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In response to the July 1993 Pesticide Reregistration Rejection
Rate Analysis for Toxicology (EPA 738-R-93-004) and realizing labs
were often having difficulty conducting acute toxicity studies to
the satisfaction of the Agency, the Precautionary Review Section
(PRS) of the Registration Division of The Office of Pesticide Programs
(OPP) decided to do a rejection rate analysis on acute toxicity studies
on pesticide products submitted to PRS. The results of the analysis
are as follows:
Acute Oral Toxicity 11%
Acute Dermal Toxicity 10%
Acute Inhalation Toxicity 23%
Primary Eye Irritation 9%
Primary Skin Irritation 11%
Dermal Sensitization 38%
It was not determined how often each study deficiency was found.
While initially PRS only conducted a rejection rate analysis, it
was later decided that information on the proper conduct on studies
was needed. PRS realized there were several deficiencies regularly
observed in acute toxicity studies not addressed in the previous
document. About the time PRS was developing a document on the conduct
of studies, the American Crop Protection Association (ACPA) informed
PRS they were also working on a similar document in preparation for
a self-certification program for acute toxicity studies. On May 23,
1995, several representatives from the Environmental Protection Agency
(EPA), ACPA, the Chemical Producers and Distributors Association
(CPDA), the Chemical Manufacturers Association (CMA), Health Canada
and the California Department of Pesticide Regulation (CPDR) met
to discuss acceptable methods for the conduct of acute toxicity studies.
The decisions made in that meeting were incorporated into the preliminary
PRS document to form the present document.
OPP realizes that, currently, complete guidance for the proper conduct
of acute toxicity studies according to EPA policy is not found in
one source, but often must be pieced together from several different
sources. It is the goal of this document to compile this information
into a supplement to the Subdivision F Guidelines so that we may
reduce the number of studies that are rejected or flawed due to incorrect
procedures or insufficient reporting. This guidance is intended to
supplement the November 1984 Subdivision F Guidelines on the
conduct of studies. To insure the correct procedures are followed
for acute toxicity studies, the laboratory should follow a copy of
Subdivision F Guidelines, November 1984 edition in addition to the
recommendations given in this PR Notice.
Contents
{The hypertext Table of Contents has been added by M. E. Mispagel
to this document.}
The studies of concern are those from the FIFRA
Subdivision F Guidelines 81-1 through 81-6: acute oral toxicity,
acute dermal toxicity, acute inhalation toxicity, primary eye irritation,
primary dermal irritation and dermal sensitization. The intent of
the six acute toxicity studies is to provide data sufficient to allow
the placement of a product into a specific toxicity category. The
toxicity category is then used for the purposes of determining precautionary
labeling.
By addressing study deficiencies observed in the past, the Agency
hopes to inform laboratories and registrants of mistakes that are
found so these mistakes may be avoided in future submissions. OPP
realizes the following list does not contain all possible
study deficiencies that may or may not lead to study rejection.
If any registrant thinks that a reviewer misunderstood a study or
otherwise incorrectly graded a study, they may submit a rebuttal.
This submission should include all information pertinent to the rebuttal:
the product's registration number, the study's MRID number, the problem(s)
identified by the Agency with the study and a clear explanation of
the registrant's disagreement. Secondary rebuttals will not be considered
unless relevant information previously not provided is forwarded.
This new information should be clarification of procedures or data,
new data, product formulations, etc.
Study deficiencies may be separated into two broad categories, general
deficiencies that may be common to many or all studies and deficiencies
specific to a particular study. Both of these categories may include
errors of conduct and/or errors of reporting. Generally, errors in
the reporting of a study are correctable, while errors in the conduct
of a study (unless they are minor) are not. Once a study has been
improperly conducted, it is not possible to correct errors without
reconducting the study. However, errors of reporting, although correctable,
can cause an avoidable delay in review time.
Industry Comment: A general principle that should
be followed in the review of acute toxicity studies should be that
all studies that conform to the current Subdivision F or OECD guidelines
must be acceptable as long as they allow a label category to be
assigned.
EPA Response : The Agency agrees.
Return to the Main Quality Assurance Page
I. General Study Deficiencies
A. Errors in Study Conduct.
1. Studies are submitted on a test material that is too dissimilar
to the registration product to be useful for making labeling
decisions. When submitting studies to support the registration
of a product, it is always preferable to use studies that were
conducted on the formulation to be marketed. When this is not
possible it is advisable to submit studies on a formulation or
product that was found to be toxicologically similar either by
a specific determination of toxicological similarity or through
the batching process of a Registration Eligibility Document (RED).
It is also acceptable to "bridge" data to support one
product that was conducted on one product to support another
product. In bridging data, a registrant would use data from a
product with either toxicity category of III or IV for the acute
oral toxicity, acute dermal toxicity, acute inhalation toxicity,
primary eye irritation and primary skin irritation studies (but
not the dermal sensitization study) to support another product
that has a similar but more dilute formulation. Or, the registrant
may use data from a product that has toxicity category I for
any of the five acute toxicity studies to support a category
I rating for a similar but more concentrated product.
2. The test animals were previously or concurrently used in
another study, or, were used to test more than one test material
at a time. As per the February 11, 1992 Robert P. Zendzian
and March 17, 1992 Penelope Fenner-Crisp memos on this subject,
OPP considers the reuse of test animals and the testing of multiple
chemicals simultaneously on test animals in acute toxicity studies
or any other toxicology studies to be scientifically unacceptable.
3. The test animals were unhealthy. The 40 CFR 160.90
(c) states that at the beginning of a study, test systems shall be
free of any disease or conditions that might interfere with the purpose
or conduct of the study.
4. The mortality exceeds more than one animal per sex for
either sex in a limit test. When there is more than one
mortality per sex, the lab should test additional dosages in
order to determine an LD50.
Industry Comment : Additional dose levels should
be tested as necessary to determine the label category (not necessarily
an LD50).
EPA Response : The Agency agrees.
5. A reduced testing scheme was used without establishing
the more sensitive sex. A reduced testing scheme allows
testing fewer than three dose levels for each sex. To qualify
for a reduced testing scheme in either the acute oral, acute
dermal or acute inhalation toxicity study, the laboratory must
first establish whether one sex demonstrates increased mortality
to the test material. This demonstration is preferably done via
the acute oral toxicity study. Dose levels must be sufficient
to clearly identify the appropriate toxicity category for the
test material.
6. Dose levels were not properly chosen . The test
did not allow the reviewer to determine the toxicity category of
the study.
