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Volume 4, Number 1, Winter 2001

Welcome...

We recently became aware of a paper published in the Journal of the American Medical Association (JAMA) concerning the reproductive toxicity of workplace solvents. The results are so stunning that we felt a need to disseminate this information as widely as possible throughout our professional community. We are not alarmists, as many of you know from our safety workshops. We take a common-sense approach to the hazards of our field, weighing the severity of the hazard with the volume of use and the level of protection afforded by reasonable workplace practices. To use the word stunning in describing this research might sound excessive, but that is truly how we felt upon reading the paper. We were literally stunned, both by the severity of the risk and the implications for histotechnologists. Please read this issue carefully and share it with all of your colleagues. Janet Dapson wrote a more extensive summary of this research for the Michigan Society of Histotechnologist's newsletter, the Mikro-Graf. If you would like reprints of that, or additional copies of this Innovator, just call. All histotechnologists should be made aware of this information.

XYLENE CONFIRMED AS A REPRODUCTIVE HAZARD

A recent reproductive study of workers exposed to workplace solvents has confirmed what safety experts have long suspected: the fetus is at risk (Khattak et al.,1999). This research is the first to combine prospective and retrospective approaches. The surprising facet of this study is the magnitude of the risk to the unborn child. While many health studies demonstrate significant differences only through statistical comparisons, these findings are so unequivocal that anyone can see clearly just how serious the threat is to human health.

The solvents were common, volatile organic liquids spanning a variety of chemical families. Importantly for us, 21 of the 125 exposed women were laboratory technicians whose likely exposure was limited to xylene (the other solvents just are not generally used in labs on a large scale). These technicians comprised the second largest group of exposed workers, so the findings are particularly relevant to us. Each exposed woman was carefully matched to a non-exposed woman of similar age, life-style, health and reproductive status. The incidence of smoking, drug use and alcohol consumption was identical in the two groups.

Women enrolled in the study at the Hospital for Sick Children in Toronto during their first trimester of pregnancy, then received a post-natal followup interview. Routine exposure occurred at least since conception, and for 117 of the exposed women, occurred also for an extended period before the enrolled pregnancy.

Table 1. summarizes a portion of the findings from reproductive histories before the enrolled pregnancy. Viewed retrospectively in comparison to the control group, women were almost 2.5 times as likely to have had a miscarriage during their years of exposure.

During the 8 year prospective study, 250 women each had one pregnancy. While control mothers had only 2 children with malformations, exposed mothers gave birth to 18 malformed babies of whom 13 had anomalies severe enough to affect the function or social acceptability of the child (Table 2). That's a rate of 11.5% compared against 1.7%, based on live births!

Further, nearly three times as many babies in the exposed group were born in distress, requiring resuscitative measures. Three times as many were born premature. Exposed mothers gave birth to babies nearly 6 ounces smaller than those in the control group. Clearly, something is terribly wrong here: workplace safety is at the root of this tragedy.

Table 1: Reproductive history of women before the study

History

Exposed Women (n=125)

Control Women  (n=125)

Average number of previous pregnancies per woman

 1.98  1.94

% of women with previous miscarriages during exposure

  46.2%  19.2%

Table 2: Incidence of fetal problems during the study

History
Exposed Women (n=125)
Control Women (n=125)
Live births 113 115
Miscarriages and abortions 12 10
Total pregnancies  125  125
Major malformations  13  1
Minor malformations 5  1
Fetal distress  17  6
Premature births  9  3
Average birth weight (kg)  3.368  3.536
Average birth weight (lb, oz)  7 lbs 6.7 oz  7 lbs 12.6 oz
Solvent toxicity has been well documented for decades. Indeed, NIOSH proposed a special standard for xylene in 1975 (NIOSH, 1975), but the political process involved with actually getting OSHA to adopt a standard failed us. Twelve years later NIOSH tried again to warn workers about the problem, this time concentrating on neurotoxicity of solvents similar to those in the Toronto study. We at Anatech Ltd. publicized that effort in 1988 (and have continued to do so in our workshops ever since). Table 3, taken from our newsletter that year, summarizes the symptoms from NIOSH's report. Disturbingly, some of these changes were deemed permanent.

