2,2',2''-(hexahydro-1,3,5-triazine-1,3,5-triyl)triethanol

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1997

Report date:

1997

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: G. Klecak: Identification of Contact Allergens (Predictive Tests in Animals) in: Advances in Modern Toxicology Vol. 4, - Dermatotoxicology and Pharmacology - ed. F. N. Marzulli, H. I. Maibach - Pages 321 - 324

Version / remarks:

1977

Deviations:

not specified

GLP compliance:

yes

Type of study:

open epicutaneous test

Justification for non-LLNA method:

Study data with guinea pigs was available that was published before the LLNA had been adopted as standard requirement under REACH.

Test material

Test material information

Constituent 1

Reference substance name:

2,2',2''-(hexahydro-1,3,5-triazine-1,3,5-triyl)triethanol

EC Number:

225-208-0

EC Name:

2,2',2''-(hexahydro-1,3,5-triazine-1,3,5-triyl)triethanol

Cas Number:

4719-04-4

Molecular formula:

C9H21N3O3

IUPAC Name:

2,2',2''-(1,3,5-triazinane-1,3,5-triyl)triethanol

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: PK 95/222

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

In vivo test system

Test animals

Species:

guinea pig

Strain:

other: (HA) BR [SPF]

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charles River GmbH - Wiga, Kisslegg, FRG
- Age at study initiation: Young adult animals
- Weight at study initiation: 333 - 395 g
- Housing: 4 animals were housed in Makrolon cages, type IV
- Diet (ad libitum): Kliba-Labordiaet 341 (Klingentakmuehle AG, Kaiseraugst, Switzerland).
- Water (ad libitum): tap water; about 2 g of ascorbic acid per 10 l water was added to the drinking water twice a week.
- Acclimation period: 6 days before the beginning of the study in the laboratory for dermal toxicity.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 12 h/12 h

Study design: in vivo (non-LLNA)

Induction

Route:

epicutaneous, open

Vehicle:

water

Concentration / amount:

15 %, 4 %, 0.4 % and 0.15 % of the test substance

Day(s)/duration:

28 days

Adequacy of induction:

other: 0.4 % is the highest non irritant concentration.

Challenge

Challenge 1

Route:

epicutaneous, open

Vehicle:

water

Challenge 2

No.:

#1

Route:

epicutaneous, open

Vehicle:

water

Concentration / amount:

15 %, 4 %, 0.4 % and 0.15 % of the test substance in aqua bidest.

Day(s)/duration:

3

Adequacy of challenge:

other: 0.4 % is the highest non irritant concentration.

Challenge 3

No.:

#2

Route:

epicutaneous, open

Vehicle:

water

Concentration / amount:

15 %, 4 %, 0.4 % and 0.15 % of the test substance

Day(s)/duration:

3

Adequacy of challenge:

other: 0.4 % is the highest non irritant concentration.

No. of animals per dose:

8

Details on study design:

Pretest:
In a pretest, the irritancy profile was determined by applying 25 µL of various concentrations (0.15 %, 0.4 %, 4 %, 15 %, and 40 %) of aqueous test substance solutions to a 2-cm² area of the shaved flanks of 6 Guinea pigs. All test concentrations were each tested on the same test animal. Test sites were visually evaluated 24 h, 48 h and 72 h after application of test solutions. The dose not causing a reaction in any animal (maximum non-irritant concentration) and the dose causing a reaction in 25 % of the animals (minimum irritant concentration) were determined.

Induction:
20 inductions were conducted. During the induction phase of the main test, groups of 8 Guinea pigs were administered 100 µL of test solution to an 8-cm² area of the right flank skin, once daily on working days (5/wk) for four weeks : Gr 4: 15 % in aqua dest.; Gr 5: 4 %; Gr 6: 0.4 %; Gr 7: 0.15 %.
The control group animals (Gr. 1, 2 and 3) were not treated since the distilled water used as formulating agent was not expected to influence the results of the study. Skin readings were performed 24 h after application.

Challenge:
Three days after the last induction treatment, each test group animal and animals from control group 1 were challenged with 25 µL of 4 different concentrations (15 %, 4 %, 1.5 %, 0.4 %) to a 2-cm² area on the previously untreated flank. Skin reactions were read on an all-or-none basis at 24, 48 and 72 h after application of the solutions.
The second challenge treatment was performed 17 days after the last induction treatment on all treatment groups and on control groups 1 and 2, while control group 3 remained untreated. Skin reactions of test animals were compared with control animals after challenge.

Evaluation:
The test was considered to be positive if at least one guinea pig of the particular concentration group exhibited positive skin reactions with a non-irritant concentration 24 and/or 48 h and/or 72 h after application of the test substance, the control animals showing a negative reaction.

