Reproductive and Developmental Toxicity of Selected QACs: A Chemical Detective Story
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Terry C. Hrubec
E. Via College of Osteopathic Medicine, Virginia Campus, Blacksburg, VA VA - MD College of Veterinary Medicine, Virginia Tech, Blacksburg, VA
DISCLOSURE
I have nothing to disclose; no financial or other ties which would pose a conflict of interest
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Stage 1. “The Crime Scene”
Control mice suddenly developed Neural Tube Birth Defects (NTDs)
Also noticed a decrease in number of mouse pups born
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Historic Observed
# of
Pup
s
Litter size
0
2
4
6
8
10
12
Historic Observed
NTD
s pe
r Litt
er (%
)
NTDs per Litter
3
Stage 2. “The Investigation”
• No change to animal husbandry, diet, source of mice, etc.
• Serology was negative for known mouse pathogens
• Toxicologic analysis of the food, bedding, enrichment materials was negative
• Rearing mice in a sterile environment had no effect
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Stage 2. “The Investigation” continued
Further questioning of the animal care supervisor revealed that they recently switched to a new disinfectant in the mouse room
• Floors, walls, and racks are foamed once a week
• Floors are mopped daily
• Mouse boxes are sprayed before opening
• Hands are wetted with disinfectant prior to handling mice
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Stage 3. “The Prime Suspect”
Quaternary Ammonium Disinfectant
Specifically a mixture of:
6.76% Alkyl dimethyl benzyl ammonium chloride (ADBAC, BAC) with 60% C - 14, 25% C - 12, 15% C - 16 chain lengths
and
10.1% Didecyl dimethyl ammonium chloride (DDAC)
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Stage 4. “Building the Case”
N = 10 - 12 litters per treatment 5 boxes per treatment
* Denotes significantly different
**
0
5
10
15
20
QAC room QAC Freeroom
QAC room QAC Freeroom
Rats Mice
% N
TDs
/ Li
tter
NTDs with Ambient Exposure
0
20
40
60
80
100
New Box 0 60 120
Det
ecte
d R
esid
ue
(ppb
)
Mouse Dose (mg/kg/day)
BAC Residues Measured in Used Mouse Boxes
05
10152025
0 60 120
% N
TDs
/ Litt
er
QAC concentration (mg/kg/day)
NTDs with Dosed Exposure*
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Stage 4. “Building the Case” continued
•NTDs persist until the offspring of the F2 generation
**
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F0generation
F0generation
F1generation
F2generation
F2generation
QACbuilding
QAC-free building QACbuilding
% N
TDs
/ Litt
er
NTDs by Generation and Building
Ambient Exposure
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Stage 4. “Building the Case” continued
Male and Female germ cells, stem cells for next generation egg and sperm, are formed by day 12 of development
About 500 cells at this stage
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Stage 4. “Building the Case” continued
*
0
5
10
15
20
25
0 30%
NTD
s / L
itter
ADBAC+DDAC (mg/kg)
NTDs with Active Ingredients
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Stage 5. “Testing the Hypothesis”
Test ResultFacilities using QAC disinfectant vs. those notusing QAC disinfectant
Reproduction and Development return to normal in the Non-QAC facility
Ambient exposure vs. oral dose No difference in result, or slight increase in dosed over ambient
Dose with cleaning product vs. a combination of the individual active ingredients (in the same proportion)
No difference in response
Oral gavage vs dose in the feed vs. dose in the water Gavage >> in feed > in water
Male exposure vs. female exposure Both are affected. Male only exposure still causes NTDs
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Stage 5. Fertility
** *
0
5
10
15
20
25
30
35
# of Ovulations # of Implants Million Sperm/mL
Female Male
Declines in Female and Male Fertility
0 mg/kg/day
120 mg/kg/day
• Significant declines in both male and female fertility
• Significant declines in blood concentrations of reproductive hormones FSH, LH
B
A
Dose Exposure
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Stage 5. Fertility continued
0%
20%
40%
60%
80%
100%
0 60 120
Perc
ent o
f Litt
ers
QAC Concentration (mg/kg/day)
Late Term Pregnancy Loss
Full Term Pregnancy Loss
05
10152025303540
0 60 120
Day
s
QAC Concentration (mg/kg/day)
Days to First Litter
