N-cyclohexylcyclohexanamine 2-[1-[[[(1R)-1-[3-[(1E)-2-(7-chloroquinolin-2-yl)ethenyl]phenyl]-3-[2-(2-hydroxypropan-2-yl)phenyl]propyl]sulfanyl]methyl]cyclopropyl]acetate

Endpoint summary

Description of key information

There was no indication that Montelukast dicyclohexylamine salt could elicit an SI ≥ 3 when

tested up to 25%, it was established that the EC3 value (the estimated test substance concentration

that will give a SI =3) (if any) exceeds 25%.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014/02/12- 2014/03/10

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

24 April 2002

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Qualifier:

according to guideline

Guideline:

other: EPA Health Effects Test Guidelines OPPTS 870.2600 Skin Sensitization EPA 712-C-03-197. March 2003.

Qualifier:

according to guideline

Guideline:

other: Interagency Co-ordinating Committee on the Validation of Alternative Methods (1999). The murine local lymph node assay: A test method for assessing the allergic contact dermatitis potential of chemicals/compounds. NIH Publication No. 99-4494

GLP compliance:

yes

Remarks:

EC Commission Directive 2004/10/EC, OECD ENV/MC/CHEM/(98)17

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA:J

Sex:

female

Details on test animals and environmental conditions:

Species Mouse, CBA/J strain, inbred, SPF-Quality. Recognized by the international guidelines as the recommended test system
Source: Janvier, Le Genest-Saint-Isle, France
Number of animals 20 females (nulliparous and non-pregnant), five females per group.
Age and body weight Young adult animals (approx. 10 weeks old) were selected. Body weight variation was within +/- 20% of the sex mean.
Identification Tail mark with marker pen.
Health inspection At least prior to dosing. It was ensured that the animals were healthy and that the ears were intact and free from any abnormality.

Conditions
Environmental controls for the animal room were set to maintain 18 to 24°C, a relative humidity of 40
to 70%, at least 10 air changes/hour, and a 12-hour light/12-hour dark cycle. Any variations to these
conditions were maintained in the raw data and had no effect on the outcome of the study.

Accommodation
Animals were group housed in labeled Makrolon cages (MIII type; height 18 cm) containing sterilised
sawdust as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG,
Rosenberg, Germany). Paper (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United
Kingdom) and shelters (disposable paper corner home, MCORN 404, Datesand Ltd, USA) were
supplied as cage-enrichment. The acclimatization period was at least 5 days before the start of
treatment under laboratory conditions. On Day 6, the animals were group housed in Makrolon MII type
cages with a sheet of paper instead of sawdust and cage enrichment.

Diet
Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).

Water
Free access to tap water.

Diet, water, bedding and cage enrichment evaluations for contaminants and/or nutrients were
performed according to facility standard procedures. There were no findings that could interfere with
the study.

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

The maximum practical concentration from the prescreen was 25%
Based on this information the following concentrations were selected for the study:
5, 10 and 25% w/v

No. of animals per dose:

Three groups of five animals were treated with one test substance concentration per group. The
highest test substance concentration was selected from the pre-screen test.
One group of five animals was treated with vehicle.

Details on study design:

Induction - Days 1, 2 and 3
The dorsal surface of both ears was topically treated (25 μL/ear) with the test substance
concentration, at approximately the same time on each day. The concentrations were stirred with a
magnetic stirrer immediately prior to dosing.

The control animals were treated in the same way as the experimental animals, except that the vehicle
was administered instead of the test substance.

Excision of the nodes - Day 6
Each animal was injected via the tail vein with 0.25 mL of sterile phosphate buffered saline (PBS)
(Merck, Darmstadt, Germany) containing 20 μCi of 3H-methyl thymidine (PerkinElmer Life and
Analytical Sciences, Boston, MA, US).

After approximately five hours, all animals were killed by intraperitoneal injection (0.2 mL/animal)
with Euthasol® 20% (AST Farma BV, Oudewater, The Netherlands). The draining (auricular) lymph
node of each ear was excised. The relative size of the nodes (as compared to normal) was estimated
by visual examination and abnormalities of the nodes and surrounding area were recorded. The nodes
were pooled for each animal in approximately 3 mL PBS.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Positive control results:

The SI values calculated for the substance concentrations 5, 10 and 25% were 1.2, 1.1 and 8.0
respectively. An EC3 value of 14.1% was calculated using linear interpolation.

The calculated EC3 value was found to be in the acceptable range of 2 and 20%. The results of the 6
monthly HCA reliability checks of the recent years were 11.9, 16.9, 14.4, 16.5, 14.5 and 13.4%.

Based on the results, it was concluded that the Local Lymph Node Assay as performed at WIL
Research Europe is an appropriate model for testing for contact hypersensitivity.

Parameter:

SI

Value:

ca. 1

Variability:

SEM 0.4

Test group / Remarks:

Group 1 0%

Parameter:

SI

Value:

ca. 1

Variability:

SEM 0.3

Test group / Remarks:

Group 2 5%v/v

Parameter:

SI

Value:

ca. 0.8

Variability:

SEM 0.3

Test group / Remarks:

Group 3 10%v/v

Parameter:

SI

Value:

ca. 0.6

Variability:

SEM 0.2

Test group / Remarks:

Group 4 25% v/v

Cellular proliferation data / Observations:

Mean DPM
Group 1 180+-45
Group 2 181+-26
Group 3 148+-34
Group 4 112+-12

No erythema/eschar formation noted in any tested animal
No significant changes to animal body weight over the course of treatment.

Interpretation of results:

GHS criteria not met

Conclusions:

Since there was no indication that Montelukast dicyclohexylamine salt could elicit an SI ≥ 3 when
tested up to 25%, it was established that the EC3 value (the estimated test substance concentration
that will give a SI =3) (if any) exceeds 25%.

The six-month reliability check with Alpha-hexylcinnamaldehyde indicates that the Local Lymph Node
Assay as performed at WIL Research Europe is an appropriate model for testing for contact
hypersensitivity (see APPENDIX 1).

Based on these results, Montelukast dicyclohexylamine salt would not be regarded as a skin sensitizer
according to the recommendations made in the test guidelines. The test substance does not have to
be classified and has no obligatory labelling requirement for sensitization by skin contact according to
the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United
Nations (2011) (including all amendments) and the Regulation (EC) No 1272/2008 on classification,
labelling and packaging of substances and mixtures (including all amendments).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Since there was no indication that Montelukast dicyclohexylamine salt could elicit an SI ≥ 3 when

tested up to 25%, it was established that the EC3 value (the estimated test substance concentration

that will give a SI =3) (if any) exceeds 25%.

Based on these results, Montelukast dicyclohexylamine salt would not be regarded as a skin sensitizer

according to the recommendations made in the test guidelines. The test substance does not have to

be classified and has no obligatory labelling requirement for sensitization by skin contact according to

the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United

Nations (2011) (including all amendments) and the Regulation (EC) No 1272/2008 on classification,

labelling and packaging of substances and mixtures (including all amendments).