Reference: Klecak G.: "Identification of contact allergens (Predictive tests in animals)", in: Advances in Modern toxicology, Vol. 4 - Dermatotoxicology and Pharmacology (eds.: Marzulli FN & Maibach HI), p. 321-324, (1977).

Results and discussion

In vivo (non-LLNA)

Results

Results 1

Reading:

rechallenge

Hours after challenge:

24

Group:

test chemical

Dose level:

15 %

No. with + reactions:

7

Total no. in group:

8

Results 2

Reading:

rechallenge

Hours after challenge:

24

Group:

test chemical

Dose level:

4%

No. with + reactions:

4

Total no. in group:

6

Clinical observations:

2 animals died 35 days after the beginning of the study.

Results 3

Key result

Reading:

rechallenge

Hours after challenge:

24

Group:

test chemical

Dose level:

0.4 %

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

3 animals died 32, 34 and 35 days after the beginning of the study.

Results 4

Reading:

rechallenge

Hours after challenge:

24

Group:

test chemical

Dose level:

0.15 %

No. with + reactions:

0

Total no. in group:

7

Clinical observations:

1 animal died 32 days after the beginning of the study.

Any other information on results incl. tables

Pretest:

In the pretest, a 15% test substance formulation (in aqua bidest) was found to be the minimum irritant concentration and the 4 % formulation the maximum non-irritant concentration. However, in the main test, 2 of 8 guinea pigs given 4 % test substance developed slight-moderate erythema and slight edema and scaling during the 3rd week of induction treatment. Thus, the maximum non-irritant concentration during induction was 0.4%.

Induction:

Individual results obtained during the induction phase:

- 3 out of 8 animals treated with 15 % test substance solution exhibited very slight to well-defined erythema during the first week of induction. After the 6th induction until the 19th induction all animals showed very slight to well-defined skin reactions, partially in addition to scaling and superficial scabbing.
- The 4 % concentration caused very slight to well-defined skin reactions and scaling in 2 out of 8 animals after the 12th induction until the 19th induction.
- No signs of skin reactions during the induction phase were seen at the application sites of the 0.4 and 0.15 % test substance concentrations.

Challenge:

- After the first challenge with the 15 % test substance concentration very slight to moderate skin reactions were caused, partially in addition to scaling and superficial scabbing in all animals induced with 15 %, 4 % and 0.4 % and in 4 out of 8 animals induced with 0.15 %. At the second challenge, all animals induced with 4 %, 0.4 % and 0.15 % showed positive reactions, as well as 7 out of 8 animals induced with 15 %.
- Challenge with 4% test substance concentration caused very slight to well-defined skin reactions in 4 out of 8 animals induced with 15 %, 4 % and 0.4 % and in 2 out of 8 animals induced with 0.15 %. At the second challenge, 6/8, 4/6, 4/5, 2/7 animals had positive reactions induced with 15 %, 4 %, 0.4 % and 0.15 %, respectively.
- Challenge with 0.4 % test substance concentration did not cause any skin changes in all groups after the first challenge. However, after the second challenge 1 out of 8 animals induced with 0.4 % had a well-defined erythema.
- Challenge with 0.15 % test substance concentration did not cause any skin changes neither after the first nor after the second challenge.

Under the experimental conditions of this study, the test substance was not sensitising in the Open Epicutaneous Test at challenge with concentrations of up to 0.4 % (max. non-irritant concentration) when induction was performed with concentrations lower than or equal to 4 %. The test substance was a skin sensitiser at 15 %.

The number of animals with skin findings after the 1st challenge and after the 2nd challenge.

	Concentration of (Test substance in aqua bidest.)
	Induction	1st challenge				2nd challenge
		15 %	4 %	0.4 %	0.15 %	15 %	4 %	0.4 %	0.15 %
Control group 1^*	-	1/8	0/8	0/8	0/8	2/6	1/6	0/6	0/6
Control group 2	-	-	-	-	-	0/8	0/8	0/8	0/8
Test group 4	15%	8/8	4/8	0/8	0/8	7/8	6/8	1/8	0/8
Test group 5^**	4%	8/8	4/8	0/8	0/8	6/6	4/6	0/6	0/6
Test group 6^***	0.4%	8/8	4/8	0/8	0/8	5/5	4/5	0/5	0/5
Test group 7^****	0.15%	4/8	2/8	0/8	0/8	7/7	2/7	0/7	0/7

 ^* 2 animals of control group 1 died 35 days after the beginning of the study.

 ^** 2 animals of test group 5 died 34 days after the beginning of the study.

 ^*** 3 animals of test group 6 died 32, 34 and 35 days after the beginning of the study.

^**** 1 animal of test group 7 died 32 days after the beginning of the study.

Macroscopic examination revealed that the cause of deaths was not substance-related.

Applicant's summary and conclusion

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria