1,6-Hexandiammonium dihydroxide, N,N'-bis-(2-hydroxypropyl)-N,N,N',N'-tetramethyl

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 10 JUL 2002 to 11 SEP 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

17.07.1992

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Version / remarks:

30.7.1996

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

August 1998

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

According to Annex VII, Column 2 from Regulation (EC) No 1907/2006 (including amendments up to Commission regulation (EU) 2016/1688 of 20 September 2016) in vivo skin sensitisation studies that were carried out or initiated before 11 October 2016, and that meet the requirements set out in Article 13(3), first subparagraph, and Article 13(4) shall be considered appropriate to address standard information requirement for the endpoint skin sensitization. This applies for the guinea pig maximization test presented here.

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

442-730-6

EC Name:

-

Cas Number:

35132-93-5

Molecular formula:

C16H38N202.2H0

IUPAC Name:

(2-hydroxypropyl)({6-[(2-hydroxypropyl)dimethylazaniumyl]hexyl})dimethylazanium propane-1,2-diol dihydroxide

impurity 1

Reference substance name:

Propane-1,2-diol

EC Number:

200-338-0

EC Name:

Propane-1,2-diol

Cas Number:

57-55-6

Molecular formula:

C3H8O2

IUPAC Name:

propylene glycol

impurity 2

Reference substance name:

N,N,N',N'-tetramethylhexamethylenediamine

EC Number:

203-842-9

EC Name:

N,N,N',N'-tetramethylhexamethylenediamine

Cas Number:

111-18-2

Molecular formula:

C10H24N2

IUPAC Name:

N,N,N',N'-tetramethylhexane-1,6-diamine

Test material form:

liquid

Details on test material:

Water added up to 100 %.

In vivo test system

Test animals

Species:

guinea pig

Strain:

other: Mol:DH (Moellegaard)

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source:M & B A/S, P.O.box 1079, DK-8680 Ry, Denmark
- Weight at study initiation: 284 - 350 g
- Housing: In transparent macrolon cages (type V) on soft wood granulate in an air-conditioned room, 5 animals per cage
- Diet: ssniffe RIM-H, ad libitum
- Water: tap water in plastic bottles, ad libitum
- Acclimation period: at leat 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 ± 3 °C
- Humidity (%): 50 ± 20 %
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

intradermal

Vehicle:

water

Concentration / amount:

0.48 % test substance (42%) in deionized water corresponding to 0.2 % active ingredient

Day(s)/duration:

single application

Adequacy of induction:

highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically

Induction 2

Route:

epicutaneous, occlusive

Vehicle:

water

Concentration / amount:

4 % test substance (42 %) in deionized water corresponding to 1.68 % active ingredient

Day(s)/duration:

48 h

Adequacy of induction:

highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically

Challenge

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

water

Concentration / amount:

4 % test substance (42 %) in deionized water corresponding to 1.68 % active ingredient

Day(s)/duration:

24 h

Adequacy of challenge:

highest non-irritant concentration

No. of animals per dose:

Number of animals in test group: 10
Number of animals in control group: 5

Pretest:
Determination of the tolerance of the intradermal injections: 2 animals
Determination of the primary non-irritating concentration: 3 animals

Details on study design:

RANGE FINDING TESTS:
DETERMINATION OF THE PRIMARY NON-IRRITATING CONCENTRATION:
In a dermal-occlusive test three animals each received 4 intradermal injections of a 50 % Freund's Complete Adjuvant emulsion (4 x 0.1 mL) into the dorsal area, since Freund's Complete Adjuvant may lower the threshold of primary irritation. Thereafter, the test substance was administered to the right or left flank of the guinea pigs. The hair on the flanks of the animals was removed mechanically. 0.5 mL of the test substance preparation was administered to a 2 x 2 cm cellulose patch, which was fixed to the flank and covered occlusively for 24 hours with a bandage and film. 24 hours after removal of the patches, the treated skin areas were examined for erythema and edema.

