4-(piperidin-4-yl)morpholine

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 Nov - 22 Dec 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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)

Version / remarks:

adopted Jul 2010

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Remarks:

The Department of Health of the Government of the United Kingdom, United Kingdom

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

4-(piperidin-4-yl)morpholine

EC Number:

627-580-3

Cas Number:

53617-35-9

Molecular formula:

C9H18N2O

IUPAC Name:

4-(piperidin-4-yl)morpholine

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS B.V., Inc., Horst, The Netherlands
- Females nulliparous and non-pregnant: Yes
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: 15 - 23 g
- Housing: Animals were housed in suspended solid floor polypropylene cages furnished with softwood woodflakes.
- Diet: 2014C Teklad Global Rodent diet (Envigo RMS (UK) Limited, Oxon, UK), ad libitum
- Water: Tap water, ad libitum
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 25
- Humidity (%): 30 - 70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

2.5, 5 and 10%

No. of animals per dose:

4

Details on study design:

PRE-SCREEN TESTS: One female per dose was treated by daily application of 25 µL of the test item at concentrations of 2.5, 5, 10, 25 or 50% to the dorsal surface of each ear for three consecutive days.
- Irritation: The animals were observed daily for local skin irritation to the application site.
- Systemic toxicity: The animals were observed twice daily on Days 1, 2 and 3 and once daily on Days 4, 5 and 6 for signs of toxicity. The body weight was recorded on Day 1 prior to dosing and on Day 6.
- Ear thickness measurements: The thickness of each ear was measured pre-dose on Day 1 and post dose on Day 3 and Day 6.
- Erythema scores: No erythema (0), very slight erythema (1), well-defined erythema (2), moderate to severe erythema (3), severe erythema to eschar formation preventing grading of erythema (4)

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: 3H-methyl thymidine incorporation determined by β-scintillation and γ-counting
- Criteria used to consider a positive response: The test item is regarded as a sensitizer if at least one concentration of the test item results in a threefold or greater increase in 3HTdR incorporation compared to control values. Any test item failing to produce a threefold or greater increase in 3HTdR incorporation will be classified as a "non-sensitizer".
The EC3 value was also calculated. The EC3 value is the concentration of the test item expected to cause a 3 fold increase in 3HTdR incorporation. The equation used for the calculation of EC3 is:

EC3 = c + [[(3 - d)/(b - d)] x (a - c)]

a = lowest concentration giving stimulation index > 3
b = actual stimulation index caused by "a"
c = highest concentration failing to produce a stimulation index of 3
d = actual stimulation index caused by "c"

The proliferation response of lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph node (disintegrations per minute/node) and as the ratio of 3HTdR incorporation into lymph node cells of test nodes relative to that recorded for the control nodes (Stimulation Index).

- Other: The animals were observed for signs of toxicity twice on Day 1, 2 and 3, and once on Day 4, 5 and 6. The body weight was recorded on Day 1 prior to dosing and on Day 6 prior to termination.

TREATMENT PREPARATION AND ADMINISTRATION: 25 µL of the test compound was applied to the dorsal surface of each ear of each mouse on Day 1, 2 and 3 in concentrations of 2.5, 5 and 10% in acetone/olive oil. 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. A single cell suspension of pooled lymph node cells was prepared per experimental group by gentle mechanical disaggregation through a 200-mesh stainless steel gauze and rinsed with PBS. The precipitates were incubated for approximately 18 h at approximately 4 °C, centrifuged, resuspended in 1 mL TCA and transferred to 10 mL scintillation fluid before β-scintillation counting.

Positive control substance(s):

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

Results and discussion

Positive control results:

A study (study dates: 14 - 20 Oct 2015; study number: 41502433) was performed to assess the sensitivity of the strain of mouse used at the testing facility to a known sensitizer and to show the sensitivity and reproducibility of the test. The positive control substance hexyl cinnamic aldehyde (25% (v/v) in acetone/olive oil 4:1) was considered to be a sensitizer under the conditions of the test (SI 6.08).

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

1.06

Test group / Remarks:

2.5%

Results 2

Key result

Parameter:

SI

Value:

3.55

Test group / Remarks:

5%

Results 3

Key result

Parameter:

SI

Value:

9.54

Test group / Remarks:

10%

Results 4

Key result

Parameter:

EC3

Value:

4.45

Cellular proliferation data / Observations:

DETAILS ON STIMULATION INDEX CALCULATION
The SI of the 2.5, 5 and 10% treatment group was 1.06, 3.55 and 9.54, respectively.

EC3 CALCULATION
The concentration of test item expected to cause a 3 fold increase in 3HTdR incorporation (EC3 value) was calculated to be 4.45%.
EC3 = 2.5 + [[(3 - 1.06)/(3.55 - 1.06)] x (5 - 2.5)] = 4.45

CLINICAL OBSERVATIONS:
No mortality and no signs of systemic toxicity were noted in the test or control animals during the test.

BODY WEIGHTS
Body weight gain of the test animals between Day 1 and Day 6 was comparable to that observed in the corresponding control group animals over the same period.

Any other information on results incl. tables

Table 1: Results of the measurement of ear thickness and mean ear thickness changes - preliminary screening test

Concentration (% w/w) in acetone/olice oil 4:1	Ear thickness measurement (mm)
	Day 1 pre-dose		Day 3 post dose		Day 6 post dose
	left	right	left	right	left	right
50	0.21	0.22	0.43	0.33	0.43	0.40
overall mean (mm)	0.22		0.38		0.42
overall mean ear thickness change (%)	na		76.74		93.02
25	0.20	0.20	0.34	0.38	0.26	0.2
overall mean (mm)	0.20		0.36		0.25
overall mean ear thickness change (%)	na		80.00		25.00
10	0.22	0.21	0.26	0.25	0.26	0.25
overall mean (mm)	0.22		0.26		0.26
overall mean ear thickness change (%)	na		18.60		18.60
5	0.24	0.22	0.24	0.24	0.24	0.24
overall mean (mm)	0.23		0.24		0.24
overall mean ear thickness change (%)	na		4.35		4.35
2.5	0.22	0.22	0.25	0.25	0.20	0.21
overall mean (mm)	0.22		0.25		0.21
overall mean ear thickness change (%)	na		13.64		- 6.82

Table 2: Individual body weights and results of the radioactivity incorporated - main test

Concentration (% w/w) in acetone/olice oil 4:1	Body weight (g)			Radioactivity incorporated
	Day 1	Day 6	Body weight change	DPM	dpm/node	SI	Result
Vehicle	20.5	21.1	0.6	18811.45	2351.43	-	-
	19.8	21.1	1.3
	21.2	21.3	0.1
	21.1	19.9	-1.2
2.5	19.3	19.2	-0.1	19998.01	2499.75	1.06	Negative
	18.4	18.9	0.5
	19.7	20.2	0.5
	19.6	20.1	0.5
5	20.5	20.7	0.2	66729.19	8341.15	3.55	Positive
	20.1	21.4	1.3
	19.4	21.4	2.0
	21.4	19.4	-2.0
10	19.3	21.0	1.7	179497.30	22437.16	9.54	Positive
	17.4	19.3	1.9
	20.5	19.8	-0.7
	18.5	18.3	-0.2

Applicant's summary and conclusion

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Conclusions:

CLP: Skin sens 1B, H317