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Specific investigations: other studies

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specific investigations: other studies
trigeminal irritation
Type of information:
experimental study
Adequacy of study:
weight of evidence
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
No details on exposure (time, concentration).

Data source

Reference Type:
Dissecting the role of TRPV1 in detecting multiple trigeminal irritants in three behavioural assays for sensory irritation
CJ Saunders, Winston Y Li, Tulsi D Patel, Jeffrey A Muday, Wayne L Silver
Bibliographic source:
F1000Research 2013, 2:74

Materials and methods

Test guideline
no guideline available
Principles of method if other than guideline:
Wildtype (C57Bl/6J) and TRPV1 knockout mice were tested in three behavioral assays for irritation to determine if TRPV1 is necessary to detect trigeminal irritants in addition to capsaicin. In one assay mice were presented with a chemical via a cotton swab and their response scored on a 5 level scale. In another assay, a modified two bottle preference test, which avoids the confound of mixing irritants with the animal’s drinking water, was used to assess aversion. In the final assay, an air dilution olfactometer was used to administer volatile compounds to mice restrained in a double-chambered plethysmograph where respiratory reflexes were monitored (modified Alarie-Test).
GLP compliance:
not specified
Type of method:
in vivo
Endpoint addressed:
eye irritation
respiratory irritation

Test material

Specific details on test material used for the study:
purchased from Sigma; >=99% pure

Test animals

Details on test animals or test system and environmental conditions:
Adult, female, wildtype (C57Bl/6J) and TRPV1 knockout mice were purchased from Jackson Laboratories in Bar Harbor, ME, USA.
Animals were given at least one week to acclimate after delivery to the animal facility.
The mice were housed in conventional polycarbonate caging.
They were provided with ad-lib water and rodent chow (Purinalab 5P00). Nesting material was provided in the form of Enrich-Nest. All animals were housed in groups of 4, provided with food and water ad libitum and were maintained on a 12 hour light cycle.

Administration / exposure

Route of administration:
unchanged (no vehicle)
Details on exposure:
Cotton swab test:
cotton applicators were saturated with the pure compound before presentation to the animal. To prevent the animals from becoming conditioned to associate the cotton swabs with aversive stimuli, several non-aversive substances were also presented to the mice. The favorable substances included chocolate powder, water and wheat flour.
Presentation was alternated between aversive and rewarding stimuli and an individual’s first three responses to the noxious substances were

Two-bottle preference test:
Felt washers were placed over the sipper tube of two water bottles and protected with a wire screen. The wire screen prevented the mouse from manipulating the washers or coming into direct contact with the compounds on the washers. One washer was soaked in undiluted
irritant. The washer on the opposite tube was saturated with distilled water in place of irritant but otherwise prepared identically. No chemicals were mixed with the drinking water in either bottle. To approach and drink from a water bottle the mouse had to inhale the vapors created as the volatile compounds evaporated from the felt washer.

Respiratory assay:
A custom built computer-controlled air-dilution olfactometer was used to administer volatile compounds to unanesthetized mice restrained in four double-chambered plethysmographs.
Analytical verification of doses or concentrations:
No. of animals per sex per dose:
16 mice


Positive control:

Results and discussion

Any other information on results incl. tables

Cotton swab test:

Wildtype mice showed aversive responses to all noxious substances. Cyclohexanone produced aversive behaviors in the wildtype mice, although

the responses were not as extreme as the response to acetic acid.

The response of TRPV1 knockout mice to most substances tested was similar to the response of wild type mice. The only substances to which TRPV1 knockout mice showed significantly less aversion to than the wildtype mice were capsaicin (p<0.01) and cyclohexanone (p<0.05). The response of the TRPV1 knockouts to capsaicin was in the non-aversive range. However, the response of the TRPV1 knockout mice to cyclohexanone was still in the aversive range.

Two bottle preference test:

To compare water consumption between wildtype and TRPV1 knockout mice, the mean and SEM water consumption values from the irritant-treated tube were normalized to the total amount of water consumed from both bottles. TRPV1 knockout mice drank significantly

more water from bottles treated with cyclohexanone (p<0.05) than wildtype mice.

Respiratory assay:

A significant interaction was detected between genotype and cyclohexanone concentration (P<0.01) by two-way ANOVA. There was also a significant difference between genotype (P<0.001) but not concentration. There was no significant difference between the respiration rate of wildtype and TRPV1 knockout mice at 1200 ppm cyclohexanone, the lowest concentration tested. At higher concentrations, the respiration rate of wildtype mice was significantly lower than TRPV1-/- mice at 1600 ppm (P<0.05), 2000 ppm (P<0.001) and 3000 ppm (P<0.001) cyclohexanone. Cyclohexanone depressed the respiration rate of wildtype mice in a dosage-dependent manner. However, cyclohexanone did not alter the respiration rate of TRPV1 knockout mice from its baseline at any concentration.

Applicant's summary and conclusion

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