ASTM D1076 Category 4 Latex and Quantifying Guayule (NRG) and Hevea (NR) Latex Protein

Katrina Cornish1, Colleen M. McMahan2, Wenshuang Xie2, Jali Williams1,

K.C. Nguyen1, David Kostyal3, Kelly Horton3, D. Thomas Marsh4, 1Yulex Corporation, 1945 Camino Vida Roble, Suite C, Carlsbad, CA 92008, USA 2USDA-ARS, Western Regional Research Center, 800 Buchanan Street, Albany, CA 92710, USA 3Laboratory of Molecular Immunology, Guthrie Foundation for Education and Research,

1 Guthrie Square, Sayre, PA 18840, USA 4Centrotrade Rubber, 5700 Cleveland Street, Suite 440, Virginia Beach, VA, 23462 USA

Abstract: Guayule latex is commercially available as a low protein natural rubber latex (Yulex® latex) which does not contain any protein that can be detected by the ASTM D6499 antigenic protein standard developed to quantify Hevea natural rubber latex (NRL) antigenic protein. In this paper, we discuss how best to quantify the proteins in guayule latex and Hevea latex using a minor modification of the modified Lowry procedure described in ASTM D5712 and compare this to quantification of proteins extracted from guayule and Hevea latex films. We also address the development of guayule-specific immunochemical methods to determine latex purity and to accurately quantify very small amounts of extractable protein from products. Introduction: Guayule latex (Yulex® latex) has entered the commercial arena as an alternative natural rubber latex suitable for the manufacture of latex medical products that will not trigger allergic reactions in patients with Type I IgE-mediated Hevea latex allergies. It is well established that guayule latex contains very little protein compared with tropical Hevea latex and far fewer different proteins1-3. Even though high ammonia levels hydrolyze Hevea latex protein and reduce the endogenous level, ammoniated guayule latex contains only about1% of this hydrolyzed level (Figure 1). The method by which guayule latex is produced ensures that soluble proteins are washed from the latex during the purification process to undetectable levels. The remaining protein is hydrophobic and associated with the rubber particle membranes. This small amount of residual hydrophobic protein is not readily leached from the rubber matrix, and so is not likely to induce Type I allergies of its own. Mouse and rabbit trials and human clinical trials, including ELISA (enzyme-liked immunosorbent assay), 1-D and 2-D immunoblots, skin-prick tests, RAST (radioallergosorbent) assays, and CAP assays of allergenic protein levels, have demonstrated that guayule latex proteins do not cross-react with anti-Hevea latex protein antibodies at concentrations at least 1000x the amount of protein sufficient to cause a response to Hevea proteins2-9. Reciprocal tests using animal antibodies (mice and rabbits) also demonstrated that antibodies specifically raised against extracted and concentrated guayule latex proteins do not cross-react with Hevea latex proteins6,9.

Natural and synthetic latex products also are known to induce contact sensitivity (or

hypersensitivity) (Type IV) immune reactions which, although not life-threatening, are undesirable. Such reactions are most commonly caused by the chemical accelerators added to latex during product manufacture. However, until recently, it was not known if guayule latex itself had any intrinsic ability to cause irritation or contact-sensitization of product users. It has now been demonstrated that guayule latex films (powder-free examination gloves) are neither irritating nor sensitizing (Repeated Patch Dermal Guinea Pig Sensitization Test - Buehler method modified for medical devices, and ISO 10993-10 2002, “Biological Evaluation of Medical Devices, Part 10, Tests for Irritation and Delayed-Type Hypersensitivity”10.

In addition, while guayule latex glove films have similar tensile properties to Hevea, guayule

latex films have higher elongation to break and lower modulus values than Hevea 11,12. Many earlier studies demonstrated excellent tensile strength and that examination glove and condom films provide effective barriers to the transmission of human pathogenic viruses13.

Thus, dipped guayule latex products have elasticity, form, fit and feel superior to existing

natural and synthetic rubber products, can meet tensile strength standards for natural rubber, exceed those for synthetics, are neither irritating nor sensitizing in Type IV contact tests, and contain no proteins that cross-react with Type I latex allergy. ASTM D1076-06 and the New Category 4 Latex The recently published ASTM D1076-06 standard (Standard Specification for Rubber – Concentrated, Ammonia Preserved, Creamed, and Centrifuged Natural Latex) retains, unchanged, the three lattices defined in D1076-02 as Types 1, 2 and 3, except that it now calls them Categories instead of Types. These first three categories all measure physical performance only, not protein content, and apply solely to Hevea-based raw materials derived from tropical rubber tree plantations mainly in Southeast Asia. In addition, D1076-06 adds a fourth Category

Figure 1. Log protein content in latex from three sources.

which includes guayule latex, and for the first time, also sets limits on the amount and type of protein that can be contained in a Category 4 latex.

