Predictors for Increased and Reduced Rat and Mouse Allergen Exposure in Laboratory Animal Facilities.

Introduction: Exposure to rat and mouse allergens during work in laboratory animal facilities represents a risk for being sensitized and developing allergic diseases, and it is important to keep the exposure level as low as possible. The objective of this study was to characterize the personal Mus m 1 and Rat n 1 exposure during work in laboratory animal facilities, and to investigate the effect of identified predictors of increased and reduced exposure.
Methods: Mus m 1 and Rat n 1 were analysed in whole day or task-based personal air samples by enhanced sensitivity sandwich enzyme-linked immunosorbent assay. Information about cage-and-rack systems, tasks, and other conditions known to influence the allergen exposure was registered. Predictors for allergen exposure were identified by multiple linear regression analyses.
Results: The median allergen exposure was 3.0 ng m-3 Mus m 1 and 0.5 ng m-3 Rat n 1, with large task-dependent variations among the samples. The highest exposed job group were animal technicians. Cage emptying and cage washing in the cage washroom represented the highest exposure, whereas animal experiments in the lab/operation room represented the lowest exposure, with laminar airflow bench being an exposure-reducing determinant. Cage changing was the highest exposed task in the animal room, where individually ventilated cages (IVCs) were predictors of reduced exposure for both Mus m 1 and Rat n 1, whereas cage-rack systems with open shelves and sliding doors were predictors of increased Rat n 1 exposure. Cages of IVC type with positive air pressure (IVC+) as well as open shelves and sliding doors were strong predictors of increased exposure during cage emptying and cage washing. Conclusions: Significant different exposure levels depending on type of work and task imply different risks of sensitization and allergy development. The fact that IVC+ cages have opposite impact on Mus m 1 and Rat n 1 exposure during different tasks may have positive clinical implications when taken into account.

Introduction

Working with laboratory animals is a well-known risk for developing allergic diseases. Typical airway symptoms include allergic inflammation in the nasal mucosa, conjunctivitis, and asthma, whereas urticaria is the most common skin reaction. Frequent contact with animals increases the probability of health effects. As many as 10–30% of the workers in laboratory animal facilities develop allergy against rat or mice (Hunskaar and Fosse, 1990; Renstrom ; Palmberg ; Simoneti ), which is considerable compared with other occupations such as bakers or dental technicians (Gautrin ). Between 5 and 40% develop allergy during the first 1–2 years of exposure (Renstrom ; Palmberg ). According to a pooled analysis of 13 studies, 80% individuals with allergic symptoms have inflammation in eyes or nasal mucosa (53–100%), 40% (13–70%) have skin reactions and 35% (13–71%) have asthma (Hunskaar and Fosse, 1990). Over 60% of individuals with laboratory animal allergy (LAA) have immunoglobulin (Ig) E antibodies specific for the animal allergens (Gordon and Preece, 2003; Palmberg ). Atopic individuals have 11 times higher risk of developing allergy (Renstrom ), and develop LAA four times faster than non-atopics (Botham ).

Allergens from mice and rats induce more allergies in humans than other animal allergens, primarily because mice and rats are used more often than other animals and not necessarily because the other animals are less allergenic (Aoyama ). The main allergens from rat and mice, Rat n 1 and Mus m 1, respectively, are lipocalin proteins produced by the liver and excreted in the urine. The excretion is hormonally controlled, and the level of Mus m 1 in urine is, due to androgen stimulation, about four times higher in male mice compared to female mice (Renstrom ). The allergens may be retrieved in hair, dandruff, and urine of the animals, as well as low levels in serum. Mice- and rat-allergens are mainly carried by particles of 3–10 µm in aerodynamic diameter (Ohman ; Pacheco ).

The number of animals killed for experiments have been reduced in some areas of science by utilizing in vitro systems as an alternative, but more recently genome-based research has led to increased use of experimental animal models, using particularly genetically modifiable mice (Feary and Cullinan, 2016). This may have resulted in both increased potential for exposure and increased number of employees in the laboratory animal facilities. On the other hand, modern equipment and facilities may have reduced the exposure risk. Although animal technicians have been the highest exposed job group, and scientists among the lowest exposed (Lieutier-Colas ; Curtin-Brosnan ), the work performed by the exposed job groups may change depending on the type of the facility. Several have expressed their concern about lacking task-based exposure assessments (Jones, 2015; Feary and Cullinan, 2016). Thus, there is need for a more detailed task-based exposure assessment of today’s laboratory animal facilities.