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10
0 60 120QAC Concentration (mg/kg/day)
Number of Pregnancies per Treatment
0
200
400
600
800
1000
0 60 120QAC Concentration (mg/kg/day)
Cumulative Number of Pups Born
Over a 6 month breeding trial
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Stage 5. Fertility continued
*
0
1
2
3
4
5
0 120Num
ber E
stru
s C
ycle
s
ADBAC+DDAC (mg/kg/day)
Number of estrus cycles per 3 week period
* *
05
1015202530354045
0 7.5 Ambient
Mill
ion
Sper
m /
mL
ADBAC+DDAC (mg/kg/day)
* *
0102030405060708090
100
0 7.5 AmbientPerc
ent m
otile
spe
rm
ADBAC+DDAC (mg/kg/day)
Sperm Count Sperm Motility
Ambient and Dose exposure
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Stage 5. Immune Function
0
20
40
60
80
100
120
cells only 0.0% 0.00001% 0.00005% 0.0001%
% o
f Pha
gocy
tic C
ells
QAC (%)
Murine Macrophage Phagocytosis
IL-6 TNF-α IL-10Cells alone 0±0 0±0 0.01±0.005Stimulated
Cells 8.36±0.16 7.05±0.58 0.32±0.04
Stimulated + QAC 13.9 ±0.23* 19.4 ±0.58* 0.15 ±0.02*
Antibody Response
Prenatal exposure: Decreased IgG response Increased IgE response
Cytokine Production (pg/L)
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Stage 5. Development
*
0
1
2
3
4
5
Control Dosed Male Dosed Female Both Dosed
% N
TDs
/Litt
er
NTDs in Dosed Males and Females
* *
0
2
4
6
8
10
Control Ambient Gavage
% N
TDs
/ Litt
er
NTDs from Dosed and Ambient Exposure
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Males received 30 mg/kg every other day for 10 days prior to breeding
Females received one dose of 15 mg/kg on day 8 of gestation.
There was no ambient exposure
Males received 7.5 mg/kg every other day for 10 days prior to breeding
Females received one dose of 7.5 mg/kg on day 8 of gestation
Mice received ambient exposure
Stage 5. Development
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Stage 6. QACs in Humans
EXPOSURE is UBIQUITOUS
• 50% of first year medical students had QAC residues on their hands ( based on a one point in time assessment) • 80 % of random participants had detectable QACs in their blood (N=43, QAC screening trial)
• Over 5,000 household products contain a QAC (based on publically available household product databases)
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Stage 6. QACs in Humans
QAC Screening Trial
• 43 random participants from a rural college town were enrolled • 2/3rds students, 1/3rd non - students (based on visual estimation of age) • 80% contained ADBAC and DDAC in their blood • All four QACs measured (C12 - BAC, C14 - BAC, C16 - BAC, DDAC) were
detected in the blood • Increased markers of inflammation • Decreased mitochondrial function • Altered cholesterol synthesis
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Stage 5. Implications
0
1
2
3
4
5
C12-BAC C14-BAC C16-BAC DDAC
QAC in Mouse Liver (µg/g of tissue)
0
2
4
6
8
10
12
C12-BAC C14-BAC C16-BAC DDAC
QAC in Mouse Brain (µg/g of tissue)
0
0.5
1
1.5
d0-C12 d0-C14 d0-C16 DDAC
QAC in Mouse Testes (µg/g of tissue)
C12-BAC C14-BAC C16-BAC DDAC
Numerous patents for use as contraceptives since the early 1970s
Licensed for use as a spermicides in Canada and Europe
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Stage 7. Implications continued
I am not saying QACs are implicated in any disease However, the possibility exists
§ Increases in Obesity Diabetes Autoimmune disorders Asthma Allergy Autism
§ Declines in Male and Female Fertility Sperm counts
§ These disorders are characterized byIncreased inflammation Mitochondrial dysfunction Alterations in cholesterol homeostasis
It behooves us to institute a biomonitoring program to better understand exposure and risk
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§ QAC use has increased dramatically in the last 30 years and will likely continue
§ This rise in use follows the same trends as:
SUMMARYIn Rodents In Humans
q QACs cause birth defects q Affects blood measurements of inflammation , mitochondrial function and
cholesterol synthesis
q QACs alter immune function (also in-vitro)
q QACs can accumulate in the tissues and cross the blood brain and blood testis barrier
in rodents
q QACs impair reproductive function
Due to the adverse findings outlined in this presentation and others, monitoring exposure is a prudent first step
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