Ideally, an acute toxicity study may be accepted if no more than
one animal of either or both sexes dies (for a total of two-out-of-ten
animals) during a limit test. The Agency has received older acute
inhalation toxicity studies that failed in their attempt to reach
the 5.0 mg/L limit test concentration. In studies of this type where
2/5 animals of one sex (and 0/5 of the other sex) die in a 5.0 mg/L
limit test, the study will be classified toxicity category III for
acute inhalation toxicity. The registrant will be given the option
of retesting (at 2.0 mg/L) to achieve toxicity category IV for acute
inhalation toxicity.
Discussion: Bracketing. If, in an acute oral toxicity study,
the lab tested the concentration of 50 mg/kg (only) and 0/10 test
animals died, this would not be sufficient to categorize the test
material. This product could conceivably have an LD50 that would
place it into toxicity category II, III or IV. The lab should then
test at least one additional dose to determine if the product meets
the limit test for Category III or Category IV.
Industry Comment : It is only necessary
to test enough doses to establish the correct label category (not
necessarily an LD50).
EPA Response : The Agency agrees. We have
modified our discussion of bracketing to note that it is not necessary
to determine an LD50. However, a test done at a very low cut-point
is not sufficient for labeling. Limit tests are useful for labeling
only if they allow placement of the product into Category III or
Category IV. Otherwise, overlabeling could result.
a. If 3-4 test animals die at the limit dose (e.g. 5000 mg/kg
for acute oral or dermal toxicity or 2 mg/L for acute inhalation
toxicity study) and no more than one animal dies at the next limit
dose level (e.g. 500 mg/kg for an acute oral toxicity, 2000 mg/kg
for acute dermal toxicity or 0.5 mg/L for an acute inhalation toxicity
study). The toxicity category for the study would be category III.
b. Another example is an acute inhalation toxicity study where
two dose levels are tested, with 7/10 animal deaths at 0.5 mg/L
and 0/10 (or 1/10) deaths at 0.03 mg/L. As the upper limit for
acute inhalation toxicity category I is 0.05 mg/L, this would not
eliminate the product from falling into toxicity category I. This
study would not be acceptable without a third dose level conducted
between 0.03 and 0.5 mg/L.
An acute oral, dermal or inhalation toxicity study where one or
more test concentrations are tested that do not allow LD50 determination
or bracketing will be rejected. Examples of this are:
c. An acute oral toxicity study is submitted with test material
concentrations of 50 and 300 mg/kg. The study had no mortalities.
This study would not allow the study to be placed into any toxicity
category as it could only be said that the LD50 is above 300 mg/kg.
This could place the toxicity category into category II, III or
IV.
d. An acute dermal toxicity study is conducted at 2000 mg/kg and
the mortality rate is 10/10. No other test material concentration
is tested. This results of this study only show that the product
could not be placed into toxicity categories III or IV. However,
this product could fall into either toxicity category I or II for
acute dermal toxicity. Thus, this study would not be acceptable.
7. The laboratory fails to follow a test method approved by
the Agency and the resulting data is questionable . Laboratories
should assure their protocols do not conflict with guidelines,
guidance or test methods approved by the Agency. They should
also ensure their protocols are followed. OECD protocols for
acute toxicity studies are accepted by the Agency.
8. An unacceptable test species was used . For the
acute oral and inhalation toxicity studies, the preferred species
is the rat. For the acute dermal toxicity study, the preferred species
is the albino rabbit, followed by the rat and the guinea pig. The
preferred species/strain for the primary eye and skin irritation
studies is the albino rabbit. The preferred species for the Buehler
Method and many of the other dermal sensitization studies is the
guinea pig. Although the species mentioned above are not the only
acceptable species for these particular studies, they are the preferred
species. Any lab conducting these studies using other than the preferred
species for that study must provide justification for the
use of another species. The Agency may reject studies based on the
test species used alone. If a lab has the intention of using a species
other than the preferred species for a particular study, they should
contact the Agency for approval or guidance beforehand.
Industry Comment : Amend the second sentence
to read ...the preferred species are the rabbit, rat and the guinea
pig.
EPA Response : The Agency believes that
the preferred species for dermal toxicity studies is the albino
rabbit. The rat and the guinea pig are also allowed. However, the
order of preference is as is given in the second sentence.
9. The animals were quarantined for an unacceptable length
of time . The minimum quarantine time is five days. Lack
of sufficient quarantine period may cause undue stress in the
test animals. Such strain may contribute to the mortality rate
and thus adversely influence the toxicity.
10. The lab failed to conduct sufficient observations of the
test animals . The lab should consult Subdivision F guidelines
for guidance on the observation of test animals.
11. The test animals used were not of an acceptable weight
and/or age. Rats to be used in acute toxicity studies
should be 6-10 weeks of age at receipt and 8-12 weeks at study
initiation. Rabbits should be at least 12 weeks of age at study
initiation. Body weights for the test animals should be in the
range of those for normal animals at that age. The weight range
should not exceed [plus or minus] 20% of the mean preexposure
weight for that sex. The lab should report the weights and ages
of the test animals for each of the six acute toxicity/irritation
studies and be able to provide a growth curve from the supplier
to show that the test animals used were of normal weight for
their age. This applies to all acute toxicity/primary irritation
studies.
Industry Comment : The requirement for the growth
curve should pertain only to rats used in the acute toxicity
studies. The age requirement for rabbits should pertain
only to acute dermal toxicity studies, not to skin and eye irritation
studies where the age and weight of the animal is not important.
EPA Response : There is no age or size requirement
for primary irritation studies. However, proper growth is an indication
of a healthy animal. Therefore, the lab should be able to demonstrate
that the test animals used were of normal weight for their age
in order to ensure that they are healthy animals.
12. A lack of equipment calibration. Although records
on equipment calibration are not required in the report, these data
must be maintained by the laboratory.
13. Incorrect replacement of animals other than at study initiation
when a dosing accident has occurred. Animals on study
should only be replaced when deaths have occurred as a result
of dosing accidents or other unrelated events. Necropsies should
be conducted to prove deaths were the result of dosing accidents
or other events.
Industry Comment : Please clarify that if a dosing
accident occurs that an animal may be replaced at study initiation.
EPA Response : If a dosing accident occurs at study
initiation, an animal may be replaced.
B. Errors in Reporting [General Study Deficiencies]
1. Failure to properly identify the test material .
Often, studies identify test materials by a pesticide manufacturer's
internal code, an obsolete name or some form of identification other
than the current product name or EPA registration number. Ideally,
the test material name should be identical to the product name.
The test material should be properly identified in the report. If
this is not possible, the registrant should include a statement identifying
the test material in the submission to the Agency. When the test
material used in a study is not the product for which registration
is sought, the registrant must include the name of the test material
(a product name if possible), the CSF of the test material and the
test material's EPA registration number (if possible), the test material's
relationship to the product (if any) and clearly explain why this
particular test material was submitted to support the product.
At times, the lab may include a copy of the product formulation
in the study report. When this reported formula is not the same as
the product CSF, this study may not be accepted. This will call for
a similarity determination based on chemical formulation.
2. The test material is not properly described . The
description of the test material should include:
a. The physical form of the test material, e.g., liquid, paste,
aerosol spray, granular, etc.
b. pH of the test material.
c. Special properties of the test material that may affect testing,
e.g., very viscous liquid, gel, encapsulated pesticide in suspension,
color, etc.
d. Manufacturer's lot number of the test substance.
Industry Comment : The pH should only be required
when appropriate, for aqueous liquids used in skin and eye irritation
studies.
EPA Response: The pH of the test material is appropriate
for many additional situations. It is appropriate for moistened
solids which will be applied to the skin and is also appropriate
for solids in solution or suspension for oral toxicity studies.
Obviously, pH would not be appropriate for a solid material instilled
in the eye.
3. Noncompliant or missing QA or GLP statements may lead to
study rejection or delayed review . Refer to the 40 CFR
part 160 and Subdivision F Guidelines for guidance. If the data
raises concerns as to its validity and the study lacks appropriate
QA and GLP statements, it will be rejected. If the data does
not otherwise raise concerns about its acceptability, deficiencies
in QA/GLP reporting will not influence the acceptance of the
study; however, such studies will be referred to the Office of
Enforcement and Compliance Assurance (OECA) for follow-up.
4. Unreported data or other missing information .
On occasion, pages are missing from the study report or the laboratory
may fail to include information such as a legend explaining abbreviations
used in reporting study results. At times reports do not include
the complete details of a study. Perhaps this is because the laboratory
assumes that certain aspects of the study are unimportant or the
laboratory has included a protocol that covers the particular type
of study. Laboratories should submit reports that address all details
of that specific study. The report should relate how that particular
study was conducted. Laboratory QA units must insure that the reports
are complete and accurate.
5. The report or parts of the report are illegible. Although
this usually concerns reprints or "blowbacks" of reports,
it is not restricted to reports submitted in the form of a reprint.
6. The report contains incorrect calculations that the reviewer
is not able to clarify. In such instances, the reviewer
must consult the registrant/lab for clarification or the submission
may have to be rejected if prompt resolution of the misunderstanding
is not attainable.
7. Dilutions of a test material are not reported or are not
adequately reported . When a test material is diluted
the dilutions must be defined and the reasons for the dilution
must be clearly explained.
8. The ages, weights and/or source of the test animals is
(are) not reported. This is a requirement for all acute
toxicity studies as per Subdivision F guidelines 81-1 through
81-6 and 80-4.
Return to the Main Quality Assurance Page
II. Acute Oral Toxicity
A. Errors in Conduct [Acute Oral Toxicity].
1. Unnecessary or improper dilution of the test material.
- Liquids should be tested undiluted.
- The highest workable test material concentration should be used
for solids and viscous materials.
- Justification must be provided for any dilution.
Caustic materials should only be diluted if it is necessary for
intubation. The dilution of caustic test materials may reduce their
corrosive effects, thus giving an inaccurate representation of their
potential threats. If it is necessary to dilute such a test material,
the report should give the pH of the diluted test material. Dilution
should be held to a minimum.
Discussion: Is the recommendation for constant dose volume or
constant dose concentration? For purposes of precautionary
labeling, constant concentration is more important than constant
volume.
Discussion: Is analytical confirmation of dosing solutions a
necessity? Analytical confirmation of dosing solutions is not
a requirement.
Industry Comment : The following guidance for acute
oral dosing was proposed by the ACPA group and is recommended to
be included as guidance:
Acute oral dosing procedures have typically employed either constant
dose volume across all dose levels (EPA, OECD guidelines) or constant
dose concentration. Systemic toxicity can usually be determined
using a constant dose volume to minimize the effects of gastric
volume on absorption. However, EPA has expressed concern that excessive
dilution of test materials may not provide a correct assessment
of the true toxicity of the test material for hazard labeling purposes,
particularly for those that are corrosive.
The following guidance is proposed:
1) Either constant volume or constant concentration administration
is acceptable, provided the guidance below on dilutions is employed.
2) When possible, liquid test material should be dosed neat.
3) If dosing with neat material is not possible, due to high
viscosity or toxicity that would preclude accurate low dose volumes,
or if constant volume has been deemed to be the more appropriate
method, the test material may be diluted. The highest concentration
possible should be administrated, although volumes less than 0.5
ml per animal would not be required. Lower dose volumes are acceptable
if they can be accurately administered.
[Note that the use of the 0.5 ml/ animal dose volume may necessitate
a 50X dilution of a highly toxic (category I, [less than or equal
to] 50 mg/kg) test material to insure accurate dosing.]
4) If possible, the maximum dose volume should not exceed 1.0
ml/ 100 grams bodyweight for all vehicles, although volumes up
to 2 ml/ 100g for aqueous vehicles are acceptable with justification.
5) Solid materials should be suspended or dissolved in the minimum
amount of vehicle and dosed at the highest concentration possible,
following the above guidance.
EPA Response: The Agency agrees with the guidance
outlined above.
3. The lab did not fast the animals before dosing. The
presence of foodstuffs in the digestive tract of the test animals
can have the effect of diluting the test material or carrying portions
of it through the alimentary canal without digestion. This action
can thereby reduce the toxicity or corrosive effects of the test
substance.
4. Lack of necropsy : Although gross necropsies are
recommended, lack of necropsy is not a reason for rejection.
B. Reporting Errors [Acute Oral Toxicity]:
1. Insufficient observation (lack of specificity). Correct
identification of symptoms in specific scientific terms and not generalized
statements or lay expressions that could mean several different things
is preferred.
2. The absence of necropsy results. When a necropsy
has been conducted, the results should be included in the report.
Return to the Main Quality Assurance Page
III. Acute Dermal Toxicity
A. Errors in Study Conduct [Acute Dermal Toxicity].
1. The dilution of liquid test materials or over-moistening
of dry test materials. All liquid test materials should
be undiluted for the acute dermal toxicity study. Dry test materials
must be moistened with water before application to ensure good
contact and no loss of the test material. The lab should state
the amount of water used to moisten dry test materials. The dry
test materials should not be moistened beyond the that point
which is necessary to assure proper contact with the skin.
Industry Comment : Can we specify that vehicles
other than water or saline such as gum arabic, ethanol + water,
carboxymethyl cellulose, glycerol, propylene glycol, PEG vegetable
oil and mineral oil can be used if water or saline cannot be used
as long as the vehicle is not irritating and the inability to use
water or saline is justified in the report.
EPA Response : The Agency agrees.
