Hexahydro-1,3,5-trimethyl-1,3,5-triazine

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 August 2009 to 21 September 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

This study was conducted in accordance with international guidelines in a GLP laboratory.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Swiss GLP Monitoring Authority

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

Study conducted as the positive result in the LLNA was suspected of being due to irritancy (false positive). Study also conducted for global regulatory use.

Test material

Test material information

Constituent 1

Reference substance name:

Hexahydro-1,3,5-trimethyl-1,3,5-triazine

EC Number:

203-612-8

EC Name:

Hexahydro-1,3,5-trimethyl-1,3,5-triazine

Cas Number:

108-74-7

Molecular formula:

C6H15N3

IUPAC Name:

1,3,5-trimethyl-1,3,5-triazinane

Test material form:

liquid

Details on test material:

Identification: R-3853Description: Clear pale yellow liquidCAS Number: 108-74-7Stability of Test Item: Stable under specified storage conditions.Stability of Test Item Dilution: Unknown in PEG 300 and in a 1:1 (v: v) mixture of Freund’s Complete Adjuvant (FCA)/ physiological saline; is excluded from the statement of compliance.Storage Conditions: At room temperature (range of 20 ± 5 °C, provided by Harlan Laboratories Ltd.), light protected.Safety Precautions: Routine hygienic procedures were used to ensure the health and safety of the personnel.Incompatibility of the test item with water or saline.

In vivo test system

Test animals

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

male

Details on test animals and environmental conditions:

Animals: Albino Dunkin Hartley Guinea Pig, CRL:(HA), SPFRationale: Skin reactions in the guinea pig are classically used for determining the potential of test items to induce delayed contact hypersensitivity. No valid nonanimal model (in-vitro) is available at present for the test of contact sensitization.Breeder: Charles River Deutschland GmbH, Stolzenseeweg 32-36, 88353 Kisslegg / GermanyNumber of Animals for Main Study / Pretest: 15 males / 4 males - Animals of either sex are acceptable for use according to guidelines Commission Regulation (EC) No 440/2008, B.6 and OECD 406.Age at Pretest Start / Beginning of Acclimatization Period: 4-6 weeksBody Weight at Pretest Start: Pretest groups: 364 - 378 gBody Weight at Beginning of Acclimatization Period: Test and control group: 357 - 395 gIdentification: By unique cage number and corresponding individual animal number.Randomization: Selected by hand at time of delivery. No computer generated randomization program.Acclimatization: Under laboratory conditions, after health examination. Only animals without any visible signs of illness were used for the study. A certificate of health was provided by the animal supplier at the animal delivery and included in the raw data.

Study design: in vivo (non-LLNA)

Induction

Route:

other: Intradermal and epidermal, occlusive

Vehicle:

polyethylene glycol

Concentration / amount:

The pretest screened intradermal concentrations of 25, 10, 5, 1, 0.5 and 0.1 % test item in polyethylene glycol and epidermal concentrations of 50, 25, 15 and 10 % test item in polyethylene glycol.For the main study, a 1 % intradermal concentration, 25 % epidermal induction concentration and a 15 % epidermal challenge concentration in polyethylene glycol were used.

Challenge

Challenge 1

Route:

other: Epidermal, occlusive

Vehicle:

polyethylene glycol

Challenge 2

Concentration / amount:

The pretest screened intradermal concentrations of 25, 10, 5, 1, 0.5 and 0.1 % test item in polyethylene glycol and epidermal concentrations of 50, 25, 15 and 10 % test item in polyethylene glycol.For the main study, a 1 % intradermal concentration, 25 % epidermal induction concentration and a 15 % epidermal challenge concentration in polyethylene glycol were used.