The formal definitions of the four categories in D1076-06 are as follows: Category 1–Centrifuged Hevea natural latex preserved with ammonia only or by formaldehyde

followed by ammonia. Category 2–Creamed Hevea natural latex preserved with ammonia only or by formaldehyde

followed by ammonia. Category 3–Centrifuged Hevea natural latex preserved with low ammonia with other necessary

preservatives. Category 4–Centrifuged, or centrifuged and creamed, guayule latex, or other natural rubber

latex, containing less than 200 µg protein per gram dry weight of latex, with ammonia or other hydroxide, with other necessary preservatives and stabilizers.

In addition, the underlying specification of a Category 4 latex also requires that such a latex

contains no protein that can be detected by ASTM D6499-03 Standard Test Method for Immunological Measurement of Antigenic Protein in Natural Rubber and its Products. This method is based on a rabbit polyclonal antibody raised against protein extracted from Hevea NRL glove films and so, at first sight, may seem somewhat anomalous appearing in the Category 4 specification. However, Category 4 is intended to provide a source of latex that is safe for people suffering from Type I latex allergy. A lack of cross-reactivity in D6499 ensures both the intrinsic safety of a Category 4 latex and that the latex has not become contaminated with Hevea latex as has occasionally happened, for example, with nitrile latex and articles produced in facilities which also produce Hevea latex articles. The low protein requirement provides the extra safety feature of very low protein exposure during product use, which should greatly reduce the chances of a user becoming allergic to a Category 4 latex or latex product. It is clear that if high levels of protein are not present in the raw material they cannot appear in the manufactured product.

At the present time, guayule latex, marketed as Yulex® natural rubber latex, is the only natural rubber latex that meets the new Category 4 standard, and, therefore, provides a new level of materials safety for medical product manufacturers. Protein Measurement Latex Protein The D1076-06 standard contains a brief description of how the protein quantification method described in the ASTM D5712-05 Standard Test Method for Analysis of Aqueous Extractable Protein in Natural Rubber and Its Products Using the Modified Lowry Method may be applied to quantify protein in latex. The intent behind measuring the amount of protein in a latex is to quantify to the total amount of protein in the raw material. This is a worst case value – no more that this amount can possibly expose an end-user through either extraction or through surface contact. However, through applying this method to a wide array of lattices we find that certain aspects of the latex protein quantification method require particular attention for a successful result. As more lattices apply for entry into Category 4 it seemed worthwhile to expound on the latex protein quantification method in this paper.

