4-methylmorpholine

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Study period:

from 1996-11-14 to 1997-09-19

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Justification for type of information:

Well, but not completely -documented GLP study, following no specific guideline, but similar to OECD guideline 429. Limited test item information is available.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1997

Report date:

1997

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

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

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

4-ethylmorpholine

EC Number:

202-885-0

EC Name:

4-ethylmorpholine

Cas Number:

100-74-3

IUPAC Name:

4-ethylmorpholine

Test material form:

liquid

Details on test material:

- Alternative names of reference substance: Jeffcat NEM; N-ethylmorpholine; 4-ethylmorpholin; Morpholine, 4-ethyl-
- Molecular formula: C6H13NO
- Molecular weight: 115.17
- SMILES notation: CCN1CCOCC1
- InChl: InChI=1/C6H13NO/c1-2-7-3-5-8-6-4-7/h2-6H2,1H3

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: CP5337 4Y506

In vivo test system

Test animals

Species:

mouse

Strain:

Balb/c

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charles River
- Age: 9 - 11 weeks
- Weight at study initiation: no data
- Housing: single housing, in wire bottom cages suspended above catch pans containing absorbent cageboard. The cages were sanitized and cage boards changed regularly in accordance with good husbandry practices.
- Diet: Purina Certified Rodent Chow, ad libitum
- Water (e.g. ad libitum): from water nipple attached to automatic water line, ad libitum
- Acclimation period: no data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 1°C
- Humidity (%): 50 ±10%
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

30% vol/vol

No. of animals per dose:

5 animals per dose group

Details on study design:

RANGE FINDING TESTS:
Each amine catalyst was tested in the LLNA at the minimal irritating concentration (MIC) determined in previous Primary Dermal Irritation Studies (PDIS). For the amine catalysts where an EC10 could not be calculated, the materials were tested at the highest concentration tested which did not trigger an increase in ear swelling.

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
This study consisted of four separate trials. Each trial contained
- a vehicle control group (5 mice treated with a solution of 4: 1 acetone:olive oil, AOO),
- three positive control groups (5 mice treated with a solution of each 1.0% dinitrochlorobenzene (DNCB), 0.17% benzalkonium chloride (BC), and 25% trimellitic anhydride (TMA), and
- five treatment groups (5 mice treated with solutions of an amine catalyst in AOO).

TREATMENT PREPARATION AND ADMINISTRATION:
-Preparation of Dosing Solutions:
The dosing solutions were prepared a maximum of 7 days prior to each test. The vehicle was prepared by mixing four parts of acetone with one part of olive oil (AOO). For the remaining solutions, the appropriate amount of material was weighed into a 16 x 100 mm glass, screw top test tube and the appropriate amount of AOO solution added using a glass pipet (wt/vol.). These solutions were diluted as necessary to obtain the desired concentration (vol. /vol.).

- Topical application:
Prior to the application of the solutions on Day I, an erythema score was assessed for each mouse. The mice were exposed to methoxyflurane. After loosing consciousness, the solutions were applied by dispensing 12.5 ul of the appropriate solution onto the dorsal and ventral side of each ear using an automatic pipettor. Each ear received a total of 25 ul of solution for a total of 50 ul per mouse. The solutions were applied once daily for three consecutive days. On the fourth day, the mice were not treated, but were observed in their cages.

EXCISION OF THE NODES
On Day 5, prior to injection with 3H-thymidine, the erythema score was assessed for each mouse. Each mouse was then injected, via the lateral tail vein, with 0.2 ml of phosphate buffered saline (PBS) containing 20 uCi of 3H-thymidine. Five hours after the injections, the mice were sacrificed by CO2 inhalation and the draining auricular lymph nodes were excised and pooled for each mouse.

TISSUE PROCESSING FOR RADIOACTIVITY
A single cell suspension of lymph node cells (LNC) was prepared as follows:
Both lymph nodes from each mouse were placed in a centrifuge tube containing PBS. This procedure was continued for each mouse. The nodes and PBS were transferred to a small petri dish and dissociated by grinding with the rough end of a syringe plunger. The resulting material was triturated
and the capsule was discarded. The lymph node cells were transferred to a labeled centrifuge tube containing PBS. This process was completed for each liet of lymph nodes. The cell suspensions were centrifuged at 200 x g at 4 °C for 10 minutes. The supernatant was carefully poured off and additional PBS was added to each tube. The tubes were inverted gently and centrifuged again at 200 x g at 4 °C for an additional 10 minutes. The supernatant was carefully poured off and 5% Trichloroacetic acid (TCA) was added to each tube. The tubes were refrigerated at 4 °C for approximately 68 hours.

RADIOACTIVITY MEASUREMENTS
The cell suspensions were centrifuged at 200 x g at 4 °C for 10 minutes and resuspended in 1 ml of 5% TCA. The resuspended cells were transferred to labeled scintillation vials containing 5 ml of Aquasol(R) scintillation cocktail. Each sample was counted for 5 minutes using a beta-scintillation counter.
Radioisotope incorporation was measured as disintegrations per minute (dpm) per mouse.

Interpretation of Raw Data:
A mean dpm value ± SE (standard error) was calculated for each experimental group.
In addition, a stimulation index (SI) was calculated using absolute dpm value for each mouse as the numerator, and the mean dpm value from the vehicle control mice as the denominator. A mean SI ± SE was calculated for each experimental group.

Criteria used to consider a positive response:
Historically, any chemical that produces a SI of> 3 in the LLNA is considered "positive" (Kimber et al., 1994)

Positive control substance(s):

other: 1.0% dinitrochlorobenzene (DNCB)... (see attached file)

Statistics:

mean dpm value ± SE (standard error) and mean SI ± SE was calculated for each experimental group where SI = dpm of mouse / mean dpm of vehicle group

Results and discussion

Positive control results:

Trial 2:
Control DNCB: 1.0 %, SI 74.16 ± 7.20
Control BC: 0.17%, SI 4.77 ± 1.19
Control TMA: 25%, SI 72.55 ± 7.65

Trial 4:
Control DNCB: 1.0 %, SI 40.90 ± 3.89
Control BC: 0.17%, SI 3.28 ± 0.45
Control TMA: 25%, SI 41.07 ± 5.27

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: not reported

Results 2

Key result

Parameter:

SI

Value:

1.18

Variability:

+/-0.15

Test group / Remarks:

30%

Results 3

Key result

Parameter:

SI

Value:

1.34

Variability:

+/-0.25

Test group / Remarks:

30%

Any other information on results incl. tables

Observations

-Viability / mortality: no data

- Clinical signs: no data

- Erythema results:

Trial 2 on 1996-11-14

Control DNCB: erythema score 1.0 (day 5), conc. 1.0%

Control BC: erythema score 0 (day 5), conc. 0.17%

Control TMA: erythema score 2.0 (day 5), conc. 25%

CAS 100 -74 -3: erythema score 0 (day 5), conc. 30%

Trial 4 on 1997-02-10

Control DNCB: erythema score 3.0 (day 5), conc. 1.0%

Control BC: erythema score 1.8 (day 5), conc. 0.17%

Control TMA: erythema score 3.0 (day 5), conc. 25%

CAS 100 -74 -3: erythema score 0 (day 5), conc. 30%

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test item elicited an SI value <3 and is concluded not be a skin sensitizer under the described conditions. Therefore, CLP classification is considered not to be appropriate.