Evaluation of a Sample Provided by Bug Bam! Products
Utilizing the Irritection Assay System
October 13, 2006

EXECUTIVE SUMMARY
A single sample was evaluated with the Irritection Assay System in order to predict its
potential for ocular and dermal irritation. The ocular results indicated that the sample of
Bug Bam Formula was a minimal ocular irritant. The dermal results demonstrated that
the sample was a dermal non-irritant.

STUDY OBJECTIVE
A single sample provided by Bug Bam! Products was evaluated with the Irritection! Assay
System in order to predict its potential to cause ocular and dermal irritation.
To achieve this objective, standard volume-dependent dose-response studies were performed
with the Ocular and Dermal Irritection test methods.

BACKGROUND
The proprietary Ocular and Dermal Irritection assays are standardized and quantitative in vitro
acute ocular and dermal irritation tests which utilize changes of relevant macromolecules to
predict acute ocular and dermal irritancy of chemicals and chemical formulations.
The Ocular Irritection assay, depicted schematically in Figure 1 below, provides significant
advances over the in vivo Draize test method. The Draize eye irritation assay has been criticized
because of the large variability of results obtained from different laboratories that have analyzed
the same specimen.


The Dermal Irritection assay, depicted schematically in Figure 2, is based on the principle that
chemical compounds will promote measurable changes in target biomolecules and
macromolecular structures. Previous studies have clearly demonstrated that the processes of
protein denaturation and disaggregation that are induced in this in vitro assay mimic the effects
that are produced when these types of irritants are applied to the skin. Consequently, this in vitro
test may be employed to predict the in vivo toxic effects of chemicals and formulations.

The quantitative Ocular and Dermal Irritection in vitro assays have been foundto be highly
reproducible. Of even greater relevance, the Ocular and Dermal Irritection assay methods can be
readily employed to evaluate multiple samples at varying volumes or concentrations. Thus,
these tests serve as extremely useful screening tools that facilitate all stages of raw material
selection, formulation development and final product selection.

MATERIALS/METHODS
The Ocular and Dermal Irritection assays are quantitative in vitro test methods that mimic acute
ocular and dermal irritation tests. To perform the Ocular Irritection standardized assay, the test
sample is applied to a synthetic biobarrier composed of a semi-permeable membrane. To
perform the Dermal Irritection standardized assay, the test sample is applied to a similar
synthetic biobarrier that is coated with a dye-containing keratin-collagen matrix. Following
application, the sample is absorbed by and permeates through this synthetic biobarrier to
gradually come into contact with a proprietary solution containing highly ordered globulins and
glycoproteins. Reaction of the test sample with these proteins and macromolecular complexes
promotes conformational changes that may be readily detected as an increase in the turbidity of
the protein solution. With the Ocular Irritection test, turbidity may be detected
spectrophotometrically at a wavelength of 405 nm. With the Dermal Irritection test, dye that has
been dissociated from the biobarrier during transit of the applied sample may be detected
spectrophotometrically at a wavelength of 450 nm.

The ocular irritancy potential of a test sample is expressed as an Irritection Draize Equivalent
(IDE), whereas the dermal irritancy potential of a test sample is expressed as a Human Irritancy
Equivalent (HIE) score. These scores are defined by comparing the increase in optical density

(OD405/450) produced by the test material to a standard curve that is constructed by measuring the
increase in OD produced by a set of Calibration substances. These Calibrators have been
selected for use in these tests because their irritancy potential has been previously documented in
a series of in vivo investigations. The predicted in vivo classification, based on these scoring
systems, is shown in Tables 1 and 2.

Table 1. Relationship of Irritection Draize Equivalent (IDE) Score to Irritancy Classification for the Ocular Irritection Test Method.

Irritection Draize Equivalent (IDE) Score	Predicted Ocular Irritancy Classification
0.0 - 12.5	Minimal Irritant
12.5 - 30.0	Mild Irritant
30.0 - 51.0	Moderate Irritant
51.0 - 80.0	Severe Irritant

Table 2. Relationship of Human Irritancy Equivalent (HIE) Score to Irritancy Classification for the Dermal Irritection Test Method.

Human Irritancy Equivalent (HIE)	Predicted Dermal Irritancy Classification
0.00 - 0.90	Non-Irritant
0.90 - 1.20	Non-Irritant/Irritant
1.20 - 5.00	Irritant

A detailed description of the Ocular and Dermal Irritection test procedures may be found in
InVitro International's Irritection! Assay System Instruction Manual. All data are calculated and
analyzed via a computer program which determines assay result acceptance based upon
qualification parameters defined in the program. In general, the program has been designed to
accept sample data as qualified if the following criteria are met: the OD values of Calibrators
and internal Quality Control samples fall within previously specified ranges; sample blanks are
less than 500 OD units; the net sample OD is greater than -15; and an Inhibition Check is
negative.

RESULTS
The results of this analysis provided a predicted in vivo classification for the test sample. The
software printouts are included in Appendix I.
Tables 3 and 4 present a summary of results for the Bug Bam! Products sample studied.

Table 3. Summary of Ocular Irritection Results

IVI NUMBER	SAMPLE DESCRIPTION	DOSE	IDE SCORE	PREDICTED OCULAR IRRITANCY CLASSIFICATION
E7413	Bug Bam Formula	25ul	10.6*	Minimal Irritant
		50ul	9.4	Minimal Irritant
		75ul	8.8	Minimal Irritant
		100ul	8.0	Minimal Irritant
		125ul	7.6	Minimal Irritant

Table 4. Summary of the Dermal Irritection Results

IVI NUMBER	SAMPLE DESCRIPTION	DOSE	HIE SCORE	PREDICTED DERMAL IRRITANCY CLASSIFICATION
E7413	Bug Bam Formula	25ul	0.77*	Non-Irritant
		50ul	0.72	Non-Irritant
		75ul	0.68	Non-Irritant
		100ul	0.68	Non-Irritant
		125ul	0.68	Non-Irritant

*Maximum Qualified Score

DISCUSSION
A single sample, provided by Bug Bam! Products, was evaluated with the Irritection Assay
System in order to predict its potential to cause ocular and dermal irritation.

A standard volume-dependent dose-response study was performed with the Ocular Irritection test
method. The following volumes of sample were applied for analysis: 50, 75, 100, and 125!l.
The results of the study indicated that the sample Bug Bam Formula was classified as a minimal
ocular irritant with an IDE score of 10.6.

A similar volume-dependent dose-response study was performed with the Dermal Irritection test
method. The results demonstrated that the sample was predicted to be a non-irritant with a HIE
score of 0.77.

In summary, the Ocular and Dermal Irritection test methods successfully classified the ocular
and dermal irritation potential of this sample.

Carroll-Loye Biological Research

Date: 22 April 2008
Test: Mosquito Spatial Repellent Efficacy Assay (Laboratory)
Material: ‘Bug Bam! Insect Repelling Grid’
Control: Untreated

Information Summary
1) Objective
The objective of this study was to test the spatial repellent characteristics of
the Test Material, with efficacy measured in terms of percent reduction in
the rate of landing on a subject stationed at three distances from the site of
installation, in comparison to an untreated environment. Freshly unpackaged
Test Material was compared to product opened for 100 hours prior to testing.

2) Test Material (with abbreviations used hereinafter)
Bug Bam! Insect Repelling Grids (‘Grids’).

3) Untreated Control
Untreated test environment (hereinafter ‘Control’ or ‘Untreated’).

Testing Materials and Methods
1) Test Site and Date
Testing was conducted in the Carroll-Loye Arthropod Behavior Laboratory
in Davis, California, and took place from 22 April, 2008.

2) Environmental Conditions
Testing was conducted in a 20’ x 20’ greenhouse laboratory in order to
mimic outdoor conditions more closely than possible in a standard indoor
laboratory setting. Temperature and relative humidity were recorded during
assessment of mosquito behavior. They ranged from 25-27°C, and 42-52%
relative humidity.

3) Mosquitoes
Yellow fever (Aedes aegypti) mosquitoes, a diurnal species, reared at Carroll-
Loye Laboratories. Approximately 750 adults were placed in the test
environment. The females had emerged from their pupal cases four days earlier
and were actively seeking blood. A small proportion of male mosquitoes, which
also approach and land on humans but do not blood-feed, were also present.

4) Materials Control
Bug Bam Company supplied the Grids by courier. The Grids were
received in good condition on 8 April, 2008 and catalogued on receipt. They
were stored in the packaging in which they were received, indoors in closed
containers at room temperature (21°C).

