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Abstract for GMM-16

Abstract

Toxicology and Carcinogenicity Study of Mixtures of 3′-Azido-3′-Deoxythymidine (AZT), Lamivudine (3TC), and Nevirapine (NVP) (CAS Nos. 30516-87-1, 134678-17-4, 129618-40-2) in Genetically Modified C3B6.129F1-Trp53tm1Brd N12 Haploinsufficient Mice (In Utero and Postnatal Gavage Studies)

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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 to load 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 hepatocellular 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 mammary 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 heterozygous 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.

Synonyms: (3′-AZIDO-3′-DEOXYTHYMIDINE) 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]
 

Synonyms: (2′,3′-DIDEOXY-3′-THIACYTIDINE) 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]
 

Synonyms: (NEVIRAPINE) NVP; 11-cyclopropyl-4-methyl-5,11-dihydro-6H- dipyrido[3,2-b:2′,3′-e][1,4]diazepin-6-one
Trade name: Viramune® 
 

 

Summary of the 45-Week Carcinogenesis and Genetic Toxicology Study of AZT, 3TC, and NVP
Doses in aqueous methylcellulose/Tween 80 by gavage Male Female
Dams (GDs 12-18) and Pups PNDs (11-28)
Vehicle Control
AZT-H
3TC-H
NVP-H
AZT/3TC-H
AZT/3TC/NVP-L
AZT/3TC/NVP-M
AZT/3TC/NVP-H

---
240 mg/kg per day
150 mg/kg per day
168 mg/kg per day
240/150 mg/kg per day
80/50/56 mg/kg per day
160/100/112 mg/kg per day
240/150/168 mg/kg per day

---
240 mg/kg per day
150 mg/kg per day
168 mg/kg per day
240/150 mg/kg per day
80/50/56 mg/kg per day
160/100/112 mg/kg per day
240/150/168 mg/kg per day
Pups (PNDs 1-3)
Vehicle Control
AZT-H
3TC-H
NVP-H
AZT/3TC-H
AZT/3TC/NVP-L
AZT/3TC/NVP-M
AZT/3TC/NVP-H

---
120 mg/kg per day
75 mg/kg per day
21 mg/kg per day
120/75 mg/kg per day
40/25/7 mg/kg per day
80/50/14 mg/kg per day
120/75/21 mg/kg per day

---
120 mg/kg per day
75 mg/kg per day
21 mg/kg per day
120/75 mg/kg per day
40/25/7 mg/kg per day
80/50/14 mg/kg per day
120/75/21 mg/kg per day
Pups (PNDs 4-10)
Vehicle Control
AZT-H
3TC-H
NVP-H
AZT/3TC-H
AZT/3TC/NVP-L
AZT/3TC/NVP-M
AZT/3TC/NVP-H

---
120 mg/kg per day
75 mg/kg per day
84 mg/kg per day
120/75 mg/kg per day
40/25/28 mg/kg per day
80/50/56 mg/kg per day
120/75/84 mg/kg per day

---
120 mg/kg per day
75 mg/kg per day
84 mg/kg per day
120/75 mg/kg per day
40/25/28 mg/kg per day
80/50/56 mg/kg per day
120/75/84 mg/kg per day
Body weights
a Combination Dose Comparison


b High Dose Comparison



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-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/3TC/NVP-M and AZT/3TC/NVP-H groups less than the vehicle control group from PND 11 to weeks 14 and 11, respectively

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

High Dose Comparison

20/25, 24/25, 22/25, 22/25

20/25, 18/25, 23/25, 22/26, 21/25, 22/25

25/25, 22/25, 19/25, 21/25

25/25, 20/25, 20/25, 20/25, 19/25, 21/25
Nonneoplastic effects
Combination Dose Comparison
High Dose Comparison

None
None

None
None
Neoplastic effects
AZT/3TC/NVP- L,M,H
AZT/3TC-H
NVP-H
3TC-H
AZT-H

Liver: hepatocellular adenoma (1/25, 8/23)
None
None
Liver: hepatocellular adenoma (1/25, 9/25); hepatocellular adenoma or carcinoma (1/25, 10/25)
Liver: hepatocellular adenoma (1/25, 7/25, 7/23, 9/23); hepatocellular adenoma or carcinoma (1/25, 9/25, 8/23, 10/23)


None
None

None
None
None
Equivocal findings
AZT/3TC/NVP- L,M,H
AZT/3TC-H
NVP-H
3TC-H
AZT-H

All organs: malignant lymphoma (1/25, 3/24)
None

None
None
All organs: malignant lymphoma (1/25, 3/25)
None

None
Mammary gland: adenoacanthoma or adenocarcinoma (1/25, 4/25)
All organs: malignant lymphoma (2/25, 5/25)
All organs: malignant lymphoma (2/25, 4/24)
All organs: malignant lymphoma (2/25, 2/25, 4/22, 4/25)
Level of evidence of carcinogenic activity
AZT/3TC/NVP
AZT/3TC
NVP 
3TC     
AZT

Clear evidence
No evidence
No evidence
Clear evidence
Clear evidence
 

No evidence
Equivocal evidence
Equivocal evidence
Equivocal 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:
Male mouse peripheral blood in vivo, PND 28:
 


Positive in AZT-H, AZT/3TC-H, AZT/3TC/NVP-M, and AZT/3TC/NVP-H groups
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

 
Report Date: October 2013

Pathology Tables, Survival and Growth Curves from NTP GMM Studies

Target Organs & Incidences from GMM Studies