Reaction mass of 7-azatridecane-1,13-diamine and hexamethylenediamine

Endpoint summary

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 31 JUL 1990 to 24 AUG 1990

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study (EU method B.6)

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Deviations:

no

Principles of method if other than guideline:

Magnusson, B. and Kligman, A.M.: The Identification of Contact Allergens by Animal Assay. The Guinea Pig Maximization Test. J. Invest. Dermatol. 52, 268 - 276 (1969).

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Species:

guinea pig

Strain:

other: Pirbright White, Dunkin Hartley

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Lippische Versuchstierzucht, Hagemann GmbH & Co. KG, Extertal, D; SPF
- Weight at study initiation: 289 - 336 g
- Housing: 5 per cage (Makrolon, type IV)
- Diet (e.g. ad libitum): Kliba 341, 4 mm; Klingentalmuehle AG, Kaiseraugst, CH; ad lib.
- Water (e.g. ad libitum): tap water (plus about 2 g of ascorbic acid per 10 L water was added to the drinking water twice a week), ad lib.
- Acclimation period: at least 7 days

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

Route:

intradermal and epicutaneous

Vehicle:

water

Concentration / amount:

intradermal induction: 0.2% in 0.9% aqueous NaCl-solution resp. in Freund's adjuvant/0.9% aqueous NaCl-solution (1 :1) resp. 0.9% aqueous NaCl-solution
epicutaneous induction: 2% in aqua bidest. (minimum irritant concentration based on results of a pretest)
challenge: 1% in aqua bidest. (maximum non-irritant concentration based on results of a pretest)

Route:

epicutaneous, occlusive

Vehicle:

water

Concentration / amount:

intradermal induction: 0.2% in 0.9% aqueous NaCl-solution resp. in Freund's adjuvant/0.9% aqueous NaCl-solution (1 :1) resp. 0.9% aqueous NaCl-solution
epicutaneous induction: 2% in aqua bidest. (minimum irritant concentration based on results of a pretest)
challenge: 1% in aqua bidest. (maximum non-irritant concentration based on results of a pretest)

No. of animals per dose:

1 test group: 10
2 control groups: 5

Details on study design:

RANGE FINDING TEST
In the preliminary test after two 24-hour epicutaneous occlusive applications within 96 hours the minimum irritant concentration was found to be a 2% aqueous test substance preparation and the maximum non-irritant concentration a l% aqueous test substance preparation.

MAIN STUDY
A. INDUCTION EXPOSURE
AA. intradermal (day 0)
- No. of exposures: - 6 intradermal injections in groups of two per animal
Injections for the test group:
a) front row: 2 injections each of 0.1 mL Freund's adjuvant without test substance emulsified with 0.9% aqueous NaC1-solution in a ratio of 1 :1
b) middle row: 2 injections each of 0.1 mL of the test substance formulation
c) back row: 2 injections each of 0.1 mL Freund's adjuvant / 0.9% aqueous NaCl-solution (1 : 1) with test substance
- Control groups: The animals were given the same injections (a,b,c) but without test substance, only with the formulating agent.
- Site: shoulder
- Frequency of applications: once
- Duration: reading after 24 h
- Concentrations: 0.2%

AB. epicutaneous (day 7)
- No. of exposures: 1
Amount applied:
2 x 4 cm filter paper strips were applied to the skin of the shoulder under an occlusive dressing. In the case of liquids the filter paper strip was soaked in the test substance formulation; thus, the animals were exposed to about 0.3 g of the test substance formulation.
- Control groups: The animals were not treated since the distilled water used as formulating agent was not expected to influence the result of the study.
- Site: shoulder (same area as in the case of the previous intradermal application)
- Frequency of applications: once
- Duration: 48 h, reading 48 h after the beginning of application = directly after patch removal
- Concentrations: 2%

B. CHALLENGE EXPOSURE (day 21)
- No. of exposures: 1
Amount applied:
2 x 2 cm filter paper strips were applied to the skin of the flank under an occlusive dressing. In the case of liquids the test filter paper strip was soaked in the test substance formulation; thus the animals were exposed to about 0.15 g of the test substance formulation.
- Exposure period: 24 h
- Control groups: treatment of the test group and of control group 1 with the test substance formulation (control group 2 remained untreated)
- Site: intact clipped flank
- Concentrations: 1%
- Evaluation (hr after challenge): 24 and 48 h after the removal of the patch

Reading:

1st reading

Hours after challenge:

48

Group:

negative control

Dose level:

0

No. with + reactions:

0

Total no. in group:

5

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 0. No with. + reactions: 0.0. Total no. in groups: 5.0.

Reading:

1st reading

Hours after challenge:

48

Group:

test chemical

Dose level:

1% test substance preparation in aqua bidest

No. with + reactions:

5

Total no. in group:

10

Clinical observations:

distinct erythema in 3/10 animals (score 2), slight erythema in 2/10 animals (score 1)

Remarks on result:

other: see Remark

Remarks:

Reading: 1st reading. . Hours after challenge: 48.0. Group: test group. Dose level: 1% test substance preparation in aqua bidest. No with. + reactions: 5.0. Total no. in groups: 10.0. Clinical observations: distinct erythema in 3/10 animals (score 2), slight erythema in 2/10 animals (score 1).

Reading:

2nd reading

Hours after challenge:

72

Group:

negative control

Dose level:

0

No. with + reactions:

0

Total no. in group:

5

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 72.0. Group: negative control. Dose level: 0. No with. + reactions: 0.0. Total no. in groups: 5.0.

