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NTP Genetically Modified Model Report on the Toxicology and Carcinogenicity Study of Mixtures of 3′-Azido-3′-Deoxythymidine (AZT), Lamivudine (3TC), and Nevirapine (NVP) (CASRNs 30516-87-1, 134678-17-4, 129618-40-2) in Genetically Modified C3B6.129F1-Trp53tm1Brd N12 Haploinsufficient Mice (In Utero and Postnatal Gavage Study)
NTP GMM 16
J.E.A. Leakey, Ph.D., W.T. Allaben, Ph.D., J.K. Dunnick, Ph.D., S.M. Lewis, Ph.D., P.C. Howard, Ph.D., C.C. Weis, B.S., D.D. Paine, B.S., B.R. Brown, B.S., J.P. Freeman, Ph.D., T.M. Heinze, P.H. Siitonen, B.S., S.J. Culp, Ph.D., J.M. Fowler, B.S., R.D. Smith, B.S., R.P. Felton, M.S., and B.T. Thorn, M.S. C.J. Cain, J.W. Carson, B.S., and A. Matson, B.S. K.A. Carroll and S.H. Green. P.W. Mellick, D.V.M., Ph.D., G.R. Olson, D.V.M., Ph.D., A.R. Warbritton, and L.P. Wiley, B.S. M.H. Hamlin, II, D.V.M., E.T. Adams, D.V.M., Ph.D., and R.A. Miller, D.V.M., Ph.D. R.A. Miller, D.V.M., Ph.D., E.T. Adams, D.V.M., Ph.D., G.P. Flake, M.D., J.R. Latendresse, D.V.M., Ph.D., and G.R. Olson, D.V.M., Ph.D. P.C. Howard, Ph.D., W.T. Allaben, Ph.D., N.J. Walker, Ph.D., and J.R. Bucher, Ph.D. S.R. Gunnels, M.A., L.M. Harper, B.S., T.S. Kumpe, M.A., E.S. Rathman, M.S., and D.C. Serbus, Ph.D.
Author Information and AffiliationsSUMMARY
Background:
3′ Azido-3′-deoxythymidine (AZT) is the most widely used chemotherapeutic agent for the treatment of people with acquired immune deficiency syndrome (AIDS) or positive for the human immunodeficiency virus (HIV). AZT treatment is also given to prevent transmission of HIV from pregnant mothers to children before or during birth, and current treatments involve combinations of drugs. Among the most commonly used are lamivudine (3TC) and nevirapine (NVP). We tested the effects of these three drugs, both individually and in combination, on the offspring of female mice (genetically modified to be sensitive to cancer induction) where the mothers were given the drugs during pregnancy.
Methods:
We exposed groups of haploinsufficient C3B6.129F1-Trp53tm1Brd N12 mice by depositing solutions containing AZT, 3TC, NVP, or combinations of the drugs in a methylcellulose solvent directly into the animals’ stomachs through a tube twice daily for 28 days following birth; in addition, their mothers were exposed to the drug for seven days during pregnancy. Other sets of mothers and pups received only the methylcellulose solvent and served as the control groups. Following the dosing period, the animals were observed until 45 weeks of age. Tissues from 34 organs were examined for every animal.
Results:
Exposure to AZT caused increases in the rates of liver cancer (hepatocellular adenoma) in the male pups after 45 weeks; groups of males receiving AZT and 3TC or AZT, 3TC, and NVP also had increased rates of hepatocellular adenomas and carcinomas. In addition, there were occurrences of a few malignant lymphomas in male pups receiving AZT or AZT, 3TC, and NVP and in female pups receiving NVP, AZT plus 3TC, or the three drugs combined.
Conclusions:
We conclude that AZT either alone or in combination with 3TC or with 3TC and NVP caused liver cancers in male pups exposed before and following birth. Malignant lymphomas in male and female pups may have been related to exposure to AZT alone or in combination with 3TC before and following birth.