No. of animals per dose:

15 males for main study4 males for pretest

Details on study design:

PREPARATION OF DOSE FORMULATIONSThe test item and vehicle or auxiliary compound were placed into a glass beaker on a tared Mettler balance and a weight/weight dilution was prepared. Homogeneity of the test item preparation was ensured and maintained during treatment using a magnetic stirrer. The preparations were made immediately prior to each dosing.TEST ITEM ADMINISTRATIONIntradermal InductionThe 1% concentration of test item ((i.e. 0.33% w/w of Hexahydro-1,3,5-Trimethyl-1,3,5-Triazine) used for the intradermal induction exposure was well-tolerated systemically and caused mild skin irritation during the pretest.Epidermal InductionThe 25% concentration of test item (i.e. 8.24% w/w of Hexahydro-1,3,5-Trimethyl-1,3,5-Triazine) used for the epidermal induction exposure was the highest concentration causing mild irritation during the pretest.Epidermal ChallengeThe 15% concentration of the test item (i.e. 4.95% w/w of Hexahydro-1,3,5-Trimethyl-1,3,5-Triazine) used for the challenge application was non-irritant.OBSERVATIONSViability / Mortality: Daily from delivery of the animals to the termination of the test.Clinical Signs / Grading of Skin Response: Daily from delivery of the animals to the termination of test. Skin responses were graded during the pretest, induction and challenge periods.Body Weights: At delivery/acclimatization start, at the end of the pretest, at test day 1 (day of treatment) and at the termination of the study.TREATMENTThe animal's fur was shaved with a fine clipper blade or equivalent just prior to the exposure. Intradermal injections or closed patches were applied to the animals as follows:0.1 mL/site for the intradermal administrations0.2 to 0.3 mL on a filter paper for the epidermal administrationsThe filter patch was covered by a strip of aluminium foil and firmly secured by an elastic plaster wrapped around the trunk of the animal and secured with impervious adhesive tape. The occlusive dressing was left in place for 24 (epidermal pretest and epidermal challenge) or 48 hours (epidermal induction).Identical patching method was used for the epidermal pretest, epidermal induction and epidermal challenge.PRETESTBased on the results, the test item concentration of 1% was selected for intradermal induction and 25 % for epidermal induction in the main study.Based on the results obtained the concentration selected for the epidermal challenge in the main study was 15% (i.e. 4.95% w/w of Hexahydro-1,3,5-Trimethyl-1,3,5-Triazine).MAIN TESTInductionIntradermal injection / Test Day 1An area of dorsal skin from the scapular region (approximately 6 x 8 cm) was clipped free of hair. Three pairs of intradermal injections (0.1 mL/site) were made just within the boundaries of a 4 x 6 cm area in the clipped region as follows:Test Group: 1) 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.2) The test item at 1% in PEG 300.3) The test item at 1% in a 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.Control Group: 1) 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.2) PEG 3003) 1:1 (w/w) mixture of PEG 300 in a 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.Epidermal Induction / Test Day 8One week after the intradermal injections, the scapular area (approximately 6 x 8 cm) was again clipped and shaved free of hair prior to the application. A 2 x 4 cm patch of filter paper was saturated with the test item at 25% in PEG 300 and placed over the injection sites of the test animals. The volume of test item preparation applied was approximately 0.3 mL. The patch was covered with aluminum foil and firmly secured by an elastic plaster wrapped around the trunk of the animal and secured with impervious adhesive tape. The occlusive dressings were left in place for 48 hours. The epidermal application procedure described ensured intensive contact of the test item. The guinea pigs of the control group were treated as described above with PEG 300 only, applied at a volume of approximately 0.3 mL. The reaction sites were assessed 24 and 48 hours after removal of the bandage for erythema andoedema according to the method of Magnusson and Kligman.Challenge / Test Day 22The test and control guinea pigs were challenged two weeks after the epidermal induction application and were treated in the same way. Two patches (3 x 3 cm) of filter paper were saturated with the test item at the highest tested nonirritating concentration of 15% (i.e. 4.95% w/w of Hexahydro-1,3,5-Trimethyl-1,3,5-Triazine, applied to the left flank) and the vehicle only (PEG 300 applied to the right flank) using the same method as for the epidermal application. The volume of test item preparation and of vehicle applied was approximately 0.2 mL. The dressings were left in place for 24 hours.Determination of Skin Reactions - Observation and ScoringThe test item skin area of the animals used for the epidermal pretest and challenge was depilated approximately 21 hours after the patches have been removed, using an approved depilatory cream (VEET Cream, Reckitt & Colman AG, 4123 Allschwil / Switzerland). The depilation was performed to clean the stratum corneum from the possible staining produced by the test item and to facilitate the reading of the possible skin reaction. The depilatory cream was placed on the patch sites and surrounding areas, and left on for up to 3-5 minutes. It was then thoroughly washed off with a stream of warm, running water. The animals were then dried with a disposable towel, and returned to their cages.The grading method used for the pretest, induction and challenge was identical. It was performed 24 ± 2 hours after removal of the patches for the intradermal and epidermal pretest and induction and for challenge and repeated 24 ± 2 hours later (48-hour grades) for the epidermal pretest, epidermal induction and challenge. The scoring system was performed by visual assessment of erythema and oedema. They were assessed as follows:0 = no visible change1 = discrete or patchy erythema2 = moderate and confluent erythema3 = intense erythema and swellingAny other gross lesions not covered by this scoring system were described.Grading of all animals was done by positioning each animal under true-light (FL-LPE OsramD16 FH 28W/840 EP).

