[The_Microbiologist]

Hi raywillping,

Unfortunately it’s true.  Even with traditional preservative packages they have to be challenge tested.  I have seen instances where formulations I expect to work fail the test, and vice versa.

Good luck!

[The_Microbiologist]

One more note…

the “common usage test” is not as good as what I proposed because there is no standardization whatsoever of the microbial inoculum.  Most mouths are sort of self-standardizing to a reasonably high concentration of bacteria (usually with nice diversity as well) and none are sterile that I know of.

[The_Microbiologist]

Hi Zink,

For a very rough preservative efficacy screen, here is what I recommend (no joke):

Make some product.  Then collect some saliva and measure it (saliva, not mucous).  I would do this at least a few hours after brushing, using mouthwash, or eating.  Then add a measured amount of the saliva to your product, say 1 ml per 10 ml product. Then mix it thoroughly, then do an APC using a dipslide.  Then let the product sit at room temp for 1 week.  Then do another APC.  If counts go up, the preservative is not working.  If counts go down substantially, it’s working at least a bit. 

Do note that oral pathogens may be present at fairly high concentrations (making them more risky) on the dipslide after incubation and should be disposed of in accordance with local laws.

That’s an inexpensive rough screen for preservative efficacy, though it lacks confirmation of preservative neutralization (which means the germs that appear to be killed may just be inhibited from growing on the dipslide). 

No ivory tower here, ha ha ha

Ben

[The_Microbiologist]

HI Zink,

You can buy germ cocktails similar to what you have outlined from a company called MicroBioLogics.  The brand name is called EZ-PEC. 

However, I agree with Mark that such testing is best left to the pros and here is why:

• The EZ-PEC organisms are lyophilized (freeze-dried).  Freeze dried germs have a tendency to disintegrate into small, ultra-lightweight, inhalable particles and go everywhere, so in my opinion lyophilized microbes should only be worked with in a Class II biological safety cabinet.
• Some of the USP germs are pathogenic, meaning they can cause disease.
• The lyophilized pellet re-hydration step is something that should be understood by the person doing it to make sure it’s done right.
• Optimally a person would verify the initial inoculum counts for each organism rather than depend on the manufacturers’ certification.  That requires serial dilution and plating.
• At the end of the test you have to know how many germs are left in the formula.  That also requires dilution and plating.
• You have to verify that any germs left in the formula can grow on the agar through a step called a neutralization/recovery validation that is rather technical.
• The whole process generates dozens of used agar plates, many of which will be loaded with bacterial and fungal colonies.  Each colony will have about 10 million germs, which means the risks posed by the organisms such as Staph aureus are greater in the case of accidental contact.
• All those agar plates, dilution tubes, etc, are considered “medical waste” in most states and must be disposed of properly, usually with some paperwork to go with it (even in Texas where my lab is located!).

Hopefully that information is helpful!

[The_Microbiologist]

Hi Compounder,

I don’t have much experience with aspen bark, sorry.  I don’t know how you are determining the 4 month shelf life but if it’s based on aesthetics the lotion is probably microbially contaminated long before you can perceive it visually. 

As a point of reference, one can put 100,000 bacteria into a milliliter of water and the water will appear to the naked eye to be crystal clear and usually won’t smell bad, either.  That all changes once you get to about 10,000,000 per milliliter.  Most of the cosmetics we test in our lab that have counts ranging into the tens of thousands or millions of cells per milliliter have subtle or no aesthetic differences from sterile samples.

You may be determining shelf life with lab tests - I am just sharing for the benefit of the forum in general.

Hopefully someone with experience with aspen bark can chime in too.

[The_Microbiologist]

It could still be microbial contamination at pH 9 in my opinion.  To quote my Ph.D. advisor Charles Gerba, “Germs never give up, they always find a way!”

pH 10 or above would be the alkalinity at which I would not anticipate much microbial growth.

The problem with germs is that they’re invisible so it’s tough to tell without lab testing.

[The_Microbiologist]

I can weigh in on micro costs:

Part of development of any formulation is ensuring microbiological stability.  That is done with preservative challenge tests like USP <51>, CTFA M-3, etc.  Each of those costs around $400-$750 depending on the lab you use.  If you use traditional preservatives at manufacturer-recommended concentrations I estimate a 90% chance you’ll pass the test on the first attempt.  If you use a more trendy preservative package (natural, etc) then it will probably take a couple or a few rounds of testing (with minor reformulations in between) to get it to pass.

