Guidance for Industry

Supplemental Guidance on Testing for 

Replication Competent Retrovirus in 

Retroviral Vector Based Gene 

Therapy Products and During 

Follow-up of Patients in Clinical 

Trials Using Retroviral Vectors 

This guidance is for immediate implementation. 

FDA is issuing this guidance for immediate implementation in accordance with 21 CFR 

10.115(g)(4)(i). Submit written comments on this guidance at anytime to the Division of 

Dockets Management (HFA-305), Food and Drug Administration, 5630 Fishers Lane, Rm. 1061, 

Rockville, MD 20852. Submit electronic comments to http://www.fda.gov/dockets/ecomments. 

You should identify all comments with the title of this guidance. 

Additional copies of this guidance are available from the Office of Communication, Training and 

Manufacturers Assistance (HFM-40), 1401 Rockville Pike, Suite 200N, Rockville, MD 20852-

1448, or by calling 1-800-835-4709 or 301-827-1800, or from the Internet at 

http://www.fda.gov/cber/guidelines.htm. 

For questions on the content of this guidance, contact the Office of Cellular, Tissues, and Gene 

Therapies at 301-827-5102. 

U.S. Department of Health and Human Services 

Food and Drug Administration 

Center for Biologics Evaluation and Research 

November 2006 
Contains Nonbinding Recommendations 

Table of Contents 

I. INTRODUCTION............................................................................................................. 1

II. BACKGROUND ............................................................................................................... 2

III. RECOMMENDATIONS FOR PRODUCT TESTING................................................. 2

A. When to Test.......................................................................................................... 2

B. Amounts for Testing ............................................................................................. 5

IV. RECOMMENDATIONS FOR PATIENT MONITORING......................................... 7

A. Testing Schedule ................................................................................................... 7

B. Recommended Assays........................................................................................... 8

V. DOCUMENTATION OF RCR TESTING RESULTS ................................................. 8

VI. CONCLUSION ................................................................................................................. 8

VI. REFERENCES................................................................................................................ 10

APPENDIX.................................................................................................................................. 12

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Contains Nonbinding Recommendations 

Guidance for Industry 

Supplemental Guidance on Testing for Replication Competent 

Retrovirus in Retroviral Vector Based Gene Therapy Products and 

During Follow-up of Patients in Clinical Trials Using Retroviral 

Vectors 

This guidance represents the Food and Drug Administration’s (FDA’s) current thinking on this 

topic. It does not create or confer any rights for or on any person and does not operate to bind 

FDA or the public. You can use an alternative approach if the approach satisfies the 

requirements of the applicable statutes and regulations. If you want to discuss an alternative 

approach, contact the appropriate FDA staff. If you cannot identify the appropriate FDA staff, 

call the appropriate number listed on the title page of this guidance. 

I. INTRODUCTION 

This guidance document applies to the manufacture of gene therapy retroviral vector products 

intended for in vivo or ex vivo use and to follow-up monitoring of patients who have received 

retroviral vector products.1 Guidance is provided for replication competent retrovirus (RCR) 

testing during manufacture, including timing, amount of material to be tested, and general testing 

methods. In addition, guidance is provided on monitoring patients for evidence of retroviral 

infection. This guidance document finalizes the draft guidance document “Supplemental 

Guidance on Testing for Replication Competent Retrovirus in Retroviral Vector Based Gene 

Therapy Products and During Follow-up of Patients in Clinical Trials Using Retroviral Vectors” 

announced in the Federal Register of November 3, 1999 (64 FR 59783). The guidance 

document also supplements the guidance and recommendations pertaining to RCR testing given 

in the following documents: 1) “Guidance for Industry: Guidance for Human Somatic Cell 

Therapy and Gene Therapy,” March 1998; and 2) a letter to Sponsors of INDs Using Retroviral 

Vectors, dated September 20, 1993. For general guidance on gene therapy refer to “Guidance for 

Industry: Guidance for Human Somatic Cell Therapy and Gene Therapy,” March 1998. 