Reading:

2nd reading

Hours after challenge:

72

Group:

test chemical

Dose level:

1% test substance preparation in aqua bidest

No. with + reactions:

5

Total no. in group:

10

Clinical observations:

distinct erythema in 2/10 animals (score 2), slight erythema in 3/10 animals (score 1)

Remarks on result:

other: see Remark

Remarks:

Reading: 2nd reading. . Hours after challenge: 72.0. Group: test group. Dose level: 1% test substance preparation in aqua bidest. No with. + reactions: 5.0. Total no. in groups: 10.0. Clinical observations: distinct erythema in 2/10 animals (score 2), slight erythema in 3/10 animals (score 1).

Induction:

1. and 2. control:

After intradermal induction distinct erythema and edema could be observed at the injection sites of the control animals and test animals at which only Freund's adjuvant/0.9% aqueous NaCl-solution (1:1) was applied.

Test substance:

Injection of the test substance preparation 0.2% in 0.9% aqueous NaCl-solution resp. in Freund's adjuvant/0.9% aqueous NaCl-solution (1:1) caused necrotic skin changes and distinct edema in the test animals.

Negative control:

The control animals which were applied with 0.9% aqueous NaCl-solution did not show any skin reactions.

The percutaneous induction was only carried out in the test group because with aqua bidest. as vehicle, that was not expected to influence the result of the study. Necrotic skin changes (caused by the intradermal induction) could be observed in addition to distinct

edema in all animals.

Challenge:

Table 1: Summary of number of animals with skin findings after the challenge/number of animals tested (21 days after intradermal induction, reading 24 h after removal of patch, i.e. 48hours after start of challenge exposure).

	1% in aqua bidest
Control 1	0/5
Test substance	5/10

x/y: number of positive reactions/number of animals tested

Table 2: Individual challenge read out of control group 1 (intradermal induction Freund's adjuvant/0.9% aqueous NaCl-solution (1:1))

	animal
h after removal of patch	1	2	3	4	5
24	0/0	0/0	0/0	0/0	0/0
48	0/0	0/0	0/0	0/0	0/0

x/y = erythema/edema

Table 3: Individual challenge read out of treatment group (intradermal induction of test substance 0.2% in 0.9% aqueous NaCl-solution resp. in Freund's adjuvant/0.9% aqueous NaCl-solution (1:1))

	animal
h after removal of patch	1	2	3	4	5	6	7	8	9	10
24	0/0	1/0	1/0	2/0	2/0	0/0	0/0	0/0	2/0	0/0
48	0/0	1/0	1/0	2/0	1/0	0/0	0/0	0/0	2/0	0/0

x/y = erythema/edema

The percutaneous challenge with the 1% test substance preparation in aqua bidest. caused distinct erythema in 3 out of 10 test animals and slight erythema in 2 out of 10 test animals.

Control group1 did not show any skin reactions.

Interpretation of results:

sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The authors of the test state that under the conditions tested and the results described above the test item has a sensitising effect on the skin of the guinea pig.

Executive summary:

Testing for sensitising properties of the test item was done using a Guinea pig maximisation test according to EU method B.6. After

intradermal induction of test substance 0.2% in 0.9% aqueous NaCl-solution resp. in Freund's adjuvant/0.9% aqueous NaCl-solution (1:1) and percutaeous induction with 2% in aqua bidest (i.e. minimal irritant concentration), the percutaenous challenge was done for 24 hours under occlusive conditions using the highest non-irritation concentration of 1% test item in aqua bidest. Concentrations applied were choosen based on the results of a preliminiary test. The first and second reading (i.e. 48 and 72 hours after start of the challenge exposure) revealed no effects on the negative control animals. In the test group 3/10 animals showed distinct erythema (score 2) and slight erythema (score 1) were seen in 2/10 animals atthe first reading. At the second reading in 2/10 animals distinct erythema were seen and in 3/10 animals slight erythema were observed. The results indicate that the test substance has sensitising properties.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

Testing for sensitising properties of the test item was done using a Guinea pig maximisation test according to EU method B.6. After

intradermal induction of test substance 0.2% in 0.9% aqueous NaCl-solution resp. in Freund's adjuvant/0.9% aqueous NaCl-solution (1:1) and percutaeous induction with 2% in aqua bidest (i.e. minimal irritant concentration), the percutaenous challenge was done for 24 hours under occlusive conditions using the highest non-irritation concentration of 1% test item in aqua bidest. Concentrations applied were chosen based on the results of a preliminary test. The first and second reading (i.e. 48 and 72 hours after start of the challenge exposure) revealed no effects on the negative control animals. In the test group 3/10 animals showed distinct erythema (score 2) and slight erythema (score 1) were seen in 2/10 animals at the first reading. At the second reading in 2/10 animals distinct erythema were seen and in 3/10 animals slight erythema were observed. The results indicate that the test substance has sensitising properties.

Migrated from Short description of key information:
Under the test conditions (as set out in EU method B.6) the results obtained indicate that the submission substance has a sensitising effect on the skin of the guinea pig.

Justification for selection of skin sensitisation endpoint:
Suitable and only one study available.

Justification for classification or non-classification

According to the results of the only available study the submission substance fulfills the criteria to be classified as skin sensitiser following criteria of Regulation (EC) No. 1272/2008 (CLP) as well as Council Directive 67/548/EEC. Given the most conservative interpretation of results reported in the key study the submission substance has to be classified in subcategory 1B for skin sensitisation. Using the criteria given in ECHA Guidance on IR and CSA, chapter R.8 -10, with an intradermal induction concentration of 0.2% and an observed incidence of 50% the substance is considered to be of moderate potency. The principal difficulty to properly identify corrosive substances as skin sensitisers and to assess its potency based on results from a GPMT is acknowledged.