Challenge controls:

The control group were also challenged with an epidermal 15 % (i.e. 4.95% w/w of Hexahydro-1,3,5-Trimethyl-1,3,5-Triazine) application.

Positive control substance(s):

yes

Remarks:

Alpha-Hexylcinnamaldehyde

Results and discussion

Positive control results:

One out of 10 test animals showed skin reactions after the first challenge treatment with ALPHA-HEXYLCINNAMALDEHYDE at 1% (w/w) in PEG 300. No skin effect was observed in the control group.Eighty to 90% of the test animals showed discrete/patchy erythema at the 24- or 48-hour reading after the second challenge treatment with ALPHA-HEXYLCINNAMALDEHYDE at 3% (w/v) in PEG 300. No skin effect was observed in the control group.

In vivo (non-LLNA)

Results

Results 1

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

15 % (4.95 % w/w test item)

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

None

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 15 % (4.95 % w/w test item). No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: None.

Results 2

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

0

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

None

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 0. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: None.

Results 3

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

15 % (4.95 % w/w test item)

No. with + reactions:

10

Total no. in group:

10

Clinical observations:

Moderate/confluent erythema in all animals, 7/10 animals observed with oedema.

Remarks on result:

other: see Remark

Remarks:

Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 15 % (4.95 % w/w test item). No with. + reactions: 10.0. Total no. in groups: 10.0. Clinical observations: Moderate/confluent erythema in all animals, 7/10 animals observed with oedema..

Results 4

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

0

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

None

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 0. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: None.

Results 5

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

15 % (4.95 % w/w test item)

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

None

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 15 % (4.95 % w/w test item). No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: None.

Results 6

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

0

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

None

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 0. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: None.

Results 7

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

15 % (4.95 % w/w test item)

No. with + reactions:

10

Total no. in group:

10

Clinical observations:

Moderate/confluent erythema in all animals, 7/10 animals observed with oedema as well as scaling.

Remarks on result:

other: see Remark

Remarks:

Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 15 % (4.95 % w/w test item). No with. + reactions: 10.0. Total no. in groups: 10.0. Clinical observations: Moderate/confluent erythema in all animals, 7/10 animals observed with oedema as well as scaling..

Results 8

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

0

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

None

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 0. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: None.