5) Test design
Grids were placed at the center of a heated, humidified room with slow
airflow, mimicking natural conditions of use. Repellency was tested with a
human subject. The subject wore protective clothing, and tested mosquitoes
by exposing a pre-washed, untreated forearm in a series of 1-min periods at
12 standard points in a spatial array (Figure 1). Sampling points (dark circles)
were in three quartets: 1–Proximate (1’ from center); 2–Margin (10’ from
center); 3–Distant (14’ from center).
Figure 1. Schematic map of testing enclosure. The enclosure was
twenty feet on each side. The large incised circle enclosed slightly
over 300 ft2. Each Bug Bam! grid was placed at the center of the
enclosure (dark square), suspended 7 ft above the floor. Samples
of the number of landing mosquitoes were taken at the 12 testing
points marked by each of the small dark circles. The arrows show
the direction of airflow through the apparatus.

Before each trial of a Grid, the subject sampled the number of mosquitoes in
the absence of the Test Material to generate Control, ‘Untreated’ values. For
testing of the product to collect ‘Treated’ values, testing began 5 minutes
after each Grid was placed in the room. Air was flushed and replaced in the
enclosure for 10 minutes between trials.
During both Treated and Untreated sampling, mosquitoes that landed on the
subject arm were captured before biting with a mechanical aspirator. Those
mosquitoes were then released at the point of capture after each exposure.
To test for an aging effect, Grids were opened at either of two times: 100
hours before being tested, or at the time of test.
Testing was replicated three times for each type of Grid (new versus 100
hours post-opening). The Grid was replaced with one of the appropriate
history for each replicate. The sequence of testing was as follow:
1) Control 1a, New Grid 1, Air Flush
2) Control 1b, 100hr Grid 1, Air Flush
3) Control 2a, 100hr Grid 2, Air Flush
4) Control 2b, New Grid 2, Air Flush
5) Control 3a, New Grid 3, Air Flush
6) Control 3b, 100hr Grid 3

6) Data collection and recording
The number of mosquitoes landing in each exposure was recorded. Sampling all
12 points in sequence required about 20 minutes. Temperature and humidity
were recorded during each exposure sequence with traceably calibrated
thermohygrometer.
Carroll-Loye Biological Research
BBAM–001 Page 6 of 12

7) Data Analyses
Descriptive statistics were calculated for counts of mosquitoes on the subject
in each replicate. The null hypothesis was that the number of mosquitoes in
Treated versus Untreated conditions, would not differ on average.
To test for any effect of treatment, we employed analysis of variance across
the Treated versus Untreated conditions.
Statistical analyses were conducted with the software program SAS JMP (v.
5.0.1.2), SAS Institute, Cary, NC.
Carroll-Loye Biological Research
BBAM–001 Page 7 of 12

Test Results
Substantial treatment effects were evident for both newly opened (‘New’)
and 100 hour old (‘Old) Grids. In each sample, four records were made at
each of three distances from the center of the enclosure (Near=1’,
Marginal=10’, and Distant=14’). Figure 2 compares the average number of
landings recorded at each of the three distances.

Control landings show the landing pattern in the enclosure in the absence of
the test product. In general, slightly more mosquitoes landed at greater
distances from the center of the enclosure (Figure 2, left panel), perhaps due
to an influence of the enclosure shape and size on their behavior. In the
presence of either New of Old Grids, the pattern was similar, except that the
mean values were much lower (Figure 2, center and right panels).
Statistical analyses support the described patterns of treatment and position
effects. Analysis of variance of the simultaneous effects of those factors
shows both to be significant, as is their interaction (Table 1).
Table 1. Analysis of variance of the effects of Treatment (including the Control)
and the three Positions (proximate, marginal and distant) , plus their interaction
(indicated by an asterisk) on the number of landing recorded. Replicates are
pooled for analysis.

Source	DF	Sum of Squares	F Ratio	Prob > F
Treatment	2	2607.1389	145.3295	<.0001
Position	2	573.2167	31.9528	<.0001
Treatment*Position	4	134.0694	3.7367	0.0065

Of the effects examined, the strength of Treatment (i.e., Grids) was most
outstanding (largest F ratio, Table 2). The interaction between treatment and
position is graphically evident in Figure 2, which shows that landing
increased with distance more strongly in the treatments (particularly 100hr
Grid) than in the controls.

Table 2 tabulates and compares the results depicted in Figure 2. Relative to
the control means, percent repellency was high (approximately 90%) near
the center in both New and Old Grid treatments, and also considerable at the
margins. It declined notably beyond the margin of the 300 ft2 area.
Within a distance, the mean number of landings was significantly lower
under treated than untreated conditions in all comparisons (Table 2). In
addition, while not tabulated, note that with the New Grid treatment, mean
landing numbers were significantly lower at the proximate and marginal
distances, while differing significantly at among all three distances with the
100-hr grid treatment.
Table 2. Mean number of mosquitoes landing under control and treated conditions
at three distances for the array center. For each distance, means followed by
different letters (‘a’, ‘b’ or ‘c’) differ significantly at P<0.05 in T-tests.

