Glycine, N-acetyl-N-[3-(trifluoromethyl)phenyl]valyl-, ethyl ester

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 13 August 2002 to 11 April 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

17th July 1992

Deviations:

yes

Remarks:

the vehicule used for epicutaneous induction is different from the vehicle used for challenge

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Version / remarks:

30 July 1996

Deviations:

yes

Remarks:

the vehicule used for epicutaneous induction is different from the vehicle used for challenge

GLP compliance:

yes (incl. QA statement)

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

Test performed before the implementation of the new requirements.

Test material

Test material information

Constituent 1

Reference substance name:

Glycine, N-acetyl-N-[3-(trifluoromethyl)phenyl]valyl-, ethyl ester

EC Number:

609-496-9

Cas Number:

379685-94-6

Molecular formula:

C18H23F3N2O4

IUPAC Name:

Glycine, N-acetyl-N-[3-(trifluoromethyl)phenyl]valyl-, ethyl ester

Test material form:

solid: particulate/powder

Details on test material:

- Other: ochre fine powder

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL- Storage condition of test material: room temperature

In vivo test system

Test animals

Species:

guinea pig

Strain:

Hartley

Remarks:

Crl: (HA) BR, Caesarian obtained, Barrier sustained - Virus Antibody Free (COBS-VAF)

Sex:

male/female

Details on test animals and environmental conditions:

TEST ANIMALS- Source: Charles River Laboratories, L'ArbresIe, France. - Females nulliparous and non-pregnant: yes- Age at study initiation: 1-2 months old- Weight at study initiation: 427 ± 27 g for the males and 390 ± 34 g for the females. - Housing: Animals were housed individually in polycarbonate cages with stainless steel lid (48 cm x 27 cm x 20 cm) equipped with a polypropylene bottle. Each cage contained autoclaved sawdust (SICSA, Alfortville, France). Sawdust was analysed by the supplier for composition and contaminant levels. - Diet: ad libitum to 106 pelleted diet (UAR, Villemoisson, Epinay-sur-Orge, France). Diet is regularly analysed by the supplier for composition and contaminant levels. The diet formula was presented in the study report. - Water: ad libitum to drinking water filtered by a FG Millipore membrane (0.22 micron). Bacteriological and chemical analyses of water were performed regularly by extemal laboratories ( include detection of possible contaminants (pesticides, heavy metals and nitrosamines)).- Acclimation period: at least 5 daysENVIRONMENTAL CONDITIONS- Temperature (°C): 22 ± 2- Humidity (%): 30 to 70%- Air changes (per hr): approximately 12 cycles/hour of filtered, non-recycled air- Photoperiod (hrs dark / hrs light): 12 /12

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

intradermal

Vehicle:

corn oil

Concentration / amount:

0.1%

Adequacy of induction:

highest technically applicable concentration used

Induction 2

Route:

epicutaneous, occlusive

Vehicle:

other: 80/20 (w/w) mixture of ethanol and purified water

Concentration / amount:

50%

Adequacy of induction:

other: highest technically applicable concentration used non-irritant substance, but skin pre-treated with 10% SDS

Challenge

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

other: acetone

Concentration / amount:

50%

Adequacy of challenge:

other: highest technically applicable concentration used highest non-irritant concentration

No. of animals per dose:

Preliminary test: 2 males and 2 females Main test: 15 males and 15 females (20 animals for the test group and 10 animals for the control group)

Details on study design:

CRITERIA FOR THE CHOICE OF VEHCILE AND DOSAGE PREPARATIONS:- The choice of the vehicle was based on tests to check the homogeneity (visual check) of the preparation (for cutaneous application and intradermal injections) and its free passage through a needle (for intradermal injections). The highest concentrations which satisfied these criteria were called the maximal practicable concentrations.- Test item was finely pulverised before being incorporated in the vehicle. All dosage form preparations were made freshly on the morning of administration and any unused material was discarded that same day.PREPARATION OF ANIMALS FOR BOTH THE RANGE FINDING AND THE MAIN TEST:The application sites of all animals were: - clipped before intradermal injections (inter-scapular region 4 cm x 3 cm), - clipped before topical applications of the induction phase (same region), - clipped and shaved before topical applications of the challenge phase (each flank 3 cm x 3 cm)- shaved before the 48h reading of the challenge phase and after the 24h reading of the induction phase (range finding test).RANGE FINDING TESTS:A. INTRADERMAL INJECTION TOLERANCE TEST- Test group: 2 males- Intradermal application: each site of the inter-scapular region was injected with 0.1 ml of a single concentration of the test item (Day1).- Concentrations: only 0.1% (w/w)- Observations: approximately 24, 48 hours and 6 days after the injectionsB. TOPICAL APPLICATION TOLERANCE TESTB.1. Under the conditions of the induction phase:- Test group: 1 male and 1 female- Dosing: a filter paper (approximately 8 cm2) was fully-loaded with a dosage form preparation and was then applied to the clipped area of the skin. The filter paper was held in place by means of an occlusive dressing for 48 h.- Concentrations: 25 and 50% (w/w) in 80/20 (w/w) mixture of ethanol and purified water - Observations: 24 and 48 hours after removal of the dressingB.2. Under the conditions of the challenge phase:- Test group: 1 male and 1 female- Dosing: the filter paper of a chamber (Finn Chamber®) was fully-loaded with a dosage form preparation. The chamber was then applied to the clipped area of the skin (one concentration per flank). The chamber was held in place by means of an occlusive dressing for 24 hours.- Concentrations: 25 and 50% (w/w) in acetone- Observations: 24 and 48 hours after removal of the dressing- On removal of the dressing, any residual test item was removed by means of a moistened cotton pad.MAIN STUDY:A. INDUCTION EXPOSURE- Test group: 10 males and 10 females- Control group: 5 males and 5 femalesA1. INTRADERMAL INDUCTION (Day 1)- Dosing: 3 pairs of intradermal injections were made at the prepared skin site of each animal. - Treatment of the Test group:* Anterior site: 0.1mL of FCA at 50% (v/v) in 0.9% NaCl * Median site: 0.1mL of test item at 0.1% (w/w) in corn oil* Posterior site: 0.1mL of test item at 0.1% (w/w) in the mixture FCA/0.9% NaCl (50/50, v/v) - Treatment of the Control group:* Anterior site: 0.1mL of 0.1mL of FCA at 50% (v/v) in 0.9% NaCl * Median site: 0.1mL of the vehicle (corn oil)* Posterior site: 0.1mL of the vehicle at 50% in the mixture FCA/0.9% NaCl (50/50, v/v)A2. TOPICAL INDUCTION (Day 8)- Preparation of the site: As the test item was shown to be non-irritant during the preliminary test, animals of both groups were treated on day 7 with 0.5 mL of sodium lauryl sulfate at the concentration of 10% (w/w) in vaseline, in order to induce local irritation. - Dosing: on Day 8, a pad of filter paper (approximately 8 cm2) was fully-loaded with the test item at the concentration of 50% (w/w) and was then applied to the inter-scapular region of the animals of the treated group. Animals of the control group received an application of the vehicle alone under the same experimental conditions. The pad was held in place for 48 h by means of an adhesive hypoallergenic dressing and an adhesive anallergenic waterproof plaster. - On removal of the dressing (Day 10), no residual test item was observed. - A local irritation was recorded in all animals of the control and treated groups.B. CHALLENGE EXPOSURE (Day 22)- Dosing: animals of treated and control groups received an application o fthe test item and vehicle. The filter paper of a chamber (Finn Chamber®) was fully-loaded with the test item at the concentration of 50% (w/w) and was then applied to a clipped area of the skin of the posterior right flank of all animals. The vehicle was applied under the same experimental conditions to the skin of the posterior left flank. The chambers were held in contact with the skin for 24 hours by means of an adhesive anallergenic waterproof plaster. - On removal of the dressing, any residual test item was removed by means of a moistened cotton pad.SCORING OF THE CUTANEOUS REACTIONS24 and 48 h after removal of the dressing of the challenge application, both flanks of the treated and control animals were observed in order to evaluate cutaneous reactions, according to the following scale: . no visible change.................................................................................................................. 0 . discrète or patchy erythema.................................................................................................. 1 . moderate and confluent erythema........................................................................................ 2 . intense erythema................................................................................................................... 3 Any observed oedema was recorded. Any other lesions were noted. OTHER 1: IN VIVO OBSERVATIONS- Mortality and morbidity: animals were observed at least once a day during the study in order to check for clinical signs and mortality. - Body weight: animals were weighed individually on the day of allocation into the groups, on the first day of the study (Day 1) and on the last day of the study (Day 25).OTHER 2: TERMINAL STUDIES- Termination and euthanasia method: At the end of the study, all the surviving animals were killed by carbon dioxide asphyxiation.- Necropsy: not performed.- Skin samples: no skin samples were takenOTHER3 : RELIABILITY CHECKThe sensitivity of the experimental technique is regularly assessed using a known moderate sensitizer, MERCAPTOBENZOTHIAZOLE. In a recent study (June 2002) performed under the testing laboratory experimental conditions, the strain of guinea pigs used showed a satisfactory sensitization response in 100% animals (results were presented in the study report).

Challenge controls:

no

Positive control substance(s):

no

Remarks:

no concomittent positive control performed but a reliability check with MERCAPTOBENZOTHIAZOLE was presented in the study report

Results and discussion

Positive control results:

In a recent study performed with MERCAPTOBENZOTHIAZOLE, under the testing laboratory experimental conditions, the strain of guinea pigs used showed a satisfactory sensitization response in 100% animals (results were presented in the study report).

