Number range CHAPTER

**FIFRA Scientific Advisory Panel Background Document**

**May 4-6, 2004 meeting**

Proposed Hazard Identification

Methodology for Assessment of Dermal Sensitization Risk

Timothy F. McMahon, Ph.D.

Jonathan Chen, Ph.D.

Antimicrobials Division

Office of Pesticide Programs

U.S. Environmental Protection Agency

Washington, D.C. 20460

**Introduction**

Among the many types of risk assessments conducted in the EPA's Office
of Pesticide Programs (OPP) is an assessment of the dermal sensitization
potential of pesticide chemicals. As noted in 40 CFR 798.4100,
"Information derived from tests for skin sensitization serves to
identify the possible hazard to a population repeatedly exposed to a
test substance." Information from this test is qualitatively assessed
and, if appropriate, precautionary language is included on the pesticide
label as well as the Occupational Safety and Health Administration's
Material Data Safety Sheets (MSDS). Occupational dermal exposures to
known or suspected dermal sensitizing pesticide chemicals can be then
dealt with appropriately, either through engineering controls or use of
personal protective equipment. Non-occupational exposures can normally
be dealt with through precautionary label statements.

Data available through the National Institute for Occupational Safety
and Health (NIOSH) indicate that occupational exposure to dermal
irritants and contact allergens accounts for a significant number of
occupational illness and that chemical agents are the most frequent
cause of such illness. Specific national occupational disease and
illness data are available from the U.S. Bureau of Labor Statistics
(BLS). The BLS conducts annual surveys of approximately 174,000
employers, selected to represent all private industries in the U.S. The
goal is to ascertain the total numbers and incidence rates of
occupational injuries and illnesses. The survey results are then
projected to estimate the numbers and incidence rates of injuries and
illnesses in the American working population. All occupational skin
diseases or disorders, including occupational contact dermatitis (OCD),
are tabulated in this survey. In 1999, of over 372,000 occupational
illnesses reported, 12% were reported as skin diseases/disorders, making
this the most common non-trauma related occupational illness (NIOSH,
2001). The economic impact of occupational skin diseases/disorders is
also significant. As measured by direct medical cost and worker
compensation, the total annual cost of occupational skin disease may
range from \$222 million to \$1 billion (NIOSH, 2001).

While pesticide chemicals can usually be labeled to warn of potential
dermal sensitization effects, there also exists the manufacture of
"treated articles or substances" (40 CFR 152.25), in which a registered
pesticide is incorporated into the article to protect the integrity of
the article or substance itself (such as paint treated with a pesticide
to protect the paint coating or wood products treated to protect the
wood against fungal or insect decay). Under such circumstances of use,
the general public may unknowingly be exposed to pesticide chemical
residue in the treated article. Therefore, prior to such use, the
pesticide chemical must first be registered under FIFRA, which requires
that the manufacturer of the pesticide demonstrate that it can be used
without unreasonable risks to humans or the environment. Treated
articles such as preserved wood, however, do not bear a pesticide label
or effectively use other communication methods to inform and protect
people against potential hazards, including the potential for dermal
sensitization

**Purpose**

The focus of this paper is on the Agency's interest in developing the
foundation of a scientifically sound approach to quantitative assessment
of dermal sensitization risk to pesticide chemicals, including pesticide
chemicals that are incorporated into other materials (i.e. treated
articles). The Agency is interested in obtaining expert advice on
methods published in the scientific literature that have been proposed
for use in determining induction thresholds and elicitation thresholds
for chemicals known or suspected of causing allergic contact dermatitis.
The Agency is also interested in seeking advice on whether there are any
susceptibility issues in the general population with respect to
development of allergic contact dermatitis, including any potential
special sensitivity of children. The Agency will present hexavalent
chromium as a case study of a known dermal sensitizer, and possible
approaches to quantitating risk of allergic contact dermatitis from
exposure to hexavalent chromium.

