National Toxicology Program

National Toxicology Program
https://ntp.niehs.nih.gov/go/36326

Abstract for TR-569 - Mixtures of AZT, 3TC, NVP and NFV (Cas Nos. 30516-87-1, 134678-17-4, 129618-40-2, 159989-65-8)

ABSTRACT

Toxicology and Carcinogenesis Studies of Mixtures of 3′-Azido-3′-Deoxythymidine (AZT), Lamivudine (3TC), Nevirapine (NVP), and Nelfinavir Mesylate (NFV) (CASRNs 30516-87-1, 134678-17-4, 129618-40-2, 159989-65-8) in B6C3F1 Mice (Transplacental Exposure Studies)

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 Abstract

With the increased administration of multidrug regimens to pregnant women who are human immunodeficiency virus type-1 (HIV-1) positive, along with the increased efficacy of these combinations, determining the long-term consequences of the antiretroviral agents in noninfected children becomes important. The goal of the current study was to determine the carcinogenicity of combinations of antiretroviral drugs in male and female B6C3F1 mouse pups exposed transplacentally and monitored for 2 years. Genetic toxicology studies were conducted in Salmonella typhimurium and Escherichia coli.

AZT

3′-Azido-3′-deoxythymidine (AZT) was synthesized initially for use as an anticancer agent and was later reported to block the infectivity and cytopathic effects, in vitro, of HIV-1, due to the inhibition (by AZT 5′-triphosphate) of viral reverse transcriptase. Pregnant women who are positive for HIV-1 are given AZT to manage the infection and to prevent maternal-to-fetal transmission of the virus.

3TC

Lamivudine (3TC) was synthesized initially as a racemate and then in enantiomerically pure forms. 3TC (as 3TC 5′-triphosphate) is thought to inhibit viral reverse transcriptase by competing with deoxycytidine 5′-triphosphate for incorporation into HIV-1 DNA. When used for the management of HIV-1 infections, 3TC is always used in combination with another nucleoside reverse transcriptase inhibitor (e.g., AZT) and either a protease inhibitor (e.g., nelfinavir mesylate, NFV) or a nonnucleoside reverse transcriptase inhibitor (e.g., nevirapine, NVP).

NVP

NVP, a nonnucleoside reverse transcriptase inhibitor, was first synthesized in 1991. NVP inhibits HIV-1 reverse transcriptase noncompetitively by binding to an allosteric site on the enzyme; this action is specific for HIV-1 reverse transcriptase. NVP is usually given as part of a three-drug regimen. Typical regimens in adults and adolescents include NVP and 3TC or emtricitabine and AZT or tenofovir.

NFV

The synthesis of NFV was reported in 1997. NFV acts by inhibiting HIV-1 protease, the enzyme responsible for cleavage of the polyprotein resulting from the gag and gag-pol genes of HIV-1. This inhibition results in an immature, noninfectious virus. NFV is always used in combination with other antiretroviral agents, typically two nucleoside reverse transcriptase inhibitors (e.g., AZT and 3TC).

2-YEAR TRANSPLACENTAL STUDY IN MICE

Female C57Bl/6N mice were bred to male C3H/HeNMTV mice, and from gestation day 12 until gestation day 18 (or until they littered), the pregnant dams were treated by gavage with AZT or mixtures of AZT and 3TC; AZT, 3TC, and NVP; or AZT, 3TC, and NFV. The high dose of each drug was 240 mg/kg body weight per day for AZT, 120 mg/kg body weight per day for 3TC, 168 mg/kg body weight per day for NVP, and 1,008 mg/kg body weight per day for NFV (ratio 1.0:0.5:0.7:4.2, respectively). The mid and low doses were 66% and 33% of these values, respectively, and maintained the same ratio among the drugs. The drugs were administered in a 0.2% methylcellulose and 0.1% Tween® 80 vehicle at a dosing volume of 20 mL/kg body weight. Control dams were administered the vehicle only. The tumor incidence in the male and female B6C3F1 offspring was monitored for 2 years after birth. The group sizes varied between 15 and 65 male or female mice per treatment.

