Registration Dossier

Administrative data

Endpoint:
respiratory sensitisation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Not reported.
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study conducted in accordance with generally accepted scientific principles, possibly with incomplete reporting or methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012

Materials and methods

Test guideline
Qualifier:
no guideline available
Principles of method if other than guideline:
There are no currently well accepted or validated methods for the identification of those chemicals with the potential to cause respiratory allergy. However, progress has been made with respect to understanding the immunobiological mechanism of respiratory allergy and there is a growing consensus that chemical respiratory allergens stimulate the preferential development of type 2 T lymphocytes. This provides the cytokine milieu (interleukin [IL]-4, IL-5, IL-10 and IL-13) necessary for the development of many of the features of chemical respiratory allergy: the production and maintenance of IgE antibody, differentiation and recruitment of eosinophils, activation of mast cells and hypersecretion of mucins in the respiratory tract.

These observations have been used to develop methods to characterise the nature of the allergic response that a chemical is likely to provoke. Thus, topical exposure of mice to chemicals that are known to cause in humans allergic sensitisation of the respiratory tract provoke the production by lymph node cells (LNC) of type 2 cytokines. In contrast, it has been found that contact allergens that lack the ability to cause respiratory sensitisation induce in mice the development of a preferential type 1 T lymphocyte response characterised by the secretion by draining LNC of interferon γ (IFN-γ) and IL-12. This experimental approach has been designated “cytokine fingerprinting”. An alternative approach, the mouse IgE test, may also be used for the identification of chemicals that have the potential to cause allergic sensitisation of the respiratory tract. The method is based upon the fact that topical exposure of mice to chemical respiratory allergens elicits a substantial increase in the total serum concentration of IgE; a response not observed with chemical contact allergens that are believed not to cause respiratory sensitisation.

Dose selection for cytokine fingerprinting and mouse IgE tests is based upon doses identified as positive in a murine LLNA test. The rationale for this is that experience to date indicates that all chemical respiratory allergens are able to induce positive responses in the LLNA and in other predictive tests for contact sensitising potential. Furthermore, previous experience in the testing laboratory is that in order to stimulate the sufficient expression of cytokines, a material must provoke a relatively vigorous response in the LLNA. Those chemicals that induce a 3-fold or greater increase in proliferation (stimulation index; SI) compared with concurrent vehicle treated controls are classified as positive in the LLNA.

A selective and step-wise approach for the characterisation of the sensitising potential of the test material was employed. The known respiratory sensitiser platinum salt ammonium hexachloroplatinate (AHCP) was utilised as a positive control. AHCP has been shown previously in the testing laboratory to stimulate a type 2 cytokine secretion profile consistent with respiratory sensitising activity when formulated in dimethylsulfoxide (DMSO). Tetraammine platinum dichloride (TPC), a material that lacks sensitising activity in experimental animals and has been used extensively in the industry for many years in the absence of occupational asthma was used as a negative control in some experiments.

Initially, sighting studies were performed using identical applications, site and the timing as the LLNA protocol to ensure lack of significant local or systemic toxicity of the test material and to identify concentrations of chemical that cause immune activation. On the basis of these studies, the activity of the test material was then examined in a modified LLNA (using BALB/c strain mice and a nonradioactive end point for the determination of proliferative activity). In parallel, a cytokine fingerprinting study was conducted to confirm that the vehicle of choice (dimethyl formamide; DMF) was without impact on the induction of a type 2 response by the positive control material AHCP. Responses provoked by the test material were compared with those elicited concurrently by the control chemicals 2,4-dinitrochlorobenzene (DNCB) and trimellitic anhydride (TMA). The former is a potent contact allergen, which lacks the ability to cause sensitisation of the respiratory tract, and provokes a Th1-type cytokine response and fails to stimulate vigorous increases in total serum IgE concentration, while the latter is a known cause of respiratory allergy and occupational asthma in humans and stimulates a Th2-type cytokine profile and marked increases in the concentration of total serum IgE. Animals were exposed topically to chemical sensitisers over a 13 day period (standard cytokine fingerprinting dosing regimen) to allow the development of a polarised cytokine phenotype. In one experiment, mice were exposed topically to chemical sensitisers over an 18 day period in order to examine the kinetics of the response to these materials. After the sensitisation period, the mice were terminated and serum collected for analysis of total serum IgE concentration and the draining (auricular) lymph nodes were excised and cultured for cytokine expression.
GLP compliance:
not specified

Test material

Constituent 1
Test material form:
other: liquid (not specified)
Details on test material:
Storage conditions: room temperature

Test animals

Species:
mouse
Strain:
Balb/c
Sex:
female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: Young adult (8 - 16 weeks).
- Housing: Groups of 5 were housed in plastic individually ventilated cages (IVC) for rodents with materials provided for environmental enrichment (nesting material).
- Diet (e.g. ad libitum): ad libitum.
- Water (e.g. ad libitum): ad libitum.
- Acclimation period: Minimum of 5 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 26 °C.
- Humidity (%): 30 - 70 %.
- Photoperiod (hrs dark / hrs light): 12 continuous hours of artificial light in each 24 hour period.

