Reaction mass of N1,N1'-(methanediyldibenzene-4,1 -diyl)diaryldiamine and 2-{4-[(4-aminophenyl)amino]benzyl}-N4-(4-{4-[(4-aminophenyl)amino]benzyl}phenyl)aryldiamine

Endpoint summary

Description of key information

The substance initiated a Stimulation Index of greater than 3 at concentrations of 5, 10 and 25 % and thus is considered to be a skin sensitizer under the conditions of the test (OECD 429 and EU Method B.42)

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 September 2008 to 13 October 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Strain: CBA
- Source: Charles River France, L'Arbresle Cedex, France
- Age at study initiation: Young adult animals (approx. 12 weeks old)
- Housing: Individually housed in labeled Macrolon cages containing sterilized sawdust as bedding material
- Diet (e.g. ad libitum): Free access to pelleted rodent diet
- Water (e.g. ad libitum):Free access to tap water
- Acclimation period: at least 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.5 to 23.9 C
-Humidity 43-85%
- Air changes (per hr): at least 15 changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours continuous light/dark cycle


IN-LIFE DATES: 24 September 2008 to 13 October 2008

Vehicle:

propylene glycol

Concentration:

5%, 10%, 25%

No. of animals per dose:

5

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: the vehicle was based on trail formulations performed at Notox. Homogeneity was obtained to visually acceptable levels.
- Irritation: At a 50% test substance concentraion severe irritation of the ears was observed and therefore the highest test substance concentration selected for the main study was a 25% concentration.
- Lymph node proliferation response: Expressed as the number of radioactive disintegrations per minute per lymph nodes from each individual animal and as the ration of ^3HTdR incorporation into lymph node cells of test nodes relative to that recorded for the control nodes (Stimulation Index).


MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: “Skin Sensitisation: Local Lymph Node Assay”
- Criteria used to consider a positive response: The test material will be regarded as a sensitiser if at least one concentration of the test material results in a threefold or greater increase in ^3HTdR incorporation compared to control values.


TREATMENT PREPARATION AND ADMINISTRATION:
The dorsal surface of both ears was epidermally treated (0.25 µL/ear). The concentrations were mixed thoroughly using a vortex mixer immediately prior to dosing. The application was repeated on days 2 and 3. On day 6 an injection of 250 µL phosphate buffered saline (PBS) containing 20 µCi of 3H-methyl thymidine (3H-TdR) was made into the tail vein of each experimental mouse. Five hours later, the draining Auricular lymph node of each ear was excised into PBS. The relative size of the nodes and surrounding area were recorded. The nodes were pooled for each animal in approximately 3mL PBS. A single cell suspension of lymph node cells (LNC) was prepared in PBS by gentle separation through stainless steel gauze. LNC were washed twice with an excess of PBS by centifugation at 200g for 10 minutes at 4C. To precipitate the DNA, the LNC were exposed to 5% trichloroacetic acid (TCA) at 4 C during the night.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Positive control results:

Concentration v/v in acetone/olive oil 4:1: 5, 10 and 25%
Stimulation Index:5%- 1.7; 10% 2.8; 25% 3.6
Result: Positive

Key result

Parameter:

SI

Value:

23.6

Test group / Remarks:

5%

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: Concentration (% v/v) Mean dpm/Animal (Standard Deviation) Vehicle 504 (± 83) 25% - 7673 (±1172) 10% - 9188 (±540) 5% - 11871 (±2958)

Key result

Parameter:

SI

Value:

18.2

Test group / Remarks:

10%

Key result

Parameter:

SI

Value:

15.2

Test group / Remarks:

25%

Individual Disintegrations per Minute and Stimulation Indices

% test substance	Animal Number	dpm/Animal a	Mean dpm/Animal (Standard Deviation)	Stimulation Index b	Result
Vehicle	1-1	587	504 (+/-83)	1.0	N/A
	1-2	790
	1-3	396
	1-4	330
	1-5	417
5	2-1	21923	11871 (+/-2958)	23.6	Positive
	2-2	14214
	2-3	6599
	2-4	11079
	2-5	5539
10	3-1	7267	9188 (+/-540)	18.2	Positive
	3-2	9319
	3-3	10093
	3-4	8956
	3-5	10305
25	4-1	8765	7673 (+/-1172)	15.2	Positive
	4-2	3752
	4-3	10686
	4-4	6631
	4-5	8533

 

Dpm = disintegrations per minutes

a = total number of lymph nodes per animals is 2

b = Stimulation Index of 3.0 or greater indicates a positive result

 

 

 

Individual Bodyweights and Bodyweight Changes

 

	Animal Number	Bodyweight (g)
		Day 1	Day 6
Vehicle	1-1	27	25
	1-2	26	23
	1-3	28	27
	1-4	26	25
	1-5	25	24
5	2-1	29	19
	2-2	27	22
	2-3	25	21
	2-4	27	22
	2-5	27	23
10	3-1	26	24
	3 -2	28	23
	3 -3	27	24
	3-4	29	25
	3-5	25	22
25	4-1	28	22
	4-2	26	23
	4-3	29	25
	4-4	25	20
	4-5	25	20

 

 

Interpretation of results:

sensitising

Conclusions:

In a Local Lymph Node Assay in the Mouse, the substance initiated a Stimulation Index of greater than 3 at concentrations of 5, 10 and 25% and thus is considered to be a skin sensitiser under the conditions of the test.

Executive summary:

In a Local Lymph Node Assay in the Mouse, conducted to Method B.42 of commission directive 440/2008 / OECD Guideline for the Testing of Chemicals No.429, the substance initiated a Stimulation Index of greater than 3 at concentrations of 5, 10 and 25% and thus is considered to be a skin sensitiser under the conditions of the test.

Reference 2

Endpoint:

skin sensitisation: in vitro

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

In a key Local Lymph Node Assay in the Mouse, conducted in accordance with OECD 429 and EU Method B.42, the dorsal surface of both ears was epidermally treated (0.25 µL/ear). The concentrations were mixed thoroughly using a vortex mixer immediately prior to dosing. The application was repeated on days 2 and 3. On day 6 an injection of 250 µL phosphate buffered saline (PBS) containing 20 µCi of 3H-methyl thymidine (3H-TdR) was made into the tail vein of each experimental mouse. Five hours later, the draining Auricular lymph node of each ear was excised into PBS. The relative size of the nodes and surrounding area were recorded. The nodes were pooled for each animal in approximately 3mL PBS. A single cell suspension of lymph node cells (LNC) was prepared in PBS by gentle separation through stainless steel gauze. LNC were washed twice with an excess of PBS by centifugation at 200g for 10 minutes at 4C. To precipitate the DNA, the LNC were exposed to 5% trichloroacetic acid (TCA) at 4 C during the night. The substance initiated a Stimulation Index of greater than 3 at concentrations of 5, 10 and 25% and thus is considered to be a skin sensitizer under the conditions of the test.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Since the test item concentration (EC3 value) resulting in a stimulation index > 3 was higher than 2 % in the Local Lymph Node Assay, classification as a skin sensitiser (category 1B) is appropriate under the criteria given by Regulation (EC) No 1272/2008 and subsequent amendments.