FDA’s guidance documents, including this guidance, do not establish legally enforceable 

responsibilities. Instead, guidances describe the FDA’s current thinking on a topic and should be 

viewed only as recommendations, unless specific regulatory or statutory requirements are cited. 

The use of the word should in FDA’s guidances means that something is suggested or 

recommended, but not required. 

 

1

 This guidance issued in October 2000, and was updated in November 2006 to cross-reference the 

recommendations contained in the “Guidance for Industry: Gene Therapy Clinical Trials – Observing Subjects for 

Delayed Adverse Events,” which supplements the recommendations in Section IV.A. of this document. 

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II. BACKGROUND 

CBER’s current recommendations for RCR testing during retroviral vector production and 

patient monitoring were developed in 1993, at a time when clinical experience with retroviral 

vectors was limited (Ref. 4). The overriding safety issues associated with the use of retroviral 

vectors are exemplified by the findings of an experiment involving administration of ex vivo 

transduced bone marrow progenitor cells that had been inadvertently exposed to high titer RCR 

contained in the retroviral vector material to severely immunosuppressed Rhesus monkeys. In 

this setting, 3/10 animals developed lymphomas and died within 200 days (Ref. 3). The RCR 

was presumed to be etiologically associated with the disease by virtue of the presence of multiple 

murine RCR sequences in the monkey lymphomas and the observed correlation between the lack 

of antiretroviral antibody response and the development of prolonged retroviremia and disease 

(Refs. 9, 11). Since 1993, accumulating experience with different vector producing cells, RCR 

detection assays and results from patient monitoring have allowed the generation of a small data 

base of information on the safety of the use of retroviral vectors in clinical applications of gene 

therapy. This information base has provided a framework for discussion of the RCR 

recommendations by Center for Biologics Evaluation and Research and the gene therapy 

community. Public discussion and development of these supplemental recommendations have 

taken place during the Retroviral Breakout Sessions at the 1996 and 1997 FDA/NIH Gene 

Therapy Conferences, with representatives of the gene therapy community, and through the 

publication of the FDA considerations on these issues (Ref. 12). 

III. RECOMMENDATIONS FOR PRODUCT TESTING 

A. When to Test 

RCR may develop at any step during manufacturing from development of the initial 

master cell bank through production of the retroviral vector supernatant. In addition, the 

growth of ex vivo transduced cells provides the potential for amplification of any RCR 

contaminant which may be below the level of detection in the retroviral vector 

supernatant. Therefore, current testing recommendations include testing of material from 

multiple stages of product manufacture (see Table 1). Use of a cell bank system is 

recommended in order to ensure an adequate and consistent supply of vector producer 

cells (VPC). The Master Cell Bank (MCB) is a collection of cells of uniform 

composition derived from a single tissue or cell. The Working Cell Bank (WCB) is 

derived from one or more ampules of the MCB, expanded by serial subculture to a 

specified passage number (refer to Points to Consider in the Characterization of Cell 

Lines Used to Produce Biologicals, 1993). 

1. Testing of Vector Producer Cell Master Cell Bank (one time testing) 

Both VPC and supernatant from production of a MCB should be tested for RCR 

using a cell line permissive for the RCR most likely to be generated in a given 

producer cell line. For example, VPC containing amphotropic Murine Leukemia 

Virus (MLV) envelope should be tested for RCR on a cell line such as Mus dunni

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that is permissive to infection by amphotropic MLV-like RCR, while VPC 

containing the gibbon ape leukemia virus envelope should be tested on a human 

cell line. Other retroviral envelopes should be tested on a cell line permissive for 

infection by the relevant RCR. 

If derivation of VPC includes use of a retroviral vector containing an envelope 

distinct from the packaging vector, for example, an ecotropic MLV, the potential 

exists for introduction of an RCR with that envelope. Even though an ecotropic 

MLV RCR may present a minimal direct safety risk to humans, the presence of 

any replication-competent genome in the VPC is problematic because of the 

increased probability of generating an RCR with a human host range by 

recombination with elements within the VPC. 