ABSTRACT
3′-AZIDO-3′-DEOXYTHYMIDINE
CAS No. 30516-87-1
Chemical Formula: C10H13N5O4 Molecular Weight: 267.24
Synonyms: 3′-azido-2′,3′-dideoxythymidine; azidodeoxythymidine; azidothymidine; 3′-azidothymidine; AZT; BW A509U; Compound S; 3′-deoxy-3′-azidothymidine; 3′-deoxy-(8CI) (9CI); ZDV; zidovudine.
Trade name: Retrovir® [Combivir® with 3TC]
2′,3′-DIDEOXY-3′-THIACYTIDINE
CAS No. 134678-17-4
Chemical Formula: C8H11N3O3S Molecular Weight: 229.26
Synonyms: 3TC; 4-amino-1-[(2R,5S)-2-(hydroxymethyl)-1,3-oxathiolan-5-yl]-1,2-dihydropyrimidin-2-one; L-2′,3′-dideoxy-3′-thiacytidine; lamivudine
Trade name: Epivir® [Combivir® with AZT]
NEVIRAPINE
CAS No. 129618-40-2
Chemical Formula: C15H14N4O Molecular Weight: 266.30
Synonyms: NVP; 11-cyclopropyl-4-methyl-5,11-dihydro-6H- dipyrido[3,2-b:2′,3′-e][1,4]diazepin-6-one
Trade Name: Viramune®
3′-Azido-3′-deoxythymidine (AZT) is the most widely used and evaluated chemotherapeutic agent for the treatment of persons with acquired immune deficiency syndrome (AIDS). Antiviral therapy is essential for treatment and prevention of AIDS in adults and children infected with human immunodeficiency virus (HIV), and to prevent mother-to-child transmission of HIV during pregnancy and labor. The studies described in this report were designed to determine possible long-term sequelae from AZT treatment, often used in combination with other antiviral drugs, such as lamivudine (3TC) and nevirapine (NVP) in preventing mother-to-child transmission of HIV.
Male and female heterozygous F1 p53+/− mice were exposed to AZT, 3TC, NVP, or combinations of the chemicals in utero on gestation days (GD) 12 through 18, then administered the same chemical or combination of chemicals by gavage from postnatal day (PND) 1 through PND 28 and then observed until 45 weeks of age. Vehicle control mice received only an aqueous solution containing 0.2% methylcellulose and 0.1% Tween 80. Mice were dosed twice daily until PND 28. Genetic toxicology studies were conducted in mouse peripheral blood erythrocytes.
The study compared three combination doses of AZT, 3TC and NVP (AZT/3TC/NVP-L, AZT/3TC/NVP-M, and AZT/3TC/NVP-H) with the vehicle controls, and compared the individual components with each other at the highest dose (AZT-H, 3TC-H, NVP-H, AZT/3TC-H and AZT/3TC/NVP-H). Because exposure to AZT/3TC/NVP-M and AZT/3TC/NVP-H reduced pup survival, additional litters were required to provide sufficient pups for the 45-week study.
45-Week Study:
In general, survival was relatively high once the pup exposure phase had been completed, with at least 75% of the mice surviving to terminal sacrifice in all groups. For males, survival was significantly greater in the AZT/3TC/NVP-L and AZT/3TC/NVP-M groups relative to the vehicle control group. There were no significant differences in survival between high dose groups of the constituent chemicals in either sex; however, survival of females in the AZT/3TC-H group was significantly less than that in the vehicle control group. Early deaths were predominantly associated with occurrences of malignant lymphoma, mammary gland tumors, and osteosarcomas.
In the combination dose comparison, males and females dosed with the AZT/3TC/NVP-H combination had significantly decreased body weights compared to the vehicle control groups from PND 11 when individual monitoring began until 20 (males) or 11 (females) weeks. In addition, mean body weights for the male and female AZT/3TC/NVP-M groups were significantly less than those of the vehicle control groups until 14 weeks. In the high dose comparison, mean body weights of the male and female AZT-H groups were significantly less than those of the vehicle control groups during some of the early weeks of dosing.
