ToxSci Advance Access originally published online on June 12, 2008
Toxicological Sciences 2008 105(1):79-85; doi:10.1093/toxsci/kfn117
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Interlaboratory Validation of 1% Pluronic L92 Surfactant as a Suitable, Aqueous Vehicle for Testing Pesticide Formulations Using the Murine Local Lymph Node Assay
* Toxicology & Environmental Research and Consulting, The Dow Chemical Company, Midland, Michigan 48674
Syngenta, Jealott's Hill International Research Centre, Bracknell, Berkshire, UK RG42 6EY
Syngenta Central Toxicology Laboratory, Alderley Park, Macclesfield, Cheshire, UK SK10 4TJ
Bayer CropScience Toxicology Research Centre, Sophia Antipolis, France
¶ The DuPont Company, DuPont Haskell Global Centers for Health & Environmental Sciences, Newark, Delaware
|| Experimental Toxicology and Ecology, BASF SE, 67056 Ludwigshafen, Germany
||| RCC Ltd, CH-4452 Itingen, Switzerland
|||| Human Health Assessment, Dow AgroSciences Limited, Abingdon, UK
1 To whom correspondence should be addressed at The Dow Chemical Company, Toxicology and Environmental Research & Consulting, 1803 Building, Midland, MI 48674. Fax: (989) 638-9305. E-mail: MWoolhiser{at}dow.com
Received April 30, 2008; accepted May 30, 2008
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ABSTRACT |
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The mouse local lymph node assay (LLNA) has become the preferred test for evaluating the dermal sensitization potential of chemicals and requirements are now emerging for its use in the evaluation of their formulated products, especially in the European Union. However, despite its widespread use and extensive validation, the use of this assay for directly testing mixtures and formulated products has been questioned, which could lead to repeat testing using multiple animal models. As pesticide formulations are typically a specific complex blend of chemicals for use as aqueous-based dilutions, traditional vehicles prescribed for the LLNA may change the properties of these formulations leading to inaccurate test results and hazard identification. The objective of this study was to evaluate the effectiveness of an aqueous solution of Pluronic L92 block copolymer surfactant (L92) as a vehicle in the mouse LLNA across five laboratories. Three chemicals with known sensitization potential and four pesticide formulations for which the sensitization potential in guinea pigs and/or humans had previously been assessed were used. Identical LLNA protocols and test materials were used in the evaluation. Assessment of the positive control chemicals, hexylcinnamaldehyde, formaldehyde, and potassium dichromate revealed positive results when using 1% aqueous L92 as the vehicle. Furthermore, results for these chemicals were reproducible among the five laboratories and demonstrated consistent relative potency determinations. The four pesticide formulations diluted in 1% aqueous L92 also demonstrated reproducible results in the LLNA among the five laboratories. Results for these test materials were also consistent with those generated previously using guinea pigs or from human experience. These data support testing aqueous compatible chemicals or pesticide formulations using the mouse LLNA, and provide additional support for the use of 1% aqueous L92 as a suitable, aqueous-based vehicle.
Key Words: toxicity; acute; LLNA; sensitization.
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INTRODUCTION |
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Determination of the contact sensitization potential of a chemical or its formulated product is an important component of the safety assessment process for registration, and the murine local lymph node assay (LLNA) has emerged as the preferred assay for this evaluation (EC, 2006
, 2007a; ECVAM, 2000; NIH, 1999). In the European Union, the LLNA has become the first choice method for all sensitization tests, and guinea pig tests can only be conducted for new materials when scientific justification is provided. (EC, 2007b). However, despite its widespread use and extensive validation, the use of this assay for directly testing mixtures and formulated products has been questioned (ICCVAM, 2008). A lack of uniform support for testing formulations could lead to duplicative sensitization testing with different animal models for global registration at a time when animal testing is trying to be minimized.
The LLNA is an immunologically based assay that assesses the sensitization potential of a substance by monitoring the induced proliferative response of lymphocytes in the draining lymph nodes during sensitization. The observed degree of lymphocyte proliferation has been shown to correlate closely with the sensitization potency of the test material (Cockshott et al., 2006; Kimber et al., 1994; McGarry, 2007). This assay has been extensively evaluated and validated and possesses several advantages over existing guinea pig assays including the reduction and refinement of animal use and the generation of quantitative dose response data which can be used to address sensitization potency (Basketter et al., 2007; Haneke et al., 2001; NIH, 1999; Sailstad et al., 2001).