Test system

Route of induction exposure:
dermal
Route of challenge exposure:
other: dermal
Vehicle:
other: dimethyl formamide
Concentration:
Induction exposure: animals received 50 µL of the test material dissolved in DMF bilaterally on each shaved flank.
Challenge exposure: 25 µL of test material was applied to the dorsum of both ears.
No. of animals per dose:
Sensitisation of mice for standard cytokine fingerprinting assay: Groups of mice (n = 3 - 10)
Sensitisation of mice for extended cytokine fingerprinting assay: Groups of mice (n = 3)
Details on study design:
SIGHTING STUDIES
Animals (n = 1 per group) received 25 µL of various doses of test material dissolved in DMF on the dorsum of both ears daily for three consecutive days. Material was applied using a Gilson variable micro-pipette. Animals were observed three times daily on the day of painting for signs of local toxicity (erythema or irritation) or systemic toxicity. Five days after the initiation of treatment, animals were terminated by asphyxiation with CO2, draining lymph nodes were excised and size/appearance recorded photographically.

MTT ASSAY FOR PROLIFERATION
Mice (n = 5 per group) received 25 µL of various doses of test material dissolved in DMF dissolved in DMF on the dorsum of both ears daily for three consecutive days. Material was applied using a Gilson variable micro-pipette. Animals were checked daily on each application day for signs of local toxicity (erythema or irritation) or systemic toxicity. Five days after the initiation of treatment, animals were terminated by asphyxiation with CO2, draining auricular lymph nodes were excised and pooled for each experimental group. A single cell suspension of lymph node cells (LNC) was prepared under aseptic conditions by gentle mechanical disaggregation through sterile 200-mesh stainless steel gauze. Viable cell counts were performed using a haemocytometer by exclusion of 0.5 % trypan blue and total cellularity per lymph node was recorded for each treatment group. LNC were resuspended in RPMI-1640 growth medium (Gibco, Invitrogen, Renfrewshire, UK) supplemented with 25 mM/L HEPES, 400 µg/mL streptomycin, 400 µg/mL ampicillin and 10 % heat-inactivated foetal calf serum (RPMI-FCS) at 10^7, 5 x 10^6, 2.5 x 10^6, 1 x 10^6 and 0.5 x 10^6cells/mL. Triplicate aliquots (100 µL) of each concentration of LNC were seeded into 96-well flat bottomed microtitre plates and cultured at 37 °C in a humidified atmosphere of 5 % CO2 in air for 72 hours in the presence of medium alone or in the presence of 2 µg/mL of the T cell mitogen concanavalin A (con A; Sigma). Control wells contained an equivalent volume of medium alone.
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma) in phosphate buffered saline (PBS; pH 7.2) at 5 mg/mL solution was added (10 µL per well), each well was mixed using a pipette and plates were incubated for 4 hours at 37 °C in a humidified atmosphere of 5 % CO2 in air followed by addition of 20 % sodium dodecyl sulfate (SDS; Sigma) in 50 % DMF. Plates were incubated overnight at 37 °C in a humidified atmosphere of 5 % CO2 in air, shaken at 300 rpm for 5 minutes and then optical density at 620 nm was measured using an automated reader (Multiskan, Flow Laboratories, Irvine, Ayrshire, UK).

CALCULATION OF STIMULATION INDEX FOR PROLIFERATION
The cellularity index (CI; fold increase in total cellularity of the lymph nodes) was calculated on a treatment group basis using the cellularity of the concurrent vehicle-treated control group as the comparator
CI = Test group cellularity / Vehicle control group cellularity

The estimated cell concentration of each treatment group (test and vehicle treated) required to reach 50 % of the maximal proliferative response (EC50 % proliferation) was calculated by linear interpolation

EC50 % proliferation = c + [(half maximal response - d) / (b - d)] (a - c)

where the maximal response was that induced by culture with the T cell mitogen con A and the data points lying immediately above and below the half maximal proliferation value have the co-ordinates (a, b) and (c, d), respectively

The proliferation index (PI) for each treatment group was derived by dividing the EC50 % proliferation value obtained for the concurrent vehicle treated control group by the EC50 % proliferation value obtained for the test material.

PI = EC50 % proliferation vehicle / EC50 % proliferation test

The overall stimulation index (SI) is a function of both the increases in cellularity of the whole lymph node and the increases in proliferation of a pre-determined number of cells, thus for each concentration of test chemical

SI = CI x PI

SENSITISATION OF MICE FOR STANDARD CYTOKINE FINGERPRINTING ASSAY
Groups of mice (n = 3 - 10) received 50 µL of test material dissolved in DMF bilaterally on each shaved flank. Material was applied using a Gilson variable micro-pipette. Further control animals were treated concurrently with 10 % TMA or 1 % DNCB dissolved in AOO (4:1 acetone:olive oil) or with 5 % AHCP in DMF. In one experiment animals were exposed to 10 % TMA in AOO or DMF in comparison with exposure to 5 % AHCP in DMF and 2 % AHCP in DMSO. Five days later this treatment was repeated. After a further 5 days, 25 µL of chemical was applied to the dorsum of both ears daily for three consecutive days.

SENSITISATION OF MICE FOR EXTENDED CYTOKINE FINGERPRINTING ASSAY
Groups of mice (n = 3) received 50 µL of test material dissolved in DMF bilaterally on each shaved flank. Material was applied using a Gilson variable micro-pipette. Further control animals were treated concurrently with 10 % TMA or 1 % DNCB dissolved in AOO or with 5 % AHCP in DMF. Five and 10 days later this treatment was repeated. After a further 5 days, 25 µL of chemical was applied to the dorsum of both ears daily for three consecutive days.

