Acute Toxicity Six-Pack
EPA-Compliant Testing Services for FIFRA and TSCA
The EPA 6-Pack is a standardized suite of six acute toxicity tests historically required by the U.S. Environmental Protection Agency (US EPA). These tests are essential for assessing the human health risks of chemicals, especially for regulatory compliance under the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) and the Toxic Substances Control Act (TSCA). The 6-Pack evaluates short-term chemical exposure hazards through both in vivo methods and increasingly adopted New Approach Methodologies (NAMs), including in vitro and non-animal testing alternatives.
With over 50 years of experience, MB Research has in-depth knowledge of the principles and practices of performing EPA six-pack studies and a strong track record of generating reliable, GLP-compliant safety data. In alignment with the EPA’s directive to reduce animal testing, MB Research’s in vitro toxicology specialists are leaders in applying validated non-animal methods that meet current regulatory expectations.
Quick Jump:
Oral Toxicity
This study evaluates the acute oral toxicity of a test substance by determining the oral median lethal dose (LD50). The testing approach follows the Up‑and‑Down Procedure (UDP), which involves staggered, sequential dosing. Based on the observed outcome (mortality or survival), the next dose is adjusted upward or downward.
The oral LD50 and 95% Confidence Limits are calculated using the AOT425StatPgm software provided by the U.S. EPA. Compared to traditional methods, this approach provides a statistically valid LD₅₀, using fewer animals.
Regulatory harmonization: This test method is harmonized with U.S. and international regulatory guidelines, allowing broad acceptance of resulting data. MB Research’s protocol supports regulatory submissions and classification under the following frameworks:
- EPA: Acute toxicity categorization (OSCPP 870.1000)
- GHS: Acute toxicity hazard classification under the Globally Harmonized System of Classification and Labelling of Chemicals
- CPSC: Toxicity determination for consumer products (16 CFR Part 1500)
- DOT: Hazardous material determination and packing group assignment (49 CFR Parts 171-180)
Dermal Toxicity
The purpose of this study is to determine the potential for toxicity of the test article when applied dermally for a 24-hour exposure. This study is designed to comply with the standards set forth in the current U.S. Environmental Protection Agency (EPA) Health Effects Test Guideline OCSPP 870.1200: Acute Dermal Toxicity and U.S. Department of Transportation (DOT) 49 CFR 173.132(b)(2).
Inhalation Toxicity
The purpose of this study is to provide information on health effects that may arise from short term exposure by the inhalation route. This study is designed to comply with the standards set forth in U.S. Environmental Protection Agency (EPA) Health Effects Test Guideline, OCSPP 870.1300 and The Organization for Economic Co-operation and Development (OECD) Guideline for the Testing of Chemicals No. 403.
Eye Irritation/Corrosion
This study assesses the potential of a test article to cause irritation or corrosion to the eye by evaluation for up to 21 days following a single exposure. The intent of this study is to evaluate test articles that have been identified as not severely irritating or corrosive to the eye according to the OECD-recommended method: the Bovine Corneal Opacity and Permeability (BCOP) Assay.
Replacement Assays For Ocular Irritation/Corrosion
The purpose of this study is to provide classification of chemicals concerning their eye irritation potential using an alternative to the Draize Rabbit Eye Test, according to the OECD Test Guideline No. 492, “Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage”. The EpiOcular™ EIT is intended to differentiate those materials that are UN GHS No Category (i.e., do not meet the requirements for UN GHS classification) from those that would require labeling as either UN GHS Category 1 or 2.
Freshly harvested bovine corneas from the eyes of cattle are utilized in the BCOP test method in order to evaluate the ocular irritancy and corrosive potential of a test chemical by assessing quantitative measurements of:
(1) Changes in corneal opacity, measured with an opacimeter
(2) Corneal permeability, measured by a visible light spectrophotometer
Both measurements are used to calculate an In Vitro Irritation Score (IVIS), which is used to assign a classification category.