Compared to the vehicle control group, none of the treatments affected the body weights of the pregnant dams. Likewise, none of the treatments affected the number of pups per litter or the ratio of male to female pups. Combinations of AZT/3TC/NVP and AZT/3TC/NFV caused dose-related decreases in body weights of male and female B6C3F1 offspring. Transplacental exposure to AZT/3TC/NVP and AZT/3TC/NFV caused dose-related decreases in survival of the B6C3F1 mice from birth until weaning at postnatal day 21.

Postweaning survival of transplacentally exposed groups of female mice was similar to that of the control group for each drug combination. Survival of all groups of male mice transplacentally exposed to AZT or AZT/3TC was similar to that of the control group; survival of male mice transplacentally exposed to AZT/3TC/NVP or AZT/3TC/NFV was decreased in a dose-related manner that was significant in the high-dose group for each of the drug combinations, relative to controls.

Mean body weights of female mice transplacentally exposed to AZT or the combination of AZT/3TC were similar to those of the controls during the 2-year transplacental exposure study. Transplacental exposure to the combination of AZT/3TC/NVP resulted in dose-related decreases in body weights in female mice; the high-dose group was significantly different from the control group at all time points, with the average decrease in weight being 18%; the low- and mid-dose combinations were significantly different from the control group at most time points, with the average decreases in weight being 8% and 5%, respectively. In female mice exposed to the combination AZT/3TC/NFV, the high-dose group was significantly different from the control group at all time points, with the average decrease in weight being 13%; the low- and mid-dose groups were significantly different from the control group at most time points, with the average decreases in weight being 5% and 6%, respectively.

Male mice exposed transplacentally to AZT showed dose-related decreases in body weight, with the differences being significant in all exposed groups at all time points. Compared to the control group, the average decrease in body weight was 9% in the high-dose group, 6% in the mid-dose group, and 5% in the low-dose group. Transplacental exposure to the combination of AZT/3TC caused dose-related decreases in body weight in male mice, with the differences being significant at all time points in the high- and mid-dose groups, and at nearly all time points in the low-dose group. The average decrease in body weight was 7% in the high-dose group, 5% in the mid-dose group, and 3% in the low-dose group. Male mice exposed transplacentally to the combination of AZT/3TC/NVP or the combination of AZT/3TC/NFV showed dose-related decreases in body weight, with the differences being significant in all exposed groups at all time points. For the AZT/3TC/NVP combination, the average decrease in body weight was 18% in the high-dose group, 9% in the mid-dose group, and 7% in the low-dose group. For the AZT/3TC/NFV combination, the average decrease in body weight was 11% in the high-dose group, 7% in the mid-dose group, and 4% in the low-dose group.

Transplacental exposure to AZT caused positive trends in the incidences of follicular cell adenoma of the thyroid gland, follicular cell adenoma or carcinoma (combined), and subcutaneous fibrosarcoma or sarcoma (combined) of the skin in female mice. The incidences of follicular cell adenoma of the thyroid gland (after adjusting for possible dam or sire effects) and follicular cell adenoma or carcinoma (combined) of the thyroid gland were significantly increased in female mice exposed to 240 mg/kg AZT.

Transplacental exposure to mixtures of AZT/3TC resulted in a positive trend in the incidences of alveolar/bronchiolar adenoma of the lung in female mice.

Transplacental exposure to mixtures of AZT/3TC/NVP caused positive trends in the incidences of subcutaneous fibrosarcoma of the skin; subcutaneous fibrous histiocytoma or fibrosarcoma (combined) of the skin; and subcutaneous fibroma, fibrous histiocytoma, or fibrosarcoma (combined) of the skin in male mice. The incidences of subcutaneous fibrosarcoma of the skin; subcutaneous fibrous histiocytoma or fibrosarcoma of the skin (combined); and subcutaneous fibroma, fibrous histiocytoma, or fibrosarcoma of the skin (combined) were significantly increased in the group of males exposed transplacentally to 240 mg/kg AZT, 120 mg/kg 3TC, and 168 mg/kg NVP. After adjusting for possible dam or sire effects, the incidences of subcutaneous fibrosarcoma of the skin; subcutaneous fibrous histiocytoma or fibrosarcoma of the skin (combined); and subcutaneous fibroma, fibrous histiocytoma, or fibrosarcoma of the skin (combined) were significantly increased in the group of males transplacentally exposed to 160 mg/kg AZT, 80 mg/kg 3TC, and 112 mg/kg NVP. The incidence of subcutaneous skin fibrosarcoma was significantly increased in female mice in the same exposed group.