2. The size of exposure area is incorrect (too small). The
exposure site should be approximately 10% of the animal's body surface
area. The exposure area is to be from the scapula (shoulder) to the
wing of the ileum (hipbone) and halfway down the flank on each side
of the animal.
Industry Comment : Less than 10% body area may
be exposed if the material is highly toxic. The second sentence
in the above paragraph should read: The prepared area for a rat
or rabbit dermal toxicity study will be defined as a shaved or
clipped area starting at the scapula (shoulder to the wing of the
ileum (hipbone) and half way down the flank on each side of the
animal.
EPA Response : The Agency does not agree with the
practice of using less than the prepared area as defined above
for highly toxic substances. We prefer some other mechanism of
reducing the amount of test substances such as a controlled dilution.
In such a case, it is easier to compare the dose to doses used
in other studies. If a lesser exposure area is used, it is hard
to calculate what the actual exposure is.
3. Improper occlusion, covering and wrapping of the test site. An
improperly wrapped test site may result in loss of test material
and thus reduce the effective dose of the pesticide. Important factors
to strive for when wrapping the test animals for the acute dermal
toxicity study are:
a. Powders or other solids should be slightly moistened (not runny)
to a paste before application to the test site.
b. It is preferred that the test material is applied to the dorsum.
c. The gauze covering is added to act as a reservoir for the test
material and to keep it localized. It is important that not too
much gauze is used. Too many plies or layers of gauze can have
the effect of absorbing the liquid test material or the water used
to moisten the solid test material thus minimizing the amount of
test material that is available to the skin.
d. The test material is further covered with an occlusive material
(such as plastic sheeting or rubber dam) or semi-occlusive material
(such as perforated plastic) to prevent evaporation of liquids
from the test site and to prevent ingestion of the test material.
Discussion: Guidance for occlusion. Although semi-occlusive
dressing is recommended, occlusive dressing is acceptable.
Industry Comment: We suggest the following
wording be added as agreed in our May meeting: When possible the
test substance should be applied directly to the skin, otherwise
it may be applied directly to a porous gauze dressing which is
immediately placed in contact with the animal's skin.
The test substance must be held in contact with the skin with
gauze and non-irritating tape for a 24-hour exposure period. The
test site must be covered in a suitable manner to retain the test
material in contact with the skin, avoid wicking of material from
the skin surface, and to ensure that the animal cannot ingest the
material. To minimize wicking, the gauze should be no more than
8-ply; fewer layers of gauze may be needed for small test volumes.
Although a semi-occlusive dressing is preferred, an occlusive dressing
will also be acceptable.
EPA Response: The Agency agrees.
B. Errors in Reporting [Acute Dermal Toxicity]:
1. Systemic toxicity, and dermal irritation are not reported
or are not reported sufficiently. This information is
required and is often helpful in evaluating other studies. Evaluations
of systemic toxicity and local irritation should be made frequently
on the day of application, and daily thereafter. Mortality checks
alone are not sufficient evaluations. If information from the
dermal toxicity study is to be used to support dermal irritation
labeling, i.e. if a waiver of the dermal irritation study may
be appropriate, observations of dermal reactions should be made
with sufficient frequency and in sufficient detail to obviate
the need for a specific dermal irritation study.
Return to the Main Quality Assurance Page
IV. Acute Inhalation Toxicity
A. Errors in Study Conduct [Acute Inhalation
Toxicity].
1. Historically, one main reason acute inhalation toxicity studies
have been rejected was that the labs were often unable to achieve
a small enough Mass Median Aerodynamic Diameter (MMAD). However,
in 1994, the Agency changed its acceptance criteria. Currently, the
Agency accepts acute inhalation toxicity studies with MMADs of 1-4
micrometers. Please refer to the 2/1/94 HED Memorandum: Interim
Policy for Particle Size and Limit Concentration Issues in Inhalation
Toxicity Studies.
a. For dry products that do not readily form aerosols with MMADs
at or below 4 microns, further attempts to reduce their particle
size must be made. Granular products should be milled (by air mill,
ball mill, hammer mill, etc.) for 24 hours if necessary. If the
product will still not form the proper aerosol and the product
is dissolved in liquid before application, the lab must attempt
to dissolve it in the vehicle to obtain an aerosol. The test material
concentration must be calculated based on the amount of test material
without the diluent. The acceptable concentration must allow for
the diluent. Water is the recommended diluent. If the diluent is
some material other than water, a vehicle control study must be
conducted.
Industry Comment: The granular products should
be milled for 24 hours as necessary or until particle size
plateaus. If the product will not form a proper aerosol
and the product is dissolved or suspended in water or another vehicle
under conditions of field use, the study should be conducted in
a suspension or solution of water or the vehicle that is used under
commercial use conditions.
EPA Response: The Agency agrees with the comment.
Granular products should be milled for a reasonable amount of time
until particle size plateaus. This may be less than 24 hours.
b. Products that do not deliver an acceptable particle size and
are not water soluble are excellent candidates for a waiver of
the acute inhalation toxicity study. Microencapsulated product
with a large percentage of capsules above four microns are also
excellent candidates for waivers. Waivers may be granted for technical
and end-use products which cannot conceivably be generated in sufficient
concentration to pose an inhalation hazard. All waiver petitions
must be considered on a case-by-case basis. The following are likely
waiver candidates:
1. Non-volatile products which cannot be readily aerosolized (e.g.
viscous liquids, waxes, and resins), and which are not heated or
diluted to an inhalable state during application.
2. Tree injections or thick liquids such as lotions, waxes, etc.,
which contain non-volatile active ingredients.
3. Corrosive or highly irritating agents (the chemical may be designated
inhalation Toxicity Category I by default). The Agency will categorize
a study by actual acute inhalation toxicity data over the corrosivity
of a product and may also use systemic toxicity data where available.
4. Slow release collars and ear tags (plasticized).
5. Products with fewer than 1% of their particles being below 50
microns under conditions of use, unless that product is toxicity
category I by the oral or dermal routes, or induces severe systemic
toxicity upon ocular dosing.
6. Non-friable granules. The registrant must demonstrate that the
granules do not produce fines when subjected to shipping and handling.
7. Microencapsulated products that are not readily fractured, dissolved,
time released, leaky or small enough to be respirable.
If an end-use product cannot be aerosolized, but the addition of
a diluent under conditions of use yields an inhalable aerosol, an
inhalation study should be performed using the most concentrated
label dilution. A vehicle control group should be included if a diluent
other than water is used.