The objective of this study is to determine the potential for ocular irritation using OECD Guidelines for the Testing of Chemicals, Nos. 492 and 437, which serve as alternatives to the traditional Draize methodology. The use of two internationally-validated, OECD-accepted non-animal tests can provide a GHS categorization for the vast majority of test articles. The combination of the Bovine Corneal Opacity and Permeability Test and EpiOcular™ Eye Irritation Test followed by a Weight-of-Evidence analysis of the results allows for the classification of GHS Category 2.
The Irritection® Assay System uses an in vitro method to determine ocular irritation and predict U.N. GHS classification for chemicals or mixtures. This study is designed to comply with the standards set forth in the OECD Guideline for the Testing of Chemicals No. 496. For more information, please click here.
Skin Irritation/Corrosion
The in vivo acute dermal irritation test (Draize rabbit test) evaluates the potential of test substances to cause skin irritation or corrosion following a single topical exposure. The test assesses the severity, duration, and reversibility of skin reactions across three time-points: 3-minute, 1-hour, and 4-hour exposures. Skin responses are observed for up to 14 days post-exposure to determine whether the effects are reversible or permanent.
MB Research’s protocol supports regulatory harmonization with U.S. and international regulatory guidelines, allowing global acceptance of resulting data and classification under the following frameworks:
- DOT: If irreversible damage (full-thickness destruction) in at least one animal at any exposure duration, the substance will be assigned to a packing group based on the following criteria set forth in DOT 49 CFR 173.137 Class 8.
- EPA : The Toxicity Category (Skin Irritation Categorization) will be determined based on the severity of the skin reactions following the four-hour exposure (OSCPP 870.1000).
- Primary Irritation Index (PII): Erythema and edema scores following the four‑hour exposure will be used to determine the PII and a response category will be assigned.
- GHS Skin Irritation Classification: The test article is classified under the Globally Harmonized System for skin irritation and corrosion labeling.
- EU CLP Classification: Results support classification under the European Union’s Classification, Labelling and Packaging (CLP) Regulation.
New Approach Methodologies (NAMs) for Skin IRRITATION/CORROSION
Non-animal methodologies and approaches have been increasingly used to assess the potential for skin irritation and corrosion. Defined approaches can be implemented using different testing strategies, such as the “top-down” approach (starting with skin corrosion testing) or the “bottom-up” approach (starting with skin irritation testing), depending on the substance’s hypothesized hazard.
In Vitro Replacement for Dermal Irritation
The purpose of this study is to provide classification of the dermal irritation potential of chemicals by using a three-dimensional human epidermis model, according to the OECD Guideline for the Testing of Chemicals No. 439, “In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method”. The EpiDerm™ SIT allows discrimination between irritants (Category 2) and non-irritants, in accordance with U.N. GHS classification.
In Vitro Replacement for Dermal Corrosion
The purpose of this study is to provide classification of the dermal corrosion potential of chemicals by using a three-dimensional human epidermis model, according to the OECD Guideline for the Testing of Chemicals No. 431, “In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method”. The EpiDerm™ SCT allows discrimination between non-corrosive and corrosive substances in accordance with U.N. GHS classification. Corrosivity classification can be further divided into either optional Sub-category 1A or a combination of Sub-categories 1B and 1C.
The Corrositex® assay is performed using a kit produced and distributed by In Vitro International. The kit contains tubes of proprietary buffers, a Chemical Detection System (CDS), and components used to make synthetic proteinaceous macromolecular bio‐barriers. After preliminary testing to determine if the test article is compatible with the Corrositex® assay (qualification) and to determine cut‐off times (categorization), the test article is applied topically on prepared bio‐barriers set atop vials of CDS. The amount of time it takes for the test article to penetrate the bio‐barriers and cause a visually detectable change in the CDS (breakthrough time) can be used to determine the UN Packing Group or the UN GHS subcategory (classification).
Dermal Sensitization
The Buehler Test is a standardized, non-adjuvant in vivo skin sensitization assay used to assess a substance’s potential to induce allergic contact dermatitis (ACD), a type IV delayed-type hypersensitivity reaction.
During the Induction Phase, the test substance is topically applied to the test group once weekly for three consecutive weeks (each application lasting six hours). After a rest period of 10 to 14 days, a challenge application is conducted on both the induced and control groups. Dermal reactions in the induced group that exceed those in the control group are considered evidence of sensitization. A test substance is considered a sensitizer if at least 15% of the induced group exhibit a positive dermal response.