PREPARATION OF CELLS FROM CYTOKINE FINGERPRINTING
Thirteen or eighteen days (extended protocol only) after the initiation of treatment, animals were terminated by asphyxiation with CO2, draining auricular lymph nodes were excised and pooled for each experimental group. A single cell suspension of LNC was prepared under aseptic conditions by gentle mechanical disaggregation through sterile 200-mesh stainless steel gauze. Viable cell counts were performed by exclusion of 0.5 % trypan blue and LNC were cultured in RPMI-FCS. Total cellularity per lymph node was recorded for each treatment group. Cells (1 mL aliquots) at 10^7 cells/mL were seeded into 24-well tissue culture plates and maintained at 37 °C in a humidified atmosphere of 5 % CO2 in air in the presence or absence of 2 µg/mL of the T cell mitogen con A. Culture was terminated after 24 - 192 hours, supernatants collected, centrifuged at 100 x gravity for 5 minutes and stored at -70 °C prior to analysis.

PREPARATION OF SERUM
Mice were exsanguinated 13 days or 18 days (extended protocol only) after the initiation of exposure. Blood was clotted overnight at 4 °C, individual serum samples were prepared by centrifugation and stored at -70 °C until analysis.

TOTAL SERUM IgE ENZYME-LINKED IMMUNOSORBANT ASSAY (ELISA)
Serum IgE was measured using a sandwich ELISA. Plastic microtitre plates (Nunc, Copenhagen, Denmark) were coated with 2.5 µg/mL of rat monoclonal anti-mouse IgE (Clone LO ME 3; Serotec, Kidlington, UK) in PBS by overnight incubation at 4 °C. The plates were blocked by incubation for a further 30 min at 37 °C with 10 % FCS in PBS. Samples were diluted in 10 % FCS in PBS and added and plates were incubated with shaking for 6 hours at room temperature. There followed a further incubation with goat anti-mouse IgE antibody (Serotec) diluted 1 in 2000 in 10 % FCS in PBS overnight at 4 °C. Plates were then incubated with a 1 in 2000 dilution of horse radish peroxidase labelled donkey anti-sheep/goat IgG antibody in 10 % FCS in PBS for 3 hours at room temperature. Enzyme substrate (o-phenylenediamine and hydrogen peroxide) was added and the reaction was stopped after 15 minutes by the addition of 0.5 M citric acid. Between each incubation stage, the plates were washed with PBS containing 0.05 % Tween 20. Substrate conversion was measured as optical density (OD) at 450 nm using an automated reader (Flow Laboratories). The concentration of IgE (ng/mL) in test samples was derived from a standard curve for monoclonal mouse anti-dansyl IgE κ (BD Biosciences, San Diego, CA, USA). Control mouse IgG (Serotec) at a 1 mg/mL concentration was routinely negative (limit of detection 0.5 ng/mL IgE; 500,000-fold differential sensitivity for IgE versus IgG).

MEASUREMENT OF CYTOKINE CONTENT OF CULTURE SUPERNATANTS
Concentrations of IL-4, IL-5, IL-10, IL-13 and IFN-γ were measured in supernatants using the Bio-Plex cytokine array system according to manufacturer’s instructions (Bio-Rad Laboratories, Hercules, CA, USA). A customised set of fluorescent anti-cytokine multiplex microbeads was supplied together with recombinant standards for the generation of a standard curve for each cytokine and the assay was performed in a 96 well microtitre plate format. The wells of the filter plate were pre-wet with 100 µL of Bio-Plex assay buffer which was then removed by vacuum filtration. Multiplex beads (50 µL per well) were added and the buffer removed by vacuum filtration. Serial (1 in 4) dilutions of the recombinant cytokines were prepared in Bio-Plex standard diluent A. Upper and lower concentrations of the standard curves were as follows:
IL-4: 5.9 to 106556 pg/mL;
IL-5: 2.8 to 46658 pg/mL;
IL-10: 1.1 to 17820 pg/mL;
IL-12: 1.1 to 21819 pg/mL;
IL-13: 2.5 to 41942 pg/mL;
IFN-γ: 1.5 to 27833 pg/mL.
Diluted standard or test sample (50 µL) was added and the sealed plate incubated at room temperature for 30 seconds (shaken at 1100 rpm) followed by a further 30 minutes (shaken at 300 rpm). Detection antibody (25 µL) was added to each well and the sealed plate incubated at room temperature for 30 seconds (shaken at 1100 rpm) followed by a further 30 minutes (shaken at 300 rpm). Streptavidin-phycoerythrin (50 µL) was added to each well and the sealed plate incubated at room temperature for 30 seconds (shaken at 1100 rpm) followed by a further 10 minutes (shaken at 300 rpm). Between each step the plates were washed 1 - 3 times with Bio-Plex wash buffer and the buffer removed by vacuum filtration. The beads were resuspended in 125 µL of Bio-Plex assay buffer, the plate was sealed and shaken at 1100 rpm for 30 s at room temperature immediately prior to analysis on a Luminex 100 multiple analyte profiler (MiraiBio Hitachi Genetic Systems, Alameda, USA).