In those cases where VPC are derived at any step by infection with an ecotropic 

retroviral vector, testing of the MCB for the presence of ecotropic RCR is 

recommended. Both cells and supernatants should be tested using a method 

validated to detect the appropriate positive control (for example: D56 (Ref. 2) or 

XC (Ref. 10)). Refer to the guidance provided in section III.B. to determine the 

amount of material for testing. 

2. Working Cell Bank Testing (one time testing) 

Either supernatant testing or cocultivation of cells for RCR is recommended using 

conditions described for master cell bank testing. 

3. Testing of Retroviral Vector Supernatant Product and End of Production 

Cells 

Both retroviral vector supernatant lots and end of production cells should be 

tested for RCR as specified in section III.B. This recommendation is based 

on data and experience reported at the 1997 FDA/National Institutes of 

Health (NIH) Gene Therapy Conference in which RCR in vector production 

lots was not consistently detected by both assays, or one assay to the 

exclusion of the other. These data support the position that dual testing 

provides a complementary approach to assuring RCR free retroviral 

supernatant. 

4. Testing of Ex Vivo Transduced Cells 

a. Cultured < 4 days after transduction 

Data presented at the 1997 FDA/NIH Gene Therapy Conference 

indicated that for ex vivo transduced cells, a minimum culture 

period of 4 days from the start of transduction is necessary for 

amplification and detection of an RCR. As a result, for ex vivo 

transduced cells cultured for a period less than four days, archiving 

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of the quantity of product needed to perform RCR testing is 

recommended in place of active RCR testing. Refer to the 

guidance in section III.B. to determine the amount of material to be 

archived. Samples should be archived with appropriate safeguards 

to ensure long-term storage (e.g., a monitored freezer alarm storage 

system) and an efficient system for the prompt linkage and 

retrieval of the stored samples with the medical records of the 

patient and the production lot records. 

b. Cultured > 4 days 

When ex vivo transduced cells are in culture for a period of time greater 

than or equal to 4 days from the start of transduction, cells and the 

appropriate volume of culture supernatant should be tested for RCR. 

Refer to guidance in section III.B. of this document for determining the 

amount of material for testing. In situations where ex vivo transduced 

cells cannot be cryopreserved during testing, and must be administered to 

patients prior to the availability of testing results, culture assays should be 

initiated at the time of patient administration. In these situations, 

alternative methods such as PCR may be appropriate to provide an initial 

analysis. Any alternative methods should be developed in consultation 

with CBER. Data on sensitivity, specificity and reproducibility should be 

provided to support the use of alternative methods. 

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Table 1. Recommendations for Product Testing 

RCR Testing for 

Expected RCR1

RCR Testing 

for Ecotropic 

MLV 

Manufacturing Step Cells Supernatant Cells Supernatant

MCB 

 -Derived by infection with 

 Ecotropic MLV vector 

 -Derived by transfection 

 of retroviral vector plasmid 

Yes 

Yes 

Yes 

Yes 

Yes 

No 

Yes 

No 

WCB Yes OR Yes No No 

End of Production Cells Yes NA2 No No 

Vector-Containing Supernatant NA Yes No No 

Ex vivo Transduced Cells 

 -Cultured <4 days after 

 Transduction 

--Cultured >4 days after 

 transduction 

No – 

archive 

Yes 

No – 

archive 

Yes 

No 

No 

No 

No 

1 RCR testing should be based on the type of packaging cell line used to derive the VPC. 

Consult text in section III.A.1. of this document for details. 

2 NA, not applicable. 

B. Amounts for Testing 

1. Supernatant Testing 

In all cases, it would be appropriate to test at least 5% of the total supernatant by 

amplification on a permissive cell line. However, for instances where supernatant 

production volumes are greater than 6 liters, and therefore, testing of 5% may not 

always be practical, an alternative approach is described below. In order to utilize 

the alternative approach, the largest volume where a single infectious RCR can be 

detected should first be determined. When high titer retroviral vector preparations 

are used, interference in RCR detection may occur. In such cases, detection of a 

single RCR may require use of such small volumes in each test that the 

application of this alternative approach may not be practical. Sponsors are 

encouraged to develop more sensitive detection methods that overcome the 

interference effect of high titer retroviral vector preparations in order to use the 

alternative approach. 