DETERMINATION OF THE TOLERANCE OF THE INTRADERMAL INJECTIONS:
To determine the tolerance of intradermal injections, each of the following preparations was administered twice by intradermal injection to 2 guinea pigs (0.1, 0.48 and 2.4 % test substance). The injection sites were all within a dorsal area measuring 2 x 4 cm in the vicinity of the shoulders. 24, 48, 72 and 96 hours after administration the injection sites were examined for local tolerance.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: Three pairs of intradermal injections and an epidermal application on test day 8
- Exposure period: 3 weeks (1 week after the inreadermal injections epidermal applicationwas done. Two weeks after that, challenge was performed)

- Exposure group:
Two intradermal injections/site (3 sites, 0.2 ml/site) were made within the shaved dorsal area of 2 x 4 cm as follows:
1) 1:1 (v/v) mixture of Freud´s Complete Adjuvant and deionized water
2) Test item at 0.48 % (corresponding to 0.2 % active ingedient) in deionized water
3) Test item at 0.48 % (corresponding to 0.2 % active ingedient) in a 50 % Freund's Complete Adjuvant emulsion


-Control group:
Two intradermal injections/site (3 sites, 0.2 mL/site) were made within the shaved dorsal area of 2 x 4 cm as follows:
1) 50 % Freund's Complete Adjuvant emulsion in water
2) deionized water
3) 50 % Freund's Complete Adjuvant emulsion in water mixed with an equal volume of water.

- Site: dorsal region
- Duration: single intradermal injection, occlusive dressing from epidermal application was left for 48 h

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: day 22
- Exposure period: 24 h
- Site: 5 x 5 cm on the left flank
- Concentrations: 4 % test substance (42 %) in deionized water corresponding to 1.68 % active ingredient in deionized water
- Evaluation: 24 and 48 h after removal of the patches, reaction sited were assessed for erythema and oedema.
- Control animals were treated with the test item as well.

Reading and Scoring:
The scoring system was performed by visual scoring of erythema, oedema and other clinical changes of skin conditions. They were assessed using the following Magnusson and Kligman gradng scale:
0 = no visible change
1 = discrete or patchy erytheman
2 = moderate and confluent erythema
3 = intense erythema and swelling

Positive control substance(s):

yes

Remarks:

alpha-hexylcinnamaldehyde (tested in a separate experiment)

Results and discussion

Positive control results:

90% (24 h and 48 h reading) of treated animals (9/10) showed a positive reaction during the observation period.

In vivo (non-LLNA)

Results

Results 1

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

1.68 % active ingredient

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

No signs of systemic toxicity were observed in the animals.

Remarks on result:

no indication of skin sensitisation

Results 2

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

1.68 % active ingredient

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

No signs of systemic toxicity were observed in the animals.

Remarks on result:

no indication of skin sensitisation

Results 3

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

1.68 % active ingredient

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

No signs of systemic toxicity were observed in the animals.

Remarks on result:

no indication of skin sensitisation

Results 4

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

1.68 % active ingredient

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

No signs of systemic toxicity were observed in the animals.

Remarks on result:

no indication of skin sensitisation

Any other information on results incl. tables

Intradermal induction treatment

Intradermal injections with Freund's Adjuvant (with and without test substance) caused well-defined erythema and edema as well as sporadically encrustations. The administration sites treated with the test substance in deionized water showed well-defined erythema and slight edema. Intradermal injections of the vehicle alone exhibited no signs of irritation.

Due to these strong irritation reactions of the skin, 10% sodium dodecylsulfate was not administered at day 7.

Dermal induction treatment

After the removal of the patches at day 10, well-defined erythema and edema, indurated and of the patches at day 10, well-defined erythema and edema, indurated and encrusted skin as well as sporadically necrosis were observed at the sites previously treated with Freund's Adjuvant. The administration sites treated with the test substance alone showed slight erythema and edema. The administration sites treated with the vehicle alone showed no signs of irritation.

Dermal challenge treatment

No skin reactions were observed in the control and the treatment group 24 and 48 hours after removal of the occlusive bandage.

The validity of the test system is confirmed by the periodically conducted positive control test using alpha-hexylcinnamaldehyde for the maximization test.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Tested in this study according to OECD test guideline no. 406 and GLP conditions the test item was not a skin sensitiser.

Executive summary:

Testing for sensitising properties of the test item was performed in female guinea pigs according to the adjuvant sensitisation test by Magnusson and Kligman (Guinea pig Maximisation Test) according to OECD TG 406 and under GLP conditions. Pretests determined the maximal intradermal and epicutaneous concentrations causing mild to moderate skin irritation while beeig systemically tolerated.

Intradermal induction was performed using 0.2 % active ingredient in deionized water or in a mixture of Freund's Adjuvant and water (1:1 (v/v)). Epidermal induction was carried out with 1.48 % active ingredient in deionized water.

Using 1.68 % active ingredient in water for the challenge treatment resulted in a 0 % incidence of animals with skin reactions at the 24 and 48 hour reading in both the test group as well as the control group.

The positive control from the test lab showed distinctive positive results under the conditions applied in the lab (up to 90 % of animals with positive skin reactions weras the control animals showed no skin reactions).

Under the conditions of the test, the test item was not a skin sensitiser.