In vivo (non-LLNA)

Results

Results 1

Key result

Reading:

1st reading

Hours after challenge:

48

Group:

negative control

Dose level:

0% Test item

No. with + reactions:

0

Total no. in group:

10

Results 2

Key result

Reading:

2nd reading

Hours after challenge:

72

Group:

negative control

Dose level:

0% Test item

No. with + reactions:

0

Total no. in group:

10

Results 3

Key result

Reading:

1st reading

Hours after challenge:

48

Group:

test chemical

Dose level:

0.1% (intrademal injection)

No. with + reactions:

0

Total no. in group:

19

Clinical observations:

a discrete erythema (grade 1) was noted in 2/19 animals

Results 4

Key result

Reading:

2nd reading

Hours after challenge:

72

Group:

test chemical

Dose level:

0.1% (intradermal injection)

No. with + reactions:

0

Total no. in group:

19

Clinical observations:

cutaneous reactions obsereved at the 48h observation time point have dully reversed

Any other information on results incl. tables

CHOICE 0F THE VEHICLE

- The test item was not soluble in 0.9% NaCl: two phases were observed.

- The vehicle chosen for intradermal injections was com oil: the dosage form preparation which could pass through a needle and into the dermis had a maximum concentration of 0.1% (w/w).

- For topical applications, the vehicles used were a mixture ethanol/water (80/20) for the induction phase and acetone for the challenge application: a homogeneous dosage form preparation was obtained at the maximum concentration of 50% (w/w).

RANGE FINDING STUDY

- Administration by intradermal route: As the concentration of 0.1% was well-tolerated systemically and locally, it was retained for the main study.

- Administration by topical route: In order to respect the criteria for the sélection of concentrations (the concentrations should be well-tolerated systemically and locally, cutaneous application for the induction should cause at most weak or moderate skin reactions or be the maximal practicable concentration, cutaneous application for the challenge phase should be the highest concentration which does not cause irritant effect), concentration chosen for the topical application of the induction phase (Day 8) and for the challenge application (Day 22) was 50% (w/w).

MAIN STUDY

- Clinical examinations: One animal of the treated group (male) was found dead on Day 6. No clinical signs were observed prior to death. As such spontaneous mortality is sometimes observed in this species, it

was not attributed to treatment with the test item. Important local reactions (ulceration) at the intradermal injection sites were recorded from Day 12 in 11/19 animals of the treated group and 2/10 animals of the control group

- Body weight: body weight gain of the treated animals was similar to that of controls (detailed results are presented in the study report).

- Challenge phase reactions: No cutaneous reactions were observed in the animais of the control group. In the treated group, a discrete erythema (grade 1) was noted in 2/19 animals, at the 48 h post challenge (24h post dressing removal) reading only. No other cutaneous reactions were recorded.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the experimental conditions of this study, the test material did not elicit a skin sensitisation response in the guinea pig. The test material is not classified for skin sensitisation according to the Regulation (EC) No 1272/2008 on classification, labelling and packaging (CLP) and the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) criteria.

Executive summary:

This GLP-compliant study was performed to assess the potential of the test material to induce and elicit delayed dermal sensitization according to the guinea pig maximization test of Magnusson and Kligman described in OECD Guideline 406 (dated July 17th, 1992) and EU Method B.6 of the E.E.C. directive n° 96/54 (dated July 30st, 1996).

Material and methods

Concentrations of the test material used in the main study were determined by performing range finding tests. The suitable concentrations were: 0.1% in corn oil for the intradermal injection, 50 % in a mixture ethanol/water (80/20) for the induction topical application and 50% in acetone for the challenge topical application (highest technically applicable concentrations conforming with guideline requirements for each phase of the test).

The main study was undertaken using 30 male and female guinea pigs (20 in the test group and 10 in the control group).

Test animals were intradermally injected with FCA at 50% (v/v) in 0.9% NaCl (anterior sites), the test material at 0.1% corn oil (middle sites), and the test material at 0.1% in FCA at 50% (v/v) in 0.9% NaCl (posterior sites). One week later, animals were treated by topical application of Sodium Lauryl Sulfate at 10% (w/w) in Vaseline in order to induce a local irritation, and then boosted by topical application of the test material at 50 % in a mixture ethanol/water (80/20) under an occlusive dressing for 48h. Control animals were treated in the same manner but using the selected vehicle instead of the test material.

Two weeks after the second induction stage, animals of both groups were challenged by topical application -under occlusive dressing for 24h- of both the vehicle and the test material at 50% in acetone (to the left and the right flanks respectively).

Skin reactions were evaluated 24 and 48 h after removal of the dressing.

Results

No systemic clinical signs and no deaths related to treatment were noted during the study.

No well-defined cutaneous reactions were observed after the challenge application.

Conclusion

Under the experimental conditions of this study, the test material did not elicit a skin sensitisation response in the guinea pig. The test material is not classified for skin sensitisation according to the Regulation (EC) No 1272/2008 on classification, labelling and packaging (CLP) and the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) criteria.