**Dermal Sensitization**

Dermal sensitization, also known as allergic contact dermatitis (ACD),
delayed contact hypersensitivity, or Type IV allergic contact
dermatitis, is defined by Marzulli and Maibach (1996) as a delayed,
immunologically mediated, inflammatory skin disease consisting of
various degrees of erythema, edema, and vesiculation. Kimber (2004) has
also defined sensitization as "stimulation by chemical allergen (in an
inherently susceptible individual) of an immune response of the quality
and vigor required to permit the provocation of an elicitation reaction
upon subsequent encounter with the same chemical." ACD is typically
characterized by two phases, termed induction and elicitation. Induction
occurs when there is an exposure of sufficient magnitude and/or duration
to activate specific immune mechanisms resulting in the acquisition of
sensitization, while elicitation occurs from a subsequent exposure to
the same chemical allergen. As it is generally recognized that a certain
level of allergen exposure must be attained to induce immune activation
in susceptible individuals, dermal sensitization is characterized as a
threshold type of response. However, dose-response relationships are
observed for both the induction and elicitation phases of ACD (Gerberick
and Robinson, 2000; Kimber et al., 2003; Scott et al., 2002), and
thresholds for induction can be reached following either a single
sufficiently high amount of exposure to the allergenic chemical, or
after contact with a large area of skin, or as a consequence of repeated
skin applications (Marzulli and Maibach, 1996). Experiments with
2,4-dinitrochlorobenzene (DNCB) and other sensitizers have shown that a
single contact can be sufficient for sensitization, but less data exist
of the relationship between lower area doses and repeated contacts over
a longer time period (Griem et al., 2003). A study summarized by Griem
et al., (2003) and conducted by Vandenburg and Epstein (1963) in which
previously non-sensitized persons were exposed to nickel chloride
suggested that subclinical sensitization occurred in some of the
subjects who responded negatively from the first test, as an increased
percentage of non-sensitized subjects responded positively upon a
repetition of the test four months later. More studies are needed in
this area.

To be capable of inducing an allergenic response, the chemical itself
must possess certain characteristics. Those chemicals able to cause
sensitization are usually low molecular weight protein-reactive
substances that can gain access to the viable epidermis via the stratum
corneum, and are also able to cause sufficient local trauma to induce
cutaneous cytokines and be inherently antigenic and recognized by
responsive T lymphocytes.

Once through the stratum corneum, the allergen makes contact with the
Langerhans cell, a member of the bone-marrow derived dendritic cell
family whose function is to act as a 'sentinel' cell and serve as a trap
for antigens entering the skin (Kimber, in Marzulli and Maibach, 1996).
Langerhans cells then direct the allergen to a regional lymph node,
where interaction with T lymphocytes occurs, followed by proliferation
of lymphocytes that have been 'primed' to react against the presented
antigen. A subsequent contact with the allergen will result in
elicitation of the sensitization response due to the reaction of
sensitized lymphocytes with the allergen. The process is illustrated in
the following diagram:

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height="3.4069444444444446in"}![](media/image4.emf){width="1.4902777777777778in"
height="0.4486111111111111in"}

![](media/image5.emf){width="2.9902777777777776in"
height="3.0875in"}![](media/image6.emf){width="2.8381944444444445in"
height="2.870833333333333in"}

From: Sailstad, D.M. (2003): Allergic Contact Hypersensitivity:
Mechanisms and Methods. In: Alternative Toxicological Methods. Harry
Salem and Sidney Katz, eds. , CRC Press, Boca Raton, Florida. Pages
193-205.

It should also be noted that in addition to Langerhans cells, epidermal
cytokines and chemokines may also play a role in the development of the
sensitization response. This is based on the observation that the
functional activity of Langerhans cells and presumably other cutaneous
antigen-presenting cells, is regulated largely by the availability of
cytokines (Kimber, in Marzulli and Maibach, 1996).