STATISTICAL ANALYSES
A one-way analysis of variance (ANOVA) was used to determine whether group means for total serum IgE content were significantly different.
Positive control substance(s):
ammonium hexachloroplatinate (HCP)
trimellitic anhydride (TMA)
other: 2,4-dinitrochlorobenzene (DNCB)
Negative control substance(s):
other: tetraammine platinum dichloride (TPC)

Results and discussion

Results:
PRELIMINARY SIGHTING STUDIES
The data from an LLNA assay indicated that up to 50 % of the test material diluted in DMF could be applied without adverse effects. The undiluted preparation could not be administered successfully topically due to its high surface tension and very poor wetting properties. Thus, doses of 75 % and 50 % in DMF were utilised in a sighting study conducted according to the standard LLNA dosing protocol (daily for three consecutive days, two days rest followed by isolation of the draining auricular lymph nodes on the 6th day). Both preparations were homogenous but possessed relatively high surface tension and were therefore difficult to apply. Both doses were tolerated well, with no evidence of local or systemic toxicity although the fur at the site of application retained some coloration from the chemical. On visual inspection of the lymph nodes there was some evidence of activation, with a treatment related increase in size compared with lymph nodes isolated from naive control animals.

PROLIFERATION ASSAYS
A nonstandard LLNA assay was used with respect to mouse strain (BALB/c rather than CBA) and the endpoint was an in vitro measurement of proliferation (a consideration of increased lymph node cellularity and the in vitro proliferative response recorded for a standard number of lymph node cells measured as a function of MTT conversion) as opposed to the standard measurement of radioactive thymidine incorporation in vivo. A dose-dependent increase in proliferation was observed following treatment with the test material (Table 1), with proliferation indices (PI) of 2.1, 4.2 and 2.9 recorded for doses of 25, 50 and 75 %, respectively. The somewhat lower PI recorded for 75 % compared with 50 % presumably reflects the difficulty in applying this concentration of material to the dorsum of the ear, given its poor wetting characteristics. These data are in general agreement with the values recorded in a conventional LLNA in which stimulation indices of 3.5 and 2.7 were recorded for 25 and 50 % respectively.
In contrast, treatment with 5 % TPC did not result in marked proliferation with a proliferation index of 1.7 recorded (Table 1).
On the basis of these studies, a dose of 50 % test material formulated in DMF was selected for the conduct of cytokine fingerprinting and total serum IgE assays.

STANDARD CYTOKINE FINGERPRINTING
Comparison of AHCP and TMA and vehicle effects
A preliminary cytokine fingerprinting experiment was conducted to determine whether DMF, the vehicle recommended for use, was appropriate for use in cytokine fingerprinting. Previously, the known respiratory sensitiser platinum salt AHCP was formulated in DMSO, and in this vehicle was show to elicit the expected type 2 cytokine secretion profile in draining LNC. An experiment was conducted using the standard cytokine fingerprinting dosing regimen in which AHCP was formulated at 2 % in DMSO (the dose and formulation used previously) and at 5 % in DMF; both preparations formed fine homogenous suspensions that were relatively easy to apply topically. Further controls were the vehicle DMF, and the reference respiratory allergen TMA, formulated at 10 % in the standard cytokine fingerprinting vehicle AOO and formulated in DMF.
Thirteen days after the initiation of exposure, draining auricular lymph nodes were excised, pooled on a treatment group basis and a single cell suspension prepared. Total cellularity per lymph node was recorded as a measure of immune activation. Topical exposure to TMA induced a higher level of lymphocyte activation when formulated in AOO compared with DMF (15 and 11 x 10^6 cells/lymph node recorded, respectively). Similarly, AHCP formulated in DMSO induced more marked stimulation than did AHCP formulated in DMF, despite the higher concentration of platinum salt (9 and 6 x 10^6 cells/lymph node recorded, respectively). Lymph node cellularity recorded following exposure to DMF was in the range observed historically for vehicle treated control groups (4 x 10^6 cells/lymph node).

Cells were cultured for 24 hours in the presence of the T cell mitogen Con A for the measurement of the type 2 cytokine IL-4. Production of this cytokine in the absence of restimulation is generally very low (less than 30 pg/mL) and it has been demonstrated previously that mitogenic stimulation enhances IL-4 expression by TMA-activated cells, but not by LNC isolated from vehicle-treated animals or from DNCB-stimulated cells. Consistent with previous experiments, LNC isolated from vehicle (DMF) treated mice expressed very low levels of IL-4 (despite activation with con A), with values less than 10 pg/mL recorded. Treatment with 10 % TMA formulated in either AOO or DMF resulted in relatively vigorous IL-4 expression by con A-stimulated LNC (values of 291 and 248 pg/mL recorded, respectively). Lower levels were recorded following treatment with 2 % AHCP in DMSO (156 pg/mL). Exposure to 5 % AHCP in DMF also induced the production of IL-4, albeit at lower levels that that observed for the same material formulated in DMSO (94 pg/mL).

