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

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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

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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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