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a. Alternative approach for determining total volume of retroviral 

vector supernatant to be tested 

A statistical approach has been applied to the determination of the 

total volume of retroviral supernatant to be tested for RCR. This 

calculation is independent of production lot size and is based on 

the application of the Poisson distribution. It is recommended that 

sufficient supernatant be tested to ensure a 95% probability of 

detection of RCR if present at a concentration of 1 RCR/100 ml. 

At this concentration, a volume of about 300 ml will have a 95% 

probability of containing an RCR. Therefore, assuming the assay 

is sensitive enough to detect a single RCR, a test volume of 300 ml 

will provide 95% probability of detecting RCR. A more detailed 

explanation of the rationale and the mathematical formulas applied 

is found in Appendix 1-1. 

To support the underlying assumption that a single retrovirus will 

be detected, one must determine a volume in which a single RCR 

can be detected by an individual RCR assay. Based on the 

determination of this volume, the total test volume should then be 

divided into replicate samples, each containing the volume 

demonstrated to detect a single RCR. An RCR standard has been 

developed, its infectious titer has been determined, and it is 

available through the American Type Culture Collection (ATCC). 

The standard can be used as a reference for determination of the 

volume in which a single RCR can be determined. Refer to 

Appendix sections 1-2 and 1-3 for detailed information about the 

RCR standard and how it can be used to determine the replicate 

size and number for RCR detection. 

b. Assays for supernatant testing 

Supernatant assays should include culture of supernatant on a 

permissive cell line (ex. Mus dunni for amphotropic MLV (Ref. 5)) 

for a minimum of 5 passages in order to amplify any potential 

RCR present. The amplified material may then be detected in an 

appropriate indicator cell assay (e.g., PG-4 S+

L- (1)). All assays 

should include relevant positive and negative controls to assess 

specificity, sensitivity and reproducibility of the detection method 

employed. Each lot of retroviral vector supernatant should be 

tested for inhibitory effects on detection of RCR by using positive 

control samples that are diluted in vector supernatant. 

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2. Cell Testing 

The current recommendation to test 1% of the total cells or 108 (whichever is less) 

pooled vector-producing cells or ex vivo transduced cells by co-culture with a 

permissive cell line will remain in place. Public consensus expressed at the 1996 

and 1997 FDA/NIH Gene Therapy Conferences was in support of the current 

recommendations for cell testing, in light of the variety of vector producing cells 

and vector backbones used, and the difficulty that is presented in development of 

a standard RCR producing positive cell stock. 

Co-culture assays should include culture with a permissive cell line (ex. Mus 

dunni for amphotropic MLV (Ref. 5)) for a minimum of five passages in order to 

amplify any potential RCR present. The amplified material may then be detected 

in an appropriate indicator cell assay (e.g., PG-4 S+L- (1)). All assays should 

include relevant positive and negative controls to assess specificity, sensitivity 

and reproducibility of the detection method employed. 

IV. RECOMMENDATIONS FOR PATIENT MONITORING 

Active monitoring for evidence of RCR infection in patients enrolled in gene therapy clinical 

trials using retroviral vectors is currently recommended in a letter to Sponsors of INDs Using 

Retroviral Vectors, dated September 20, 1993. Based on input from the gene therapy 

community, problematic aspects of the current recommendations were defined as the number 

of time points for testing, the recommendations for life-long annual testing, and the types of 

assays recommended. 