3. Studies that do not demonstrate that the particle concentration
was consistent over the period of the exposure are not acceptable. Studies
that do not conduct the particle size analysis at least twice
during the exposure are not acceptable. Studies that conduct
particle size analysis once during the four hour exposure have
not demonstrated that the particle size was consistent during
the exposure. Chamber concentration and particle size measurements
should be made at least twice during the study at time points
spaced well apart. Chamber concentrations may be measured using
gravemetric analysis or by analytical measurement. If these are "reasonably
consistent" ([plus or minus] 10% for liquid aerosol, gas
or vapor, [plus or minus] 20% for dry aerosol), then two measurements
should be sufficient. If the measurements are not consistent,
then further measurements (a total of 3 or 4) should be considered.
Pretest measurements may be helpful.
4. Studies with MMADs that are not within an acceptable range
will not be acceptable. All particle size analysis should
show the MMAD to be within the acceptable range of 1-4 microns.
Otherwise, the test animals will not have been exposed to a respirable
particle distribution throughout the exposure.
Discussion: Studies where the MMAD exceeds four microns. The
Agency will evaluate such studies on a case-by-case basis. If the
MMAD is only slightly above four microns and there are no other reasons
to question the acceptability of the study, the Agency will accept
the study.
Industry Comment: Studies should not be
rejected, if reasonable efforts to minimize particle size are employed.
EPA Response: The Agency agrees.
5. The lab did not obtain a concentration sufficient to determine
an LC50 or conduct a proper limit test. At times, OPP
has received acute inhalation toxicity studies that tested one
or two aerosol concentrations, the concentration tested was below
the limit test concentration and no explanation was given. If
the lab can demonstrate to the satisfaction of the Agency that
the test was actually conducted at the maximum attainable concentration,
and there is no evidence of toxicity at that concentration, the
product will be assigned toxicity category IV. The report must
include a description of the physical and chemical nature of
the test material, and a justification for the acceptance of
the study. Otherwise, this study will not be acceptable.
Often times, labs had problems attaining limit test concentrations.
An acute inhalation toxicity limit test was originally a four hour
exposure to a test atmosphere at a concentration of 5.0 mg/L. However,
in 1994 the Agency changed the limit test concentration to 2.0 mg/L.
Acceptable acute inhalation toxicity studies which show no mortality
at concentrations above 2.0 mg/L will be placed into toxicity category
IV.
Corrosive test materials that cannot achieve appropriate particle
distributions and/or particle concentrations may be placed into toxicity
category I if there is no other evidence indicating low toxicity.
Other evidence would likely be the results of an acute oral toxicity
study.
6. The limit test concentration was too low. OPP may
also reject studies that test more than one concentration below the
limit test concentration and fail to determine the LC50 (or LD50).
If a lab tests product concentrations below the limit test without
determining an LC50, they should include an explanation of this as
part of their submission. This has overwhelmingly been a problem
of acute inhalation toxicity studies.
7. Studies that do not measure the test material concentrations
from the breathing zone of the animals will not be accepted. Studies
not measuring the test material concentration from the breathing
zone will not be taking representative samples.
8. Studies that do not provide a continuous four hour
test material exposure. The exception to this rule is
studies with a 100% mortality before the end of the exposure.
If a 100% mortality level is found and the study does not allow
the placement of the product into toxicity category I,
other concentrations must be tested. A mean or time weighted
average should be used if more than two measurements were taken
and significant variability exists between the measurements.
9. Studies that report analytic or gravimetric concentrations
that are higher than the nominal concentration. In these
situations, the lab appears to be measuring more test material
than was introduced into the test chamber.
10. Studies displaying mortality with the oxygen concentrations
below 19% or fewer than ten chamber air changes per hour. A
study may have an insufficient oxygen level due to the concentration
of the oxygen in the supplied air or due to an insufficient number
of air changes. If such a study reported mortality, it could
not be said that the mortality was solely attributable to exposure
to the test material. Studies whose only deficiency is the lack
of reporting the oxygen concentration or number of chamber air
changes per hour may be accepted.
11. Failure to remove excess test material from animal's coats
after exposure. An effort should be made to remove excess
test material. Removal of excess material eliminates concerns
of exposure to the test material from ingestion or dermal absorption.
Delayed deaths cannot be solely attributed to inhalation of test
material if this precaution is not taken.
12. Animals (by volume) occupy more than 5% of the
exposure chamber. This requirement is stated in the Subdivision
F guidelines. Animal volume is calculated assuming that each
gram of animal occupies one cc of volume within the chamber.
13. Lack of necropsy. Although it is not a requirement,
gross necropsies are recommended.
B. Reporting Errors [Acute Inhalation Toxicity].
1. A lack of information of the study conditions. The
following is a list of data that must be included in the inhalation
toxicity study report. The information to be included in the report
is not limited to the list below. This list should be used
in conjunction with the Subdivision F guidelines. Failure to report
any of the following factors may cause a study to be classified supplementary
data and thus be rejected until receipt of the information by the
Agency.
a. The length (of time) of the exposure of the test animals to
the aerosol.
b. The method of exposure of the test animals, e.g., whole body
or nose only exposure.
c. The chamber rate of airflow, temperature, humidity, and how
each was measured.
d. The actual concentrations of test material in the chamber atmosphere
measured from the breathing zone of the test animals. This must
be in tabular form.
e. The chamber concentration must be reported in mg/L, not ppm
or mg/m3.
f. Reports should state whether concentrations were measured from
the breathing zone.
g. The rate of air flow, volume of air taken and length of time
the air was measured for aerosol concentration measurements.
h. The oxygen content of the chamber air during the exposure.
Industry Comment: Oxygen content should not be
required as long as ten air changes are done according to EPA guidelines.
EPA Response: The Agency agrees.
i. The Mass Median Aerodynamic Diameter (MMAD) and Geometric Standard
Deviation (GSD).
j. The equipment used to determine the MMAD and GSD, the sampling
zone, the rate and volume of air measured, and the actual values
and percentages obtained from each stage of the impactor.
k. The chamber volume.
2. Failure to identify the test atmosphere generation system.
3. The failure to report the time taken to reach 90% or 99%
of the desired particle concentration. (The time needed
to reach 99% is more desirable.)
Return to the Main Quality Assurance Page
V. Primary Eye Irritation
A. Errors in the Conduct of the Study [Primary
Eye Irritation]
1. Failure to test aerosol products by spraying test material
into the eyes. Aerosols should be sprayed into an eye
held open for one second from a distance of 10 cm. It is necessary
for aerosol products to be sprayed so that the physical damage
that may occur because of the force of the spray may be evaluated.
The exception to this rule is total release foggers. In testing
most total release fogger sprays, instillation of the liquid
from the fogger spray will have to be carried out. CRP will be
required for such products and labeling identifying the physical
hazard will be required.
The Agency will allow industry to develop a system to determine
the ocular damage that would be caused by the physical force of various
aerosol sprays.
2. The use of fluorescein stain stopped before resolution
of opacity. Fluorescein aids in sighting lesions that
otherwise may not be apparent.