Once you’re manufacturing, you’ll want to do micro on each batch to the tune of about $50 a batch.

[The_Microbiologist]

Hello labtechnician,

I notice the “n/a” for Candida and Aspergillus at day 7.  I presume those organisms were just not analyzed at that timepoint.  Is that correct?  Was this by chance an ISO method? 

Nevertheless this appears to be a well-preserved formula in general.  I see the same “odd” data that you do with regard to Candida.  It’s strange for that organism to have what we would call a “shoulder” die-off curve, meaning slight reduction followed by precipitous reduction.  Usually Candida reduction is much more linear, meaning you’ll loose a log (90%) or so each week on the way down to a final result of non-detection.  It’s possible that the 14 day data was artificially increased by chance, by clumps of organism within the test material or something else.

If USP <51> criteria were applied it would “pass” so you are in good shape.  If you want to be extra diligent, you could re-test just the Candida and see if next time you see a more linear (sort of more predictable and thus more dependable) die-off.  If you wanted to be even more diligent, you could do that at a different lab.  If two labs get the same result, it’s almost certainly “real.”

Here is my last note, please verify with your lab that they did separate controls to verify neutralization and recovery from the test sample.  Ask them for the data for that, too.  They should have it and it should be quantitative.  If they don’t, then they’re not doing the test right and it calls into question much of the data.  Feel free to email me privately if that comes up and I’ll help you work through it and re-interpret the data.

[The_Microbiologist]

FrankTankNitty,

What do you mean by “CHG compatible?”  Do you mean CHG can be put in as an active ingredient, or do you mean that the lotion won’t inactivate or interfere with CHG skin antiseptics? 

There was a big thing about this a couple years ago…basically the medical field realized that many ordinary hand lotions are not compatible with CHG-based skin antiseptics.  If that’s what you’re talking about, get in touch with Antimicrobial Test Laboratories.  They can talk you through testing options and are specialists in such studies.

[The_Microbiologist]

Perry

Sorry, was just catching up on the thread and realized that your question was about chemistry. and not chemistry, biology, etc..oops!  Sorry to hijack your chemistry thread with micro info.

[The_Microbiologist]

This thread wouldn’t be complete without a mention of microbiology.  One doesn’t need a degree or anything, but DOES need to spend about 1-2 hours familiarizing themselves with the two basic micro tests done on cosmetics, the APC and preservative challenge test.  To safely and sustainably make cosmetics one should know what each test tells them and when each test is appropriate.

Here’s a crash course (for more info there are several educational articles at http://www.CosmeticTestLabs.com):

• APCs are cheap and give “snapshots” of germ counts over time.  <100 cfu/ml is ideal.  They are often done several times in a product’s life cycle (usually after each round of production).
• Challenge tests cost more and tell a formulator whether or not the formulation has some “built in” resistance to microbes.  They are usually done once, after a formulation is complete and before manufacturing begins.

Good luck!

Ben

[The_Microbiologist]

Polymergirl,

I haven’t heard of that practice before…probably means I spend too much time in the lab

Anyhow, I am sure it’s fine for the day or two.  Then after that I’m pretty sure it would turn into germ soup unless as Bob says the pH is very high. 

[The_Microbiologist]

Hi milliachemist,

At our lab we usually do 10 weeks @ 45C and call that roughly equivalent to one year at room temp.

[The_Microbiologist]

Hi milliachemist,

Good point about the duration of the stability study.  What would you recommend as a “default” time and temperature?

[The_Microbiologist]

Hi love,

The results from an APC test should be expressed as “CFU/ml” or “CFU/g,” not as a percentage.  I think there must be some confusion, either at your end or at the lab’s end.  If you want, you can email me the report and I’ll check it out to see if I can make sense of it.  If you’re interested, please email the main lab address (available on the website).

vjay is spot on.  Do the challenge test to see if it can withstand microbial “insults,” then do stability to make sure it doesn’t undergo physical changes over time that could be off-putting, then make batches of the product and make sure that your APC counts are <10 (or some companies use <100).

[The_Microbiologist]

Isaiah

The only micro tests typically done on finished product are aerobic plate counts for bacteria and fungi.  Those tests tell a person how many bacteria and how many fungi are present in a given gram or milliliter of product, which in turn indicates whether or not the finished product meets a company’s specifications for acceptable quality for release/sale.  Less than 100 bacteria or fungi per gram or milliliter is a common specification, though less than 10 bacteria or fungi per milliliter or gram is more typical for eye-area cosmetics.