GENETIC TOXICOLOGY

AZT, 3TC, NVP, and NFV (the same lots that were used in the 2-year animal studies) were tested for bacterial mutagenicity in S. typhimurium strains TA98 and TA100 and in E. coli strain WP2 uvrA/pKM101. Only AZT was found to be mutagenic; the other three compounds showed no evidence of mutagenicity in bacteria. With AZT, significant increases in mutant colonies were seen in the E. coli strain, with and without induced rat liver metabolic activation enzymes. No evidence of mutagenicity was seen with AZT in S. typhimurium strains TA98 or TA100.

CONCLUSIONS

AZT

Under the conditions of this transplacental exposure study, there was no evidence of carcinogenic activity of AZT in male B6C3F1 mice whose dams were exposed to 80, 160, or 240 mg/kg by gavage. There was equivocal evidence of carcinogenic activity of AZT in female B6C3F1 mice based on increased incidences of thyroid gland neoplasms (primarily adenoma) and subcutaneous skin fibrosarcoma or sarcoma.

AZT and 3TC

Under the conditions of this transplacental exposure study, there was no evidence of carcinogenic activity of mixtures of AZT and 3TC in male B6C3F1 mice whose dams were exposed to 80/40, 160/80, or 240/120 mg/kg by gavage. There was equivocal evidence of carcinogenic activity of mixtures of AZT and 3TC in female B6C3F1 mice based on increased incidences of lung alveolar/bronchiolar adenoma.

AZT, 3TC, and NVP

Under the conditions of this transplacental exposure study, there was some evidence of carcinogenic activity of mixtures of AZT, 3TC, and NVP in male B6C3F1 mice whose dams were exposed to these chemicals by gavage based on increased incidences of subcutaneous skin neoplasms (fibroma, fibrous histiocytoma, or fibrosarcoma). There was equivocal evidence of carcinogenic activity of mixtures of AZT, 3TC, and NVP in female B6C3F1 mice based on an increased incidence of subcutaneous skin fibrosarcoma.

AZT, 3TC, and NFV

Under the conditions of this transplacental exposure study, there was no evidence of carcinogenic activity of mixtures of AZT, 3TC, and NFV in male or female B6C3F1 mice whose dams were exposed to 80/40/336, 160/80/672, or 240/120/1,008 mg/kg by gavage.

Synonyms: AZT; zidovudine; 3′-azido-2′,3′-dideoxythymidine; azidodeoxythymidine; azidothymidine; 3′-azidothymidine; 3N-deoxy-3′-azidothymidine; 3′-deoxy-(8CI)(9CI); BW A509U; Compound S; ZDV 

Trade name: Retrovir®

Synonyms: 3TC; (-)2′,3′-dideoxy-3′-thiacytidine; (2R-cis)-4-amino-1-[2-(hydroxymethyl)-; 1,3-oxathiolan-5-yl]-2(1H)-pyrimidinone; (-)-BCH-189; GR-109714X

Trade names: Epivir®, Zeffix®

Synonyms: NVP; BIRG-587; 11-cyclopropyl-5,11-dihydro-4-methyl-6H-dipyrido-[3,2-b:2′,3′-e][1,4]diazepin-6-one

Trade name: Viramune®

Synonyms: NFV; AG1343; (3S,4aS,8aS)-N-(1,1-dimethylethyl)decahydro-2-[(2R,3R)-2-hydroxy-3-isoquinoline carboxamide] methane sulfonate