This method aligns with OECD Test Guideline 406, facilitating global acceptance of data. MB Research’s validated protocol supports regulatory submissions and hazard classification.
Contact dermal sensitization is an immunological process where the host animal, through repeated skin exposure, acquires a specific allergic sensitivity to a substance. In the Guinea Pig Maximization (GPMT) model, contact dermal sensitivity is manifested as increased erythema.
The purpose of this study is to determine the sensitizing potential of topically applied test articles. This LLNA protocol, utilizing the BrdU ELISA method, is designed to be a stand-alone assay for dermal sensitization as described in the NIH report “The Murine Local Lymph Node Assay: A Test Method for Assessing the Allergic Contact Dermatitis Potential of Chemicals/Compounds,” NIH No. 99-4494, and the LLNA test guidelines as defined in EPA OCSPP 870.2600 and OECD Guideline for the Testing of Chemicals No. 442B.
New Approach Methodologies (NAMs) for Skin Sensitization
OECD-approved non-animal test guidelines support the evaluation of skin sensitization by targeting Key Events in the Adverse Outcome Pathway (AOP). These New Approach Methodologies (NAMs) are used in an Integrated Testing Strategy (ITS) that follows a “2 out of 3” approach; at least two out of the three validated assays must provide concordant results to classify a substance as a skin sensitizer.
Key Event 1 (KE 1): The DPRA is an in chemico method used to evaluate the skin sensitization potential of chemicals. Specifically, the assay measures the binding of test substances to synthetic peptides containing either cysteine or lysine, mimicking the molecular initiating event in the skin sensitization adverse outcome pathway (AOP). The test substance is incubated with synthetic peptides for 24 hours. High-performance liquid chromatography (HPLC) with UV detection is then used to quantify peptide depletion. The percentage of peptide depletion is calculated and interpreted using a defined prediction model that categorizes the chemical into one of four reactivity classes, which supports hazard identification as a potential skin sensitizer.
Key Event 2 (KE 2): The KeratinoSens™ assay is an in vitro method that measures activation of Keap1-Nrf2-ARE pathway. These tests use an immortalized, adherent, human keratinocyte cell line (HaCaT) that was transfected with a plasmid to monitor luciferase gene induction, which can provide quantitative measurement of gene expression changes.
Key Event 3 (KE 3): The human Cell Line Activation Test (h-CLAT) is an in vitro assay designed to assess skin sensitization potential of chemicals. It measures the expression of cell surface markers CD86 and CD54 on THP-1 cells, a human monocytic leukemia cell line, following exposure to a test substance. Changes in the expression levels of these dendritic cell activation markers are quantitatively evaluated using flow cytometry.
Frequently Asked Questions
What are the EPA Toxicity Categories?
The EPA Toxicity Categories are a system used by the U.S. Environmental Protection Agency (EPA) to classify the acute hazards of pesticide products and chemicals. They help determine labeling requirements, precautionary statements, and first aid instructions under FIFRA (Federal Insecticide, Fungicide, and Rodenticide Act) and 40 CFR Part 156.
There are four categories:
Category Signal Word
Category I DANGER
Category II WARNING
Category III CAUTION
Category IV None or “CAUTION”
Does the EPA allow any of the 6-Pack toxicity tests to be waived?
Yes. The EPA may allow waivers for certain six-pack acute toxicity tests when the endpoint is not relevant, such as omitting an acute oral toxicity study for a pesticide that exists solely as a vapor or gas. Waivers may also be granted if existing data from similar formulations can be scientifically justified through bridging, read-across, or weight-of-evidence approaches. All waiver requests must be submitted in writing to the EPA, following the format and requirements outlined in PR Notice 2011-3 and 40 CFR § 158.45.
Learn more about the EPA’s Bridging or Waiving Data Requirements.
Does the EPA require GLP testing?
Yes, the U.S. Environmental Protection Agency (EPA) generally requires GLP-compliant testing under 40 CFR Part 160 (for FIFRA) and 40 CFR Part 792 (for TSCA) to support pesticide registration, chemical safety evaluations, and risk assessments.