Industry Comment: Fluorescein does not measure
opacity and therefore it may be stopped before the resolution of
opacity. Opacity is recorded as a separate observation. Lack of
fluorescein staining is not a reason for study rejection; however
the use of this method is encouraged.
EPA Response: The Agency agrees that the use of
fluorescein is encouraged, but not required. Although fluorescein
may not measure opacity, if it is used and anomalies are observed,
the use should be continued until the anomalies are resolved. Otherwise,
the results are ambiguous and difficult to interpret.
3. The study ended before resolution of all irritation. For
eyes showing irritation at 72 hours, scoring should be continued
and as frequently as needed until all irritation has subsided or
for 21 days, whichever comes first. The exception to this rule is
irritation so severe that the test material will obviously be placed
into toxicity category I whether the study is continued or not.
4. Incorrect volume of test material used. The correct
volume is 0.1 ml for liquids, and for solid test substances, the
weight equivalent of 0.1 ml or a weight of not more than 100 mg should
be used. Aerosols should be sprayed into an eye held open for one
second from a distance of 10 cm. The Agency prefers the use of
the weight equivalent of 0.1 ml be used for solid substances, even
if the weight if more than 100 mg. For most test substances, 0.1
mg and 0.1 ml will be fairly close. But if a material is very heavy
and 100 mg is a very small volume, this may not represent the "worst-case" eye
irritation potential.
5. The test material was a solid consisting of large granules
or other large solid particles, and was not ground before instillation. Where
possible, test materials should be ground to a fine powder before
instillation into the eye. If the solid test material can not
be readily ground, but can still be placed into the conjunctival
sac, then it should be tested as is.
Industry Comment: If the granules are large and
not amenable to grinding then a waiver may be obtained.
EPA Response: The Agency agrees.
6. The data submitted was only on animals whose eyes had been
rinsed immediately after test material instillation. The
report must include data on test animals whose eyes were not
washed out before 24 hours after instillation.
7. The test material was diluted before instillation into
test animals' eyes.
8. Studies using animals that display
irritation, "background
opacity" or "naturally occurring opacity" before
test material administration. Such animals should not
be selected for testing.
9. The method of observation was incorrect. Evaluations
should be made using white light, resembling day light. Pen lights
are not acceptable for reading ocular irritation. The use of magnification
and fluorescein is encouraged. The slit lamp is considered the most
accurate way of detecting lesions in the eyes. Absence of fluorescein
staining may not always signify healing of all corneal lesions, because
the eye heals from the corneal epithelium inward.
Industry Comment: The above comment is not acceptable,
and not as we agreed at our meeting. We recommend the following:
The use of bright room light or a pen light with or without the
use of magnification is acceptable.
If observation of the reactions is aided by the use of a binocular
loupe, hand slit lamp, biomicroscope or other suitable device,
these findings should be thoroughly described, recorded and reported.
EPA Response: The Agency does not agree that bright
room light or a pen light with or without the use of magnification
is acceptable.
10. Discussion: How many animals are required for primary eye
irritation study? Although the Agency prefers six test animals
for this study, it will accept three in accordance with OECD protocol.
11. Discussion: Are scores of "1" considered
to be positive? Scores of 1 for conjunctival redness, discharge and/or
swelling are not considered to be positive and do not influence
precautionary labeling.
Industry Comment: Animals with only these scores
do not need to be examined past 72 hours.
EPA Response: The Agency agrees.
B. Errors in Reporting [Primary Eye Irritation].
1. The ocular grading scale used is not defined in the report. The
grading system should be the Draize Scale and must be included in
the report.
2. The method of examination was not reported. The
use of fluorescein, anesthetics, the type of light used, the frequency
of observations, etc., must be reported.
3. Disregarding the presence of stippling when it is stained. The
Agency may count stained stippling as positive ocular irritation
and it may affect the toxicity category of a study.
Return to the Main Quality Assurance Page
VI. Primary Skin Irritation
A. Errors in Study Conduct [Primary Skin Irritation].
1. The size of the exposure area is incorrect. The
appropriate size is 1 in2 (6 cm2).
2. An improper amount of test material was used. The
proper quantity is 0.5 milliliters for liquids and 0.5 grams for
solids.
3. The dry (solid, powder) test material was not moistened
for application. The moistening of the test material
helps it to penetrate the dermal membrane. The test material
must be moistened before application to assure
uniformity. The dry test material should be slightly moistened
(not runny) to a paste using water. The amount of water used
to moisten the test material should be stated in the report.
4. The test material was diluted rather than moistened without
giving an explanation. Dilution (over-moistening) of
the test material may reduce the irritation obtained.
5. The exposure site was not properly occluded. The
exposure site should be semi-occluded. In cases where the test material
contains a significant amount of an alcohol or petroleum distillate,
care should be taken to preclude a reaction between the adhesive
and the test material. Interaction between some test materials (containing
an alcohol or petroleum distillate) and the adhesive could easily
change the outcome of the study. It is important that the lab not
cover the test material with enough gauze to pull liquid test material
or a moistening agent away from the skin site. Other considerations
in dermal application are discussed under acute dermal toxicity.
Industry Comment: Please see the specific
wording for semi-occlusion under the industry comments for dermal
toxicity. At the industry meeting in May it was also agreed that
use of an occlusive dressing would not be a criteria for study
rejection.
EPA Response: The Agency agrees.
6. The exposure period was not four hours. The study
may be accepted if the exposure period was greater than four hours
and minor irritation (toxicity category III or IV) was noted.
7. The exposure site was not rinsed with water or another
suitable vehicle after the four hour exposure and severe irritation
was observed. Without wiping the exposure site, the residual
test material may continue to cause irritation and fallaciously
increase the toxicity category of the product.
8. Preparation of the exposure site is incorrect. One
example could be that the exposure site was not depilated.
9. Abrasion of test sites. This is thought to exacerbate
the irritation. The study will be rejected if the abrasion may have
placed the study in a higher toxicity category (I or II).
10. Observations were not conducted until irritation had subsided.
Observations should be continued until irritation has subsided (no
scores other than "1" for dermal erythema and/or edema)
or 14 days has been reached. If there is still irritation at 14 days
the study may be ended. The exception to this rule would be ending
a study early when it is evident that it will be placed into toxicity
category I.
11. Test animals were not young adult. Healthy adults
should be used. The test animals should be of normal weight for their
age.
Industry Comment: The requirement
should read "Healthly
adult animals" (not necessarily young) should be used. As
long as animals are healthy, their weight is not important, it
was agreed in the industry/regulator's meeting in May. There will
be no weight restrictions since there is no scientific evidence
that age or weight impacts the results of eye or skin irritation
studies. Reference EPA guidelines.
