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Administrative data

Description of key information

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Test item is identified as Montelukast dicyclohexylamine; Read-across is based upon a commonality of functional groups, constituents and predicted skin metabolites between the surrogate and target substances. A detailed description of the metabolic pathways and predicted metabolites is included in section 13.
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA
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 (e.g. OECD, EC, EPA).
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.

6.3. Animal husbandry
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:
0, 5, 10 and 25% w/w
No. of animals per dose:
5 (five)
Details on study design:
6.5. Pre-screen test
A pre-screen test was conducted in order to select the highest test substance concentration to be
used in the main study. In principle, this highest concentration should cause no systemic toxicity, may
give well-defined irritation at the most (maximum grade 2 (see section 6.7) and/or an increase in ear
thickness < 25%) and is the highest possible concentration that can technically be applied.
Two test substance concentrations were tested; a 10% and 25% concentration. The highest
concentration was the maximum concentration that could technically be applied.
The test system, procedures and techniques were identical to those used in the main study except
that the 25% formulation was vortexed prior to dosing and the assessment of lymph node proliferation
and necropsy were not performed. Two young adult animals per concentration were selected (in the
range of 8 to12 weeks old). Each animal was treated with one concentration on three consecutive
days. Animals were group housed in labeled Makrolon cages (MII type, height 14 cm). Ear thickness
measurements were conducted using a digital thickness gauge prior to dosing on Days 1 and 3, and
on Day 6.
Animals were sacrificed after the final observation.

6.6. Main study
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.

6.6.1. Allocation
Group1 animal numbers induction (test substance; % w/w)
1 01 - 05 0 (Acetone/Olive oil (4:1 v/v))
2 06 - 10 5
3 11 - 15 10
4 16 - 20 25
1. five females each group

6.6.2. 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.

6.6.3. 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.

6.6.4. Tissue processing for radioactivity - Day 6
Following excision of the nodes, a single cell suspension of lymph node cells (LNC) was prepared in
PBS by gentle separation through stainless steel gauze (diameter 125 μm). LNC were washed twice
with an excess of PBS by centrifugation at 200g for 10 minutes at 4ºC. To precipitate the DNA, the
LNC were exposed to 5% trichloroacetic acid (TCA) (Merck, Darmstadt, Germany) and stored in the
refrigerator until the next day.

6.6.5. Radioactivity measurements - Day 7
Precipitates were recovered by centrifugation, resuspended in 1 mL TCA and transferred to 10 mL of
Ultima Gold cocktail (PerkinElmer Life and Analytical Sciences, Boston, MA, US) as the scintillation
fluid. Radioactive measurements were performed using a Packard scintillation counter (2800TR).
Counting time was to a statistical precision of ± 0.2% or a maximum of 5 minutes whichever came first.
The scintillation counter was programmed to automatically subtract background and convert Counts
Per Minute (CPM) to Disintegrations Per Minute (DPM).

6.7. Observations
Mortality/Viability Twice daily.
Body weights On Day 1 (pre-dose) and Day 6 (prior to necropsy).
Clinical signs Once daily on Days 1-6 (on Days 1-3 between 3 and 4 hours after
dosing).
Irritation Once daily on Days 1-6 (on Days 1-3 within 1 hour after dosing)
according to the following numerical scoring system. Furthermore, a
description of all other (local) effects was recorded.

Grading Irritation Reactions:
Erythema and eschar formation:
No erythema ..............................................................................……………………………………….. ......... 0
Very slight erythema (barely perceptible) ....................................................……………………………… .... 1
Well-defined erythema ...................................................................…………………………………… .......... 2
Moderate to severe erythema (beet redness) to slight eschar formation (injuries in depth) .........……… ... 3
Severe erythema (beet redness) to eschar formation preventing grading of erythema .........……… ......... 4

Necropsy No necropsy for gross macroscopic examination was performed
according to protocol.

6.8. Electronic data capture
Observations/measurements in the study were recorded electronically using the following programs:
REES Centron Environmental Monitoring system version SQL 2.0 (REES scientific, Trenton, NJ,
USA); Quantasmart 2.03 (PerkinElmer Life Sciences, Boston, MA, USA): LSC software.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
DPM values are presented for each animal and for each dose group. A Stimulation Index (SI) is
calculated for each group using the individual SI values. The individual SI is the ratio of the
DPM/animal compared to DPM/vehicle control group.
If the results indicate a SI ≥ 3, the test substance may be regarded as a skin sensitizer.
The results were evaluated 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 of the European Parliament and of the Council of 16 December 2008
on classification, labelling and packaging of substances and mixtures, including all amendments.
Consideration was given to the EC3 value (the estimated test substance concentration that will give a
SI =3) (Ref. 1).
Positive control results:
group % Hexylcinnamaldehyde mean DPM ± SEM SI ± SEM
2 5% 464 ± 58 1.2 ± 0.3
3 10% 447 ± 63 1.1 ± 0.3
4 25% 3185 ± 427 8.0 ± 1.8
1 0% ((Acetone:Olive oil (4:1 v/v))) 396 ± 71 1.0 ± 0.3

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.
The raw data, protocol and report from this study are kept in the WIL Research Europe archives. The
test described above was performed in accordance with WIL Research Europe Standard Operating
Procedures and the report was audited by the QA-unit.
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
test substance concentration Mean SI± SEM 0% 1.0±0.4 5% 1.0±0.3 10% 0.8±0.3 25% 0.6±0.2
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see Remark
Remarks:
test substance concentration Mean DPM± SEM 0% 180±45 5% 181±26 10% 148±34 25% 112±12

7. RESULTS

7.1. Pre-screen test : No irritation and no signs of systemic toxicity were observed in any of the animals examined. Variations in ear thickness during the observation period were less than 25% from Day 1 pre-dose values. Based on these results, the highest test substance concentration selected for the main study was a 25% concentration.

