Guidance for Industry and FDA Reviewers/Staff
Guidance for Over-the-Counter
(OTC) Human Chorionic
Gonadotropin (hCG) 510(k)s
Document issued on:  July 22, 2000
U.S. Department Of Health and Human Services
Food and Drug Administration
Center for Devices and Radiological Health
Clinical Chemistry and Clinical Toxicology Branch
Division of Clinical Laboratory Devices
Office of Device Evaluation Preface
Public Comment
Comments and suggestions may be submitted at any time for Agency consideration to, Veronica
Calvin, Division of Clinical Laboratory Devices, HFZ-440, 9200 Corporate Blvd, Rockville,
MD 20850.  Comments may not be acted upon by the Agency until the document is next
revised or updated.  For questions regarding the use or interpretation of this guidance contact
Veronica Calvin at 301-594-1243, extension 151.
Additional Copies
World Wide Web/CDRH home page:  http://www.fda.gov/cdrh/ode/guidance/1172.pdf  or
CDRH Facts on Demand at 1-800-899-0381 or 301-827-0111, specify number 1172 when
prompted for the document shelf number.
2 1
Guidance  for Over-the-Counter (OTC)
Human Chorionic Gonadotropin (hCG)
510(k)s
This document presents current approaches and concerns regarding over-the-counter hCG devices.  It
is based on: 1) current science; 2) clinical experience; 3) previous submissions by manufacturers to the
Food and Drug Administration (FDA); 4) the FDA Modernization Act of 1997 (FDAMA); and 5)
FDA regulations in the Code of Federal Regulations (CFR).  As advances are made in science and
technology, and as changes in implementation of legislation occur, these Review Criteria will be re-
evaluated and revised as appropriate.  So that the draft may be revised as necessary, please send
comments as instructed in the Preface.
DEFINITION:  This generic type device is intended for home use as an in vitro diagnostic (IVD) test
for the qualitative measurement of hCG.
PRODUCT CODE:  LCX
PANEL:  75 - Clinical Chemistry
REGULATION NUMBER:  21 CFR 862.1155
                        
(a) Identification.  An hCG test system is a device intended for the early detection of pregnancy.
 It is intended to measure hCG, a placental hormone, in serum, plasma or urine.
(b) Classification.  Class II
REVIEW REQUIRED:  510(k)
Refer to 21 CFR 807.87 for information to be provided in a 510(k).
PURPOSE:  The purpose of this document is to provide guidance on information to be submitted in
510(k) submissions.  It is anticipated that this document will assist the Center for Devices and
Radiological Health (CDRH) in rendering consistent decisions based on reliable, reproducible and
standardized commercial tests.
                    
