Ambient and Dosed Exposure to Quaternary Ammonium Disinfectants Causes Neural Tube Defects in Rodents

Abstract

Background: Quaternary ammonium compounds are a large class of chemicals used for their antimicrobial and antistatic properties. Two common quaternary ammonium compounds, alkyldimethylbenzyl ammonium chloride (ADBAC) and didecyldimethyl ammonium chloride (DDAC), are combined in common cleaners and disinfectants. Introduction of a cleaner containing ADBAC+DDAC in the vivarium caused neural tube defects (NTDs) in mice and rats.

Methods: To further evaluate this finding, male and female mice were dosed in the feed at 60 or 120 mg/kg/day, or by oral gavage at 7.5, 15, or 30 mg/kg ADBAC+DDAC. Mice also received ambient exposure to ADBAC+DDAC from the disinfectant used in the mouse room. Embryos were evaluated on gestational day 10 for NTDs, and fetuses were evaluated on gestational day 18 for gross and skeletal malformations.

Results: We found increased NTDs with exposure to ADBAC+DDAC in both rats and mice. The NTDs persisted for two generations after cessation of exposure. Notably, male exposure alone was sufficient to cause NTDs. Equally significant, ambient exposure from disinfectant use in the vivarium, influenced the levels of NTDs to a greater extent than oral dosing. No gross or significant axial skeletal malformations were observed in late gestation fetuses. Placental abnormalities and late gestation fetal deaths were increased at 120 mg/kg/day, which might explain the lack of malformations observed in late gestation fetuses.

Conclusion: These results demonstrate that ADBAC+DDAC in combination are teratogenic to rodents. Given the increased use of these disinfectants, further evaluation of their safety in humans and their contribution to health and disease is essential. Birth Defects Research 109:1166-1178, 2017. © 2017 The Authors. Birth Defects Research Published by Wiley Periodicals, Inc.

Keywords: QAC disinfectants; QACs; abnormal development; environmental contaminants; neural tube defects; teratogenesis.

FIGURE 1

Diagram depicting the animal facilities and rooms used in experiments. In all, six experiments were conducted, numbered 1 to 6. Three animal facilities were used, indicated as Facility A, B, or C. Within Facility A, three rooms were used, indicated as Room A1, A2, or A3. The numbers within the light blue rectangles represents the experiment number. The location of the numbers corresponds to the housing location of a treatment group. For example, in Experiment 5, control mice were held in Facility B and exposed mice in Facility A room 3.

FIGURE 2

Neural tube defects (NTDs) in GD10 mouse embryos exposed to ADBAC+DDAC. A: Control unexposed. B–D: Incomplete closure of the rostral face, B also has two spinal NTDs. E,F: Cranial NTDs demonstrating a range in defect size. G: Spinal NTD. H: Embryo with altered morphology in the absence of open NT.

FIGURE 3

Experiment 1: NTDs in GD 11 rat and GD 10 mouse embryos exposed to ambient ADBAC+DDAC disinfectant from normal husbandry practices and after being raised in a room using ethanol and chlorine dioxide. NTDs were reduced after being removed from the ADBAC+DDAC environment (* indicates significant difference between the two rooms, ANOVA p ≤ 0.05). Values represent the mean ± SEM percent per litter with N = 20 rats and 13 mice in the QAC room, and N = 9 rats and 9 mice in the QAC-free room.

FIGURE 4

Experiment 2: NTDs in GD 10 mouse embryos dosed in the feed with ADBAC+DDAC disinfectant for 8 weeks. The numbers of NTDs were significantly greater in the 120 mg/kg/day exposed group compared with controls (* indicates significant difference from control, ANOVA p ≤ 0.05). Values represent the mean ± SEM percent per litter with N = 10 to 12 dams per treatment.

FIGURE 5

ADBAC residues detected in mouse boxes. ADBAC residues from new or used mouse boxes were extracted with methanol and analyzed by liquid chromatography with ultra violet detection. New boxes did not contain ADBAC residues. Residues were detected in all used boxes. Residues in the boxes of control mice (0 mg/kg/day) indicated inadvertent exposure in the room. N = 2 samples for each treatment. Each sample contained the residue from five boxes. Each sample was run in duplicate and averaged.

FIGURE 6

Experiment 2: Average fetal weight and placental weight of GD 18 fetuses in mice dosed in the feed with ADBAC+DDAC disinfectant for 8 weeks. Fetal and placental weights were significantly lower in 60 and 120 mg/kg/day exposed mice compared with controls (* indicates significant difference from control, ANOVA p ≤ 0.05). Values represent the mean ± SEM with N = 10 to 11 dams per treatment.

FIGURE 7

Experiment 2: Distribution of average fetal–placenta ratio for GD 18 litters dosed in the feed with ADBAC+DDAC disinfectant for 8 weeks. While the mean ratios were quite similar in each treatment group, the range was significantly greater in the 60 and 120 mg/kg/day exposed litters (F-test, N = 10–11 dams per treatment).

FIGURE 8

Experiment 3: NTD malformations in GD 10 embryos followed for three generations after moving to a facility that did not use QAC containing disinfectants. F0 mice were exposed to ambient disinfectant in the QAC facility. F0 females moved to a QAC-free facility, had fewer NTDs than the same cohort remaining in the QAC facility. Subsequent generations in the QAC-free facility exhibited a decline in NTDs (* indicates significant difference from F2 Dams, ANOVA p ≤ 0.05). Values represent the mean ± SEM percent per litter, N = 12 to 15 dams per generation.

FIGURE 9

Experiment 4: The role of maternal and paternal ADBAC+DDAC exposure on NTDs in GD 10 in offspring. One or both parents were dosed with 120 mg/kg ADBAC+DDAC in the feed for 8 weeks. Mice were moved to a non-QAC facility for breeding and gestation except one group that continued exposure of both parents throughout breeding and gestation. NTDs were significantly higher when only a single parent was dosed; however, husbandry use of ADBAC+DDAC disinfectant was discontinued in between evaluation of single parent and both parent groups. Removal of the ambient exposure likely resulted in lower NTDs in the groups with both parents dosed. (* indicates significant difference from controls, ANOVA p ≤ 0.05). Values represent the mean ± SEM percent per litter, N = 12 to 15 dams per treatment group.

FIGURE 10

Experiment 5: The role of maternal and paternal ADBAC+DDAC exposure on NTDs with no ambient exposure from disinfectant use in the mouse room. Males were dosed every other day for 10 days with 30 mg/kg, and females were dosed once on GD 8 with 15 mg/kg ADBAC+DDAC. NTDs were significantly higher when both parents were dosed (* indicates significant difference from controls, ANOVA p ≤ 0.05). Values represent the mean ± SEM percent per litter, N = 12 to 15 dams per treatment group.

FIGURE 11

Experiment 6: NTDs in mice exposed to ambient husbandry use of the disinfectant or ambient plus 7.5 mg/kg ADBAC+DDAC by gavage. Males were dosed every other day for 10 days, and females were dosed once on GD 8. NTDs were significantly higher than controls. There was no difference between mice receiving ambient or ambient+gavage exposure (* indicates significant difference from controls, ANOVA p ≤ 0.05). Values represent the mean ± SEM percent per litter, N = 12 to 15 dams per treatment group.