Methods and Materials Protein quantification using a Lowry modified from ASTM 5712 Several different guayule latex samples were provided from the Yulex pilot plant, and compared with a sample of synthetic polyisoprene latex and samples of ammoniated Hevea latex. To determine the amount of protein in the latex, the latex proteins are first solubilized in 1% SDS and 50 mM sodium phosphate buffer (final concentration) and then quantified using the modified Lowry test according to ASTM D 5712. As described in D 1076, three latex samples (500 µl each are mixed with 450 µl 100 mM sodium phosphate buffer in a microfuge tube; 50 µl 20% SDS is added to each tube and mixed. The mixture is then incubated at 25 °C for 2 hours with shaking at 200 rpm, spun for 5-15 minutes, the rubber pad removed, then the aqueous phase transferred into new tubes and spun again until clarified. Each sample is then divided into 3 x 0.6 ml aliquots in microfuge tubes (these can be stored at 4°C overnight). Bovine serum albumin protein standards are prepared in extraction buffer at 0, 5, 10, 15, 25, 50, 100, 200, 300, 400 ug/ml. Sixty µl 1.5 mg/ml sodium deoxycholate is added to samples and standards, mixed, and allowed to stand for 10 minutes. Then 120 µl of 72% freshly mixed trichloroacetic acid and phosphotungstic acid (1:1) are added to each sample, mixed, and incubated for 30 minutes at room temperature. The samples are spun 15 minutes at 6000 xg, and the supernatant removed. The pellet is air dried and resuspended in 250 µl 0.2 M sodium hydroxide. These samples can be stored at 4°C until assayed. Samples should be assayed within 24 hours using the modified Lowry test according to ASTM D 5712. High solids content lattices present a challenge in obtaining the clean extracts needed for accurate results. Dilution of the high solids latex 10-fold (with DDW) prior to extraction results in lower solids material in the supernatant following centrifugation. D 1076 does not specify the centrifuge speed to be used; we have found high speed 12,000-14,000 rpm produces a clear subnatent without evidence of precipitate. Nevertheless, multiple transfers (2-3) of subnatent to fresh tubes yields the best, cleanest extract. The diluted latex (500 µl) can be mixed with 450 µl buffer and 50 µl SDS and tested per the stated method with good results, especially for low protein level materials. The specified standard curve concentration series can, however, be problematic because, as D 5712 points out, there is significant nonlinearity beyond 100 µg/ml protein. We have found addition of standards at 1 ug/ml and 2 µg/ml give better definition to the low concentration end of the curve; typically the 200-400 µg/ml standards are not needed, or can be introduced for measurement of high protein concentration materials. Because of the high concentration of protein in Hevea latex, we determined that the quantification methods worked better, and were more reproducible when the latex was first diluted 10-fold – by either 0.5 ml latex with 4.5 ml buffer or by 1 ml latex in 10 ml buffer in 50 ml centrifuge tubes - and shaken for 2 hours at 23-25 °C at 200 rpm. 3 x 1 ml of each sample were taken and spun at 14,000 rpm for 5 minutes and the aqueous protein solutions were placed into new tubes. 400 µl of each were precipitated as previously described (each with triplicates). When these samples were quantified and compared to the BSA standard, or to the guayule or polyisoprene latex, after each protein pellet was suspended in 480 µl NaOH, 240 µl (1/2 volume)

of each non-Hevea sample was assayed but only 24 µl (1/20 vol.) of each Hevea sample, in order to keep the results on the linear portion of the standard curves. Per D 5712, the NaOH solvent volume used for samples vs standards must be accounted for in the calculations. It is also necessary to assure the extract/Reagent C and extract/Reagent D ratios remain constant. In other tests Hevea latex samples were treated with different solubilization methods (final concentrations stated): water only, 50 mM sodium phosphate buffer only (pH 7.4), 50 mM PBS (sodium phosphate buffer +150 mM sodium chloride) with 1%SDS, and compared with the cytosolic fraction in water. In these experiments, 0.5 ml latex samples were mixed with 4.5 ml diluent and shaken for 2 hours at 200 rpm at 21-22°C. The cytosolic fraction was prepared as described above. Protein quantification using Lowry ASTM 5712 Cis-1,4-Polyisoprene lattices from Hevea, guayule, and synthetically-produced materials were analyzed for protein content by both the standard Lowry D 5712, and by the new test, D 1076/D 5712. Thin (~0.5 mm) film specimens were prepared by pouring uncompounded latex into clean glass petri dishes. The films were allowed to dry overnight, then extracted with PBS buffer per D 5712. The same lattices were diluted 10:1 and extracted with sodium phosphate buffer/SDS per the D 1076 method described above. SDS-PAGE Latex samples were mixed with gel loading buffer (10% glycerol, 141 mM Tris, 106 mM Tris-HCI, 2% LDS, 0.51 mM EDTA, 0.22 mM SERVA Blue G250, 0.175 mM Phenol Red) and 50 mM DTT, then denatured at 95°C for 10 minutes, then vortexed and clarified by centrifugation. The proteins in aqueous phase were loaded onto gels. Protein samples from latex cytosolic fractions (i.e. the soluble latex proteins, also known as C-serum protein) were prepared as follows. Latex was mixed with water (1:1) and centrifuged immediately at 14,000 rpm for 5 minutes to float and coagulate the rubber particles, and the aqueous phase was passed through a 0.2 µm filter to remove remaining particulates. A cytosolic sample was mixed with gel loading buffer, denatured at 95°C for 10 minutes, and then loaded and run on the gel. The volume loaded onto the gel was equivalent to the comparable latex sample, i.e. the same amount of cytosolic protein would have been in the cytosolic fraction and in the matched latex lane. Protein samples were run on 4-12% Bis-Tris gel from Invitrogen. Running conditions were approximately 35 minutes at 200 volts, using a 1x running buffer of 50 mM MES, 50 mM Tris, 0.1% SDS, 1 mM EDTA, pH 7.3. The gels were then fixed and stained with either Coomassie overnight and then destained in water, or with silver (Bio-Rad, Hercules, CA, silver stain kit).