Table 3. Symptoms of xylene exposure

  • Impaired neurological functions
    • Reaction time
    • Manual dexterity
    • Coordination
    • Body balance
    • Learning ability
    • Peripheral nerve function
  • Sleepiness
  • Mental and physical fatigue
  • Headaches
  • Irritability
  • Personality changes/mood swings
  • Diminished motivation

Notice that many of these symptoms mirror the changes associated with aging, PMS or menopause. If histotechnology was a male-dominated career, perhaps these symptoms would have raised more alarm, not because of sex discrimination but rather because similar problems are so prevalent in the general population of women. Realize, however, that NIOSH was able to show that these symptoms occur with greater frequency and severity in exposed populations.

Despite the publicity, the vast majority of laboratories still use xylene. Monitoring the workplace or its personnel rarely demonstrates dangerous levels of exposure, in part because the PEL of 100 ppm is probably way too high. More importantly, skin exposure is a significant route for histotechs.

There is no adequate protective device for your skin. ACGIH recommends against the use of butyl, Neoprene, latex, and nitrile gloves, regardless of thickness (Schwope et al., 1987). Certain of these materials will not dissolve in xylene, and they may prevent your fingers from getting wet, but they afford no protection against the passage of xylene into your skin.

If you are concerned about your level of exposure, have your urine tested at the end of your shift for methylhippuric acids, the breakdown products from the metabolism of xylene. According to ACGIH (1999), there should be no more than 1.5 g per g of creatinine. Such Biological Exposure Determinants do not demarcate between safe and unsafe levels of exposure (unlike PEL's), but when you or a group of workers repeatedly have high readings, corrective action is indicated.

In the light of the Toronto study and NIOSH's findings, we believe that routine exposure in a lab using xylene for processing and staining is unacceptable. And so we want to talk about removing xylene from the laboratory, or at least reducing its use to a bare minimum. We want to help put health back into health care, and there is no better place to start than your workplace. If you are ready for life-saving change, here are some answers.

Xylene substitutes have been around since 1981 when limonene was introduced to the field. Unfortunately, that chemical has cast a shadow over the subject because of several issues. Limonene is oily and cannot be recycled reliably (the recycled solution is different from the original product). Its odor is overpowering and quickly pervades neighboring rooms and halls. Most troublesome is its ability to cause serious sensitization reactions in exposed workers. While some laboratories do use it successfully, we strongly advise against it because of its poor safety record. Sensitization creeps up on you and then is permanent; unlike irritation, there are no warning signs until it is too late.

Many of you have said you would rather stay with a known hazard (xylene) than risk using something new, and point to the problems of limonene as justification. We understand that sentiment. However, the hazards of limonene were known from the start. Further, the health issues of xylene are far too serious. Finally, the alternative is not new or unknown.

In 1983, short-chain aliphatic hydrocarbon clearants were introduced to our field, and today are by far the most popular xylene substitute. At least three major companies, Anatech included, sell these products. To our knowledge, no health-related problems have ever been raised. These solvents have been used for many years outside the lab, and from actual industrial experience have been deemed non-irritating to normal human skin, non-sensitizing and non-carcinogenic. The permissible exposure limit.

What are aliphatic hydrocarbons? You know many of them: methane, ethane and propane are gases, octane is a liquid, petroleum jelly is a gel, and paraffin wax is a solid. These chemicals differ only in their number of carbon atoms. By judiciously choosing a mixture of the liquid forms, a solution having the evaporative properties of xylene can be obtained. We call them short-chain to distinguish these commercial products from longer-chain varieties that do not evaporate readily. The latter are also sold as clearing agents, and have the same safety profile as their smaller cousins, but they do not penetrate tissues as easily, contaminate the wax more, and cause coverslips to dry very slowly. Thus, we do not recommend them.

We know that some of you had tried short-chain aliphatics and were not satisfied. Please try again: we are deeply concerned about your health. These products do work, but you have to understand how to use them. Here is a summary of tips which we have used to address all of the problems known to us. Aliphatics are more gentle solvents than xylene, so they need additional time to clear tissues properly.

That does not mean adding time to your processing schedule; rather, use three stations of clearant instead of two. Your blocks will probably cut better with this change. Xylene hardens tissues, especially with heat on the processor; aliphatics do not. This change will help remove fat from larger specimens while keeping the more delicate tissues soft.

Figure 1. Keratinized epithelium, vagina(oppossum), Prefer, H&E.20x

Figure 2.Tonsil (human),PreferUCHL-1, without antigen retreival. 20x
Processing with aliphatics will not harm immunoreactivity or receptivity of the tissue to special stains and H+E. Heat might, and short fixation with formalin certainly will have adverse effects. Too many labs have been using these clearing agents successfully for almost 2 decades now for this to be a real issue. Figures 1 and 2 are typical examples of results.