In male and female mice, absolute brain weights of the combination dose groups decreased with increasing dose and, except in low dose males, the absolute brain weights of the dosed groups were significantly less than those of the vehicle control groups. When the high doses of the constituent chemicals were compared, absolute brain weights of the male and female AZT-H and AZT/3TC/NVP-H groups were significantly less than those of the vehicle control groups. However, relative brain weights were not significantly altered. Relative liver weights of male combination dose groups followed a positive trend with dose. When the high dose groups were compared, increases in relative liver weights of male mice appeared to be associated with AZT exposure. In combination dose groups, the absolute heart weight of AZT/3TC/NVP-H females was significantly greater than that of the vehicle control group, and there was a positive trend in absolute heart weights. There was also a positive trend for relative heart weights in these combination dose groups, though no individual group relative weight was significantly greater than that of the vehicle control group. In females, absolute heart weight was also significantly increased in the AZT/3TC-H group relative to the vehicle control group.
A small but statistically significant increase in serum alanine aminotransferase activity was observed in the male AZT/3TC/NVP-H group compared to the vehicle control group.
In the combination dose comparison, the incidences of hepatocellular adenoma and hepatocellular adenoma or carcinoma (combined) in the liver of all groups of males dosed with AZT/3TC/NVP were significantly increased compared to the vehicle control group.
In the high dose comparison, the incidences of hepato-cellular adenoma in males in the AZT-H group and hepatocellular adenoma and hepatocellular adenoma or carcinoma (combined) in males in the AZT/3TC-H and AZT/3TC/NVP-H groups were significantly greater than those in the vehicle control group; the incidences of these lesions in the 3TC-H and NVP-H groups were significantly less than those in the AZT/3TC/NVP-H group.
The incidences of malignant lymphoma in males administered AZT-H or AZT/3TC-H and in females administered AZT/3TC/NVP-M, AZT/3TC/NVP-H, NVP-H, or AZT/3TC-H were slightly greater than those in the vehicle control groups. The incidence of mam-mary gland adenoacanthoma or adenocarcinoma (combined) in females administered 3TC-H was slightly greater than that in the vehicle control group.
Genetic Toxicology:
In the peripheral blood of 1-day-old male and female mice, the percentage of total reticulocytes (RETs) was significantly decreased in groups exposed to doses that contained AZT. In addition, the percentages of micronucleated normochromatic erythrocytes (NCEs) and micronucleated RETs were generally significantly increased in groups exposed to doses containing AZT, but not in the 3TC-H or NVP-H groups. The percentages of micronucleated NCEs in the AZT/3TC/NVP-H groups were greater than in the AZT-H and the AZT/3TC-H groups. In peripheral blood of male pups evaluated at PND 28, both the percentage of micronucleated RETs and the percentage of micronucleated NCEs were significantly increased in the group where 3TC was coadministered with AZT compared to the group administered only AZT.
Conclusions:
Under the conditions of this gavage study, there was clear evidence of carcinogenic activity* of AZT alone in male heterozygous F1 p53+/− mice based on increased incidences of hepatocellular adenoma. There was clear evidence of carcinogenic activity of AZT in combination with 3TC, and AZT in combination with 3TC and NVP in male heterozygous F1 p53+/− mice based on increased incidences of hepatocellular adenoma and hepatocellular adenoma or carcinoma (combined). The occurrence of malignant lymphoma may have been related to treatment with AZT alone and with AZT in combination with 3TC.
There was no evidence of carcinogenic activity of 3TC alone in male heterozygous F1 p53+/− mice administered 150 mg/kg. There was no evidence of carcinogenic activity of NVP alone in male heterozygous F1 p53+/− mice administered 168 mg/kg.
There was equivocal evidence of carcinogenic activity of NVP alone, AZT in combination with 3TC, and AZT in combination with 3TC and NVP in female heterozy gous F1 p53+/− mice based on the occurrence of malignant lymphoma. There was equivocal evidence of carcinogenic activity of 3TC alone in female heterozygous F1 p53+/− mice based on the occurrence of mammary gland adenoacanthoma or adenocarcinoma (combined).
