dermosifiliofr
105(9): 840-846.
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provide reliable data for the specific exposure conditions investigated and will assist in the risk
assessment process for establishing appropriate and practical elicitation thresholds. The Board
suggests that EPA carefully consider relationships among the studies and their findings, where
vehicle effects, inductions thresholds, application methods, and average days to elicitation
response all play a role.
In addition, the HSRB recommends that EPA consider the following scientific issues:
1)	Different population subgroups may have different sen si l i \ ities. Mi-sensitized
individuals are the main focus for these ROAT elicitation studies. However there may be
other individuals affected by low levels of MI in products, such as those with eczema and
sensitive skin problems. Data on eczema and contact dermatitis would be useful in a risk
assessment and related to their influence 011 both induction and elicitation thresholds.
Cumulative effects from multiple products containing Ml or mixtures of MCI/MI will
also need to be considered.
2)	In a risk assessment, longer lb I low up of Ml allergic subjects may be needed to determine
a safe level. Is it possible that a \ cry low dose (below those tested in any of these
studies) could produce a response in .1 or 4 months of continued use (beyond the times
used in these ROAT studies)
3)	The HSRB also recommends considering warning labels on MI products and whether
they adequately ad\ ise consumers about the risk of skin sensitization.
4)	I'inally. the I ISRIi recommends careful coin ersion of dosing calculations to determine
how product concentration and usage patterns determine actual dosing applied in practice
e. In the /achariae et al article, ppm must be converted to % (converted to jug/g) and
combined w iih the \ olunie applied and area of skin to determine a |ig/cm2, of MI. It is
also necessary to know the g/|il of the product (entire product) to make an assessment of
|ig/cm2 of MI, where volume application of products are used. In the Lundov study, the
weight of the product is not reported, but there was an application of 4.2 mg
cream/cm2/day reported so this also corresponds to a total of 4.2 mg/cm2 x 9 cm2 = 37.8
mg/day, so 40|il of the product (two applications over the entire 9 cm2 area), telling us
the weight of the product 37.8 mg/40|il or 0.945 mg/|il (i.e., weight of product per
volume). Also, 100 ppm MI is equivalent to 100 |ig Ml/g in cream, therefore one
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application of this concentration would mean (100 |ig Ml/g cream x 0.945 mg/|il x 20 |il
x lg/100mg)/9cm2 = 0.21 |ig Ml/cm2. There are other perhaps more direct ways to make
this calculation, but this agrees with the 0.21 |ig Ml/cm2 reported for the highest dose of
MI applied in the ROAT (Table 1), of the lOOppm MI product. The Board recommends
that EPA confirm how concentrations are determined and eliminate any confusion about
concentrations applied in these studies.
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References
Paradis et al., 2014 "Developing a Framework for the Quantitative Risk Characterization of
Dermal Sensitizers in the Workplace, 3M Company, St, Minnesota, found at
https://schc.memberclicks.net/assets/meetings/spring2014/schc_poster_developing_framework.p
df
EPA, 2004, FIFRA Scientific Advisory Panel Background Document: Proposed Hazard
Identification Methodology for Assessment of Dermal Sensitization Risk, Washington, D.C.
Anderson and Meade, 2014 "Potential Health Effects Associated with Dermal Exposure to
Occupational Chemicals" Environmental Health Insights. K( SI)
World Health Organization, 2008 "Skin Sensitization in Chemical Risk Assessment"
Harmonization Project Document 5, found at
http://www.inchem.org/documents/harmproj harm proj/harmproj 5.pdf
Ezendam, J., Braakhuis, H., and Vandebriel (20 K\) "State of the Art in Non-Animal Approaches
for Skin Sensitization Testing: From lndi\ idnals Test Methods Towards Testing Strategies",
Arch Toxicol., 90:2861-2883.
Leiva-Salinas et al., (2014) "Update on Allergic Contact Dermatitis due to
Methylchloroisothiazolinon Mclhylisolhiazolinone and Methylisothiazolinone" Actas
Dermosifiliogr, 1 < >5( ) X4<)-X4(->
Zangetal., (201 7) "Prediction of Skin Sensitization Potency Using Machine Learning
Approaches".! Appl Toxicol l)oi l<> |(>(>2 jat3424
Griem et al . 2<)i)3 "Proposal lor a Risk Asessment Methodology for Skin Sensitization Potency
Data", Regulatory Toxicology and Pharmacology. 38 2O9-290
Alves et al., 2<)| 5 "Predicting Chemically-Induced Skin Reactions. Part II: QSAR Models of
Skin Permeability and the Relationship between Skin Permeability and Skin Sensitization",
Toxicol Appl Pharmacol 2S4(2) 273-280.