A. Testing Schedule 

Note: This section IV.A. immediately below has been supplemented by the 

“Guidance for Industry: Gene Therapy Clinical Trials – Observing Subjects for 

Delayed Adverse Events.” 2

 

This guidance presents alternatives to the time points for monitoring originally 

described in a letter to Sponsors of INDs Using Retroviral Vectors, dated 

September 20, 1993. These recommendations are based on data accumulated in 

on-going gene therapy clinical trials using retroviral vectors (Refs. 6, 7). The 

monitoring schedule recommended here should include analysis of patient 

samples at the following time points: pre-treatment, 3 months, 6 months, 1 year 

after treatment, and yearly thereafter. If all post-treatment assays are negative 

during the first year, the yearly samples should be archived. Samples should be 

archived with appropriate safeguards to ensure long-term storage (e.g., a 

monitored freezer alarm storage system) and an efficient system for the prompt 

linkage and retrieval of the stored samples with the medical records of the patient 

and the production lot records. 

 

2

 See http://www.fda.gov/cber/genetherapy/gtpubs.htm. 

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Contains Nonbinding Recommendations 

If any post-treatment samples are positive, further analysis of the RCR and more 

extensive patient follow-up should be undertaken, in consultation with CBER. It is 

further recommended at the time of collection of the yearly patient specimen, that a brief 

clinical history should be obtained. This history should be targeted towards 

determination of clinical outcomes suggestive of retroviral disease, such as cancer, 

neurologic disorders, or other hematologic disorders. Suspect clinical outcomes may 

trigger additional analysis of archived samples, in consultation with CBER. If patients 

die or develop neoplasms during a gene therapy trial, every effort should be made to 

assay for RCR in a biopsy sample of the neoplastic tissue or the pertinent autopsy tissue. 

B. Recommended Assays 

Two methods are currently in use and recommended for detecting evidence of RCR 

infection in patients: 1) detection of RCR-specific antibodies; and 2) analysis of patient 

peripheral blood mononuclear cells by polymerase chain reaction (PCR) for RCRspecific DNA sequences. The choice of assay may depend on the mode of vector 

administration and the clinical indication. For example, it has been shown that direct 

administration of VPC or repeat direct injection of a vector can result in vector-specific 

antibodies which do not correlate with the presence of RCR (Refs. 6, 7). Therefore, in 

cases where vector or vector-producing cells are directly administered, a PCR assay may 

be preferable over serologic monitoring. Additional instances where monitoring of 

patients by PCR may be preferred over serologic monitoring, are those cases where the 

patients are immunocompromised to an extent that antibody production may be minimal 

or not at all. In either situation, all positive results should be pursued by direct culture 

assay to obtain and characterize the infectious viral isolate. 

V. DOCUMENTATION OF RCR TESTING RESULTS 

RCR testing results from production lots and patient monitoring should be documented in an 

amendment to the investigational new drug (IND). Positive results from patient monitoring 

should be reported immediately as an adverse experience in the form of an IND safety report 

(21 CFR 312.32). Negative results should be reported by way of the IND annual report (21 

CFR 312.33). In addition, CBER encourages members of the gene therapy community to 

publish data and/or provide permission in the IND for FDA to discuss data publicly in order 

to enhance the cumulative data base on RCR testing assays, experience with different vector 

producer cell lines, patient monitoring and safety. 

VI. CONCLUSION 

This guidance provides additional guidance for testing for RCR associated with the use of 

gene therapy retroviral vectors. These supplemental recommendations are based on data and 

analyses generated by CBER and by members of the gene therapy community. For safety 

testing of retroviral vectors or vector-transduced cells, IND sponsors may either follow the 

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recommendations previously provided in the “Guidance for Industry: Guidance for Human 

Somatic Cell Therapy and Gene Therapy,” or follow the recommendations outlined here. 

Application of this supplemental guidance: 1) effectively reduces the volume of supernatant 

required for testing, especially in the case of large volume retroviral supernatant production 

lots; 2) revises the time points tested and types of assays which should be used to monitor 

patients who are treated in gene therapy clinical trials which involve the use of retroviral 

vectors; and 3) changes the recommendation for life-long monitoring from active monitoring 

on an annual basis to collection and archiving of patient samples and tracking of relevant 

clinical history on an annual basis. 

A retroviral vector supernatant standard has been developed to aid in measurement of assay 

sensitivity. Availability of this standard supports the use of a statistical approach for 

determination of volume of retroviral supernatant to be tested. In addition, the retroviral 

supernatant standard will provide a tool for comparing the sensitivity of RCR detection by 

different labs and/or testing methods and may lead to improvements in assay sensitivity. 