It is well recognized that the vehicle in which a material is administered can have profound effects on the induction of sensitization due to potential alterations in test material physiochemical properties, penetration or metabolism (Andersen et al., 1985; Basketter et al., 2001a; Kligman, 1966; McGarry, 2007). These effects have been observed in mice, guinea pigs and humans and are therefore important considerations for the conduct of any skin sensitization assay. Guidelines for the conduct of the LLNA state that the vehicle should be selected to maximize the test concentrations while also maintaining a preparation suitable for dosing (OECD, 2002). Recommended vehicles in order of preference include acetone/olive oil (AOO; 4:1 vol/vol), dimethylformamide (DMF), methyl ethyl ketone, propylene glycol and dimethyl sulfoxide (DMSO) (OECD, 2002). Unfortunately, these vehicles may change the properties of end-use products that have been carefully and specifically formulated to be used as aqueous dilutions. Introduction of another chemical such as acetone or DMSO could lead to significant changes in these materials including altered solubility or physiochemical properties. These effects could compromise the ability of the assay to accurately predict the true hazard potential of the substance when compared with the actual human exposure scenario. However, test guidelines state that alternate vehicles may be utilized provided there is sufficient scientific rationale and care is taken to ensure that the vehicle adequately wets the skin and does not immediately run off as observed for wholly aqueous vehicles such as water (OECD, 2002). Previously, researchers have evaluated Pluronic L92 block copolymer surfactant (L92) as a potential alternative aqueous based vehicle for use in the LLNA (Ryan et al., 2002). L92 was chosen based on its skin wetting characteristics (ability to promote test material retention on the ear by preventing run-off), low acute toxicity profile and low irritation potential. After various performance evaluations in the LLNA, these researchers recommended the use of 1% L92 in deionized water as a vehicle for the assessment of aqueous-based test materials and indicated that it may provide a testing vehicle that is more relevant to anticipated human exposure situations.
Questions surrounding the use of the LLNA for testing the skin sensitization potential of chemical mixtures and formulations need to be resolved with scientific evidence (McGarry, 2007). As plant protection products are typically formulated for application as aqueous dilutions, testing in an aqueous vehicle is relevant for the evaluation of the human hazard potential. In this regard, 1% L92 represents a vehicle that facilitates evaluation in an aqueous solvent while providing prolonged dermal contact through its wetting properties. The objective of this study was to conduct an interlaboratory evaluation to further examine the performance of an aqueous dilution of L92 as a vehicle in the LLNA while also illustrating the performance of the LLNA with pesticide formulations. Test materials consisted of three chemicals with known sensitization potential (positive controls), and four pesticide product formulations for which the sensitization potential in guinea pigs and/or humans had previously been determined.
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MATERIALS AND METHODS |
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Participating laboratories.
Participating laboratories included The Dow Chemical Company (Midland, MI), Bayer CropScience (Sophia Antipolis, France), The DuPont Company (Wilmington, DE), BASF SE (Ludwigshafen, Germany), and Syngenta (Alderley Park, UK) whose evaluation was conducted through RCC, Ltd (Itingen, Switzerland).
Animals.
The Dow Chemical Company obtained female CBA/J mice from Harlan laboratories (Indianapolis, IN or Bar Harbor, ME). The Bayer CropScience Company obtained female CBA/JHsd mice from R. Janvier (Le Genest St Isle, France). The DuPont Company obtained female CBA/JHsd mice from Harlan Sprague Dawley (Frederick, MD). BASF used CBA/CaOlaHsd mice supplied by Harlan Winkelmann GmbH (Borchen, Germany). Female CBA mice for RCC Ltd were obtained from either Harlan Netherlands (AD Horst, The Netherlands; CBA/CaOlaHsd) or RCC, Ltd (Füllinsdorf, Switzerland; CBA/CaHsdRcc [SPF]). For studies conducted at Dow and DuPont, mice were housed, fed and handled in compliance with standards set forth by the U.S. animal welfare act or recommendation in the National Institutes of Health "Guide for the Care and Use of Laboratory Animals." The procedures performed on the mice were reviewed and approved by a veterinarian and the Institutional Animal Care and Use Committee. Studies at Bayer were conducted in accordance with the regulations of the "Guide for the Care and Use of Laboratory Animals" (Public Health Service, NIH publication N° 86-23, revised 1985) and "Le Guide du Journal Officiel des Communautés Européennes L358, 18 December 1986, n° 86/09/CEE du 24 November 1986." BASF conducted the studies in an AAALAC-approved laboratory in accordance with the German Animal Welfare Act and the European Council Directive 86/609/EEC. Studies conducted at RCC were performed in an AAALAC-approved laboratory in accordance with the Swiss Animal Protection Law under license no. 114.