While it is generally accepted that the necessary exposure for induction
of dermal sensitization is greater than the exposure needed to elicit
sensitization, it is important to recognize that thresholds are largely
determined by the potency of the chemical allergen and that induction
and elicitation thresholds vary among individuals (Kimber et al., 2003).
It is thus necessary to consider dose-response relationships in
establishing "safe" levels for prevention of induction and elicitation.
A recent investigation by Scott et al. (2002) examined the quantitative
relationship between sensitization and elicitation concentrations and
the ability to elicit an ACD reaction in murine models. Using two
established sensitizers (DNCB and squaric acid dibutyl ester (SADBE),
dose-response relationships were determined using the LLNA to derive EC3
values for both chemicals. Then, for DNCB, various elicitation
concentrations were tested in mice that had been induced to various
concentrations of DNCB. As the sensitizing concentration of DNCB
increased, it was observed that the concentration required to elicit
measurable sensitization in sensitized mice decreased. That is, mice who
had been sensitized to higher concentrations of DNCB required less DNCB
to elicit a measurable sensitization response. For SADBE, two different
elicitation concentrations were compared over a range of induction
concentrations. The group given the higher elicitation concentration
showed measurable sensitization at lower induction concentrations. The
results of this study suggested that, as the induction dose was
increased (using DNCB), the concentration required for elicitation was
decreased. Correspondingly, as the elicitation concentration was
increased (using SADBE), the concentration required for induction was
decreased.

**Hazard Identification**

It is desirable to be able to conduct quantitative assessments for
dermal sensitization in order to prevent consumers especially from
developing ACD when dermal contact, including repeated dermal contact,
could not be completely avoided (Griem et al., 2003). In the case of
hexavalent chromium as a component of treated wood, this is important,
as there would be repeated dermal contact with the treated wood surface
when this treated wood is used in residential decking and playground
structures.

There are several accepted methods for hazard identification of dermal
sensitization, including the Buehler occluded patch test, the guinea pig
maximization test, and the murine local lymph node assay (LLNA). The
guinea pig maximization test and the Buehler test, while providing
reliable information on whether a substance is a skin sensitizer or not,
are best suited only for hazard identification.

By contrast with the Buehler test and maximization test, the Mouse Local
Lymph Node Assay (LLNA) is a more recent test method for assessing the
allergic contact dermatitis (skin sensitization) potential of chemicals,
specifically the induction phase of sensitization. Using the
incorporation of radiolabeled thymidine or iododeoxyuridine into DNA,
the LLNA measures lymphocyte proliferation in the draining lymph nodes
of mice topically exposed to the test article. The stimulation index
(ratio of lymphocyte proliferation in treated mice compared to controls)
is used as the indicator of potential sensitization. In 1998, following
review by the FIFRA Scientific Advisory Panel (SAP), the LLNA was
incorporated as a screening test in OPPTS Test Guideline 870.2600 Skin
Sensitization. In 1999, the Interagency Coordinating Committee on the
Validation of Alternative Methods (ICCVAM) Immunotoxicity Working Group
(IWG) recommended the LLNA as a stand-alone alternative for contact
sensitization hazard assessment, provided that certain protocol
modifications were made. Following additional studies to validate the
method, the FIFRA SAP agreed with the Agency proposal that the LLNA was
applicable for testing chemicals to elicit contact sensitization and
should be considered a preferred, stand-alone assay. The OPPTS guideline
870.2600 (Skin Sensitization) has been revised to include the LLNA as a
stand-alone assay for appropriate applications. The OPPTS guideline has
also been harmonized with OECD\'s Guideline 429 for LLNA, which was
adopted in April 2002.

Although the LLNA has not been validated for determination of
sensitization potency of chemical allergens and sensitization to metals
has not been extensively studied , approaches for determination of
quantitative assessment of sensitization induction thresholds have been
proposed in the scientific literature using LLNA data (Gerberick 2000,
2001; Griem et al., 2003). It may therefore be useful to consider data
from the LLNA as a starting point for quantitation of induction
thresholds, as dose response data can be generated from this assay, and
a NOAEL can potentially be identified (Felter et al., 2003). However,
while the LLNA has been validated and accepted as a stand-alone test
method for assessment of dermal sensitization potential by the
Interagency Coordinating Committe on the Validation of Alternative Test
Methods (ICCVAM , 1999), the test itself has not been validated for its
utility in dermal risk assessment (Felter et al., 2003). It has been
proposed to group sensitizing chemicals according to relative potency as
determined in the LLNA, and then compare these categories with the
relative sensitization potency in humans on the basis of clinical
experience and/or prevalence of ACD in the population (Griem et al,
2003). While a good correlation between LLNA results and sensitization
potency in humans has been reported (Griem et al., 2003), there is not
yet general agreement on categories and ranges that should be used in
classification of relative potency.