There was no evidence of carcinogenic activity of AZT alone in female heterozygous F1 p53+/− mice administered 240 mg/kg.
Summary of the 45-Week Carcinogenesis and Genetic Toxicology Studies of AZT, 3TC, and NVP
| Male | Female | |
|---|---|---|
| Doses in aqueous methylcellulose/Tween 80 by gavage | ||
| Dams (GDs 12–18) and Pups PNDs (11–28) | ||
| Vehicle Control | — | — |
| AZT-H | 240 mg/kg per day | 240 mg/kg per day |
| 3TC-H | 150 mg/kg per day | 150 mg/kg per day |
| NVP-H | 168 mg/kg per day | 168 mg/kg per day |
| AZT/3TC-H | 240/150 mg/kg per day | 240/150 mg/kg per day |
| AZT/3TC/NVP-L | 80/50/56 mg/kg per day | 80/50/56 mg/kg per day |
| AZT/3TC/NVP-M | 160/100/112 mg/kg per day | 160/100/112 mg/kg per day |
| AZT/3TC/NVP-H | 240/150/168 mg/kg per day | 240/150/168 mg/kg per day |
| Pups (PNDs 1–3) | ||
| Vehicle Control | — | — |
| AZT-H | 120 mg/kg per day | 120 mg/kg per day |
| 3TC-H | 75 mg/kg per day | 75 mg/kg per day |
| NVP-H | 21 mg/kg per day | 21 mg/kg per day |
| AZT/3TC-H | 120/75 mg/kg per day | 120/75 mg/kg per day |
| AZT/3TC/NVP-L | 40/25/7 mg/kg per day | 40/25/7 mg/kg per day |
| AZT/3TC/NVP-M | 80/50/14 mg/kg per day | 80/50/14 mg/kg per day |
| AZT/3TC/NVP-H | 120/75/21 mg/kg per day | 120/75/21 mg/kg per day |
| Pups (PNDs 4–10) | ||
| Vehicle Control | — | — |
| AZT-H | 120 mg/kg per day | 120 mg/kg per day |
| 3TC-H | 75 mg/kg per day | 75 mg/kg per day |
| NVP-H | 84 mg/kg per day | 84 mg/kg per day |
| AZT/3TC-H | 120/75 mg/kg per day | 120/75 mg/kg per day |
| AZT/3TC/NVP-L | 40/25/28 mg/kg per day | 40/25/28 mg/kg per day |
| AZT/3TC/NVP-M | 80/50/56 mg/kg per day | 80/50/56 mg/kg per day |
| AZT/3TC/NVP-H | 120/75/84 mg/kg per day | 120/75/84 mg/kg per day |
| Body Weights | ||
| Combination Dose Comparison a | AZT/3TC/NVP-M and AZT/3TC/NVP-H groups less than the vehicle control group from PND 11 to weeks 14 and 20, respectively | AZT/3TC/NVP-M and AZT/3TC/NVP-H groups less than the vehicle control group from PND 11 to weeks 14 and 11, respectively |
| High Dose Comparison b | AZT-H group less than the vehicle control group during the early weeks of dosing; AZT/3TC/NVP-H group less than the vehicle control group from PND 11 to week 20 | AZT-H group less than the vehicle control group during the early weeks of dosing; AZT/3TC/NVP-H group less than the vehicle control group from PND 11 to week 11 |
| Survival rates | ||
| Combination Dose Comparison | 20/25, 24/25, 22/25, 22/25 | 25/25, 22/25, 19/25, 21/25 |
| High Dose Comparison | 20/25, 18/25, 23/25, 22/26, 21/25, 22/25 | 25/25, 20/25, 20/25, 20/25, 19/25, 21/25 |
| Nonneoplastic effects | ||
| Combination Dose Comparison | None | None |
| High Dose Comparison | None | None |
| Neoplastic effects | ||
