Registration Decision for the New Active Ingredients, Nitrogen dioxide 

and Dinitrogen tetroxide 

Date: 4 (rs /!G. -- ~-~~~~------
Registration Decision for the New Active Ingredients, Nitrogen dioxide 

and Dinitrogen tetroxide 

Summary 

The U.S. Environmental Protection Agency (EPA) is registering a new end-use product 

containing two new active ingredients, dinitrogen tetroxide and nitrogen dioxide (N02), under 

the Federal Insecticide, Fungicide, and Rodenticide Act (FJFRA), as amended. 

Nitrogen dioxide and dinitrogen tetroxide are formulated into an end-use product, Noxilizer N02 

Sterilant, for use as a gaseous sterilant in industrial, commercial, and institutional settings on 

laboratory equipment, isolators, medical devices, pharmaceuticals, and pharmaceutical 

equipment. oxilizer 0 2 Sterilant is not labeled for use in sterile reprocessing of reusable 

medical devices. 

Background 

On August 6, 20 14, EPA received an application from Noxili zer, Inc. to register an end-use 

product (Noxilizer N02 Sterilant) containing the new active ingredients, dinitrogen tetroxide and 

nitrogen dioxide (CAS Numbers I 0544-72-6 & I 0 I 02-44-0), for use as a sterilant. Noxilizer 

N0 2 Sterilant may only be used with Noxilizer licensed N02 generators or Noxilizer branded 

Sterilizers. These two devices have a standard operating manual which calls for a 90 minute 

exposure time. Once instruments have been exposed, then the chamber has to aerate before the 

doors can be opened. 

The company submitted product chemistry, efficacy, toxicology, human exposure, ecological 

effects, and environmental fate data in support of its application for registration of the one enduse product containing the new active ingredients. 

Public Comments 

As required by Flf-RA, the Agency published a notice of receipt of an application to register a 

pesticide product containing the new acti ve ingred ients, nitrogen dioxide and dinitrogen 

tetroxide, in the Federal Register on December 04. 2013 (78 FR 25596), and a 30 day public 

comment period commenced. One anonymous comment was received in response to the Federal 

Register notice. 

Comment: The sole commenter stated opposition to the registration '·of these new active 

ingredients." The commenter stated that it was crucial to assess "all possible complications with 

agriculture related chemicals due to synergy between these new ingredients and other chemicals 

present in both our terrestrial and aquatic world." 

EPA Response: The proposed use of nitrogen dioxide is as an antimicrobial pesticide for sterilant 

use in medical, pharmaceutical, and laboratory settings. Environmental exposure is minimal and 

is mitigated through the use of scrubbers that remove most of the nitrogen dioxide during the 

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discharge process. The amount discharged after passing tlu-ough the scrubbers is below the 

ambient levels of nitrogen dioxide naturally occuning in the environment. The agency is not 

aware of any data regarding the synergy ofN02 with agricultural or other chemicals, but if such 

information becomes available, the agency would review and assess any impact of such data on 

terrestrial and aquatic organisms. 

The Agency sought public comment under the public participation process, FIFRA section 3(c) 

4, for the proposal to issue an unconditional registration for Noxilizer N02 Sterilant on March I 0, 

2016, and a 30 day public comment period commenced. No comments were received. 

Evaluation 

In evaluating a pesticide registration application, the EPA assesses a wide variety of exposure 

information, (i.e., where and how the pesticide is used), environmental fate (i.e., how the 

chemical will move in the environment), and toxicity studies and waiver rationales (i.e., effects 

on humans and other non-target organisms) to determine the likelihood of adverse effects (i.e., 

risk) from exposures associated with the proposed use of the product. Risk assessments are 

developed to evaluate the environmental fate of the compound as well as how it might affect a 

wide range of non-target organisms including humans, terrestrial and aquatic wildlife (plants and 

animals). On the basis of these assessments, EPA evaluates and approves language for each 

pesticide label to ensure the directions for use and safety measures are appropriate to mitigate 

any potential risk. The pesticide's label communicates directions for use that are necessary to 

prevent unreasonable adverse effects. It is a FIFRA violation to use a registered pesticide in a 

manner inconsistent with its labeling. 