Cells were cultured for 72, 96 and 120 hours in the absence of further restimulation for the measurement of the additional type 2 cytokines IL-5, IL-10 and IL-13 and the type 1 cytokines IL-12 and IFN-γ. In general, peak cytokine expression was recorded at 120 hours. LNC isolated from vehicle (DMF) treated mice produced very low levels of all three type 2 cytokines. LNC isolated from mice exposed to the reference respiratory allergen TMA secreted relatively high levels of each of the three type 2 cytokines, with the AOO formulation generally provoking more vigorous cytokine secretion than the DMF formulation. Exposure to AHCP also resulted in increased type 2 cytokine secretion compared with LNC isolated from vehicle-treated mice. Levels were considerably lower than those induced by the reference respiratory allergen TMA (formulated in AOO or DMF), and in general the platinum salt formulated in DMSO provoked more vigorous cytokine secretion than did the material formulated in DMF. LNC derived from vehicle (DMF) treated mice secreted relatively high levels of the type 1 cytokine IL-12 whereas the respiratory allergens TMA and AHCP, regardless of the vehicle utilised, expressed comparatively low levels of this cytokine. Furthermore, none of the treatments resulted in vigorous expression of the type 1 cytokine IFN-γ, with levels of less than 150 pg/mL recorded. Such levels are considerably lower than those reported previously following topical exposure to the chemical contact allergen DNCB, with values above 1000 pg/mL recorded.
Thus, type 2 cytokine secretion profiles consistent with the potential to cause respiratory allergy were observed for both TMA and AHCP when formulated in AOO and DMF, or in DMF and DMSO. In general, cytokine responses were less vigorous when chemical was formulated in DMF compared with AOO or DMSO, however, these differences were quantitative not qualitative, with the type 2 inducing properties of the chemicals unaffected by the vehicle. These data demonstrate that 5 % AHCP formulated in DMF is an appropriate control respiratory sensitising material for subsequent cytokine fingerprinting assays.

CYTOKINE FINGERPRINTING OF TEST MATERIAL
Mice were exposed to 50 % test material formulated in DMF, or to 5 % AHCP in DMF and an additional group of mice was treated with TPC which was dissolved in water at 10 % and diluted 1:1 with DMF to improve wetting characteristics (final concentration 5 % TPC in water:DMF). Further control groups were treated with the vehicle DMF alone, or with the reference respiratory allergen TMA or the reference contact allergen DNCB formulated at 10 % or 1 %, respectively, in the standard cytokine fingerprinting vehicle AOO. Thirteen days after the initiation of exposure, draining auricular lymph nodes were excised, pooled on a treatment group basis and a single cell suspension prepared. Total cellularity per lymph node was recorded as a measure of immune activation (Table 2). Topical exposure to TMA and DNCB induced a marked increase in lymph node cellularity (values of 15 and 8 x 10^6 cells/lymph node recorded, respectively). AHCP induced modest increases in lymph node cellularity (6 x 10^6 cells/lymph node) whereas TCP and the test material were without marked effect on LNC numbers (4x 10^6 cells/lymph node recorded in both cases). Lymph node cellularity recorded following exposure to DMF was in the range observed historically for vehicle treated control groups (3 x 10^6 cells/lymph node).

Cells were cultured for 24 hours in the presence of the T cell mitogen Con A for the measurement of the type 2 cytokine IL-4 (Table 2). Consistent with previous experiments, LNC isolated from vehicle (DMF) treated mice and from DNCB treated mice expressed very low levels of IL-4 (despite activation with con A), with values less than 50 pg/mL recorded. Treatment with 10 % TMA resulted in relatively vigorous IL-4 expression by con A-stimulated LNC (2729 pg/mL). Exposure to 5 % AHCP and to 50 % test material also induced the production of IL-4, albeit at lower levels than that provoked by TMA (289 and 99 pg/mL, respectively) whereas treatment with 5 % TCP failed to stimulate IL-4 production above the levels recorded for LNC derived from DMF-treated mice.

Cells were cultured for 72, 96 and 120 hours in the absence of further restimulation for the measurement of the additional type 2 cytokines IL-5, IL-10 and IL-13 and the type 1 cytokines IL-12 and IFN-γ (Table 2). Consistent with the previous experiment, LNC derived from DMF-treated animals expressed relatively low levels of all cytokines with the exception of the type 1 cytokine IL-12. A similar pattern of cytokine production was recorded for LNC isolated from mice treated with TCP: very low levels of IL-5, IL-10, IL-13 and IFN-γ and relatively high expression of IL-12. LNC isolated from DNCB-treated mice exhibited a selective type 1 cytokine profile with relatively vigorous IFN-γ production (1668 pg/mL) and IL-12 expression (281 pg/mL) but relatively low levels of type 2 cytokines IL-5, IL-10 and IL-13. As observed above, TMA-activated LNC expressed the converse type 2 cytokine profile with relatively high levels of IL-5, IL-10 and IL-13 but low levels of the type 1 cytokines IFN-γ and IL-12. A similar type 2 pattern of cytokine expression was recorded following topical exposure to AHCP and the test material, albeit at lower levels compared to those recorded for LNC derived from TMA-exposed mice. However, each type 2 cytokine was expressed at higher levels by LNC isolated from test material exposed mice compared with those treated with AHCP. Peak type 2 cytokine expression was recorded at 120 hours for the test material, and therefore the kinetics of cytokine production was somewhat delayed compared with that induced by TMA-stimulated LNC. Thus, for TMA-activated LNC, similar levels of type 2 cytokines (particularly IL-10 and IL-13) were recorded after both 96 and 120 hours of culture, whereas for the test material very little cytokine expression was detected at the 96 hour timepoint.