**Induction Threshold Methods**

Gerberick (2000, 2001) proposed a methodology for determination of a
'sensitization reference dose' for sensitizers in consumer products. The
lower boundary of the potency category for a sensitizing chemical is
used as the starting point, with application of uncertainty factors for
interindividual variability, product matrix effects, and use pattern.
This approach was applied to the fragrance component cinnamic aldehyde
and the preservative methylchloroisothiazolinone/methylisothiazolinone
for which both LLNA and human sensitization potency were available
(Griem et al., 2003).

A use for the LLNA in quantitative risk assessment was investigated by
Griem et al (2003). Identification of known human sensitizing chemicals
for which both an EC3 value from an LLNA test and a NOAEL or LOAEL from
human repeat insult patch tests (HRIPT) or the human maximization test
(HMT) were available were identified. The reported concentrations were
converted into specific and molar area doses. Comparison of the area
doses of the LLNA and human test results indicated that sensitization
thresholds were similar in mice and humans despite the fact that the
area doses for different chemicals ranged over several orders of
magnitude (Griem et al., 2003). It was concluded from this analysis that
the LLNA EC3 value is a useful measure of sensitizing potency in humans,
and that the EC3 value can be used as a surrogate value for the human
NOAEL which, in turn, can be used as a starting point in quantitative
risk assessment.

**Elicitation threshold Methods**

The above approaches describe methods that may be used to estimate
thresholds for safe area doses that are considered protective against
induction of sensitization. There are also proposed approaches for
estimation of safe area doses that are considered protective against
elicitation of sensitization in already sensitized persons. By
inference, protection against elicitation would also be protective of
induction, as thresholds for induction are generally higher than those
for elicitation (Kimber et al., 2003).

An approach to estimate an acceptable area dose for protection against
elicitation is the concept of the Minimum Elicitation Threshold, or MET.
This approach has been discussed in previous publications (Nethercott et
al., 1994; Zewdie, 1998; NJDEP, 1998; Basketter et al., 2003)
specifically with respect to hexavalent chromium. The concept behind the
MET is that there is an 'elicitation threshold' below which no
sensitization reaction is expected; thus, the MET is analogous to an RfD
(Horowitz and Finley, 1994). The estimation of an MET is usually based
on the results of tests in previously sensitized individuals; thus, the
MET is considered protective of elicitation reactions. However, there
has not been an extensive discussion of the criteria for employing this
concept for purposes of risk assessment. Nethercott (1994) calculated a
value of 0.089 ug/cm^2^ as a 10% MET (i.e. the concentration at which
10% of the study group responded) based on results of a human repeat
insult patch test in 54 chromium-sensitized volunteers and claimed that
this value should be protective against ACD for hexavalent chromium in
at least 99.99% of the population exposed to contaminated soil.
Paustenbach (1992) estimated a 10% threshold response of 54 parts per
million for hexavalent chromium in soil, but no discussion of the
relevance of the 10% response level was presented. Two states
(Massachusetts and New Jersey) have proposed soil cleanup standards
based on an ACD endpoint using either the human patch test data of
Nethercott (1994) or the human forearm water exposure data of Fowler
(2000).

Griem et al. (2003) also proposed an approach for derivation of a safe
area dose at which ACD would not be manifest in sensitized individuals.
As several of the factors that influence induction of sensitization
(skin penetration, uptake by antigen-presenting cells, metabolism) are
also relevant for elicitation, it was postulated that a correlation
between the induction potency and elicitation potency of a chemical
could be established. However, comparison of induction and elicitiation
area doses from limited data in humans did not show an obvious
correlation. While induction threshold doses spanned five orders of
magnitude, values for elicitation were mainly within one order of
magnitude. Using the log transformation of the ratio of induction/
elicitation to elicitation, it was proposed that the ratio of the
induction/sensitization threshold could be predicted on the basis of an
established sensitization (induction) threshold. Examples of the
derivation of safe area doses for three chemicals (MCI/MI, a
preservative in many cosmetics and household products; cinnamic
aldehyde, a fragrance and flavor ingredient; and nickel) were presented.
For strong sensitizers such as MCI/MI, it was demonstrated that the safe
area doses for induction and elicitation were close together, while for
relatively weaker sensitizers such as nickel, the safe area doses for
induction and elicitation were farther apart, consistent with the
mathematical relationship of the ratio of induction/elicitation
threshold vs. sensitization threshold. That is, for lower potency
sensitizers, a relatively high area dose may be needed to cause
induction of sensitization, but elicitation may be possible with much
lower area doses, while for a potent sensitizer, the area dose needed
for induction is close to the area dose that will elicit the reaction in
a sensitized individual.