Distance from center	Control	New Grid Mean	New Grid % repelled	100hr Grid mean	100hr Grid % repelled
Proximate	11.1a	1.1b	90	1.3b	88
Margin	12.1a	1.7b	86	3.8c	70
Distant	13.5a	6.0b	56	9.0c	43

Results are presented in additional detail in Appendix 1.

Conclusions
Bug Bam! Insect Repellent Grids substantially and consistently reduced the
rate at which captive, aggressive yellow fever (Aedes aegypti) mosquitoes
landed on the otherwise unprotected arm of the test subject. Mosquito
landing pressure in the testing environment was higher than that which
consumers would be likely to encounter under typical conditions of use. The
results obtained are excellent for an area repellent, in which reductions
greater than 50% are regarded as substantial.
Repellency approaching 90% is more typical of topical repellents, and near
the Grid, repellency was that great for the product at 100 hr after being
removed from the packaging. Spatial effects were also pronounced, with
strong repellency being consistent within a 10 ft radius of the grid, but
dropping substantially beyond that distance within the test enclosure.
In summary, Bug Bam! Insect Repelling Grids were effective in
repelling Mosquitoes at a rate of 70-90% within an area of 300 square feet
for a duration of 100 hours after opening. That performance is likely to
substantially exceed that of area repellents such as citronella candles, and is
likely attributable to the higher concentration of active ingredient, which
includes Geranium oil, in a time-released matrix.

Appendix 1.
Figure 3A-C shows the raw values and means for number of
landings that were recorded at point at each distance from the center of the
enclosure. For each of the three replicated Treated or Control samples at
each distance, there are four records (one taken from each of the four
sampling points arrayed at each distance; see Figure 1 of text).
The figure shows that at both the proximate and marginal distances, the
number of landings was consistently reduced by Bug Bam! Grids, regardless
of age. At the distant points, the influence of Bug Bam Grids was also
evident, but not as pronounced. Only in distant samples, that is, those made
beyond the margin of the 300ft2 circle, was there strong indication of
product failure (Old Grid Trial 3, Figure 3C).

Figure 3A–C. Number of landings in all trials; diamond centers show means of
each. The gray horizontal line is the grand mean for trials at each distance.


The University of New South Wales
August 2004

AIM
To evaluate by exposure to mosquitos (Culex quinquefasciatus), the repellency effects of a
test formulation applied as an insect repelling wristband.
FORMULATION
The formulation evaluated in this report, were supplied by Bug Bam Insect Repellent Wrist
Bands Pty Ltd, and was labelled as follows:
• Bug Bam Insect Repelling Wrist Bands
Active Ingredients: Citronella, Lemongrass & Geranium oils
In this report the formulation is referred to as Bug-Bam. Each sample supplied was regarded
as a blind sample; CERIT cannot verify the content of the samples.
The formulation was a plastic wrist band individually sealed in a "vapour barrier bag"; the
samples were stored at room temperature (not exceeding 30°C).
METHOD
The methods used follow those described in the protocol IN-VITRO REPELLENCY
PROTOCOL USING HOUSEHOLD FLYING INSECTS CERIT/HF-HM/REP 1.0
27/11/91 and was carried out on 14/7/04 and 21/7/04.
Modifications to Standard Protocol CERIT/HF-HM/REP 1.0 27/11/91
Formulations
For these tests: plastic insect repelling wrist bands individually sealed in a vapour barrier
packaging were used.
The standard protocol: allows for pump action spray-on formulations.
In-vitro Repellency Trial vs Mosquitos
(Bug Bam Insect Repellent Wrist Bands Pty Ltd: 1 formulation)
IRB/01/04 REPORT Treated Surface
For these tests: the plastic insect repelling wrist bands were the treated surface.
The standard protocol: allows for a 10cm x 10cm square of absorbent paper towel to be
treated with the formulations.
Control
For these tests: a white plastic rectangle 45mm x 115mm was used as an untreated control for
the wrist bands. Additionally, a 10cm x 10cm square of untreated paper towel attached to a
cardboard backing was used as a control for normal mosquito activity.
The standard protocol: allows for a 10cm x 10cm square of untreated paper towel to be used.
Treatment Method
For these tests: the insect repelling wrist bands were removed from their vapour barrier bags.
The wrist bands were cut in half and stapled together to create a rectangular shape
approximately 45mm x 115mm. Insect exposures commenced immediately.
The standard protocol: allows for the formulation to be applied to large sheets of paper towel,
then cut into squares and attached treated side down to a cardboard square 10cm x 10cm.
Analysis of Results
For these tests: paired t-test was used to compare the insect repelling wrist band against the
plastic control. This is more appropriate for a single treatment versus a control trial.
The standard protocol: allows for Analysis of Variance and Student-Newman Keuls test to
allow multiple comparisons between formulations.