EPA Response: As previously noted, there are not
weight restrictions. However, the health of animals that are not
of normal weight for their age will be questioned.
12. Unjustified use of a moistening agent other than water
(or saline). Water is the preferred agent to be used
for moistening dry test materials for primary skin irritation
studies because it resembles sweating by humans. Other vehicles
may cause irritation by themselves or exacerbate irritation caused
by the test material. Other moistening agents are not acceptable.
The study may be upgraded upon subsequent explanation of the
choice in vehicle.
Industry Comment: Although water or saline is the
preferred moistening agent, other agents may be used providing
the use is justified. Acceptable alternatives include: gum arabic,
ethanol + water, carboxymethyl cellulose, polyethylene glycol,
glycerol, vegetable oil and mineral oil. These can be used if water
or saline cannot be used as long as the vehicle is not irritating
and the inability to use water or saline is justified in the report.
EPA Response: The Agency agrees.
13. Too few animals were used. The Agency prefers
that six animals be used in the primary skin irritation study; however,
in accordance with OECD criteria, the Agency will accept primary
skin irritation studies conducted with only three animals.
14. Discussion: Waivers. The primary skin irritation study
may be waived for products with pHs of 2 or less, or, 11.5 or above.
These products will be placed into toxicity category I by default.
If the registrant wishes to have such a product placed into a toxicity
category other than I, he must have a primary skin irritation study
conducted to prove that a toxicity category I is inappropriate for
this product.
The primary skin irritation study may also be waived if the material
was no more than slightly irritating after the 24-hour acute dermal
toxicity test exposure. The sites will have to have been scored using
the Draize Scale. Such products will be placed into toxicity category
IV.
B. Reporting Errors [Primary Skin Irritation].
1. The irritation scoring scale used by the lab is not reported. Dermal
irritation is usually reported numerically. When the scale used for
grading is not identified or included in the report, the Agency cannot
be sure of the irritation observed.
2. The method of preparation of the exposure site is not reported. The
report should state how the hair was removed from the exposure site,
etc.
3. The location of the exposure site is not reported. The
report should state where on the animal's body the testing site was
located.
4. Insufficient detail or imprecise reporting of observations. For
example, stating that the exposure site is exfoliated, and
giving no further information is vague. Exfoliation can be desquamation
or sloughing. Quite often, reports fail to go into detail or fail
to attempt to give a visual representation of the irritation observed
when such irritation goes beyond erythema or edema.
Industry Comment: For consistency the Agency should
provide a definition of the descriptive terms for exfoliation,
desquamation and sloughing.
EPA Response: The laboratory should define the
terms used in the reporting of observations.
5. Failure to report the condition of the skin after sloughing. Sloughing
very often leaves the skin thickened and denuded (hyperplasia or
hyperkeratosis and dead skin follicles).
Observations should clearly indicate whether
the skin has returned to "normal" or whether the skin
has been irreversibly changed, i.e., scarred, blanched or denuded.
Reversibility of skin reactions
is as important as the primary skin irritation index.
Return to the Main Quality Assurance Page
VII. Dermal Sensitization
Discussion: When is the study required? The dermal sensitization
study is required when there will be opportunity for repeated exposure
to the product. EPA will waive the dermal sensitization study for
formulations which are classified as toxicity category I for dermal
toxicity or dermal irritation. Products that are placed into toxicity
category I for dermal toxicity or dermal irritation with use dilutions
that do not require the use of protective clothing will also require
a dermal sensitization study. The sensitization study is always required
for the technical materials, regardless of the toxicity or irritation
properties of that technical. In Canada, the sensitization study
is always required unless the use dilution is also irritating. The
California Department of Pesticide Regulation does not routinely
require skin sensitization studies to register pesticides. Products
may also be categorized as sensitizers if components of the formulation
are known sensitizers.
The dermal sensitization study is the only one of the six acute
toxicity/irritation studies that may be conducted using one of several
techniques. Subdivision F Guidelines offer a choice of methodologies.
The seven testing methods accepted by the Office of Pesticide Programs
for dermal sensitization studies are:
1. The Buehler Method/the Modified Buehler Method. This is by far
the method submitted most often to OPP for dermal sensitization.
Recommended references for this study are:
a. Ritz, Harry L. and Buehler, Edwin V. "Planning, Conduct,
and Interpretation of Guinea Pig Sensitization Patch Tests",
in Current Concepts in Cutaneous Toxicity, V.A. Drill
and P. Lazar (eds.), Academic Press, New York, 1980, p. 25-40.
b. Robinson, et al. "A Review of the Buehler Guinea Pig Skin
Sensitization Test and Its Use in a Risk Assessment Process for
Human Skin Sensitization", Toxicology, vol.
61, 1990, p. 91-107.
c. "Experimental Skin Sensitization in the Guinea Pig and
Man", Animal Models in Dermatology, H.I. Maibach
(ed.), Churchill, Livingstone, Edinburgh, 1975, p. 56-66.
d. "A Rationale for the Selection of Occlusion to Induce
and Elicit Delayed contact Hypersensitivity in the Guinea Pig",
in Current Problems in Dermatology, E.V. Buehler,
vol. 14, Karger, Basel, 1985 p. 39-58.
e. Buehler, E.V. "Occlusive Patch Method for Skin Sensitization
in Guinea Pigs: The Buehler Method", Food and Chemical
Toxicology, vol. 32, no. 2, 1994, p.97-101.
It is recommended that the laboratory personnel familiarize themselves
with each of the five citations listed above before conducting Buehler
Method studies.
2. The Guinea Pig Maximization Test is another
method accepted by OPP for the determination of dermal sensitization.
A recommended
reference for this study is: "The Identification of Contact
Allergens by Animals Assay. The Guinea Pig Maximization Test", The
Journal of Investigative Dermatology, vol. 52, no. 3, 1969,
p. 268-276.
3. The Split Adjuvant Technique. A recommended
reference for this study is "The Bioassay of Contact Allergens in the Guinea Pig", J.
Sac. Cosmet Chem., vol. 24, March, 1973, p. 151-162.
4. The Open Epicutaneous Test. A recommended
reference for this method is "Screening of Fragrance Materials for Allergenicity
in the Guinea Pig", Journal of the Society of Cosmetic
Chemists, vol. 28, February 1977, p. 53-64.
5. The Mauer Optimization Test. A recommended
reference is Mauer, T., et al. "The Optimization Test in The Guinea Pig: Method
for the Predictive Evaluation of the Contact Allergenicity of Chemicals", International
Congress Series, Excerpta Medica, #376, 1975.