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VI. REFERENCES 

1. Bassin, R. H., S. Ruscetti, I. Ali, D. K. Haapala, and A. Rein. 1982. Normal DBA/2 

mouse cells synthesize a glycoprotein which interferes with MCF virus infection. Virology. 

123:139-151. 

2. Bassin, R. H., N. Tuttle, and P. J. Fischinger. 1971. Rapid cell culture assay technique for 

murine leukemia viruses. Nature. 229:564-566. 

3. Donahue, R. E., S. W. Kessler, D. Bodine, K. McDonagh, C. Dunbar, S. Goodman, B. 

Agricola, E. Byrne, M. Raffeld, R. Moen, J. Bacher, K. M. Zsebo, and A. W. Nienhuis.

1992. Helper virus induced T cell lymphoma in nonhuman primates after retroviral mediated 

gene transfer. J. Exp. Med. 176:1125-1135. 

4. Gunter, K. C., A. S. Khan, and P. D. Noguchi. 1993. The safety of retroviral vectors. 

Human Gene Therapy. 4(5):643-645. 

5. Lander, M. R., and S. K. Chattopadhyay. 1984. A Mus dunni cell line that lacks sequences 

closely related to endogenous murine leukemia viruses and can be infected by ecotropic, 

amphotropic, xenotropic, and mink cell focus-forming viruses. J. Virol. 52(2):695-698. 

6. Long, Z., L.-P. Li, T. Grooms, C. Lockey, K. Nader, I. Mychkovsky, S. Mueller, I. 

Burimski, P. Ryan, G. Kikuchi, D. Ennist, S. Marcus, E. Otto, and G. McGarrity. 1998. 

Biosafety monitoring of patients receiving intracerebral injections of murine retroviral vector 

producer cells. Human Gene Therapy. 9:1165-1172. 

7. Martineau, D., W. M. Klump, J. E. McCormack, N. J. DePolo, E. Kamantigue, M. 

Petrowski, J. Hanlon, D. J. Jolly, S. J. Mento, and N. Sajjadi. 1997. Evaluation of PCR 

and ELISA Assays for screening clinical trial subjects for replication-competent retrovirus. 

Human Gene Therapy. 8:1231-1241. 

8. Miller, A. D., M.-F. Law, and I. M. Verma. 1985. Generation of helper-free amphotropic 

retroviruses that transduce a dominant-acting, methotrexate-resistant dihydrofolate reductase 

gene. Molecular and Cellular Biology. 5(3):431-437. 

9. Purcell, D. F. J., C. M. Broscius, E. F. Vanin, C. E. Buckler, A. W. Nienhuis, and M. A. 

Martin. 1996. An array of murine leukemia virus-related elements is transmitted and 

expressed in a primate recipient of retroviral gene transfer. Journal of Virology. 70(2):887-

897. 

10. Rowe, W. P., W. E. Pugh, and J. W. Hartley. 1970. Plaque assay techniques for murine 

leukemia viruses. Virology. 42:1136-1139. 

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11. Vanin, E. F., M. Kaloss, C. Broscius, and A. W. Nienhuis. 1994. Characterization of 

replication-competent retroviruses from nonhuman primates with virus-induced lymphomas 

and observations regarding the mechanism of oncogenesis. Journal of Virology. 68(7):4241-

4250. 

12. Wilson, C. A., T.-H. Ng, and A. E. Miller. 1997. Evaluation of recommendations for 

replication competent retrovirus testing associated with use of retroviral vectors. Human 

Gene Therapy. 8(7). 

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APPENDIX 

1-1. Derivation of Recommendation for Test Volume for RCR Detection 

Assuming the RCR are present in the production lot at a concentration (c) and 

that an assay will detect a single retrovirus in the sample, the probability (p) of 

detecting retrovirus in a volume (Vt) is given by the formula: p= 1-exp(-cVt), 

because the number of RCR in Vt follows a Poisson distribution with a parameter 

cVt. Solving for Vt, one gets the following equation: 

Vt= - (1/c) ln (1-p), 

where ln denotes the natural logarithm. 