**Uncertainty**

After the appropriate level of concern has been identified (e.g. NOAEL
determination, for example), areas of uncertainty need to be considered
in extrapolating the result to conditions relevant to the human exposure
of interest. Areas of uncertainty that have been identified for dermal
risk assessment include (1) interspecies variability/susceptibility
(i.e. extrapolation from animals to humans); (2) intra-species
variations in response within humans; (3) vehicle or product matrix
effects; and (4) exposure considerations (i.e., area of the body
exposed, repeated exposures). Briefly, and as discussed in more detail
by Felter et al. (2002) and Griem et al. (2003), the inter-individual
variation in response to induction and elicitation of dermal
sensitization must be taken into account, as there may be differences in
response based on age, sex, and genetic factors, or health status of the
skin. In addition, formulation of chemical allergens into product
matrices that may result in an enhancement or inhibition of ACD must be
considered in the risk assessment paradigm, as must also extrapolating
from experimental conditions to real-world exposure conditions, i.e.
site of the body exposed , effects of occlusion, and environmental
conditions (temperature, humidity, and repeated dermal exposures). For
each of the 4 areas, a range from 1-10 has been suggested for
uncertainty in each area.

**Populations of Concern**

Approaches to estimating safe area doses for ACD have been proposed
using EC3 values derived from the murine LLNA (Gerberick and Robinson,
2000; Felter et al., 2003, and MET values from human repeat insult patch
tests (Nethercott et al., 1994; Basketter et al., 2001; Hansen et al.,
2003). Approaches using the results of murine LLNA data are intended to
estimate area doses that are protective against induction of ACD, while
use of the MET approach is intended to be protective against elicitation
of ACD in sensitized persons. Griem et al. (2003) have also proposed an
approach for calculation of safe area doses designed to be protective
against both induction and elicitation of ACD through use of appropriate
uncertainty factors applied to the results of murine LLNA or human patch
test study results. This proposal is worth examining, as it has been
acknowledged in the past for sensitizing chemicals such as nickel and
hexavalent chromium that it is difficult to protect individuals who are
already sensitized (Felter et al., 2003; USEPA, 1998).

In addition, consideration should be given to whether there are
potentially susceptible subpopulations who may be more susceptible to
the induction and/or elicitation of ACD. Paustenbach et al. (1992) and
Felter et al. (2002) have discussed the issue of whether children are
more or less at risk for development of ACD. Paustenbach et al.
addressed this issue specifically for hexavalent chromium, and it was
concluded that risk to children ages 3 to 8 is not likely to be greater
than risk to adults as there is no evidence that repeated exposures to
hexavalent chromium places a person at greater risk of sensitization.
Felter et al. suggested that infants and children may actually be at
lower risk for development of ACD based on data gathered from
dinitrochlorobenzene and pentadecylcatechol (poison ivy allergen). These
views are somewhat counter to the opinion of Griem et al. (2003) who
suggested a possible higher sensitizing potency of a chemical upon
repeated exposures. This would make sense in the case of hexavalent
chromium, as the significant irritancy of the chemical could lend itself
to an increased sensitizing potency by allowing more chemical to
penetrate the stratum corneum.

**Case Study- Hexavalent Chromium**

As noted in the 1998 IRIS Toxicological Review for hexavalent chromium
(USEPA, 1998) as well as in numerous publications, hexavalent chromium
is one of the most common and potent contact sensitizers. Exposures to
hexavalent chromium occur in a number of occupational settings including
including chrome plating baths, chrome colors and dyes, cement, tanning
agents, wood preservatives, anticorrosive agents, welding fumes,
lubricating oils and greases, cleaning materials, and textiles and furs
(USEPA, 2003). Non-occupational exposures to hexavalent chromium have
also been noted in household detergents (Basketter et al., 2003; Stern
et al., 1993) as well as in cement.