Steiling (2016) "Safety l-\ aluation of Cosmetic Ingredients Regarding their Skin Sensitization
Potential", Cosmetics, 3. doi 10.3390/cosmetics3020014.
Kimber et al., (2002) "Allergic Contact Dermatitis", International Immunopharmacology, 2: 201-
211.
Jaycock (1998) "Risk Assessment of Contact Allergens", American Journal of Contact
Dermatitis, 9(3): 155-161.
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Dearman and Kimber (2003) "Factors Influencing the Induction Phase of Skin Sensitization",
American Journal of Contact Dermatitis, 14:188-194.
Gerberick and Robinson (2000) "A Skin Sensitization Risk Assessment Approach for Evaluation
of New Ingredients and Products", American Journal of Contact Dermatitis, 11:65-73.
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US ENVIRONMENTAL PROTECTION AGENCY
HUMAN STUDIES REVIEW BOARD
Chair
Liza Dawson, Ph.D., Research Ethics Team Leader, Division of AIDS National Institutes of
Health (NIH), National Institute of Allergy and Infections Disease (NIAID), Bethesda, MD
Vice Chair
Edward Gbur, Jr., Ph.D., Professor, Agricultural Siali sties Laboratory. I nivcrsity of Arkansas,
Fayetteville, AR
Members
Jennifer M. Ca\u1Uii'i. Sc D . C TTT. .\ssislanl Professor. l)i\ision of Occupational and
Environmental Medicine. Department of Community Medicine. I niversity of Connecticut
Health Center, I 'arminuton. CT
Gary I. Cliaduick. Pliarni I). M P 11 . C I P . Senior Consultant, HRP Consulting Group, Inc.
Fairporl. \Y
Alesia C. Ferguson. MS . MPII . PhD., Associate Professor, Department of Environmental and
Occupational I lealth. College of Public Health, University of Arkansas Medical Sciences,
Little Rock, AR
George Fernandez, Ph.D., Statistical Training Specialist, SAS Institute, Statistical Training and
Technical Services, Sparks, NV
Kyle L. Galbraith, Ph.D., Research Integrity Officer, University of Illinois at Urbana-Champaign
Office of the Vice Chancellor for Research, Champaign, IL
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Jewell H. Halanych, M.D., Assistant Professor, Internal Medicine Residency Program,
Montgomery Regional Campus, University of Alabama at Birmingham, Montgomery, AL
Walter Thomas Klimecki, D.V.M., Ph.D., Associate Professor and Department Head (Interim),
Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona,
Tucson, AZ
Randy Maddalena, Ph.D., Physical Research Scientist. Indoor I-n\ ironment, Lawrence Berkeley
National Laboratory, Berkeley, CA
Jun Zhu, Ph.D., Professor of Statistics and of I jiloniology. Department of Statistics, University
of Wisconsin - Madison, Madison, WT
Consultant to the Board:
KendraL. Lawrence. I'll I). IK'I-. PMP, I leal ill Sciences Product Manager, US Army Medical
Materiel Development \cti\ity. It Detrick. Ml)
Human Studies Ke^eu Board Stall'
Jim Downing, L\eaili\ e Director. Human Studies Review Board Staff, Office of the Science
Advisor, United States I ji\ iron mental Protection Agency, Washington, DC
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NOTICE
This report has been written as part of the activities of I lie I ¦ l\\ I In man Studies Review Board, a
Federal advisory committee providing advice, information and recommendations on issues
related to scientific and ethical aspects of hum an subjects research This report has not been
reviewed for approval by the Agency and, hence, the contents of this report do not necessarily
represent the view and policies of the I-n\ ironmenlal Protection Agency, nor of other agencies in
the Executive Branch of the Federal uo\ eminent, nor does the mention of trade names or
commercial products constitute a recommendation lor use
In preparing this document, the Board carefully considered all information provided and
presented by the Agency presenters, as well as information presented by public commenters.
This document addresses the inlbnmilion pro\ ided and presented within the structure of the
charge by the Agency
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