Chemicals.
-Hexylcinnamaldehyde (HCA), potassium dichromate (PDC), and formaldehyde (FORM; 37% solution) were purchased from Sigma Chemical Co. (St Louis, MO). Oxyfluorfen emulsion concentrate (EC), Dinocap EC, Quinoxyfen/Cyproconazole suspension concentrate (SC), and Trifluralin EC were provided by Dow AgroSciences LLC (Indianapolis, IN). Pluronic L92 block copolymer surfactant (L92) was purchased from BASF (Mount Olive, NJ).
Local lymph node assay.
The LLNA was conducted as described previously (Gerberick et al., 2000; Kimber et al., 1995; Loveless et al., 1996). Doses for all test materials were selected to avoid overt toxicity including moderate/severe irritation and significant effects on body weight. Groups of mice (n = 5) were treated by topical application on the dorsal surface of both ears with 25 µl of each test material or with vehicle alone for three consecutive days. The mice were allowed to rest for two days. On day 6, the mice were injected via the tail vein with 250 µl of phosphate-buffered saline (PBS) containing 20 µCi (7.4 x 105 Bq) of [methyl-3H]thymidine (3H-TdR). Five hours later the mice were euthanized via carbon dioxide inhalation and the draining auricular lymph nodes were excised and placed in 10 ml of PBS for each individual mouse. Single-cell suspensions of LNCs were prepared, cell suspensions were washed twice with an excess of PBS, precipitated in 3 ml of 5% trichloroacetic acid (TCA) and refrigerated at 4°C for at least 18 h. The samples were then centrifuged, resuspended in 1 ml of 5% TCA and mixed with scintillation cocktail. Incorporation of 3H-TdR was measured by β-scintillation counting and expressed as disintegrations per minute (dpm) per mouse. Mean dpm with standard deviation was calculated for each experimental group. The stimulation index (SI) was calculated using the absolute dpm value for each mouse as the numerator and the mean dpm value from the vehicle control mice as the denominator; the mean SI and standard deviation was calculated for each experimental group. A test material that, at one or more concentrations, caused a three-fold or greater increase in proliferation was considered to be positive in the LLNA. An estimate of the sensitizing potency of the test material formulations was determined by calculation of an EC3 value (the estimated concentration required to induce a threshold positive response). The calculation of the EC3 values was carried out by linear interpolation according to the equation:
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RESULTS |
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Positive Control Chemicals
FORM, HCA, and PDC were chosen as positive control chemicals for this evaluation due to their extensive use as positive controls in previous LLNA evaluations. In addition, these chemicals are largely compatible with aqueous solvents and therefore their responses in the assay will not be influenced by altered solubility or changes in physiochemical properties when diluted in aqueous 1% L92. The LLNA results for these chemicals with L92 as a vehicle are presented in Table 1. FORM was evaluated at concentrations of 1, 5, and 20% and yielded positive responses (SI > 3) in each of the five laboratories. Evaluation of the sensitization potency of FORM across the laboratories was conducted through the comparison of EC3 values which revealed consistent potency determinations with values ranging from 3.8 to 12.3%. HCA was evaluated at concentrations of 3, 10, and 30% and also yielded positive responses in each of the five laboratories with EC3 values ranging from 6.7 to 17.6%. Finally, PDC was evaluated at concentrations of 0.02, 0.1, and 0.5% and tested positive for sensitization in each laboratory with EC3 values ranging from 0.1 to 0.3%. The positive response for each of these chemicals and the similarity of the EC3 values indicate the reliability and consistency of the LLNA across independent laboratories when L92 is used as a vehicle.