Different methods for allergen measurements hamper direct comparisons of concentrations reported between studies. However, with reasonable consistency, the allergen exposure has been shown to vary with the facility, the cage systems, the number of animals in the room, and the type and duration of performed tasks (Eggleston ; Ohman ; Nieuwenhuijsen ; Hollander ; Thulin ; Glueck ). Use of equipment such as individually ventilated cages (IVCs) has been shown to reduce the allergen exposure in animal rooms (Gordon ; Renstrom ), whereas handling dirty cages is a high-exposed task (Lieutier-Colas ; Thulin ; Korpi ).

It is difficult to establish occupational exposure limits for allergens, partly due to the lack of standardized measurement methods and partly because of complex exposure-response relationships (Jones, 2015) and the influence of genetic susceptibility, such as atopy (Palmberg ). To reduce the incidence of allergen sensitization and LAA, it is therefore important to keep the allergen exposure in laboratory animal facilities as low as possible. It is thus necessary to identify the conditions and tasks that can give the lowest possible exposure as well as predictors of high exposure that may provide a basis for priority setting of preventive measures. The aim of this study was to characterize the personal exposure to rat and mice allergens, and to investigate the effect of identified predictors of increased and decreased exposure.

Methods

Study population

The study population consisted of employees in laboratory animal facilities of Norway’s four largest universities with contact with mice and rats, and included animal technicians, laboratory technicians, scientists, and PhD students. Laboratory technicians, scientists, and PhD students were working at the lab/operation room, whereas animal technicians were working in animal room and cage-washing room.

Work environment

The laboratory animal facilities consisted of clean and dirty zones with a sluice between the zones. Clean zone consisted of corridors, offices, lunchroom, wardrobes, and clean side of the washing room, where the preparation of new cages took place. Work clothes, consisting of laboratory coat or coveralls, hairnet, gloves, shoes, and respiratory protective equipment, were put on in the sluice. The dirty zone consisted of animal rooms for unpacking of animals, laboratories, storage rooms, and cage washrooms including station for emptying of used bedding from cages. Strains, and number of mice and rats that were present during the study, varied between the facilities. All facilities had separate stables for mice and rats. There were separate breeding rooms and various contamination regimes. The animal cages were either open or IVCs with positive (+) or negative (−) air pressure. IVCs were connected to separate ventilation units that controlled air exchange, air humidity, and temperature in the cages, and filtered the exhaust air through high efficiency particulate air (HEPA) filters. In some facilities, the air was recirculated because the exhaust air was not always connected to the general ventilation in the room. The animal rooms had balanced ventilation and negative pressure relative to the surroundings, except rooms with stricter isolation regimes, which had positive pressure to protect the animals. Exhaust channels from the stables had HEPA filters to prevent emission of allergens outside the animal facility. When open cages were used in open shelves, sliding doors of plexiglass were mounted in front of the shelves to prevent the release of allergens out in the room, and the rooms had special ventilation that extracted the air from the room, past and out behind the shelves. All facilities used aspen bedding. A description of the work in each room, as well as the type of workbench, cage, and racks registered in this study, is presented in Table 1.

Table 1.

Work descriptions.