All aliphatics are fully compatible with all tissue processors. They may even be used in the purge cycle (we recommend it). If you use a high-polymer wax, you may want to do a double purge every now and then to remove the additives. Most customers do not need to do that. The buildup of salts from neutral buffered formalin are more likely to be the cause of deposits in the chamber or lines.
The textbooks suggest deparaffinizing slides with xylene using 3 changes, 3 minutes each. You can get by with less when using xylene, but don't cut corners here with aliphatics. Sections up to 6 µ will deparaffinize nicely in that time. Do you have clumps of wax on one edge of your slide? That's from having the drying oven too hot; you should not melt the wax, only drive out the water beneath the section. The aliphatics may not dissolve those clumps off entirely, but they will probably float off in the water rinses. At any rate, staining of the section will not be affected.

The biggest problem with aliphatics occurs during humid weather. Eosin may be seen bleeding out of the section hours or days after coverslipping. Popular opinion to the contrary, the clearant is not contaminated with water, the last alcohol is. It is almost impossible to keep anhydrous alcohol dry during the summer in certain regions. You partially empty a gallon bottle, and the fluid removed is replaced by moisture-laden air which is very effectively dried out by the remaining alcohol. Next time you pour from that bottle, you dispense alcohol that is no longer anhydrous. Xylene can tolerate that but aliphatic clearants cannot. Alcohol that is not replaced properly during clearing of the sections is left to extract eosin from the tissue. Circumvent the problem by putting plain (non-indicating) Dririte in your last alcohol to remove the moisture.

Aliphatics are not solvents for the resins in mounting media, although they are miscible in certain proportions with toluene- or xylene-based solutions of a few of those resins. Buy only approved mounting media (each major manufacturer has one for its aliphatic clearant, but they are compatible with all brands of aliphatics). These media are fast-drying, nearly colorless solutions; some contain anti-oxidants to further protect your stains. Other types of media are not compatible.

If you have a coverslipping device that uses a bath of xylene, do not replace that solution with an aliphatic. This is one of the few areas where xylene should be retained. The other is for removing coverslips or thinning mounting media. Your exposure to these sources of xylene vapors would be insignificant.

If you coverslip by hand, by all means use an aliphatic. Nitrile or Neoprene gloves will provide some protection from the defatting action of the clearant. Most of us who use aliphatics find that coverslipping "upside down" works better than the more conventional approach. Simply put the mounting medium on the coverslip, then lower a well-drained slide over it.

What about price? Aliphatics will cost slightly more than xylene. If you recycle you will hardly notice it, and even if you don't, the savings in health-related expense could be incalulable.

Well, that's it. Nothing complicated, nothing time consuming, only a few minor changes in protocol. But what a difference in safety! If you are a woman of childbearing age, or have coworkers who are, you owe it to yourself and them to eliminate this terrible reproductive risk. If you and they do not fall into this category, consider the neurological damage attributable to xylene exposure.

We would be delighted if you choose our Pro-Par Clearant and Refrax Mounting Medium for your replacements. We would be equally pleased to learn that as a result of this Innovator, you replaced xylene with one of our competitor's reasonably equivalent products. Keep us posted.

References

ACGIH. 2000. TLV's and BEI's. American Conference of Governmental Industrial Hygienists, Inc., Cincinnati, OH. 192 pp

Dapson, JN. 2000. Organic solvents as reproductive hazards. Mikro-Graf 29 (2):1-4. Reprints available from Anatech Ltd.

Dapson, JN, RW Dapson. 1995. Hazardous materials in the histopthology laboratory: regulations, risks, handling and disposal, 3rd edition. Anatech Ltd., Battle Creek, MI. 255 pp Detailed discussions on protective clothing, Permissible Exposure Limits, Biological Exposure Indices, ventilation and many related topics.

Khattak, S, G K-Moghtader,K McMartin, M Barrera, D Kennedy, G Koren. 1999. Pregnancy outcome following gestational exposure to organic solvents. J Amer Med Assoc 281:1106-1109 (March 24/31). Available for a limited time on JAMA's website: www.ama-assn.org/special/womh

Schwope, AD, PP Costas, JO Jackson, JO Stull, DJ Weitzman. 1987. Guidelines for the selection of chemical protective clothing. American Conference of Governmental Industrial Hygienists, Inc., Cincinnati, OH