7.2. Main study

7.2.1. Skin reactions / Irritation: No irritation of the ears was observed in any of the animals examined. White staining of test substance remnants on the dorsal surface of the ears did not hamper scoring for erythema.

7.2.2. Systemic toxicity: No mortality occurred and no clinical signs of systemic toxicity were observed in the animals of the main study. Body weights and body weight gain of experimental animals remained in the same range as controls over the study period.

7.2.3. Macroscopy of the auricular lymph nodes and surrounding area: The majority of auricular lymph nodes were considered normal in size, except for the nodes in four animals treated at 25% which were reduced in size. No macroscopic abnormalities of the surrounding area were noted in any of the animals.

7.2.4. Radioactivity measurements and SI values: Mean DPM/animal values for the experimental groups treated with test substance concentrations 5, 10 and 25% were 181, 148 and 112 DPM respectively. The mean DPM/animal value for the vehicle control group was 180 DPM. The SI values calculated for the substance concentrations 5, 10 and 25% were 1.0, 0.8 and 0.6 respectively.

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Montelukast backbone diol is non-sensitising based upon a read-across to Montelukast Dicyclohexylamine salt. The rationale for the read-across is a commonality of protein binding mechanisms between the two substances, predicted using the skin metabolism simulator within the OECD toolbox version (3.2.0.103). 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.
Based on the study results, Montelukast dicyclohexylamine salt would not be regarded as a skin sensitizer
according to the recommendations made in the test guidelines. Consequently the target substance, Montelukast backbone diol would also be considered a non-sensitiser and the 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).
Executive summary:

Assessment of Contact Hypersensitivity to Montelukast dicyclohexylamine salt in the Mouse (Local Lymph Node Assay). The study was carried out based on the guidelines described in:

OECD, Section 4, Health Effects, No.429 (2010),

EC, No 440/2008; B42: "Skin Sensitization: Local Lymph Node Assay"

EPA, OPPTS 870.2600 (2003) “Skin Sensitization”.

Test substance concentrations selected for the main study were based on the results of a pre-screen test.

In the main study, three experimental groups of five female CBA/J mice were treated with test substance concentrations of 5, 10 or 25% w/w on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with vehicle alone (Acetone/Olive oil (4:1 v/v)).

Three days after the last exposure, all animals were injected with 3H-methyl thymidine and after five hours the draining (auricular) lymph nodes were excised and pooled for each animal. After precipitating the DNA of the lymph node cells, radioactivity measurements were performed. The activity was expressed as the number of Disintegrations Per Minute (DPM) and a stimulation index (SI) was subsequently calculated for each group.

No irritation of the ears was observed in any of the animals examined. White staining of test substance remnants on the dorsal surface of the ears did not hamper scoring for erythema.

The majority of auricular lymph nodes were considered normal in size, except for the nodes in four animals treated at 25% which were reduced in size. No macroscopic abnormalities of the surrounding area were noted in any of the animals.

Mean DPM/animal values for the experimental groups treated with test substance concentrations 5, 10 and 25% were 181, 148 and 112 DPM respectively. The mean DPM/animal value for the vehicle control group was 180 DPM. The SI values calculated for the substance concentrations 5, 10 and 25% were 1.0, 0.8 and 0.6 respectively.

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.

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).

Subsequent read-across was made from Montelukast DCHA salt to the montelukast backbone diol based upon a commonality of protein binding mechanisms between the two substances, predicted using the skin metabolism simulator within the OECD toolbox version (3.2.0.103).

Based on the study results, Montelukast dicyclohexylamine salt would not be regarded as a skin sensitizer according to the recommendations made in the test guidelines. Consequently the target substance, Montelukast backbone diol would also be considered a non-sensitiser and the 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)
Additional information:

Montelukast backbone diol is non-sensitising based upon a read-across to Montelukast Dicyclohexylamine salt. The rationale for the read-across is a commonality of protein binding mechanisms between the two substances, predicted using the skin metabolism simulator within the OECD toolbox version (3.2.0.103). 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. Based on the study results, Montelukast dicyclohexylamine salt would not be regarded as a skin sensitizer according to the recommendations made in the test guidelines. Consequently the target substance, Montelukast backbone diol would also be considered a non-sensitiser and the 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).


Migrated from Short description of key information:
Montelukast backbone diol is non-sensitising based upon a read-across to Montelukast Dicyclohexylamine salt. The rationale for the read-across is a commonality of protein binding mechanisms between the two substances, predicted using the skin metabolism simulator within the OECD toolbox version (3.2.0.103)

Justification for selection of skin sensitisation endpoint:
Montelukast backbone diol is non-sensitising based upon a read-across to Montelukast Dicyclohexylamine salt. The rationale for the read-across is a commonality of protein binding mechanisms between the two substances, predicted using the skin metabolism simulator within the OECD toolbox version (3.2.0.103).

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification