Reaction mass of 2-aminoethanol and 6-[(p-tosyl)amino]hexanoic acid, compound with 2-aminoethanol (1:1)

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 29 January 2016 to 29 February 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study was conducted by a GLP accredited laboratory using OECD Testing Guideline 429. The study was conducted on the registered substance.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

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)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of 2-aminoethanol and 6-[(p-tosyl)amino]hexanoic acid, compound with 2-aminoethanol (1:1)

IUPAC Name:

Reaction mass of 2-aminoethanol and 6-[(p-tosyl)amino]hexanoic acid, compound with 2-aminoethanol (1:1)

Constituent 2

Reference substance name:

Reaction mass of 2-aminoethanol and 6-[(p-tosyl)amino]hexanoic acid, compound with 2-aminoethanol (1:1)

EC Number:

942-732-1

Molecular formula:

C13H19NO4S.C2H7NO/C2H7NO/H2O

IUPAC Name:

Reaction mass of 2-aminoethanol and 6-[(p-tosyl)amino]hexanoic acid, compound with 2-aminoethanol (1:1)

Test material form:

other: Liquid

Details on test material:

- Name of test material (as cited in study report): Reaction mass of 2-aminoethanol and 6[(p-tosyl)amino] hexanoic acid, compound with 2-aminoethanol (1:1)- Physical state: Pale yellow liquid- Storage condition of test material: room temperature in the dark

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS- Source: Envigo RMS B.V. Inc- Age at study initiation: 8-12 weeks- Weight at study initiation: 15-23g- Housing: Suspended solid floor polypropylene cages furnished with softwood woodflakes- Diet: ad libitum- Water: ad libitum- Acclimation period: 5 daysENVIRONMENTAL CONDITIONS- Temperature (°C): 19-25- Humidity (%): 30-70- Air changes (per hr): 15- Photoperiod (hrs dark / hrs light): 12 hours dark/ 12 hours light

Study design: in vivo (LLNA)

Vehicle:

other: 1% pluronic L92 in distilled water

Concentration:

25%, 50%, and 100% v/v

No. of animals per dose:

4

Details on study design:

Groups of four mice were treated with the test item at concentrations of 50%, or 25% w/w in propylene glycol. The preliminary screening test suggested that the test item would not produce systemic toxicity or excessive local skin irritation at the highest suitable concentration. The mice were treated by daily application of 25 μL of the appropriate concentration of the test item to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The test item formulation was administered using an automatic micropipette and spread over the dorsal surface of the ear using the tip of the pipette.A further group of four mice received the vehicle alone in the same manner.Five days following the first topical application of the test item or vehicle (Day 6) all mice were injected via the tail vein with 250 μL of phosphate buffered saline (PBS) containing 3H-methyl thymidine (3HTdR: 80 μCi/mL, specific activity 2.0 Ci/mmoL, ARC UK Ltd) giving a total of 20 μCi to each mouse.Clinical Observations: All animals were observed twice daily on Days 1, 2 and 3 and on a daily basis on Days 4, 5 and 6. Any signs of toxicity or signs of ill health during the test were recorded.Body Weights: The body weight of each mouse was recorded on Day 1 (prior to dosing) and Day 6 (prior to termination).Termination: Five hours following the administration of 3HTdR all mice were killed by carbon dioxide asphyxiation followed by cervical separation. The draining auricular lymph nodes from the four mice were excised and pooled for each experimental group. For each group 1 mL of PBS was added to the pooled lymph nodes.Preparation of Single Cell Suspension: A single cell suspension of pooled lymph node cells was prepared by gentle mechanical disaggregation through a 200-mesh stainless steel gauze. The lymph node cells were rinsed through the gauze with 4 mL of PBS into a petri dish labeled with the study number and dose concentration. The lymph node cell suspension was transferred to a centrifuge tube. The petri dish was washed with an additional 5 mL of PBS to remove all remaining lymph node cells and these were added to the centrifuge tube. The pooled lymph node cells were pelleted at 1400 rpm (approximately 190 g) for 10 minutes. The pellet was re-suspended in 10 mL of PBS and re-pelleted. To precipitate out the radioactive material, the pellet was re-suspended in 3 mL of 5% Trichloroacetic acid (TCA).Determination of 3HTdR Incorporation: After approximately 18 hours incubation at approximately 4°C, the precipitates were recovered by centrifugation at 2100 rpm (approximately 450 g) for 10 minutes, re-suspended in 1 mL of TCA and transferred to 10 mL of scintillation fluid. 3HTdR incorporation was measured by β-scintillation counting. The "Poly Q™" vials containing the samples and scintillation fluid were placed in the sample changer of the scintillator and left to stand in darkness for approximately 20 minutes. The purpose of this period of time in darkness was to reduce the risk of luminescence, which has been shown to affect the reliability of the results. After approximately 20 minutes, the vials were shaken vigorously. The number of radioactive disintegrations per minute was then measured using the Beckman LS6500 scintillation system (Beckman Instruments Inc, Fullerton, CA, USA).

Positive control substance(s):

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

Statistics:

EC3 value calculated using the following equation: EC3=c+[[(3-d)/(b-d)]x(a-c)]

Results and discussion

Positive control results:

Concentration (% v/v) in 1% pluronic L92 in distilled water: 25Stimulation Index: 4.47Result: Positive

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: 0% - N/A25% - 1.7050% - 2.39100% - 3.68

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: 0% - 1726.58 dpm/215.82 dpm per node25% - 2941.25 dpm /367.66 dpm per node50% - 4129.02 dpm/516.13 dpm per node100% - 6362.32dpm /795.29 dpm per node

Any other information on results incl. tables

Individual clinical observations and mortality data

Concentration (%v/v) in 1% pluronic L92 in distilled water	Animal number	Day 1		Day 2		Day 3		Day 4	Day 5	Day 6
		Pre-dose	Post-dose	Pre-dose	Post-dose	Pre-dose	Post-dose
Vehicle	1 -1	0	0	0	0	0	0	0	0	0
	1 -2	0	0	0	0	0	0	0	0	0
	1 -3	0	0	0	0	0	0	0	0	0
	1 -4	0	0	0	0	0	0	0	0	0
25	2 -1	0	0	0	0	0	0	0	0	0
	2 -2	0	0	0	0	0	0	0	0	0
	2 -3	0	0	0	0	0	0	0	0	0
	2 -4	0	0	0	0	0	0	0	0	0
50	3 -1	0	0	0	0	0	0	0	0	0
	3 -2	0	0	0	0	0	0	0	0	0
	3 -3	0	0	0	0	0	0	0	0	0
	3 -4	0	0	0	0	0	0	0	0	0
100	4 -1	0	0	0	0	0	0	0	0	0
	4 -2	0	0	0	0	0	0	0	0	0
	4 -3	0	0	0	0	0	0	0	0	0
	4 -4	0	0	0	0	0	0	0	0	0

0= No signs of systemic toxicity

Individual body weights and body weight changes

Concentration (% v/v) in 1% pluronic L92 in distilled water	Animal number	Body weight (g)		Body weight change (g)
		Day 1	Day 6
Vehicle	1 -1	20.4	20.9	0.5
	1 -2	19.1	19.9	0.8
	1 -3	17.9	18.6	0.7
	1 -4	19.4	20.2	0.8
25	2 -1	20.0	21.1	1.1
	2 -2	19.3	19.8	0.5
	2 -3	19.4	19.7	0.3
	2 -4	17.5	18.2	0.7
50	3 -1	18.4	19.8	1.4
	3 -2	16.9	17.7	0.8
	3 -3	17.6	18.6	1.0
	3 -4	19.5	20.3	0.8
100	4 -1	18.3	19.4	1.1
	4 -2	19.7	20.4	0.7
	4 -3	17.5	18.2	0.7
	4 -4	17.4	18.8	1.4

The LLNA study on Reaction mass of 2-aminoethanol and 6-[(p-tosyl)amino] hexanoic acid, compound with 2-aminoethanol (1:1)

Concentration	dpm	dpm/node	Test/Control Ratio	Result
Vehicle	1726.58	215.82	N/A	N/A
25%	2941.25	367.66	1.70	Negative
50%	4129.02	516.13	2.39	Negative
100%	6362.32	795.29	3.68	Positive

dpm= disintegrations per minute

dpm/node= disintegrations per minute/node obtained by dividing the disintegrations per minute value by 8 (total number of lymph nodes)

Simulation index of 3.0 or greater indicates a positive result

N/A= not applicable

Applicant's summary and conclusion

Interpretation of results:

sensitising

Remarks:

Migrated informationCategory 1BCriteria used for interpretation of results: EU

Conclusions:

The test item produced a positive result when tested at 100% under a LLNA. The test item was classified as a sensitiser (category 1B) according to Regulation (EC) No.1272/2008 on the Classification, Labelling and Packaging of Substances and Mixtures.

Executive summary:

The in vivo skin sensitisation of the test substance was determined in accordance with the OECD Guideline for Testing of Chemicals 429. A Local Lymph Node Assay (LLNA) was performed to assess the skin sensitization potential of the test item in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear.

Following a preliminary screening test in which no clinical signs of toxicity were noted at a concentration of 100% w/w, this concentration was selected as the highest dose investigated in the main test of the Local Lymph Node Assay. Three groups, each of four animals, were treated with 50 μL (25 μL per ear) of the test item as a suspension in propylene glycol at concentrations of 100%, 50%, or 25% w/w. A further group of four animals was treated with 1% pluronic L92 in distilled water.

The Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group provided a positive result when tested at 100%. The test item was classified as a sensitiser (category 1B) according to Regulation (EC) No.1272/2008 on the Classification, Labelling and Packaging of Substances and Mixtures.