Value for p 

For the use of this formula, it is recommended that the value for p be set 

at 0.95. With the recommended replicate size and number defined in 

Appendix 1-3, p becomes the probability of detecting an RCR in the 

production lot. 

Value for c 

It is recommended that the value for c be set no higher than 0.01 RCR/ml 

or 1 RCR/100 ml. If the concentration of RCR in the production lot is 

0.01 RCR/ml or greater, then the probability of detection is at least 0.95. 

If the production lot contains RCR at a concentration of <0.01 RCR/ml, 

the RCR may not be detected and would be administered to the patient. 

Value for Vt 

With the recommended value for p and c, the total volume of retroviral 

supernatant to be tested, independent of lot size, is calculated as follows: 

Vt = - (1 / 0.01 RCR/ml) ln (1 -0.95) ≅ 300 ml 

Proposals to use smaller volumes should be developed and reviewed in 

consultation with CBER. 

1-2. Empirical Determination of Assay Sensitivity 

In collaboration with the ATCC, a standard retroviral stock (ATCC # VR1450) has been established for use in determination of sensitivity and 

validation of assays used to detect the presence of replication competent 

retrovirus which would be produced from VPC containing amphotropic 

envelope. This stock can be used to determine the relative assay sensitivity 

for detecting RCR. This information can subsequently be used to determine 

the size of replicates of retroviral supernatant to be tested that will ensure 

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detection of a single retrovirus and thus, the number of replicates to ensure an 

adequate total volume, Vt, as specified in this guidance (see Appendix 1-3). 

The virus stock is derived from a cell line which has been transfected with a 

molecular clone encoding Moloney murine leukemia virus (MoMLV) with a 

substitution of the envelope coding region from the 4070A strain of 

amphotropic murine leukemia virus (A-MLV) (Ref. 7). Therefore, this virus 

stock represents a typical recombinant virus that could be generated in a 

retroviral packaging cell line containing coding sequences for a MLV 

envelope. The infectious titer of the viral stock prepared by ATCC was 

determined using the direct S+L- PG-4 assay (Ref. 1). The stock was 

independently assessed for infectious titer by several different laboratories. 

The result of this analysis established the infectious titer +/- SD of the first lot 

of virus stock to be 6.9 x 107/ml (standard deviation for three experiments is 

2.0 x 107

/ml). Thawing and refreezing of the material appeared to result in a 

lower detectable infectious titer of 3.7 x 106

/ml (standard deviation of 4.7 x 

106

/ml). Periodically, the vector stock will be replenished and the infectious 

titer of the new stock evaluated in comparison to the first lot. 

The standard virus stock and its infectious titer can be used as a positive 

control to empirically determine the relative sensitivity of assay methods used 

for detection of RCR in retroviral vectors. In particular, this stock will allow 

investigators to determine the largest test volume in which a single RCR can 

be detected. The determination should be performed in the presence of a 

retroviral vector supernatant typical of a production lot in order to control for 

inhibitory effects of the retroviral vector particles on detection of RCR. 

Availability of this standard should allow individual investigators to establish 

this methodology in their own laboratories, as well as allow exploration of 

alternative methods for detection of RCR. 

1-3. Formula to Determine Replicate Size and Number 

Depending on the volume in which a single RCR can be detected by an 

individual RCR assay (as determined by use of the RCR standard, Appendix 1-

2), it may be necessary to divide the total test volume into several replicate 

samples to ensure the detection of RCR in the sample. The number of replicates 

(r), can be determined using the formula, 

r = Vt / Vs, 

where Vs is the volume in which one RCR can be consistently detected (see 

Appendix 1-2 for determination of Vs). For example, if 1 RCR can be detected 

in 2 ml, then the total test volume of 300 ml may be tested in 300/2 = 150 

replicates of volume Vs or 150 2-ml replicates. 

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