1This document is intended to provide guidance.  It represents the Agencys current thinking on the above.   It does
not create or confer any rights for or on any person and does not operate to bind FDA or the public.  An alternative
approach may be used if such approach satisfies the requirements of the applicable statute, regulations, or both.
3 I. Background
Pregnancy tests are based on the detection of the hormone hCG primarily in urine or serum. 
HCG, which is thought to be produced by trophoblastic tissue, appears around the 8-9th day
after ovulation where fertilization has occurred, or around the 4th day after conception.  In a 28
day cycle with ovulation occurring at day 14, hCG can be detected in urine or serum in minute
quantities around day 23, or 5 days before the expected menstruation.  Its function includes
facilitation of implantation as well as maintenance and development of the corpus luteum.  HCG
levels rise rapidly, doubling approximately every two days, and peak around 100,000-200,000
mIU/mL in the latter part of the first trimester of pregnancy.  In normal subjects, hCG in urine
provides an early indication of pregnancy.  Since elevated hCG levels are also associated with
trophoblastic disease and certain nontrophoblastic neoplasms, the possibility of having these
diseases must be eliminated before a diagnosis of pregnancy can be made.(1)(2)
HCG is a glycoprotein composed of an alpha subunit and a beta subunit.  HCG is closely
related to two other gonadotropins, luteinizing hormone (LH) and follicle stimulating hormone
(FSH), as well as thyroid stimulating hormone (TSH), all three of which are glycoprotein
hormones. The alpha subunits of these various glycoprotein hormones are structurally very
similar, but the beta subunits differ in amino acid sequences.  These differences are responsible
for their biological and immunological specificity.(3)
HCG circulates as the intact molecule in the serum of women who have an uncomplicated
pregnancy.  Subunits are cleaved rapidly and cleared promptly by the kidney.  As a result, the
urine contains intact hCG, and alpha and beta subunits; however, only intact hCG and beta
subunits retain immunologic specificity in urine.  The relative concentrations of intact and subunit
hCG in serum and urine vary a great deal in patients with trophoblastic disease because of
unbalanced synthesis of subunits.(4)
The International Reference Preparation for Chorionic Gonadotropin (1st IRP for CG), officially
designated the 3rd International Standard (3rd IS), contains only pure intact hCG.  The 2nd IS
contains a mixture of intact hCG and its beta subunit. The 1st IRP is also available as pure beta
subunit and pure alpha subunit.  It is now possible for manufacturers to characterize and define
the specificity of their assays for intact hCG and/or its subunits.  It is not possible to obtain
uniformity in the reporting of results solely through the use of a common standard, since hCG is
a complex molecule.  Different immunoassays may use different antibodies that recognize
different components (epitopes) of the molecule.(5)
4 II. Historical Background and Description
Clinically useful bioassays were introduced by Ascheim (1927), Zondek (1931), the Friedman
test (1931), and the Xenopus laevis test (Shapiro, 1934).  The first tests could confirm
pregnancy approximately two months after a missed period.(6)  In addition to the fact that they
were not very sensitive, bioassays had other problems limiting their usefulness as routine
pregnancy tests.  Because of this, they were replaced by immunoassays with the first being
reported in 1960.  Sensitive radioimmunoassay (RIA), immunoradiometric (IRMA), and
enzyme immunoassay (EIA) techniques now available allow an accurate and precise quantitation
of hCG through the use of highly specific antibodies. 
Presently, all of the home pregnancy tests available use monoclonal or polyclonal antibodies in
an enzyme-linked immunoassay format.  Monoclonal antibodies are highly sensitive to one
specific site along the hCG molecule.  If hCG is present in urine, it will be trapped by the anti-
hCG antibody that is bound to a solid surface.  A second antibody is utilized in the device; it is
linked to an enzyme that reacts with the anti-hCG complex to cause a color change, producing a
positive result.  These tests can detect pregnancy by the first day of the missed menstrual
period.  The development of monoclonal antibodies to hCG provides a supply of homogeneous
antibodies and improves the specificity of the assay. (7)(8)  Consequently, commercially
available pregnancy tests are much less susceptible to interference from other substances than
were the earlier immunoassays.
III.  Performance Characteristics/Laboratory Evaluation
A. Comparison Study/OTC Study
1.  Study Design
FDA recommends that at least 100 fresh, human urine specimens be used. Each
specimen should be split between the subject and the professional, who should