Guayule-specific ELISA Development Proteins were prepared from guayule plant homogenate and from purified guayule latex as follows.

1. 50 ml of homogenate was centrifuged to remove residual solids. After spinning, the liquid was decanted from the tube

2. 25 ml each of spun homogenate and latex were pipetted into 50 ml tubes, to which 25 ml PBS buffer was added

3. Each solution was shaken on an orbital shaker for 2-hours 4. Both extracts were centrifuged to clarify, and any phase separated rubber was removed

from the liquid surfaces. This process was repeated for further clarification 5. 35 ml of each spun extract wase transferred to new tubes, to which 3 ml of 1.5 mg/ml

DOC solution was added. This was vortex mixed for 5-seconds and allowed to stand for 10 minutes

6. Following the 10-minutes stand period, 5 ml each of phosphotungstic acid and trichloroacetic acid were added to each tube. Each was vortex mixed for 5-seconds then allowed to stand for 30 minutes

7. Following the 30-minutes stand period both tubes were centrifuged to collect the precipitated protein

8. The supernatant was decanted, and the remaining liquid was removed by blowing nitrogen gas over the top of each tube

9. To each tube was added 15 ml of 0.2 N sodium hydroxide, followed by sonication until the protein precipitate was resolubilized

10. Final protein concentrations were determined using ASTM D 5712 Pre-bleeds from two sets of three rabbits were first tested against protein polyclonal antibodies using ELISA’s and immunoblots using both the antibodies employed in ASTM D 6499. This was to ensure that they hadn’t already raised antibodies against Hevea latex proteins, as does sometimes occur during rearing. The pre-bleeds also were tested against the guayule homogenate protein. Negative results were obtained for all six rabbits for all tests. Three rabbits were immunized with guayule homogenate proteins as follows. Three mg of the homogenate protein was lyophilized and resuspended in 1.0 mls dH20 and an equal volume of TitreMax adjuvant. The phases were vortexed to achieve a homogeneous solution which was then injected into the backs of NZ white rabbits at four different sites subQ (100 µl/site). Three rabbits were immunized with protein from purified guayule latex as follows. 1.2 mg of the purified protein was lyophilized and resuspended in 0.6 mls dH20 and an equal volume of TitreMax adjuvant. The phases were vortexed to achieve a homogeneous solution which was then injected into the backs of NZ white rabbits at four different sites subQ (100 µl/site).

Film Preparation and Testing Films were prepared from a proprietary formulation developed by Yulex Corporation. Films were produced per the following process flow:

Films were chlorinated, rinsed, and dried. Physical testing was performed in accordance with ASTM D 412, (Test Methods for Vulcanized Rubber and Thermoplastic Rubbers and Thermoplastic Elastomers-Tension) . Results Quantification of Latex Proteins The method described in D 1076 was used in conjunction with the Lowry described in D 5712 taking care to remain within the linear portion of the protein calibration curves (Figure s 2 and 3), which avoids the need for a quadratic fit as described in D 5712.

Figure 2. Calibration curve used to calculate protein concentration in samples with low concentrations.

Figure 3. Calibration curve used to calculate protein concentration in samples with high concentrations.

When the modified D 5712 method was used to quantify the protein levels in a range of latex samples, it became apparent that some interference was occurring – for example, the synthetic polyisoprene sample gave a positive result (Figure 4). However, if this is used as a control (as indicated by the dashed horizontal line on this figure) it is apparent that almost half of the guayule samples are indistinguishable from the polyisoprene control, and that most meet the Category 4 protein standard.

We are still working to determine if the samples giving significantly higher values reflect genuinely higher values, and if so why, or if they reflect variation in the quantification method. Nonetheless, the guayule latex consistently contains very low protein levels compared with Hevea latex and we do not anticipate difficulties meeting the protein requirements of the Category 4 standard using the modified D 5712 methods. Visualization of Latex Proteins Proteins were visualized in 6 mg (dw) of ammoniated Hevea and guayule latex using Coomassie Blue staining and compared with the amount of protein in the same amount of latex from which the rubber particles (with the rubber particle-bound proteins) had been removed by a combination of centrifugation and filtration (Figure 5). Coomassie Blue stains proteins in a quantitative manner. Thus, it is very clear that Hevea latex (lane 2) contains far more protein than guayule latex (lane 1). Compare this also with the marker lanes, which contain 250 ng protein. Similarly, in the cytosolic fraction (sometimes also called C-serum) considerable blue