Results 9

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Dose level:

3%

No. with + reactions:

9

Total no. in group:

10

Results 10

Reading:

2nd reading

Hours after challenge:

48

Group:

positive control

Dose level:

3%

No. with + reactions:

9

Total no. in group:

10

Any other information on results incl. tables

SKIN REACTIONS IN THE EPIDERMAL INDUCTION

Table 1. Control Group Treated with PEG 300 (Scapular Area)

Animal No.	Sex	Reactionafter Removal of Bandage
		24 hours	48 hours
2657 2658 2659 2660 2661	M M M M M	0 0 0 0 0	0 0 0 0 0

Table 2. Test Group Treated with Test Item (25 %) in PEG 300 (Scapular Area)

Animal No.	Sex	Reactionafter Removal of Bandage
		24 hours	48 hours
2662 2663 2664 2665 2666 2667 2668 2669 2670 2671	M M M M M M M M M M	1 0 1 1 1 2 1 1 1 0	0 0 0 1 0 1 1 0 1 0

SKIN REACTIONS IN THE CHALLENGE APPLICATION

Table 3. Control Group Treated with PEG 300 (Right Flank)

Animal No.	Sex	Reactionafter Removal of Bandage
		24 hours	48 hours
2657 2658 2659 2660 2661	M M M M M	0 0 0 0 0	0 0 0 0 0

Table 4. Control Group Treated with Test Item (15%) in PEG 300 (Left Flank)

Animal No.	Sex	Reactionafter Removal of Bandage
		24 hours	48 hours
2657 2658 2659 2660 2661	M M M M M	0 0 0 0 0	0 0 0 0 0

Table 5. Test Group Treated with PEG 300 (Right Flank)

Animal No.	Sex	Reactionafter Removal of Bandage
		24 hours	48 hours
2662 2663 2664 2665 2666 2667 2668 2669 2670 2671	M M M M M M M M M M	0 0 0 0 0 0 0 0 0 0	0 0 0 0 0 0 0 0 0 0

Table 6. Test Group Treated with Test Item (15 %) in PEG 300 (Left Flank)

Animal No.	Sex	Reactionafter Removal of Bandage
		24 hours	48 hours
2662 2663 2664 2665 2666 2667 2668 2669 2670 2671	M M M M M M M M M M	2* 2 2* 2* 2* 2* 2* 2 2* 2	2*^ 2 2*^ 2*^ 2*^ 2*^ 2*^ 2 2*^ 2

* Oedema present

^ Scaling present

Applicant's summary and conclusion

Interpretation of results:

Category 1A (indication of significant skin sensitising potential) based on GHS criteria

Conclusions:

Based on the results of this study, the test item should be classified as a skin sensitiser.

Executive summary:

In order to assess the cutaneous allergenic potential of R-3853, the Maximization-Test was performed in 15 (10 test and 5 control) male albino Dunkin Hartley guinea pigs, in accordance with OECD Guideline No. 406 and the Commission Regulation (EC) No 440/2008, B.6. The intradermal induction of sensitization in the test group was performed in the nuchal region with a 1% (i.e. 0.33% w/w of Hexahydro-1,3,5-Trimethyl-1,3,5-Triazine) dilution of the test item in PEG 300 and in an emulsion of Freund's Complete Adjuvant (FCA)/physiological saline. The epidermal induction of sensitization was conducted for 48 hours under occlusion with the

test item at 25% (i.e. 8.24% w/w of Hexahydro-1,3,5-Trimethyl-1,3,5-Triazine) in PEG 300 one week after the intradermal induction. The animals of the control group were intradermally induced with PEG 300 and FCA/physiological saline and epidermally induced with PEG 300 under occlusion.

Two weeks after epidermal induction the control and test animals were challenged by epidermal application of the test item at 15% (i.e. 4.95% w/w of Hexahydro-1,3,5-Trimethyl-1,3,5-Triazine) in PEG 300 and PEG 300 alone under occlusive dressing.

Cutaneous reactions were evaluated at 24 and 48 hours after removal of the dressing.

No deaths occurred. All 10 test animals showed skin reactions after the challenge treatment with R-3853 at 15% (i.e. 4.95% w/w of Hexahydro-1,3,5-Trimethyl-1,3,5-Triazine) in PEG 300. No skin effect was observed in the control group.