perform testing on the predicate device (for professional use or OTC use) and
the new device.  In addition to comparing the new device to a predicate device,
the new device can also be used to compare the subjects results to the
professional users results.
The portion of the comparison study performed by the subject should mimic
actual use.  FDA recommends that the subject collect the sample and perform
the test without assistance.  We recommend using freshly voided first-morning
urine specimens because the hCG concentration is highest at this time.  If
specimens cannot be tested immediately, they should be stored at 2-8C for up
to 48 hours.(9)  If claims are being made to "use any time of day", then samples
collected any time of day should be used in the study.
5 Alternatively, the home users may be provided with coded positive and negative
specimens.  For spiked (urines with hCG added) specimens, FDA recommends
including specimens around (e.g., 10-20% above and below) the sensitivity
level. Also, the FDA requests that the concentrations of spiked specimens be
provided in the 510(k) submission.
For devices utilizing more than one testing procedure, e.g., urine stream and dip
procedure, we strongly recommend that data be provided to validate the
equivalency of both procedures.  Fifty or  more OTC users (subjects) should
perform the urine stream method.
OTC users (subjects) can be selected on a random basis as they present
themselves at clinics and/or physicians offices or via advertisements.  They
should represent diversity of age, background, and education.  When the testing
is complete, users should complete a questionnaire.  The questionnaire is to
generate information such as, feedback on the test and ease of use, and clarity
and readability of the package insert. FDA requests that a summary of the
responses to the questions be provided in the 510(k) submission. 
Include a summary of the OTC study protocol in the 510(k) submission. 
2.  Data Analysis
FDA recommends expressing the data in terms of percent accuracy, which
should never exceed >99%.  Accuracy, per the NCCLS labeling guideline, is
based on test efficiency, i.e., true positive plus true negative results divided by
the total number of samples tested.
This performance can be described in text or tabular form.  In addition,
misleading statements such as, virtually 100% accurate, nearly 100% accurate,
100% accurate, etc., should be avoided.
B. Specificity
Specificity studies should be performed on specimens with high physiological
concentrations of luteinizing hormone (LH), follicle stimulating hormone (FSH), and
thyroid stimulating hormone (TSH).  Ideally, high levels of LH will not significantly
cross-react with the hCG antibody used.  Spiking samples with LH, FSH or TSH is
useful in demonstrating the absence of cross-reactivity at high LH, FSH or TSH levels.
C. Interfering Substances
Interference studies should be performed on urine containing prescription/OTC drugs,
elevated levels of chemical analytes (e.g., caffeine, ascorbic acid), and elevated levels of
6 biological analytes (e.g., glucose, protein, albumin, bilirubin, hemoglobin).  It is also
useful to evaluate the effects pH may have on the results.
D. Sensitivity/Detection Limit
For qualitative assays, the sensitivity/detection limit is the analyte concentration at which
95% of the test results are positive.  Assay sensitivity should be such that small
quantities of hCG will be detected while false-positive results due to the presence of LH
will be minimized.(2)  Additionally, it is expected that all positive results will have
reacted within the specified time frame.
Sensitivity can be evaluated by spiking at least 30 clinical samples from normal,
nonpregnant females or males with six different concentrations of hCG below (including
one at 50% of the sensitivity and one at 25% of the sensitivity), at and above the
stated sensitivity.  For example, for a test with a detection limit of 25 mIU/mL,
concentrations of approximately 0, 12.5, 18.75, 25, 50, and 100 mIU/mL may be
used.  FDA requests that the concentrations tested be provided in the 510(k)
submission.
   E. Imprecision or Reproducibility Studies
These studies may be performed if desired.  FDA recommends following a scientifically
valid protocol when evaluating imprecision.
F. Expected Values
A section entitled as such need not appear in an OTC insert.  However, within the text
the manufacturer may state that the test is capable of detecting pregnancy by the first
day of the missed period and no sooner, unless validated by clinical data. (Note:  We
recommend discussing clinical study proposals with Division representatives before
performing studies.)
G. Calibration
The source of reference material that the standards or test are calibrated against (1st
IRP, 2nd IS, or 3rd IS) for hCG should be stated in the submission only.                      
                               