6. Freund's Complete Adjuvant Technique
7. The Footpad Technique in the Guinea Pig.
Freund's Complete Adjuvant Test, the Guinea
Pig Maximization Test, The Split Adjuvant Technique, the Buehler
Test and the Open Cutaneous
Test are discussed in "Identification of Contact Allergens:
Predictive Tests in Animals", Dermatotoxicology and Pharmacology,
F. Marzulli and H.I. Maibach, eds.
Below are study deficiencies that have been found
in dermal sensitization studies conducted by the Buehler Method.
The Agency chose to focus on the Buehler Method because it is by
far the dermal sensitization study submitted most often to the Agency.
The following Buehler Method study deficiencies are separated into
errors in the conduct of the study and reporting errors.
A. Errors in Study Conduct [Dermal Sensitization].
1. Failure to select the proper induction and/or challenge
concentrations from the primary irritation screening. An
improper induction concentration (too low) may fail to evoke
sensitization with a substance that may have otherwise brought
about sensitization.
Too high a challenge concentration is a problem that is frequently
seen in dermal sensitization studies. Labs sometimes choose a challenge
concentration that elicits irritation in unsensitized animals. When
the lab chooses an irritating concentration of test material for
challenge, it may not be possible to determine whether the irritation
noted was a result of a sensitization reaction or simply dermal irritation.
2. Failure to report the results of the primary irritation
screening. Without the results of the primary irritation
screening, it is not possible (or at least difficult) to assess
the choice of induction and challenge concentrations.
3. The lab used the wrong vehicle for the test material. Although
water or saline is a the preferred moistening agent, other agents
may be used providing the use is justified. Acceptable alternatives
include: gum arabic, ethanol + water, carboxymethyl cellulose, polyethylene
glycol, glycerol, and ethanol for induction with acetone for challenge.
These can be used if water or saline cannot be used as long as the
vehicle is not sensitizing and the inability to use water or saline
is justified in the report. Mineral oils and petrolatum should be
avoided when possible because they may induce irritation independent
of any test materials.
4. Use of ethanol as a vehicle for both induction and challenge. Buehler
has stated that ethanol alone can induce sensitization. When sensitization
reactions occur with the use of ethanol as the vehicle for both induction
and challenge, it is not possible to decide whether the reactions
were a result of the test material or the vehicle. Please refer to "A
Review of the Buehler Guinea Pig Skin Sensitization Test and Its
Use in a Risk Assessment Process for Human Skin Sensitization", Toxicology,
Robinson et al., vol. 61, 1990, p. 91-107.
5. The study contained a vehicle control instead of a naive
control. Vehicle controls are necessary when one is using
a vehicle other than water, saline, carboxymethylcellulose, ethanol
or acetone; however, they may not be used to replace naive controls.
Vehicle controls are recommended when corn oil or glycerol are
used as a vehicle.
6. Induction and challenge performed on the same exposure
site. Skin fatigue may cause irritation in this situation
that would falsely appear to be sensitization.
7. Use of the same concentration (other than 100% for nonirritating
test materials) for both induction and challenge. The
lab must conduct the induction at an at least minimally irritating
concentration. Challenge should be conducted at the highest nonirritating
concentration. The only time the same concentration may be used
for both induction and challenge is if testing a nonirritant
and a 100% concentration is selected for both induction and challenge,
or if the lab is testing a moistened solid.
A preliminary irritation screening study should be conducted to
determine a mildly to moderately irritating concentration for induction
as well the maximum non-irritating concentration to be used at challenge.
Non-irritants should be tested undiluted. Solid non-irritants should
be tested at the most concentrated dilution possible. Although it
is possible to induce sensitization at a non-irritating concentration,
a dermal reaction causing mild to moderate irritation (scores of
1 to 2) in the induction phase is preferred. Moderate or severe irritation
in the induction phase will not cause a study to be rejected. Concentrations
of 100%, 75%, 50% and 25% are acceptable for the irritation screen.
Significant irritation at the lowest concentration would necessitate
a second range-finding study.
The challenge concentration should be lower than the induction concentration,
unless the undiluted test material is non-irritating. Ideally, this
concentration should cause no more than 50% +/- scores (if the Buehler
method is used).
8. The chosen protocol was not followed. The test
deviated from the procedures as recommended. Refer to VII (1) above.
9. Rechallenge was not conducted when appropriate. Rechallenge
is needed when the challenge results are equivocal. For example,
5/10 +/- or .5 scores in the test group without reactions in the
naive control group, or 9/10 +/- or .5 scores and 1/10 grade 1 scores
in the test group with 5/10 +/- or .5 scores in the naive control
group.
10. A different exposure time was used for induction and challenge
and a naive control group was not included. The preferred
exposure time is six hours. Preliminary screening exposure must
also be six hours. Some labs tend to use exposure concentrations
of 24 hours. While this is generally acceptable, it may lead
to problems if there was a 24 hours prescreen with too little
irritation during a six-hour exposure induction.
11. Using different scales to grade different parts of a study. It
is recommended that labs use the Buehler Scale (for the Buehler Method).
It is also acceptable to use the 0, 1, 2, 3 scale from the Magnusson & Kligman
testing method. As there is more than one version scale, reference
to which scale was used should be provided. This is acceptable as
the EPA has agreed to accept OECD protocols. The rating scale should
be described in the report.
12. The solid test material was not moistened or was not properly
moistened. Solids must be slightly moistened (not runny)
to a paste, preferably with water or saline. Other vehicles,
including corn oil, may be used if justified and if they do not
cause irritation. Suspensions and emulsions may be diluted with
water provided that an acceptable irritation dose-response relationship
can be demonstrated and provided that the induction and challenge
concentrations fulfill the appropriate criteria. If not, then
the test material should be dissolved, preferably in acetone
or alcohol, although other non-irritating vehicles may be used
if justified.
13. The volume of test material was too small. The
recommended dosage is 0.4 ml or 0.5 ml for liquids and 0.4 g or 0.5
g for solids. A volume of 0.3 ml may be appropriate if a Hilltop
Chamber is used.
Industry Comment: The references listed above should
be followed. Exact volume may vary, since the references specify
that the patch should be saturated so that there is a maximum concentration
at the interface of the patch and the skin.
14. Lack of a naive control group
- No naive control animals were tested. (This is acceptable if
the test material was non-irritating and the study induced and
challenged at a 100% test material concentration.)
- A second set of naive controls is needed if a rechallenge is
conducted.
15. Deficiencies in the positive control study
- Lack of a positive control study.
- Positive control study was not conducted within six months of
the main dermal sensitization study.
- The positive control study did not prove sensitization.
- Submission of only summary data for the positive control study.
- Use of the same concentration for both induction and challenge.
The positive control study is used to prove a laboratory's ability
to properly conduct a dermal sensitization study. The positive control
study should be conducted in the same manner as the main sensitization
study. The Buehler Method should be followed if that was the method
of the main sensitization study.
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