1. Assessment of Risks to Human Health 

EPA requires a wide range of information in order to assess human health risk from exposure to 

a pesticide. The human health effects and risks of N02 from its use as a sterilant are assessed in 

the February 1 I, 2016 document entitled, "Revised Risk Assessment for the New Active 

Ingredient Nitrogen Dioxide for use as a Steri/ant" (see Docket 10: EPA-HQ-OPP-20 13-0792). 

The toxicity database to assess risk from exposure to nitrogen dioxide is adequate to support the 

use of nitrogen dioxide as a gaseous sterilant. For the use ofN02 as a gaseous sterilant, exposure 

is expected to occur only for workers handling the product in an occupational setting. Residential 

use is not allowed and there is no potential for food or drinking water exposure from the sterilant 

use. 

Inhalation occupational risk was assessed for two exposure scenarios associated with its use as a 

sterilant: I) peak (one hour) exposures to N02 during opening of sterilization chamber doors or 

isolation units after treatment; and 2) average dail y (8 hour time weighted average) exposure to 

N02 potentially released into the room air resulting from multiple cycles of treatments and 

opening of sterilization chamber door(s) and isolation units during a single day. 

Toxicity endpoints were selected for N02 to perform exposure and risk assessments for 

inhalation occupational exposure (see Table 2). For the human health assessment, human 

epidemiology studies that were most relevant to the exposure scenarios for the use were selected, 

and provide the basis for selection of inhalation endpoints, with animal studies providing support 

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in the weight of evidence. Epidemiology studies were used as the basis for endpoint selection 

because the data showed a strong association between exposure to N02 and respiratory effects, 

particularly the exacerbation of asthma. Epidemiology studies also provided information on 

responses in susceptible subpopulations. 

T a e bl 2 - S ummary o fT OXICO og1ca IE • n d 1pom . t s t or N"t 1 rogen o· IOXI "d e 

Exposure Dose used in Uncertainty Study and Toxicological Effects 

Scenario risk assessment Faetor(s) 1 

Acute (I hour) I 00 ppb I Ostro et al. (200 I) and Linnet al. (2000; 

TWA astluna-related effects) 

8 hour TWA 34 ppb I Strak et al. (20 12); Delfino et al. (2002) 

(increase in respiratory symptoms) .. 1In detennmmg uncertamty factors for nsk assessment, EPA consrders both the reiatronsh1p of the response m 

experimental animals to the human response (interspecies extrapolation), and variability in response among the 

human population (intraspecies variation). In the absence of information suggesting otherwise, a I 0-fold uncertainty 

factor for interspecies extrapolation and a I 0-fold uncertainty factor for inrraspecies variation is used by EPA, for a 

total uncertainty factor of I 00, to account for these uncertainties. In this risk assessment for N02, the uncertainty 

factors were reduced based on the use of human epidemiology data as the basis for the endpoints of concern. There 

was no need for an interspccies extrapolation uncertainty factor to extrapolate from animals to humans (the 

interspecies extrapolation factor was reduced to I x), and the intraspecies variation factor (variability within the 

human population) was reduced to I x, as the endpoints of concern selected for the acute and daily TWA inhalation 

hazard were conservative, representing responses in the susceptible subpopulations of concern (asthmatics and 

ch ildren). The total uncertainty factor for this risk assessment was therefore I x. 

Human health risk conclusions from the use ofNoxilizer are summarized as follows: 

Peak Exposure. Sterilization worker inhalation exposures to N02 from opening of the 

isolator or sterilization chamber door(s) are not like ly to be of risk concern. The 

maximum measured concentration during the period of highest exposure (i.e. door 

opening) was 177 ppb for the isolator and 158 ppb for the sterili zation chamber. 

Although these values are above the acute endpoint value of 1 OOppb, they represent 

maximum exposures occuning over a span of only a few minutes from opening of the 

isolator or sterilization chamber doors. By comparison, the endpoint va lue of I OOppb 

represents a 1 hour TWA. In light of the fact that exposure from opening of isolator or 

sterilization chamber doors occurs for only a few minutes after each sterilization cycle of 

90 minutes, the average exposure over any one hour period would not likely exceed 

lOOppb. 

Average Daily Exposure. Sterilization worker 8 hour TWA exposures for the isolator unit 

is 35 ppb, which exceeded EPA's LOC of34 ppb. Potential exposure can be reduced to 

29 ppb (below EPA's LOC) ifthe room ventilation rate (air changes per hour [ACH]) is 

increased to 5 ACH on the product labeling. 