H. Quality Control
If a device contains a procedural or design control, the function of the control should be
described in detail.  Examples of descriptions are listed below. 
  The control determines if chemicals are working properly.
  The control determines if an adequate amount of sample was added.
  The control determines if the proper procedure was followed.
7 FDA also requests that you identify the components of the internal control and state
where applicable, the sensitivity (detection limit) of the reference line.
I. Stability Data
The data showing stability of the reagents need not be submitted to the FDA, but may
be kept on file by the manufacturer in accordance with Good Manufacturing Practices
(GMP).
A summary of data to support the stability of the results (i.e., maximum time for
interpreting result after it first appears) should be provided in the 510(k) submission.
J. Antibody Information
Information on characterization and purification of the monoclonal antibody(ies) in the
device is usually provided in the 510(k) submission.  If these procedures are performed
by another manufacturer, a copy of the Certificate of Analysis from that manufacturer
may be included in the 510(k) file.
K. Limitations
The list presented here is not all-inclusive.  Include limitations in the labeling (i.e.,
package insert) as needed.
1. The test cannot be reused.
2. Do not use this test past the expiration date.
3. Pain relievers, oral contraceptives, antibiotics, and other commonly used
medications (for example) should not interfere with the test.  (Studies should be
performed to validate this claim.)
4. Certain health conditions, such as an ovarian cyst or ectopic pregnancy
(pregnancy outside the uterus), can cause a false or irregular result.
5. The procedures should be followed precisely for accurate results.
6.  A false negative result (negative when pregnancy exists) may occur if the urine is
too dilute or with a very early stage pregnancy.  If pregnancy is still suspected,
retest using a first-morning urine.
7. For in vitro diagnostic use (not for internal use).
IV. Labeling
The package insert should be concise, easy to understand, and contain clear illustrations and
drawings.  The labeling format should conform to the 21 CFR 809.10 labeling regulations. 
However, for OTC products, sections such as,
  Intended Use
  Summary and Explanation of the test
8   Principle of the Procedure
  Reagents
  Quality Control
  Expected Values
  Performance Characteristics
need not be presented with the formality of professional use labeling.  Information relative to
these sections can be simplified and contained within the text without such subheadings.  This is
to eliminate "technical" or incomprehensible language. 
For example, include in the beginning of the package insert:
  the analyte being measured
  type of specimen used
  a description of how the test works
When listing materials provided, the test device could be described as a test strip containing
chemicals, a test strip containing antibodies, etc, and not identify the specific biological and
chemical components. 
As a part of quality control, describe in detail the function of the internal control.  This is often
included under Interpretation of the Results or in the Question and Answer section. 
We also recommend that a maximum time for interpreting results or how long the results are
stable be included in the insert, particularly for negative results, which may become positive over
time.  A sentence relative to the sensitivity of the test, i.e., how early pregnancy can be detected,
should be included as well as the accuracy of the device determined by laboratory studies and in
the hands of OTC users.  For additional guidance, refer to the NCCLS labeling guideline
GP14-P (10), DCLD's Points to Consider for Home Use IVDs (11), and CDRH's Write It
Right guidance (12).
9 V. Bibliography
1.  Braunstein GD, Grodin JM, Vaitukaitis J and Ross GT.  Secretory rates of human chorionic
gonadotropin by normal trophoblast.  American Journal of Obstetrics and Gynecology,
115:447-50, 1973.
2.  Brody S and Carlstrom G.  Immunoassay of human chorionic gonadotropin in normal and
pathologic pregnancy.  Journal of Clinical Endocrinology and Metabolism, 22:564, 1962.
3.  Ross GT. Clinical relevance of research on the structure of human chorionic gonadotropin. 
American Journal of Obstetrics and Gynecology 1977; 129:795.
4.  Lab Report for Physicians.  Standardization of human chorionic gonadotropin.  December
1985; 7:12.
5.  Jeffcoat, SL.  Standardisation of hCG immunoassays and pregnancy kits.  Lancet 1982;
1:803.
6.  Chayen J, Daly JR, Loveridge N.  The cytochemical bioassay of hormones.  Recent Prog.
Horm. Res. 1976; 32:33-72.
7.  Henry JB.  Clinical Diagnosis and Management by Laboratory Methods, 18th edition, 1991,
WB Saunders and Co.
8.  Wide L, Gemzell CA.  An immunological pregnancy test. Acta Endocrinology 1960;
35:261.
9.  Krieg AF.  Pregnancy tests and evaluation of placental function.  Clinical Diagnosis and
Management by Laboratory Methods, 18th edition, Henry JB, editor, 1979; 680.
10.  National Committee for Clinical Laboratory Standards.  Labeling of Home-Use In Vitro
Testing Products.  NCCLS Document GP14-P, Vol. 9, No. 8, 1989.
11.  Report of the CDRH Home-Use IVD Working Group Assessing the Safety and
Effectiveness of Home-Use IVDs:  Guidance Regarding Premarket Submissions.
12.  Write It Right.  Recommendations for Developing User Instruction Manuals for Medical
Devices Used in Home Health Care.
10 ABBREVIATIONS
CDRH Center for Devices and Radiological Health
DCLD Division of Clinical Laboratory Devices
FDA Food and Drug Administration
FSH Follicle Stimulating Hormone
HCG Human Chorionic Gonadotropin
IRP International Reference Preparation
IS International Standard
IVD In Vitro Diagnostic
LH Luteinizing Hormone
NCCLS National Committee for Clinical Laboratory Standards
OTC Over-the-Counter
QC Quality Control
TSH Thyroid Stimulating Hormone
11 CHECKLIST
Instructions:  Use this checklist for premarket submissions for human chorionic gonadotropin
(hCG)/pregnancy tests intended for over-the-counter use.  Please check the box next to the items
below that you have included in the premarket notification.

CDRH Premarket Submission Cover Sheet

Truthful and Accurate Statement verbatim as required by 21 CFR 807.87(j).  Additions and
deletions are not permitted.

510(k) Summary or Statement as required by 21 CFR 807.92 or 21 CFR 807.93 respectively.

Indications for Use Form

Sensitivity/Detection Limit Data

Specificity Data including LH, FSH, and TSH

Interfering Substances Data

Comparative Study Data including the following:  summary of study protocol, concentrations of
spiked specimens used, data to validate all testing procedures (e.g., urine stream and cup
method), summary of responses to OTC  study questionnaire.

Antibody Characterization/Purification Information

Components and Function of Internal Control

Predicate Device Labeling
12