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Plant Protection Products
Dinocap EC, Oxyfluorfen EC, Quinoxyfen/Cyproconazole SC, and Trifluralin EC, which represent herbicides and fungicides that are applied as aqueous dilutions, were used in the evaluation of L92 as a vehicle in the LLNA with plant protection products. Results of the interlaboratory evaluation with these materials are presented in Table 2. As was observed with the positive control chemicals, there was good agreement across the five laboratories for the identification of the sensitization potential and potency for each of the four plant protection products. Dinocap EC was positive for sensitization (EC3 > 3) in each of the laboratories with EC3 values ranging from 0.85 to 3%. Trifluralin EC and Quinoxyfen/Cyproconazole SC also tested positive for sensitization in all five laboratories with EC3 values ranging from 5.8 to 16% and 9.7 to 50%, respectively. Oxyfluorfen EC was tested at concentrations of 1, 7, and 33% and yielded positive results in three of five laboratories with EC3 values of 18.1, 18.9, and 31%. Although this product did not test positive in the remaining two laboratories, the DPM values were dose responsive and the stimulation indices at the high dose were 2.9 and 2.3 indicating that they were approaching the threshold for a positive response. By comparison, examination of the stimulation indices at the top dose for the three laboratories that yielded positive results for Oxyflurofen EC reveals values of 3.1, 4.9, and 5.4 which are also near the threshold for a positive assay response. Therefore, the similarity of the response to this material is still evident and supports the overall consistency of the data between the five laboratories for these four plant protection products.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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As certain chemicals cause contact dermatitis in humans, evaluation for dermal sensitization potential remains an important component of the hazard assessment process for registration of chemicals or their formulated products. Unfortunately, the accurate prediction of this potential cannot yet be performed using in silico or in vitro methods; therefore, the use of laboratory animals remains necessary. The LLNA has emerged as the preferred assay for assessing the contact sensitization potential of chemicals due to its reduction and refinement of animal use, its quantitative, dose-response endpoint, and its extensive evaluations for reliability in interlaboratory trials (Cockshott et al., 2006
; McGarry, 2007). Despite this, the use of this assay for directly testing mixtures and formulated products has been questioned (ICCVAM, 2008). However, as a mechanistically-based assay with a quantitative endpoint, the LLNA possesses robustness and flexibility which allow it to be used for researching parameters which may influence the potential for contact sensitization as well as for the assessment of complex materials or formulations (Basketter et al., 2002). In addition, the dose-response component of the assay offers the ability to estimate the relative potency of test materials which presents opportunities for more extensive and accurate human hazard evaluations (Basketter et al., 2005, 2007). A number of reports have already examined vehicle effects in the LLNA and have indicated that the results of the assay may be influenced by the vehicle used (Andersen et al., 1985; Basketter et al., 2001a; Lea et al., 1999), an observation which is consistent with guinea pig responses, as well as data from human experience (Kligman, 1966). Furthermore, it is realized that organic vehicles may alter the physiochemical properties of the test material thereby yielding results that do not accurately reflect the true human hazard potential (Ryan et al., 2002).
Regulatory guidance currently requires sensitization data for active ingredients as well as formulated products; therefore sensitization testing is required for complex test materials or formulations. In these instances, characterization of the sensitization potential for individual chemical components may not necessarily define the hazard of a final product or its aqueous dilutions. As plant protection products are typically complex formulations that are applied as aqueous dilutions, these materials ideally require an aqueous vehicle in the LLNA. The advantages of the mouse LLNA make it very desirable to test formulated products while offering the benefits of a consistent testing paradigm between individual components and the final formulation (Kimber et al., 2001). Therefore, to address the need for an aqueous vehicle in the LLNA and to assess assay performance with formulations, an interlaboratory evaluation was conducted to with 1% L92 in deionized water as the vehicle.