Figure 4. Protein content of ten different lots of guayule latex produced by Yulex in the pilot plant and semi-works facilities from June 2005 to May 2006 (white bars), compared with a synthetic polyisoprene protein free control (PI, black bar, and dashed line), and two Hevea latex samples (grey bars).

staining was apparent in the Hevea sample (lane 4) but very little could be detected in guayule (lane 3). The same results were obtained when the lower sample loading of 1.2 mg rubber was used cf. lane 6 and 5, and lane 8 and 7.

It is known that Coomassie Blue, although a good quantitative stain, does not detect all proteins, especially glycoproteins and lipoproteins, and that many rubber particle bound proteins are not detected by this test. In contrast, Silver stain is both more sensitive than Coomassie and detects a much broader range of proteins, although it is perhaps a less quantitative stain across different proteins. Thus, in an additional comparison, 1.5 mg dry weight of ammoniated latex samples from Hevea and guayule were run of matched SDS-PAGE gels and stained with either Coomassie Blue (Figure 6A) or silver (Figure 6B). Also, in these gels, two different amounts of marker protein were included for comparison, 900 ng in lane M1 but only 45 ng of protein in lane M2. Figure 6A, the Coomassie stained gel, shows a comparable picture to that already shown and discussed in Figure 5, and we can see that the 45 ng of protein in the M2 marker lane is barely detectable. However, the sensitive silver stain clearly shows up the M2marker proteins, and also clearly shows that the guayule latex (lane 1) contains much less than this amount, whereas the Hevea latex sample contains far more. Similarly, these gels demonstrate that the latex purification process has left very little soluble protein in the cytosolic fraction of the guayule latex, especially compared with Hevea latex (compare lane 3 with lane 4, lane 7 with lane 8, and lane 9 with lane 10).

Figure 5. SDS-PAGE gel (4-12%) of Hevea and guayule latex proteins, stained with Coomassie Blue.

A.

B.

Figure 6. SDS-PAGE gel (4-12%) of Hevea and guayule latex proteins, stained with (A) Coomassie Blue and (B) Silver.

Solubilization of Hevea Latex Proteins Using different buffers to solubilize the proteins from Hevea latex, it immediately became apparent that the extraction solvent has a tremendous impact on the results obtained (Figure 7). For example, use of a sodium phosphate buffer actually underestimates the amount of protein contained within the latex, giving a value substantially below the soluble protein level. The water control, as is not unexpected gave a protein level comparable to the cytosolic protein level and does not extract much protein from the hydrophobic rubber particles. The SDS/PBS extraction gave the greatest value although it is unknown whether even this extraction method extracts all of the protein from the rubber phase.

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Figure 7. Proteins were quantified in Hevea latex after extraction in different buffers and compared with the cytosolic protein. Top panel: A single experiment in which each value is the mean of three determinations + s.e. Bottom Panel: The experiment in the top panel was repeated five times (except for the SDS/PBS treatment which was only repeated in three of the experiments). Although each experimental treatment was again in triplicate, the bars represent the experimental means + s.e. (i.e. the between experiment variation).

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Immunochemical determination of Latex Purity The first of the rabbit polyclonal antibodies is being raised against a total guayule homogenate protein preparation. These antibodies are intended for use in-house to rapidly monitor our latex purification process so that we can ensure that as much soluble protein as possible has been eliminated from the final latex. All three rabbits, immunized with the homogenate protein mounted a satisfactory immunological response (Figure 8). Antibodies also were successfully raised against the purified guayule latex proteins. As expected, because these proteins represent only a small subset of the total protein in the homogenate protein preparation, the anti-latex protein polyclonal antibodies reacted only weakly against the homogenate protein preparation.