Elicitation thresholds in persons sensitized to hexavalent chromium have
been described in the literature (Nethercott, 1994; Fowler, 2000).
However, there are no recent data on induction thresholds for hexavalent
chromium. Some investigation has been performed on the question of
induction thresholds in general, as it has been stated (Marzulli and
Maibach, 1996; Griem et al., 2003) that repeated dermal contact over a
longer time period may also result in a threshold for induction.
Although more work is needed in this area, Griem (2003) proposed an
uncertainty factor be applied for repeated dermal contact with chemical
allergens, as there may be a higher sensitizing potency of a chemical
upon repeated exposure.

The Antimicrobials Division of OPP is concerned with the risk from
dermal exposure that may occur from dermal contact with hexavalent
chromium on the surface of wood treated with a wood preservative product
containing hexavalent chromium, as hexavalent chromium is known to be a
potent dermal irritant and sensitizing agent. The Antimicrobials
Division, using existing science policies in OPP, currently performs
hazard and risk assessments for non-cancer endpoints through selection
of a level of concern (e.g., a NOAEL or LOAEL value) and compares this
level of concern to estimated or actual exposures to derive a Margin of
Exposure. The Margin of Exposure is then weighed against the
"acceptable" Margin of Exposure, which takes into account uncertainties
in the risk assessment (e.g., inerspecies differences in response,
intraspecies differences in sensitivity).

**Hazard Identification** **for hexavalent chromium**

Murine LLNA hazard data reported by Kimber et al. (1995) from five
different laboratories reported EC3 values for hexavalent chromium using
potassium dichromate as the test substance. These data are shown below:

+---------+------+--------+--------+--------+--------+--------+--------+
| lab     | A    | B      | C      | D      | E      | Avg.   | US Avg |
| oratory |      |        |        |        |        |        |        |
+---------+------+--------+--------+--------+--------+--------+--------+
| country | UK   | US     | UK     | US     | US     |        |        |
+---------+------+--------+--------+--------+--------+--------+--------+
| area    | 5.12 | 11.56  | 13.24  | 10.77  | 11.2   | 10.36  | 11.15  |
| dose    |      |        |        |        |        |        |        |
|         |      |        |        |        |        |        |        |
| F       |      |        |        |        |        |        |        |
| g/cm^2^ |      |        |        |        |        |        |        |
+---------+------+--------+--------+--------+--------+--------+--------+

Several published studies (Nethercott et al., 1994; Basketter et al.,
2001; Hansen et al., 2003) have reported elicitation thresholds in
persons previously sensitized to chromium. In the study by Nethercott et
al., a 10% MET of 0.089 Fg/cm^2^ was reported from results of patch
testing using 54 human volunteers known to be sensitized to hexavalent
chromium. The lowest dose tested in this study, 0.018 Fg/cm^2^, also
showed evidence of elicitation in one subject.

Basketter et al. (2001) reported elicitation reactions to potassium
dichromate in 17 volunteers giving fully informed consent using
closed-patch and open appliation techniques. Skin pre-treated with 0.2%
sodium lauryl sulfate showed reaction to potassium dichromate at 1 ppm,
with a dose-response evident at the higher concentrations. Using open
application techniques, 3 of 15 volunteers reacted to a level of 5 ppm
potassium dichromate. To protect those already sensitized to chromium as
well as to prevent development of additional chromium-sensitive
subjects, a chromium contamination level of 1 ppm was suggested on the
basis of this study.

Hansen et al. (2003) compared the 10% MET for both hexavalent and
trivalent chromium in 18 volunteers known to have chromium allergy. The
results of this study indicated a 10% MET of 0.03 Fg/cm^2^ for
hexavalent chromium and 0.18 Fg/cm^2^ for trivalet chromium, and suggest
that both trivalent and hexavalent chromium should be taken into
consideration when characterizing chromium skin allergy.