Trade name: Viracept®

Summary of the 2-Year Transplacental Carcinogenesis and Genetic Toxicology Studies of Mixtures of AZT, 3TC, NVP and NFV
  Male B6C3F1 Mice Female B6C3F1 Mice
Concentrations in utero
AZT 0, 80, 160, or 240 mg/kg 0, 80, 160, or 240 mg/kg
AZT/3TC 0, 80/40, 160/80, or 240/120 mg/kg 0, 80/40, 160/80, or 240/120 mg/kg
AZT/3TC/NVP 0, 80/40/56, 160/80/112, or 240/120/168 mg/kg 0, 80/40/56, 160/80/112, or 240/120/168 mg/kg
AZT/3TC/NFV 0, 80/40/336, 160/80/672, or 240/120/1,008 mg/kg 0, 80/40/336, 160/80/672, or 240/120/1,008 mg/kg
Body Weights
AZT 80 mg/kg group 5% less, 160 mg/kg group 6% less, and 240 mg/kg group 9% less than the control group Exposed groups similar to the control group
AZT/3TC 160/80 mg/kg group 5% less, 240/120 mg/kg group 7% less than the control group Exposed groups similar to the control group
AZT/3TC/NVP 80/40/56 mg/kg group 7% less, 160/80/112 mg/kg group 9% less, and 240/120/168 mg/kg group 18% less than the control group 80/40/56 mg/kg group 8% less, 160/80/112 mg/kg group 5% less, and 240/120/168 mg/kg group 18% less than the control group
AZT/3TC/NFV 160/80/672 mg/kg group 7% less, 240/120/1,008 mg/kg group 11% less than the control group 80/40/336 mg/kg group 5% less, 160/80/672 mg/kg group 6% less, and 240/120/1,008 mg/kg group 13% less than the control group
Survival rates
AZT 46/65, 39/48, 38/48, 35/48 45/64, 38/48, 28/47, 37/48
AZT/3TC 46/65, 39/51, 35/48, 34/48 45/64, 32/48, 35/51, 35/48
AZT/3TC/NVP 46/65, 37/48, 35/48, 25/50 45/64, 31/48, 34/48, 39/49
AZT/3TC/NFV 46/65, 37/48, 36/51, 6/15 45/64, 30/50, 37/49, 16/26
Nonneoplastic effects
AZT None None
AZT/3TC None None
AZT/3TC/NVP None None
AZT/3TC/NFV None None
Neoplastic effects
AZT None None
AZT/3TC None None
AZT/3TC/NVP Skin (subcutaneous tissue): fibroma, fibrous histiocytoma, or fibrosarcoma (2/65, 2/47, 7/48, 12/48) None
AZT/3TC/NFV None None
Equivocal findings
AZT None Thyroid gland (follicular cell): adenoma (0/59, 1/46, 0/46, 3/47); adenoma or carcinoma (0/59, 1/46, 0/46, 4/47)
Skin (subcutaneous tissue): fibrosarcoma or sarcoma (2/63, 0/46, 4/47, 5/48)
AZT/3TC None Lung: alveolar/bronchiolar adenoma (2/62, 1/48, 3/50, 6/48)
AZT/3TC/NVP None Skin (subcutaneous tissue): fibrosarcoma (1/63, 0/47, 7/47, 0/49)
AZT/3TC/NFV None None
Level of evidence of carcinogenic activity
AZT No evidence Equivocal evidence
AZT/3TC No evidence Equivocal evidence
AZT/3TC/NVP Some evidence Equivocal evidence
AZT/3TC/NFV No evidence No evidence
Genetic toxicology Bacterial gene mutations:
AZT Negative in S. typhimurium strains TA98 and TA100, with and without S9; positive in E. coli strain WP2 uvrA/pKM101 with and without S9
3TC Negative in S. typhimurium strains TA98 and TA100, with and without S9; negative in E. coli strain WP2 uvrA/pKM101 with and without S9
NVP Negative in S. typhimurium strains TA98 and TA100, with and without S9; negative in E. coli strain WP2 uvrA/pKM101 with and without S9
NFV Negative in S. typhimurium strains TA98 and TA100, with and without S9; negative in E. coli strain WP2 uvrA/pKM101 with and without S9

 

Date: January 2013


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