Contribution of Fragrances to ASD 179tested [41]. Also, during pregnancy, the use of fragrances and other cosmetics
may expose the developing fetus to DEP, a common fragrance solvent that can
cause abnormal development of reproductive organs in infant males, attention
deficit disorder in children, and sperm damage in adults [7,29–33].
Compared with genetic studies of ASD, where DNA or RNA can be easily
extracted, sequenced and analyzed, studies of environmental risk factors are
very difficult to carry out. The primary reason is the lack of a suitable in vitro
model that can mimic the in vivo condition. Although animal models have
been used extensively, one major drawback is that rodent and other mamma
lian models (i.e., wolves, meerkats, and small primates) lack the ability to com
municate and convey to the observers what is happening in their brains.
Furthermore, most of them have very small brain size or lack the complexity of
the Homo sapiens brain to discern qualitative information. The industrial test
ing services that some of the chemical industries employ generally utilize
mouse models and look for toxicity by determining if the animals injected with
a particular chemical(s) have died or have specific adverse effects. These are
giant companies that have vast profit margins and work for the EPA or the
chemical manufacturers. Some of the testing companies have been criticized
and some of their evaluation methods and results are questionable, according
to published data available for public review [44].
The Ames test, originally described by Ames et al. in 1973 [45], is a well‐
established and widely applied biological assay to assess the mutagenic poten
tial of chemical compounds. A positive test indicates that the chemical is
mutagenic; therefore, it may act as a carcinogen, since cancer is often linked to
mutations [3]. The test serves as a convenient screening assay to evaluate the
carcinogenic potential of a compound; standard carcinogen assays on mice or
in vitro tests are time‐consuming and expensive. The test utilizes specific
strains of Salmonella typhimurium that carry mutations in genes involved in
histidine synthesis. This strain is called an auxotrophic mutant and requires
histidine for growth and is unable to produce histidine. Histidine is an amino
acid. The method tests the capability of mutagenic agents to create mutations
that can result in a reversion back to a nonauxotrophic state, which permits
cells to grow in a histidine‐free medium (meaning that this strain now can
make its own histadine). In order to study the mutagenic mutagenic/carcino
genic potential of perfumes, one can mix 1 μl of the bacteria in an agar tube
containing 1.5 ml of agar at 46 °C with 1 μl of histidine (final dilution 1:15,000)
in duplicates. This mixture is pipetted on a plate and allowed to culture at 37 °C
for 48 h. The small amount of histidine added in the growth medium allows
the bacteria to grow for an initial time, and have the opportunity to mutate.
With histidine depletion, only those bacteria that have an “induced gain‐of‐
function mutation” that allows them to produce their own histidine will
survive. The mutagenicity of a substance is proportional to the number of colo
nies produced on an agar plate.