OSHA requirements are set by statute, standards and regulations. Our interpretation letters explain these requirements and how they apply to particular circumstances, but they cannot create additional employer obligations. This letter constitutes OSHA's interpretation of the requirements discussed. Note that our enforcement guidance may be affected by changes to OSHA rules. Also, from time to time we update our guidance in response to new information. To keep apprised of such developments, you can consult OSHA's website at https://www.osha.gov.

January 18, 1991

Mr. Harvey Shapiro
Assistant Regional Administrator for Technical Support
Occupational Safety and Health Administration
201 Varick Street
New York, New York 10014

Dear Mr. Shapiro:

In regards to your January 2, 1991 letter concerning sampling for benzidine congener dyes, we give the following responses.



  1. Is there one preferred method for air sampling of benzidine based dyes? If so, what is it?

    Answer: For the Benzidine Based Dyes which we have received so far the preferred method has been: Sample Media: Glass Fiber Filter Air Volume: 100 Liters Flow Rate: 1.0 L/min.

    To date, we have not found any benzidine based dyes which can not be collected by the above way. Azo dyes have a tendency to be light and heat sensitive. So far we have only found a problem with Direct Black 38 being light and heat sensitive and therefore we recommend protecting Direct Black 38 samples from light and heat and shipping them on blue ice.

  3. Can we air sample to determine worker exposure to various benzidine based dyes, that may be present at the same time, on one filter?

    Answer: We have not had a problem with analysis when several benzidine based dyes are present on the same filter. If many were present on the same filter then we might have problems in obtaining analytical separation and therefore problems in accurate quantitation due to interferences.

  5. What analysis should be requested?

    Answer: Wherever possible the specific benzidine based dye should be requested, one gram (approximately 1 teaspoon equivalent...not more than this.... of the dye submitted as a bulk which we may use as an analytical standard if necessary, the approximate % of dye present in the bulk noted on the paperwork, if known) and any material safety data sheets submitted at the same time. If a specific dye is not requested then a modified NIOSH method 5013 which is non specific will be used for analysis.

  7. Can the lab analyze for free benzidine and benzidine based dyes on the same filter?

    Answer: Yes. It is best to request the analysis that way or to call and specify what is needed. If "free benzidine" and "benzidine based dyes" are requested the analysis will be performed to show any free benzidine present as a contaminant in addition to the analysis of the intact dyes.

  9. Can the lab analyze for both benzidine based dyes and o-tolidine based dyes and/or o-dianisidine based dyes on the same filter?

    Answer: Yes. It is best to specify which dyes are requested for analysis and follow the procedure listed under # 3 above. Otherwise a modified NIOSH method 5013 will be used for a non specific analysis.

  11. What is the minimum amount of time needed to sample (or minimum volume needed to be collected) to just show the presence of benzidine based dyes? How about for o-tolidine and o-dianisidine?

    Answer: Guideline: 60 Liters. This may vary depending upon detector sensitivity to specific dye and presence or absence of interferences.

  13. Do air sampling filters need to be protected from light when sampling for benzidine congener dyes?

    Answer: Guideline: No, except for Direct Black 38. Although azo compounds can be light and heat sensitive, so far we have only had a problem with Direct Black 38. There may be problems with dyes we have not received yet for analysis. The ones we have analyzed and found no problems with light sensitivity are listed in the Chemical Information File. Only Direct Black 38 has a note on it regarding collection, storage, and shipping to protect from light and heat.

  15. What is the preferred method of taking wipe samples for benzidine based dyes, o-tolidine based dyes, and o-dianisidine based dyes?

    Answer: Glass Fiber Filter. Wipe may be taken dry or moistened with deionized water or methanol.

  17. Are bulk samples needed to be taken if we request an analysis, for example, of just the percentage of benzidine in benzidine based dyes that we sampled for?