Room type	Work	Work description	Bench type, cages, and racks
Lab/operation room	Experiments	Minor surgery on animals under anaesthesia, behaviour experiments with registration of brain activity, blood sampling, and euthanization	Bench type: ventilated, LAF, other
Animal room	Changing cages	Transfer of animals from used cages with soiled beddings to clean cages with new bedding was primarily done in changing stations with LAF, but some places on ordinary tables/benches	Bench type: ventilated, ordinary, LAF, other/not providedCage type: open, filter top, IVC+, IVC−, other/not providedCage-racking system: open shelves, open shelves + sliding doors, IVC, other/not provided
	Animal caring and feeding	Checking general condition of animals, refill of food and water, medication, separation of offspring. Almost all tasks implied opening of cages
Washing room	Emptying cages	Dirty cages with used bedding were emptied in particular emptying stations, mainly with bedding transport band and LAF, in the unclean zone of the washing room. Cage tops were removed, feeders were emptied in the cages, and the cages were stacked on the floor or trolleys at the side of the emptying station. One cage at a time was emptied by turning the cage upside down on the transport band, and subsequently knocking and scraping the rest of the bedding out	Bench type: ventilated, otherCage type: open, IVC+, IVC−, other/not providedCage-racking system: open shelves, open shelves + sliding doors, IVC, other/not provided
	Washing cages	Cage washing was mainly an automatic process where the cages were washed either by stacking on a washing stand that was rolled into the washing machine, or by placing cages on a transport band that transported the cages through a tunnel washer
	Preparation of new cages	Adding new bedding, food, and water in clean cages were done on the clean side of the washing room
All rooms	Cleaning	Cleaning of surfaces was done successively after work operations throughout the day. IVC units were cleaned regularly with HEPA vacuum cleaning of the filters, and dismantling and cleaning of the ventilation channels. Cleaning of the LAF stations was done after each cage changing session in addition to a weekly main cleaning with HEPA vacuum cleaning. The emptying station and the floor around it were vacuum cleaned after each session of emptying. Main cleaning of all rooms in the facilities was done weekly in some facilities. Existing routines for control and service of IVC ventilation units, emptying station, and the washing machines were followed

Sampling strategy

Ten series of allergen sampling at six different laboratory animal facilities were performed during 2009–2015 by occupational hygienists employed at the universities, according to a standardized protocol for this study. The allergens were collected on Teflon filters (diameter 25 mm, pore size 1.0 µm, Millipore FALP 02500), placed in IOM cassettes (SKC Inc., Valley View, PA, USA) connected to a battery-powered pump with an airflow of 2 l min−1. Personal sampling (n = 184) of airborne allergens was performed either during the whole workday or during specific tasks (task-based strategy). The filter was placed in the breathing zone of the employees, outside of any respiratory protective equipment. The time of sampling was 15–380 min (median 133 min). Information about type of room, cage and cage rack, number of animals, sex of the animals, ventilation, work tasks and routines, job title, and use of personal protective equipment was registered in the sampling protocol.

Determination of Mus m 1 and Rat n 1 allergen

After sampling, filters were transferred to 3 ml Nunc Minisorp tubes with the exposed surface rolled in towards the centre of the tube. Allergens were extracted by incubating the exposed filters in 2-ml phosphate buffered saline (PBS) containing 0.5% Tween-20 and 0.15% ProClin (Sigma Aldrich) for 2 h with rotation. After 10 min centrifugation at 1000 g, the eluate was transferred into a fresh Minisorp tube containing 20-mg heat-fractionated Bovine serum albumin [BSA (Sigma-Aldrich)]. The BSA was dissolved by rotation for 5 min, and the eluate was aliquoted into 5 tubes and stored at −20°C until analysis. The samples were slowly thawed before analysis. Of the 184 collected samples, Mus m 1 and Rat n 1 allergen were analysed in 136 and 139 extracts, respectively. Both allergens were analysed in 91 of the samples. The allergens were detected by enzyme-linked immunosorbent assay (ELISA) using commercial Mus m 1 and Rat n 1 ELISA kits (Indoor Biotechnologies Inc., Wiltshire, UK) as basis for sandwich ELISAs with enhanced sensitivity, as previously described (Korpi ). In brief, flat-bottomed 96-well microtiter plates were coated overnight at 4°C with polyclonal rabbit anti-Mus m 1 at 1 µg ml−1 or monoclonal mouse IgG1 anti-Rat n 1 antibody (RUP-6) at 5 µg ml−1 diluted in 50 mM carbonate-bicarbonate buffer, pH 9.6. After washing with PBS containing 0.05% Tween-20 and blocking with Casein buffer (Fitzgerald Industries Int., Concord, MA, USA) at 1:20, a 9-step dilution row from 5 to 1280 pg ml−1 of Mus m 1 or Rat n 1 standards (Indoor Biotechnologies Inc., Wiltshire, UK) and samples diluted 1:2 or 1:20 in casein buffer were added and incubated for 90 min at room temperature. After washing, biotinylated rabbit anti-Mus m 1 polyclonal antibody at 1:10 000 dilution or monoclonal IgG1 anti-Rat n 1 antibody (RUP-1) diluted 1:2000 was added and incubated for 60 min at room temperature. The plates were washed, and streptavidin-polyHRP 80 conjugate (Fitzgerald Industries International, North Acton, MA, USA) was added at 1:20 000 dilution for Mus m 1 and 1:10 000 for Rat n 1, and incubated for 60 min at room temperature, before washing again. Thereafter, 1-step ultra tetramethylbenzide substrate (Thermo Scientific) was added and the reaction stopped after 15 min by adding 1N H2SO4. The optical density (OD) of the resulting colour was read at 450 nm in a Spectramax i3 spectrophotometer (Molecular Devices LLC., Sunnyvale, CA, USA) and adjusted for background signal read at 650 nm. A standard curve was obtained by 4- or 5-parameter best-fit evaluation, and upper and lower detection limit (DL) as well as the sample values were determined based on the standard curve. Determinations with coefficient of variation (CV) <10% of the response value for the duplicates, with CV <20% for different dilution of the same samples, and with background signal with OD <0.2, were accepted. Samples with OD <0.2 were treated as below the DL. The DL was between 18 and 80 pg ml−1 for both Mus m 1 and Rat n 1. Sample values were adjusted for dilution and air volume and expressed as allergen exposure in ng m−3.