Sterilization worker 8 hour time weighted average (TWA) exposures for the sterilization 

chamber is 23 ppb, which is less than the LOC of 34 ppb and is not of concern. 

2. Assessment of Ecological Risk 

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Based on the use pattern of Noxilizer N02 Sterilant, N02 will not result in any additional 

exposure to the environment because the sterilant will be contained in the sterilization 

equipment. In addition, air scrubbers are required to be used as a safety measure to remove N02 

during the discharge process which will preclude accidental release to the environment and 

exposure to non-target organisms. The amount discharged after passing through the scrubbers is 

below ambient levels ofN02 naturally occurring in the environment. Therefore, N0 2 exposure 

from the use of this product should not cause any adverse effects to non-target organisms. All 

aquatic, avian, and terrestrial plant ecological effects studies have been waived. 

3. Assessment of Efficacy 

EPA requires efficacy data to support antimicrobial pesticide product registrations that make 

specific public health claims against microorganisms that arc infectious or pathogenic to man on 

labe ling such as sanitizer, steril ant, virucide, or disinfectant. The efficacy of Nitrogen Dioxide is 

assessed in the January 23, 20 15 document entitled, "Efficacy Review for Noxilizer N0 2 

Sterilant, EPA File Symbol 89265-R .. (see Docket 10: EPA-HQ-OPP-20 13-0792). 

The 1\0AC Sporicidal Test is required for validating sterilizing claims. Any product making 

sporicidal or sterilizing claims must submit data against spores of both Bacillus subtilis (A TCC 

19659) and Clostridium sporogenes (A TCC 35 84) on 3 product samples representing 3 different 

batches. one of which is at least 60 days old (240 carriers per sample; a total of 720 carriers). 

Any sterilizing agent (liquid, vapor, or gas) that is recommended for use in a specific device 

must be tested by the AOAC Sporicidal Test in that specific device in accordance with the 

directions for use. Sixty carriers, representing each of 2 types of surfaces (porcelain 

penicylinclers and silk suture loops), must be tested. A product must demonstrate killing on all 

720 carriers; no failures are pe1mitted. 

Validation data confim1ing that the product is efficacious as a sterilant must be conducted using 

a second, independent laboratory (other than the laboratory that developed the original data). The 

confirmatory data requires the utilization of one sample of the product to be tested against both 

Bacillus subtilis (A TCC 19659) and Clostridium sporogenes (ATCC 3584). Thirty carriers, 

representing each of the 2 types of surfaces (silk suture loops and porcelain peni cylinders) must 

be tested. A product must demonstrate killing on all 30 carriers; no failures are pennitted. 

This steril ant may only be used with Noxilizer licensed N0 2 generators or Nox ilizer branded 

Sterilizers. These two devices have a standard operating manual which calls for a 90 minute 

exposure time. Once instruments have been exposed, then the chamber has to aerate before the 

doors can be opened. 

The submitted AOAC Sporicidal Activity Test data and the Sporicidal Activity Confirmatory 

Test data demonstrated that Noxilizer N0 2 Sterilant is an effecti ve sterilant by showing 100 % 

kill or no grovvth of B. subtilis and C. sporogenes in 750 carriers (720 carriers standard 

sporicidal test data and 30 carriers from the confirmatory test data) when used only with 

Noxilizer produced or li censed equipment at 20 mg/L in N02, in a 75% pre-humidified closed 

system, following 90 minutes exposure time or greater, at room temperature, followed by an 

aeration time. 

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Regulatory Decision 

Under Section 3(c)(5) of the Federal Insecticide, fungicide and Rodentic ide Act (FIFRA), the 

Agency is unconditionally registering a new end-use product, Noxilizer N02 Sterilant, comprised 

of new active ingredients, nitrogen dioxide and dinitrogen tetroxide. The product label has been 

revised to increase the room ventilation rate to 5 ACH to mitigate the risk to humans from 

inhalation exposure to N02. The registration is summari zed below: 

89265-1 (Noxlizer N02 Sterilant) 

• 99.5% Nitrogen dioxidc/dinitrogen tetroxide 

• End-use Product, use as a gaseous sterilant in industrial, commercia l, and institutional 

settings on, laboratory equipment, isolators, medical devices, pharmaceuticals, and 

pharmaceutical equipment 

The risk assessment supporting this decision can be found in the regulatory docket (Docket ID: 

EPA-HQ-OPP-20 13-0792). 

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