The use of 1% L92 as a vehicle in the LLNA has previously been investigated and was chosen for further evaluation as it possesses good skin wetting characteristics, low acute toxicity, low irritation potential, and did not induce lymph node proliferation at concentrations up to 50% (Ryan et al., 2002). In the case of the positive control chemicals, comparison of the EC3 values and their variability across laboratories reveals notable consistency to previously conducted LLNA evaluations for these substances including interlaboratory evaluations that employed organic vehicles such as AOO, DMF, and DMSO (Table 3) (Basketter et al., 2001b, 2007; Haneke et al., 2001; Hilton et al., 1998; Kimber et al., 1991, 1995; Loveless et al., 1996). Ryan et al. (2002) previously evaluated both FORM and PDC in 1% L92 and reported EC3 values of 4.2 and 0.17%, respectively, which are similar to the determinations of 7.6 and 0.2% observed in this study. Furthermore, comparison to published data conducted with organic solvent vehicles reveals similar potency and variability of the EC3 response for both HCA and PDC (Basketter et al., 2007; Kimber et al., 1995). In the case of FORM, previously published reports using organic solvent vehicles report an EC3 near 1% indicating a somewhat higher potency determination compared with the present evaluation (Basketter et al., 2001b; Hilton et al., 1998), however, this difference is consistent with the data of Ryan et al. (2002). It is well recognized that vehicles such as AOO and DMSO may enhance the skin penetration of chemicals, which may account for the higher EC3 values for FORM. Other factors may also influence the response including induction of danger signals, alterations in metabolism and altered evaporation from the skin surface (Basketter et al., 2001a). Therefore, although the potency may be greater with organic vehicles, the response with the aqueous vehicle may more accurately represent the end-use sensitization potential, provided the intended use involves aqueous-based applications. Overall, these data indicate the general similarity in potency and variability for these materials when compared with previously published reports using either L92 or organic solvents as vehicle.
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The results for the four plant protection products were largely consistent with that reported in guinea pig evaluations and from human experience (Table 4). The positive sensitization results for Dinocap EC and Quinoxyfen/Cyproconazole SC in the interlaboratory evaluation are consistent with the positive responses in both guinea pigs and humans (Chowdhury et al., 2001
; Larson et al., 1959). Oxyflurofen EC is a borderline negative for sensitization classification using a guinea pig maximization test and no evidence has been reported to suggest sensitization potential in humans. However, the results in the guinea pig assay are near the threshold for a positive response (26% responding versus the 30% required for a positive classification), suggesting the formulation may not be void of sensitization potential which is consistent with the borderline results for this material in the LLNA interlaboratory evaluation (SI values ranging from 2.3 to 5.4). In the case of Trifluralin EC, although a negative response was observed in the Buehler guinea pig assay, a positive response was noted in the current LLNA interlaboratory evaluation which is consistent with human evidence for sensitization potential (Pentel et al., 1994). Furthermore, the Buehler assay for Trifluralin EC yielded a 10% response which is approaching the 15% required for a positive response in this assay. Therefore, when objective comparisons are made, which go beyond the simple descriptive classification criteria, the responses across the LLNA and guinea pig assays are comparable for these formulations, and are consistent with the human experience when available. Collectively, these data confirm the consistency of the LLNA response with formulations across laboratories when L92 is used as a vehicle and highlight the concordance with results observed in guinea pig assays and with human experience.
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In summary, the present study has further evaluated the utility of 1% L92 as an alternative vehicle in the LLNA while also illustrating the performance of the LLNA with pesticide formulations. The results with the positive control materials and plant protection products support the use of L92 for testing aqueous compatible materials, including plant protection products. The use of this vehicle is consistent with the provisions outlined in the OECD guidelines which allow for alternate vehicles to be utilized provided there is sufficient scientific rationale and care is taken to ensure that the vehicle adequately wets the skin and does not immediately run off (OECD, 2002
). Ultimately, if the goal is to accurately assess the sensitization potential of a material or formulation for a particular application, the vehicle used should closely simulate the anticipated exposure situations. In this regard, this study demonstrates that 1% L92 provides a valuable alternative vehicle for the LLNA that is more relevant for the accurate assessment of the hazard potential of aqueous test materials. Furthermore, this study provides data to support the use of the LLNA for assessing the contact sensitization potential of pesticide formulations. |
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European Crop Protection Association.
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