The data reported above using human volunteers are from subjects
previously sensitized to hexavalent chromium and give some indication of
an elicitation threshold, while the LLNA data reported in Kimber et al.
(1995) show induction thresholds in the murine LLNA test.

Estimation of 'safe' area doses for protection from hexavalent chromium
ACD can be performed in both cases using published methdologies. For the
LLNA study results, uncertainty factors for interspecies extrapolation,
intraspecies variation, product matrix, and exposure considerations
should be taken into account. For the interspecies uncertainty factor,
Griem et al. (2003) proposed a factor of 3 based on comparison of human
and murine data showing that sensitizing doses are within a factor of 3
of each other, and that skin penetration tends to be higher in rodents
than in humans. An uncertainty factor of 10 is proposed for intraspecies
variation in humans, as there are few data on induction thresholds in
humans. A product matrix uncertainty factor of 10 is proposed for
hexavalent chromium. The LLNA assay is performed using an acetone/olive
oil vehicle, while the wood preservative formulation containing
hexavalent chromium is likely more complex and could affect the
availability and potency of the allergen. An exposure uncertainty factor
of 10 is proposed based on uncertainty regarding the repeated dermal
exposure that could occur to the treated wood and how this would affect
the potency of hexavalent chromium as a dermal sensitizer. The total
uncertainty factor from this analysis is 3000 applied to the calculated
area dose from the average of 5 LLNA assays to yield a 'sensitization
reference dose.' This is illustrated below:

+---------+------+--------+--------+--------+--------+--------+--------+
| lab     | A    | B      | C      | D      | E      | Avg.   | US Avg |
| oratory |      |        |        |        |        |        |        |
+---------+------+--------+--------+--------+--------+--------+--------+
| country | UK   | US     | UK     | US     | US     |        |        |
+---------+------+--------+--------+--------+--------+--------+--------+
| area    | 5.12 | 11.56  | 13.24  | 10.77  | 11.2   | 10.36  | 11.15  |
| dose    |      |        |        |        |        |        |        |
|         |      |        |        |        |        |        |        |
| F       |      |        |        |        |        |        |        |
| g/cm^2^ |      |        |        |        |        |        |        |
+---------+------+--------+--------+--------+--------+--------+--------+
| inter   | 3    | 3      | 3      | 3      | 3      | 3      | 3      |
| species |      |        |        |        |        |        |        |
| extrap  |      |        |        |        |        |        |        |
| olation |      |        |        |        |        |        |        |
| UF      |      |        |        |        |        |        |        |
+---------+------+--------+--------+--------+--------+--------+--------+
| intra   | 10   | 10     | 10     | 10     | 10     | 10     | 10     |
| species |      |        |        |        |        |        |        |
|         |      |        |        |        |        |        |        |
| va      |      |        |        |        |        |        |        |
| riation |      |        |        |        |        |        |        |
| UF      |      |        |        |        |        |        |        |
+---------+------+--------+--------+--------+--------+--------+--------+
| matrix  | 10   | 10     | 10     | 10     | 10     | 10     | 10     |
| UF      |      |        |        |        |        |        |        |
+---------+------+--------+--------+--------+--------+--------+--------+
| e       | 10   | 10     | 10     | 10     | 10     | 10     | 10     |
| xposure |      |        |        |        |        |        |        |
| UF      |      |        |        |        |        |        |        |
+---------+------+--------+--------+--------+--------+--------+--------+
| S-RfD   | 0.   | 0.0038 | 0.0044 | 0.0035 | 0.0037 | 0.0034 | 0.0038 |
| F       | 0017 |        |        |        |        |        |        |
| g/cm^2^ |      |        |        |        |        |        |        |
+---------+------+--------+--------+--------+--------+--------+--------+

The S-RfD can then be compared to estimated or measured human exposure
to determine if the Margin of Safety is adequate. If, as suggested by
Griem et al. (2003) that the maximum uncertainty factor should be no
greater than 1000, then the S-RfD values shown above, using the maximum
uncertainty factor of 1000, would be 0.005, 0.01, 0.013, 0.01, and 0.01
Fg/cm^2^ respectively for the five studies, with an average U.S. value
of 0.01 Fg/cm^2^.