[The_Microbiologist]

Hi MakingSkinCare and Tasiashton,

Perhaps I can kill two birds with one stone with this statement about “natural” preservatives (including Leucidal):

Can a cosmetic formulation be microbiologically stabilized using one or more “natural” preservatives like Leucidal, sorbic acid, and lactic acid?  Yes.  Is it easy?  No!  Is it cheap?  No!  Will it always work with “tough” formulations? No!

And about grapefruit seed extract and vitamin E, I have never been wowed by them.  That’s not to say they don’t work, just that I don’t recall ever seeing it happen, and I vaguely recall some scandal where grapefruit seed extract appeared to have been spiked with quaternary ammonium compounds.

We get a lot of samples through the lab for preservative challenge testing, many from customers we haven’t worked with before who are submitting formulations to us that are as new to them as they are to us.  When they use traditional preservatives (even retailer-preferable traditional preservatives like phenoxyethanol), their success rate on challenge tests is very high.  I estimate they fail only one in every 5 or 10 tests.  That’s great, because challenge tests take a long time to conduct and are relatively spendy. 

On the other hand, when they use natural preservatives it often takes a few rounds of testing and reformulation for them to pass.  That’s good for the lab but bad for them, and my heart goes out to them when I see them give up altogether rather than throw in a tiny amount of synthetic preservative and go to market   

So have I seen Leucidal work, yes.  Do I think it is as efficacious as most of the traditional preservatives, no.  But on the other hand, many traditional preservatives are being unfairly vilified.  So unfair or not, there’s a great big and trendy marketing trade-off to be considered.

Frequently we see customers use antifungal preservatives such as sorbic acid in combination with Leucidal.  That seems to be a pretty good place to start and I’m sure the folks at Active Micro Technologies (makers of Luecidal) could give some other suggestions, too.

MakingSkinCare:  As for polysorbate 60, I doubt it interferes but am not certain.  As for the proposed formulation 1% GC/D GMCY and p-Anisic/Dermostoft, I just don’t know.  I am pretty sure that your formulation expertise - even as related to preservation - exceeds mine!  I am a “test it and let’s see” sort of guy, which I suppose is a good fit for a testing lab owner

Tasiaashton:  Thanks for giving us a shot!

Thanks all for the great discussions.

[The_Microbiologist]

Wow, what a nice welcome back to the forum!  Thanks everybody, and thanks Mark for the referrals.  We have sent some folks your way, too.  Hopefully they have found you well.

MakingSkinCare - That article is a gem.  I looked over it briefly and will read it in full later.  In the meantime, I can confirm that polysorbate 80 does neutralize certain classes of preservatives.  In fact, we use it in our all-purpose neutralizing broth for that very purpose in the lab.  Here is the product insert and formula if you are interested.  In that document (and in lots of scientific literature), it says the purpose of polysorbate 80 is to neutralize substituted phenolics.  I don’t think phenoxyethanol is technically a substituted phenolic (it would seem to me to be more like a monophenyl ether), but it is structurally similar to many substituted phenolics so the two chemicals probably should not be present in the same cosmetic product if the goal is to make it microbiologically stable.

I am not as familiar with adsorption of preservatives onto plastics, though I know some long-time formulators who consider the plastic type of their packaging to be effectively one more formulation variable.

As for the cellulosics, I would anticipate some interference with preservative effectiveness, but in a minor way.  We see this with almost all thickening agents.  Maybe the best example comes from a contact lens solution maker several years ago.  I don’t have all the information, but I think that basically what happened in that instance is they added a thickening agent to their lens solution to aid in eye feel that bound up some portion of their preservative, thereby knocking out some efficacy and allowing some pathogenic fungi to gain a foothold. 

Most antimicrobial agents carry a positive charge - and this is just a broad theory of mine - which physically leads them to the negatively charged cell wall, where the rest of the molecule does the destruction.  Since cellulose is a huge molecule with lots of charge variation over its surface, it would not surprise me to see it bind up some preservatives. However I would not expect them to be too tightly bound so the effect may not be a formula-killer.

milliachemist - some non-ionic surfactants do interfere with antimicrobial efficacy but usually in a minor way and the effect can be expected to vary depending on other aspects of the formulation such as pH.  I don’t have a list in my head of which ones should be avoided, but I have definitely seen instances where removal, substitution, or scale-down of a non-ionic surfactant improves antimicrobial activity.