    Answer: Usually yes. When you say the percentage of benzidine in benzidine based dyes we need you to specify whether you mean "free benzidine" present as a contaminant or if you are requesting a non-specific analysis such as a modified NIOSH 5013. The answer is usually yes because if you request a specific dye we may not have a bulk we can even use as a standard and if we do have a bulk it may not be identical to your bulk. If you want the dyes of interest to be individually identified and quantitated, then we need your bulks. Please also send in any material safety data sheets you may have.

  19. Once wipe and/or bulk samples are taken, do they need to be protected from light?

    Answer: So far, only Direct Black 38 has been a problem.

  21. After sampling has been completed for air monitoring, wipes and/or bulks, what should be done with the samples as far as storage is concerned?

    Answer: No special handling except for Direct Black 38 for the ones that we have analyzed and that are listed in the Chemical Information File. Others may also be all right.

  23. If it is necessary to use dry ice, is it necessary that samples taken that day be packed in dry ice at the company (on-site)?

    Answer: So far only Direct Black 38 has needed this. Do not use dry ice if shipping by airplane. See #15.

  25. Should benzidine based dye samples, o-tolidine based dye samples and o-dianisidine all be separated from one another when shipping to the lab?

    Answer: No special precautions need to be taken.

  27. How should the samples be shipped? Should they be sent "overnight delivery"?

    Answer: Normal sample shipment (except for Direct Black 38). Overnight shipment recommended for Urine Samples. See #15 for more information.

  29. In regards to urine samples how should we proceed in as far as collection, storage, and shipping?

    Answer: Collection: Contact John Linkletter at the OSHA lab for bottles containing the citric acid stabilizer. You need to tell us how large a bottle you want. Are you taking total urine output for 24 hours? Are you taking only one sample at the beginning or end of the workday? You may want to talk to others who have collected urine samples on the best strategy for this. If you need names we can generate a list from our computer files. Take precautions in handling any biological samples to prevent exposure to diseases.

    Storage: Leave at least 15% space in bottle and freeze contents as soon as possible and store that way (water and therefore urine) expands when it freezes. Urine does not keep well and needs to be frozen. It is also best to have samples frozen before actual shipment. No spillage will occur during shipment and usually arrive still frozen if they start frozen.

    Shipping: Call the lab and speak to an Organic Service Branch Chief and tell him/her when you are shipping urine samples, how you are shipping them (airplane, etc. flight # when they are due to arrive, etc. or other means so we will watch for them and we will get them to lab as soon as possible). Ship the urine samples in an insulated container (many air lines will not take dry ice because animals may be in the same cargo area and the dry ice gives off CO2 and can cause a problem for the animals). Blue ice works well if frozen first.

  31. Can the lab analyze for all metabolites in urine (i.e. benzidine monoacetyl benzidine, n-acetyl benzidine) in the same sample?

    Answer: We have analytical standards and the capability to do the individual metabolites. Our recommendation is that instead of asking for the individual metabolites that you request "free benzidine" and "metabolized" benzidine. We do a twofold analysis in this case. First, we analyze for the "free" benzidine. Then we hydrolyze the sample so that all benzidine "metabolites" are converted back to the "free" amine (benzidine). We reanalyze the sample for the total of original "free" benzidine and free benzidine that was originally "metabolized". The difference in the two answers is the original "metabolized" benzidine. This makes the analysis simpler and gives a meaningful answer. If you do not request "metabolized" and "free" benzidine, then the sample will be hydrolyzed and "total" benzidine reported rather than a twofold analysis.

  33. If an employee is exposed to either benzidine based dyes and/or o-tolidine based dyes and/or o-dianisidine based dyes can the lab analyze for metabolites of all three in the same urine sample?

    Answer: Yes. However, our recommendation is the same as in #16.

If you have any questions, please call [the Salt Lake Technical Center at (801) 233-4900].


Lois F. Septon Chief,
Organic Service Branch II

[Correction 2/13/04]