CYTOKINE FINGERPRINTING ASSAY: EXTENDED KINETICS IN VITRO
Given the somewhat delayed kinetics of the cytokine response for the test material compared with the reference TMA, the kinetics in culture of the cytokine expression profile was examined in more detail. In order to provide for sufficient cells to examine cytokine secretion at the additional time points of 144, 168 and 192 hours, between 3 and 10 animals per treatment group were dosed. Mice were exposed to 50 % test material formulated in DMF, or to 5 % AHCP in DMF. Control groups were exposed to the vehicle DMF alone, or to TMA or DNCB formulated at 10 % or 1 %, respectively, in the standard cytokine fingerprinting vehicle AOO. Thirteen days after the initiation of exposure, draining auricular lymph nodes were excised, pooled on a treatment group basis and a single cell suspension prepared. Total cellularity per lymph node was recorded as a measure of immune activation (Table 3). Topical exposure to TMA and DNCB induced a marked increase in lymph node cellularity (values of 14 and 12 x 10^6 cells/lymph node recorded, respectively). Exposure to AHCP and the test material induced modest increases in lymph node cellularity (between 6 and 8 x 10^6 cells/lymph node recorded). Lymph node cellularity recorded following exposure to DMF was in the range observed historically for vehicle treated control groups (3 x 10^6 cells/lymph node).

Cells were cultured for 24 hours in the presence of the T cell mitogen Con A for the measurement of the type 2 cytokine IL-4 (Table 3). LNC isolated from vehicle (DMF) treated mice and from DNCB treated mice failed to express detectable levels of IL-4 (despite activation with con A). Treatment with 10 % TMA resulted in relatively vigorous IL-4 expression by con A-stimulated LNC (1492 pg/mL). Exposure to 5 % AHCP and to 50 % test material also induced the production of IL-4, albeit at lower levels than that provoked by TMA (97 and 146 pg/mL, respectively).
Cells were cultured for 72, 96, 120, 144, 168 and 192 hours in the absence of further restimulation for the measurement of the additional type 2 cytokines IL-5, IL-10 and IL-13 and the type 1 cytokines IL-12 and IFN-γ (Table 3). Consistent with the previous experiment, LNC derived from DMF-treated animals failed to express detectable levels of all cytokines with the exception of the type 1 cytokine IL-12 at any time point examined. Consistent with previous experiments, TMA-activated LNC expressed the converse type 2 cytokine profile with relatively high levels of IL-5, IL-10 and IL-13 but low levels of the type 1 cytokines IFN-γ and IL-12. The extended kinetics in culture revealed that the expression of the type 2 cytokines by TMA-stimulated LNC continued to increase with time reaching maximal levels at 192 hours.

As observed previously, a similar type 2 pattern of cytokine expression was recorded following topical exposure to AHCP and the test material, albeit at lower levels to those recorded for LNC derived from TMA-exposed mice. In this experiment, higher levels of type 2 cytokines were produced by AHCP-stimulated LNC than test material-activated LNC with the exception of IL-5. The kinetics of cytokine production was similar to that observed for TMA, with levels of type 2 cytokines tending to increase with time, reaching maximal levels at later time points in culture.

CYTOKINE FINGERPRINTING: EXTENDED PRIMING PERIOD IN VIVO
A further experiment was conducted in which animals were exposed to an extra priming dose of chemical in vivo, thereby extending the exposure period from 13 days to 18 days. As the previous experiments had demonstrated that cytokine production by platinum salt activated LNC reached maximal levels after 120 hours of culture, in this experiment the cells were cultured for 120 to 168 hours. Mice were exposed to 50 % test material formulated in DMF, or to 5 % AHCP in DMF. Control groups were exposed to the vehicle DMF alone, or to TMA or DNCB formulated at 10 % or 1 %, respectively, in the standard cytokine fingerprinting vehicle AOO. Eighteen days after the initiation of exposure, draining auricular lymph nodes were excised, pooled on a treatment group basis and a single cell suspension prepared. Total cellularity per lymph node was recorded as a measure of immune activation (Table 4). Topical exposure to TMA and DNCB induced a marked increase in lymph node cellularity (values of 16 and 10 x 10^6 cells/lymph node recorded, respectively). Exposure to AHCP and the test material induced in each case modest increases in lymph node cellularity (6 x 10^6 cells/lymph node recorded). Lymph node cellularity recorded following exposure to DMF was in the range observed historically for vehicle treated control groups (4 x 10^6 cells/lymph node).

Cells were cultured for 24 hours in the presence of the T cell mitogen Con A for the measurement of the type 2 cytokine IL-4 (Table 4). LNC isolated from vehicle (DMF) treated mice and from DNCB treated mice failed to express detectable levels of IL-4 (despite activation with con A). Treatment with 10 % TMA resulted in relatively vigorous IL-4 expression by con A-stimulated LNC (92 pg/mL). Exposure to 5 % AHCP and to 50 % test material also induced the production of IL-4, albeit at lower levels than that provoked by TMA (42 and 61 pg/mL, respectively).

Cells were cultured for 120, 144 and 168 hours in the absence of further restimulation for the measurement of the additional type 2 cytokines IL-5 , IL-10 and IL-13 and the type 1 cytokines IL-12 and IFN-γ (Table 4). Consistent with the previous experiment, LNC derived from DMF-treated animals expressed low to undetectable levels of all cytokines with the exception of the type 1 cytokine IL-12 at each time point examined. LNC isolated from DNCB-treated mice exhibited a selective type 1 cytokine profile with relatively vigorous IFN-γ production peaking after 120 hours of culture (2017 pg/mL) and IL-12 expression (94 to 109 pg/mL) but relatively low levels of type 2 cytokines IL-5, IL-10 and IL-13. Consistent with previous experiments, TMA-activated LNC expressed the converse type 2 cytokine profile with relatively high levels of IL-5, IL-10 and IL-13 but low levels of the type 1 cytokines IFN-γ and IL-12.
As observed previously for type 2 cytokines, expression by TMA-stimulated LNC continued to increase with time reaching maximal levels at 192 hours of culture. A similar type 2 pattern of cytokine expression was recorded following topical exposure to AHCP and the test material, albeit at lower levels to those recorded for LNC derived from TMA-exposed mice. In this experiment, higher levels of type 2 cytokines were produced by AHCP-stimulated LNC than test material-activated LNC with the exception of IL-5. The kinetics of cytokine production was similar to that observed for TMA, with levels of type 2 cytokines tending to increase with time, reaching maximal levels at later time points in culture.

IgE ASSAY: STANDARD EXPOSURE PROTOCOL
A total of three independent experiments were conducted using the standard 13 day exposure regimen and at termination mice (n = 3 per treatment group) were bled and serum prepared for analysis of total IgE content by ELISA. Mice were exposed to 10 % TMA in AOO, 50 % test material or 5 % AHCP in DMF or DMF alone. In one experiment, mice were also treated with 5 % TPC in water:DMF (1:1). Mean and SE for the combined data for the three experiments for each treatment group are displayed in Table 5. There was some inter-animal variation in IgE content in the DMF control group. In the majority of serum samples tested in the control group, levels were below 300 ng/mL, however in one sample very high levels were detected (2500 pg/mL). When the high sample was included in the data set, the mean value recorded for the vehicle control group was 465 pg/mL (Table 5). The levels of IgE recorded in serum isolated from mice treated with 50 % test material or 5 % TPC were considerably lower than those recorded for the vehicle treated control group (means less than 250 pg/mL). Exclusion of the serum sample with the abnormally high levels of total serum IgE from the vehicle control group resulted in a mean value of 223 pg/mL (Table 5).

Treatment with TMA resulted in elevated levels of total serum IgE (mean 1040 pg/mL), however, this result did not achieve statistical significance in comparison to the vehicle (DMF) control group. Treatment with AHCP, in contrast, resulted in a very vigorous and significant elevation in total serum IgE concentration (mean 2008pg/mL; p<0.001). The exclusion of outlier control value from the data set was without effect on the outcome of statistical analyses: i.e. AHCP caused a significant elevation in total serum IgE (p<0.001), whereas the results for the test material and TPC were not statistically elevated. However, exclusion of this sample from the vehicle group did result in the mean for TMA achieving statistical significance (p<0.05).

IgE ASSAY: EXTENDED KINETICS IN VIVO
Two independent experiments were conducted using the extended in vivo 18 day exposure regimen and at termination mice (n = 3 per treatment group) were bled and serum prepared for analysis of total IgE content by ELISA. Mice were exposed to 10 % TMA in AOO, 50 % test material or 5 % AHCP in DMF or DMF alone. Mean and SE for the combined data for the two experiments for each treatment group are displayed in Table 6. Total serum IgE levels in the DMF vehicle control treated group varied between 136 and 432 pg/mL (mean 256 pg/mL). Treatment with TMA and 50 % test material resulted in elevated levels of total serum IgE (mean 1698 and 737 pg/mL, respectively), however, these elevations did not achieve statistical significance in comparison with the control. Treatment with AHCP, in contrast, resulted in a very vigorous and significant elevation in total serum IgE concentration (mean 2424 pg/mL; p<0.01).

Any other information on results incl. tables

Table 1 Lymph Node Proliferative Responses and Changes in Lymph Node Cellularity Provoked by Treatment with the Test Material

Treatment

Cellularity Cells/node (x 10^6)

Cellularity Index (CI)

EC50 Proliferation (x 10^6)

Proliferation Index (PI)

Stimulation Index (SI) CI x PI

75 % Test material

5.2

1.4

1.7

2.1

2.9

50 % Test material

5.6

1.5

1.3

2.8

4.2

25 % Test material

4.3

1.1

1.9

1.9

2.1

DMF

3.8

1

3.6

1

1

5 % TPC

2.9

1.1

2.2

1.5

1.7

 

Table 2 Influence of the Test Material Following Standard Cytokine Fingerprinting Protocol: Comparisons with AHCP and TMA

Treatment

Cellularity Cells/node (x 10^6)

IL-4 (pg/mL)

IL-5 (pg/mL)

IL-10 (pg/mL)

IL-13 (pg/mL)

IL-12 (pg/mL)

IFN-γ (pg/mL)

72 h

96 h

120 h

72 h

96 h

120 h

72

h

96

h

120 h

72 h

96 h

120 h

72 h

96

h

120 h

10 % TMA/AOO

15

2729

102

157

238

290

334

345

1191

1777

1911

154

103

132

69

267

272

50 % Test Material/DMF

4

99

-

83

209

-

27

92

-

309

740

-

96

69

-

71

98

5 % TCP/DMF

4

6

-

3

5

-

0

3

-

0

11

-

113

133

-

11

27

5 % AHCP/DMF

6

289

-

16

35

-

47

67

-

152

417

-

130

112

-

114

203

DMF

3

24

-

2

3

-

1

1

-

0

0

-

168

202

-

19

12

1 % DNCB/AOO

8

36

17

63

85

13

29

21

314

743

641

275

289

281

303

1561

1668

- = no sample (insufficient cells)

 

Table 3 Influence of the Test Material Following Cytokine Fingerprinting Protocol with Extended Kinetics in vitro: Comparisons with AHCP and TMA

Treatment

Cellularity Cells/node (x 10^6)

IL-4 (pg/mL)

IL-5 (pg/mL)

IL-10 (pg/mL)

72 h

96 h

120 h

144 h

168 h

192 h

72 h

96 h

120 h

144 h

168 h

192 h

10 % TMA/

AOO

14

1492

0

0

71

84

61

321

74

175

342

430

414

625

50 % Test Material/DMF

8

146

0

14

88

30

101

23

2

16

42

40

63

26

5 % AHCP/

DMF

6

97

0

10

19

31

76

129

7

81

63

129

121

134

DMF

3

0

-

0

0

0

0

-

-

0

0

0

0

-

1 % DNCB/

AOO

12

0

0

0

0

0

0

0

0

0

14

12

5

19

Treatment

IL-13 (pg/mL)

IL-12 (pg/mL)

IFN-γ (pg/mL)

72 h

96 h

120 h

144 h

168 h

192 h

72 h

96 h

120 h

144 h

168 h

192 h

72 h

96 h

120 h

144 h

168 h

192 h

10 % TMA/AOO

173

341

537

525

575

972

88

166

171

241

225

192

16

10

4

0

0

0

50 % Test Material/DMF

34

80

110

60

123

34

163

195

218

248

282

264

0

0

0

0

0

0

5 % AHCP/DMF

45

195

160

195

195

155

120

103

175

115

113

138

0

28

0

0

38

2

DMF

-

0

0

0

0

-

-

121

164

184

194

-

-

0

0

0

0

-

1 % DNCB/AOO

0

0

65

34

0

0

249

317

210

275

354

317

0

9

487

243

30

67

- = no sample (insufficient cells)

 

Table 4 Influence of the Test Material Following Cytokine Fingerprinting Protocol with Extended Kinetics in vivo: Comparisons with AHCP and TMA

Treatment

Cellularity Cells/node (x 10^6)

IL-4 (pg/mL)

IL-5 (pg/mL)

IL-10 (pg/mL)

IL-13 (pg/mL)

IL-12 (pg/mL)

IFN-γ (pg/mL)

120 h

144 h

168 h

120 h

144 h

168 h

120 h

144 h

168 h

120 h

144 h

168 h

120 h

144 h

168 h

10 % TMA/AOO

16

92

293

433

515

542

717

718

2173

2653

3246

73

73

58

166

140

49

50 % Test Material/DMF

6

61

69

129

167

23

31

52

39

75

175

115

123

128

0

0

0

5 % AHCP/DMF

6

42

71

117

213

175

110

147

783

1039

729

93

95

92

23

6

12

DMF

4

0

0

0

-

15

28

-

0

0

-

165

162

-

2

0

-

1 % DNCB/AOO

10

0

48

133

138

11

23

18

85

343

455

109

94

102

2017

1120

1791

- = no sample (insufficient cells)

 

Table 5 Influence of the Test Material on Total Serum IgE Levels Following Standard Cytokine Fingerprinting Protocol: Comparisons with AHCP and TMA (Statistical Analyses)

Treatment

IgE Concentration (ng/mL)

Statistical Significance

Mean

SE

Versus DMF

Versus ACHP

10 % TMA/AOO

1040

279

p>0.05

p<0.01

50 % Test Material/DMF

224

22

p>0.05

p<0.001

5 % TCP/DMF

173

50

p>0.05

p<0.001

5 % AHCP/DMF

2008

280

p<0.001

-

DMF

465

243

-

p<0.001

 

Table 6 Influence of the Test Material on Total Serum IgE Levels Following Cytokine Fingerprinting Protocol with Extended Kinetics in vivo: Comparisons with AHCP and TMA (Statistical Analyses)

Treatment

IgE Concentration (ng/mL)

Statistical Significance

Mean

SE

Versus DMF

Versus ACHP

10 % TMA/AOO

1698

529

p>0.05

p>0.05

50 % Test Material/DMF

737

204

p>0.05

p<0.05

5 % AHCP/DMF

2424

420

p<0.01

-

DMF

256

44

-

p<0.01

Applicant's summary and conclusion

Interpretation of results:
sensitising
Conclusions:
Under the conditions of this study, the test material provokes the quality of immune response associated with sensitisation of the respiratory tract.
Executive summary:

Studies were conducted to assess the inherent potential of the test material to cause allergic sensitisation of the respiratory tract. Results were compared with those obtained with both positive and negative reference standards.

Two experimental paradigms were deployed. Firstly, cytokine fingerprinting which identifies chemicals with the potential to cause respiratory allergy as a function of their induction in mice of a selective T helper 2 (Th2) response, associated with the production of type 2 cytokines such as interleukin 4. The second experimental approach was to measure changes induced in the total serum concentration of IgE immunoglobulin following exposure of mice to the test material and reference controls.

 

On the basis of both cytokine fingerprinting and measurement of changes in serum IgE levels, the test material displayed the characteristics associated with chemical respiratory allergens. The reference chemicals behaved as expected.

Therefore, the investigation revealed that the test material has the potential to cause allergic sensitisation of the respiratory tract.