| AZT-H | Liver: hepatocellular adenoma (1/25, 8/23) | None |
| 3TC-H | None | None |
| NVP-H | None | None |
| AZT/3TC-H | Liver: hepatocellular adenoma (1/25, 9/25); hepatocellular adenoma or carcinoma (1/25, 10/25) | None |
| AZT/3TC/NVP- L,M,H | Liver: hepatocellular adenoma (1/25, 7/25, 7/23, 9/23); hepatocellular adenoma or carcinoma (1/25, 9/25, 8/23, 10/23) | None |
| Equivocal findings | ||
| AZT-H | All organs: malignant lymphoma (1/25, 3/24) | None |
| 3TC-H | None | Mammary gland: adenoacanthoma or adenocarcinoma (1/25, 4/25) |
| NVP-H | None | All organs: malignant lymphoma (2/25, 5/25) |
| AZT/3TC-H | All organs: malignant lymphoma (1/25, 3/25) | All organs: malignant lymphoma (2/25, 4/24) |
| AZT/3TC/NVP-L,M,H | None | All organs: malignant lymphoma (2/25, 2/25, 4/22, 4/25) |
| Level of evidence of carcinogenic activity | ||
| AZT | Clear evidence | No evidence |
| 3TC | No evidence | Equivocal evidence |
| NVP | No evidence | Equivocal evidence |
| AZT/3TC | Clear evidence | Equivocal evidence |
| AZT/3TC/NVP | Clear evidence | Equivocal evidence |
| Genetic toxicology | ||
| Total reticulocytes | ||
| Male and female mice peripheral blood in vivo, PND 1: | Negative in groups exposed to AZT | |
| Micronucleated normochromatic erythrocytes and reticulocytes | ||
| Male and female mice peripheral blood in vivo, PND 1: | Positive in AZT-H, AZT/3TC-H, AZT/3TC/NVP-M, and AZT/3TC/NVP-H groups | |
| Male mouse peripheral blood in vivo, PND 28: | Positive in AZT/3TC-H group | |
- a
Vehicle control, AZT/3TC/NVP-L, AZT/3TC/NVP-M, AZT/3TC/NVP-H
- b
Vehicle control, AZT-H, 3TC-H, NVP-H, AZT/3TC-H, AZT/3TC/NVP-H
- *
Explanation of Levels of Evidence of Carcinogenic Activity is on page 13. A summary of the Peer Review Panel comments and the public discussion on this Report appears on page 15.
Contents
- FOREWORD
- CONTRIBUTORS
- EXPLANATION OF LEVELS OF EVIDENCE OF CARCINOGENIC ACTIVITY
- NATIONAL TOXICOLOGY PROGRAM TECHNICAL REPORTS PEER REVIEW PANEL
- SUMMARY OF PEER REVIEW PANEL COMMENTS
- INTRODUCTION
- MATERIALS AND METHODS
- RESULTS
- DISCUSSION AND CONCLUSIONS
- REFERENCES
- APPENDIX A. SUMMARY OF LESIONS IN HETEROZYGOUS F1 p53+/− MICE IN THE IN UTERO/POSTNATAL GAVAGE STUDY OF AZT, 3TC, AND NVP
- APPENDIX B. GENETIC TOXICOLOGY
- APPENDIX C. CLINICAL PATHOLOGY RESULTS
- APPENDIX D. BODY WEIGHT ANALYSES
- APPENDIX E. ORGAN WEIGHTS AND ORGAN-WEIGHT-TO-BODY-WEIGHT RATIOS
- APPENDIX F. CHEMICAL CHARACTERIZATION AND DOSE FORMULATION STUDIES
- APPENDIX G. LITTER EFFECTS ON SURVIVAL, BODY WEIGHT, AND PATHOLOGY
- APPENDIX H. LITTER SUCCESS AND SURVIVAL
- APPENDIX I. TOXICOKINETIC STUDIES
- APPENDIX J. HISTORICAL CONTROL INCIDENCES
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