A similar approach can be applied to the MET values from the Nethercott
et al., Hansen et al., and Basketter et al. studies. As the data are
from human studies, the interspecies extrapolation factor could be
reduced to 1. An intraspecies uncertainty factor of 3 is proposed based
on the use of sensitized persons, as elicitation thresholds have been
found to be less variable than induction thresholds. An uncertainty
factor of 3 is also applied for the use of LOAEL values, as the studies
were not designed for specific determination of a NOAEL. An uncertainty
factor of 1 is proposed for exposure considerations, based on the use of
a sensitized study group. The total uncertainty factor of 10 applied to
the reported human LOAEL valuesof 0.018 Fg/cm^2^ , 0.01 Fg/cm^2^, and
0.03 Fg/cm^2^, results in 'safe' area doses of 0.0018, 0.001, and 0.003
Fg/cm^2^ for persons previously sensitized to hexavalent chromium.

Comparison of the values derived from the induction studies using the
LLNA and the patch test data shows under the current scheme that
induction and elicitation does for hexavalent chromium do not differ
appreciably, consistent with the fact that hexavalent chromium is a
potent sensitizer and that induction and elicitation doses will not
differ widely, as discussed by Griem et al (2003). This factor could
change from application of uncertainty factors of differing magnitude; a
reasonable case has been presented using the available data.

**Environmental Exposures**

Application of the experimental data to environmental exposures is also
a significant aspect of the risk assessment, as for hexavalent chromium,
there will be dermal contact not only with the treated wood product, but
with soil in contact with or in proximity to the treated wood structure.

As the experimental hazard dose metric (i.e., the area dose) for
induction and/or elicitation of dermal sensitization may differ
according to the matrix of exposure, it is desirable to characterize as
accurately as possible the influence of the exposure matrix variables on
determination of an acceptable area dose level. For contact with a wood
matrix into which a chemical allergen is incorporated, the Office of
Pesticide Programs estimates a safe area dose from available scientific
data and compares that level of concern to the estimated or measured
level of exposure to calculate an acceptable Margin of Exposure.
Variables that influence the calculation of the exposure to the chemical
on the surface of wood include transfer efficiency of the chemical from
the wood to the skin, number of dermal contact events, surface area of
skin exposure, and level of the chemical on the surface of the wood.
Approaches to assessing treated wood exposures have been considered
recently by the FIFRA SAP in a December, 2003 meeting in which the SHEDS
model was presented and considered as a probabilistic approach to
assessing exposures to arsenic and chromium in treated wood, including
children's exposures.

For determination of an acceptable area dose from contact with a
chemical in a soil matrix, many of the variables are similar to the wood
matrix. However, other soil matrix properties may have a greater impact
on the transfer of the chemical from the soil to the skin. Additional
variables are taken into account in calculating skin contact dose, such
as skin surface area of contact, soil adherence to skin, contact
frequency (important for determination of children's potential hazard
and risk), bioavailability of the chemical in soil, physio-chemical
properties of the soil (i.e. moisture content, soil type), valence or
complex state of the chemical, and chemical solubility in sweat.
Guidance for determination of acceptable soil levels for Superfund
cleanup sites is found at the following web address:
[[http://www.epa.gov/superfund/resources/soil/ssgmarch01.pdf]{.underline}](http://www.epa.gov/superfund/resources/soil/ssgmarch01.pdf).
It is noted here that soil cleanup values estimated for dermal contact
in the Superfund program are based upon systemic effects resulting from
dermal exposures, although dermal sensitization effects should also be
considered in the assessment.

Water also represents a matrix of exposure that is different than wood
or soil, in that it represents a three-dimensional matrix of contact
(i.e. Fg/cm^3^) vs. a two-dimensional matrix of contact ( i.e.
Fg/cm^2^). Activities such as showering represent exposure scenarios
where potential dermal sensitization hazard may need to be
characterized, but as with soil, other variables may influence the
estimation of a safe area dose in this matrix. Thus, while the
experimental hazard data for hexavalent chromium indicate that low
levels are adequate to induce and/or elicit sensitization, the actual
concentration necessary for such reactions may differ according to the
matrix in which the chromium is found.

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