Data analyses

The exposure values were skewed to the right and were ln-transformed to obtain near normal distribution before statistical analyses. Measurements below the DL of the ELISA were substituted by the DL/√2 for inclusion in the statistical analyses. The general exposure concentrations are presented by arithmetic mean (AM), median, minimum (min), and maximum (max). In addition, the 90 and 95 percentiles are presented for different room categories. Group differences in the ln-transformed mean of the personal measurements were tested by analysis of variance and subsequent post hoc pairwise tests with Bonferroni correction for multiple comparison, and by independent sample t-test for comparison of two groups. A P-value of 0.05 was regarded as statistically significant. Samples were grouped into exclusive room categories suitable for regression modelling of room-dependent exposure determinants (Fig. 1). Measurements not exclusive for any of the room categories were not included in regression analyses, but the exposure level was for the record collectively presented as a separate category. The most important determinants for allergen exposure in each room were identified by backwards selection in multiple linear regression analyses with the concentration of Mus m 1 or Rat n 1 as dependent variables and different determinants of exposure as independent variables. General linear regression models were built based on this, and the strength of the determinants is given as a factor related to the constant in the model. Exposure levels of different combinations of determinants can be computed from the regression models as follows:

Figure 1.

Flow chart of the grouping of personal samples into exclusive room categories suitable for regression modelling of determinants of Mus m 1 and Rat n 1 exposure. Work in lab/operation room was tantamount with experiments in the exclusive room category. ‘Other tasks’ consisted of one measurement of vacuum cleaning of rack aggregate and filter, one measurement of floor cleaning and wash/disinfection of cage changing station, one measurement of floor cleaning and filter change, and one measurement of floor cleaning and garbage handling.

where E = exposure, c = intercept of the regression model, and b1 and b2 = regression coefficients of the determinants 1 and 2.

The IBM software package SPSS version 24 was used for the statistical analyses (IBM Corp, Armonk, NY, USA).

Results

Allergen levels in laboratory animal facilities

The median allergen exposure was 3.0 ng m−3 Mus m 1 and 0.5 ng m−3 Rat n 1. The variation among the samples was large (Table 2). The mean Mus m 1 exposure among animal technicians was much higher than among scientists/students (P = 0.003) and laboratory technicians, although the difference to the latter was not statistically significant. The number of samples from scientists and laboratory technicians was too few for meaningful statistical analyses of the Rat n 1 exposure differences. However, when scientists and laboratory technicians were combined, their exposure levels were statistically significantly different from animal technicians, regarding both rat and mouse allergens (P = 0.03 and P < 0.001, respectively).

Table 2.

Exposure to rat and mouse allergens in all personal samples and by job title.

	Mus m 1 (ng m−3)						Rat n 1 (ng m−3)
	n	n < DL	AM	Median	Min	Max	n	n < DL	AM	Median	Min	Max
All personal samples	136	8	74	3.0	0.02	2261	139	48	8.4	0.5	0.01	413
Animal technician	120	4	83.7	5.0	0.02	2261	126	37	9.3	0.6	0.1	413
Scientist and students	11	4	1.2	0.3	0.05	6.7	7	7	0.3	0.1	0.1	1.0
Laboratory technicians	5	0	3.6	0.3	0.06	11	6	4	0.2	0.2	0.08	0.5

The median of the samples when including non-exchanged values for samples

The median of the samples when including non-exchanged values for samples