OBJECTIVE OF TEST
The aim of the test was to evaluate by exposure to mosquitos the repellency effects of a test formulation
applied as an insect repelling wrist band.
INSECTS
Species: The insect species tested was the mosquito (Culex quinquefasciatus). The strain used was
laboratory bred and insecticide susceptible.
Number of insects: a minimum of 250 mosquitos per cage (20 x 20 x 25cm) were used.
Sex and life stage of insects: mixed sex adults aged between 5 and 10 days post emergence were used.
Details of rearing: Mosquitos were maintained at a temperature of 26.0+1.0°C and humidity of
75%+5%. Mosquito larvae were reared in deionised water and were fed fish food sticks. Adult
mosquitos were kept in mesh cages with 10% sugar water provided ad. lib.
FORMULATIONS
The formulation, which was a plastic insect repelling wrist band, individually sealed in vapour barrier
packaging, was supplied by the client. Formulation samples were stored at room temperature (not
exceeding 30°C).
TREATED SURFACE
The plastic wrist bands measuring 22mm x 230mm were the treated surface.
A white plastic rectangle 45mm x 115mm as well as an untreated paper towel 10 x 10cm square were
used as a control.
TREATMENT METHOD
The insect repelling wrist bands were removed from their vapour barrier bags. The wrist bands were cut
in half and stapled together to create a rectangular shape approximately 45mm x 115mm. Insect
exposures commenced immediately.
CONDITIONS DURING TEST
Tests were carried out in a large room with good ventilation to avoid saturation of formulation in the air.
Temperature - 25.0+1.0°C; Humidity - Ambient
TESTING PROCEDURE - REPELLENCY ASSAY
The trial commenced at 0 hours when the newly prepared wrist band was clipped against one side of the
cage. Before each count, mosquitos were 'activated' by disturbing the air and brushing the sides of the
cage until a majority of the mosquitos were flying. The cage was left undisturbed for 30 seconds to allow
the mosquitos to land. The number of mosquitos, which had landed upon the cage over the area where
the treated surface was clipped, were recorded at 0, 4, 24, 40 and 48 hours post treatment.
After each count, the treated surface was removed and taken to another room to prevent saturation of the
air with the formulation. The test was to be terminated prematurely if repellency action had expired.
The test was replicated eight times over a period of three days for the test formulation and the controls.
ANALYSIS OF RESULTS
A paired t-test was applied to the landing results for the test formulation and the plastic control, (Sokal
and Rohlf "Biometry", Freeman 1981).
RESULTS
Table 1: Means and 95% Confidence Limits calculated from 'mosquito landings' results for
eight replicates of each formulation at the indicated assessment times.

		Assessment time
Treatment		0 hours	4 hours	24 hours	40 hours	48 hours
Bug-Bam	-95% CL	-0.2	-0.1	0.4	0.0	0.3
	Mean	0.3	0.1	0.8	0.5	0.8
	+95%	0.7	0.4	1.1	1.0	1.2
Untreated plastic control	-95% CL	2.4	0.9	4.1	3.1	4.1
	Mean	3.4	2.3	5.1	4.4	4.6
	+95%	4.4	3.6	6.1	5.7	5.1
Untreated paper control	-95% CL	3.8	1.9	7.0	5.0	6.5
	Mean	5.5	2.5	9.5	7.1	8.6
	+95%	7.2	3.1	12.0	9.3	10.8

Graph 1: Means and 95% Confidence Limits calculated from 'mosquito landings' results for


DISCUSSION
Mosquitoes were deterred from landing on the cage area where the Bug-Bam treatment was
attached, with little variation in mosquito landings over the 48 hour period for which the
exposure continued. The t-test comparisons indicate that for every assessment time, the
mosquito landings on the Bug-Bam treatment were significantly lower than the landings on
the plastic control (5% level of significance). The landings on the paper control were within
the range normally expected for this protocol, indicating normal mosquito activity.