Extractable Protein from Films Uncured films When latex was simply poured into a dish and dried, without vulcanization, and then proteins were extracted with SDS buffer, concentrated and quantified, far less protein (10.2%) could be pulled from an uncured guayule latex film than from a Hevea one (data not shown). However, this result does also indicate that a significant amount of the rubber particle bound protein in the Hevea film remained trapped within the film matrix. This experiment was repeated in a comparison with a synthetic polyisoprene film, two lots of high ammonia Hevea NR latex and two lots of guayule latex, one of which was tested

Figure 8. Antibody titers for three rabbits immunized with proteins extracted from ammoniated guayule homogenate (third bleed).

twice (Figure 9). It is clear that a small amount of protein can be pulled from the guayule films but much less than that from the Hevea films. It is interesting to note that the polyisoprene film gives a small but positive result, as we saw in the modified Lowry results for the latex (Figure 4). This activity is probably not due to the presence of protein but rather to chemical interference with the assay itself. However, it is also possible that these results may be caused by bacterial or dust contamination of the polyisoprene emulsion or the surface of the plate used to cast the film may provide protein material. It is also noteworthy that the two Hevea samples gave very different although both large results (Figure 9) although the amount of protein in the starting latex was not very different (see Figure 4). This suggests that differences in film formation or thickness can cause significant differences in the subsequent protein extraction. Measurements of the films revealed that the film yielding the lower protein value was 0.32 mm thick whereas the second film was 0.49 mm thick. Thus, at least in a non-vulcanized film, thickness has considerable influence on the amount of extractable protein and this is not solely a function of film area.

Figure 9. Protein was extracted from dried, uncured, unleached latex films and quantified. Each value is the mean of three determinations + se.

Cured films When we have applied ASTM D 5712 to finished products (cured glove films) made from guayule latex, we obtained the result “below detection limit”. This is reassuring from a product perspective but uninformative. Although we would be able to label a product with a “less than 50 µg protein/g” label it would be preferable to be able accurately to determine the actual level of extractable protein that might expose a product user. Because the Lowry, and other available protein quantification tests, lacks the sensitivity necessary to quantify guayule proteins, we are developing a guayule-specific immunochemical ELISA test analogous to the ASTM D 6499 test. However, unlike the ASTM D 6499, which uses a polyclonal antibody raised against latex C-serum proteins (the water-soluble latex proteins) from high and low ammoniated latex, and which represents a subset of the proteins actually in a latex product, we are using a total guayule latex protein preparation as the antigen, which makes no assumptions about the extraction ability of an end-user. Thus, all of the antigenic guayule latex proteins should be detectable by this ELISA. Our intention is to develop and validate this guayule-specific method sufficiently to submit a method to ASTM for approval.

Tensile Strength and Stress-Relaxation Studies

Guayule latex surgical and examination glove films have previously been shown to have high elongation to break values and low 500% modulus values, while still being able to achieve the ASTM standards for tensile strength (ASTM D 3577-01 Standard Specification for Rubber Surgical Gloves and ASTM D 3578-01 Standard Specification for Rubber Examination Gloves), see also Figure 10. These data suggested that the guayule latex films might have favorable properties compared to both synthetic and natural latex. Detailed stress relaxation studies have indicated that guayule latex gloves would cause less stress and fatigue to the hand over time (Figure 11).

Figure 10. Tensile strength plotted against elongation for Hevea and guayule chlorinated, powder-free, latex glove films.

ASTM D 1418 Standard Practice for Rubber and Rubber and D 1566 Standard Terminology

Relating to Rubber

ASTM International also coined a new term for guayule natural rubber (GNR) (in D 1488-06) and guayule natural rubber latex (GNRL) and added a definition for guayule latex (in D 1566-06) because it recognized that natural rubber (NR) and natural rubber latex (NRL) should be limited to Hevea products and would not serve to describe rubber and latex made from guayule.

Conclusions The low protein content of guayule latex and latex products is substantially lower than that of well-leached Hevea latex products, which were used safely for many decades. The low protein levels, coupled with the hydrophobic nature of the proteins, make it unlikely that large-scale use of guayule latex medical products will cause the wide-spread development of guayule latex allergies. Nonetheless, Yulex Corporation is maintaining an active research program in this area, and has a Scientific Advisory Board which focuses on latex allergies, both Type I and Type IV, to ensure that products made from Yulex® latex remain as safe as possible. The new ASTM D1076-06 Category 4 latex standard positions Yulex® latex (or NRGL) as a viable and leading alternative to traditional NRL sources for the medical device industry. Use of a minor modification of the Lowry described in ASTM D 5712, coupled with ASTM D 6499, will ensure the continued qualification and safety of guayule latex as a Category 4 latex under D-1076-06 and sets out the requirements that other lattices must meet in order also to qualify under this new natural rubber latex safety standard.

Figure 11. Stress relaxation curves of Hevea and Guayule chlorinated, powder-free, latex glove films. (A) MPa, (B) % Load retained._

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