2-Propanamine, compd. with boron trifluoride, reaction products with Bu glycidyl ether

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

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:

2-Propanamine, compd. with boron trifluoride, reaction products with Bu glycidyl ether

EC Number:

270-846-5

EC Name:

2-Propanamine, compd. with boron trifluoride, reaction products with Bu glycidyl ether

Cas Number:

68478-97-7

Molecular formula:

C10H23BF3NO2

IUPAC Name:

2-propanamine, compd. with boron trifluoride, reaction products with butyl glycidyl

Test material form:

liquid: viscous

Details on test material:

Manufacturer Air Products and Chemicals, Inc.
Batch# / Lot # 1726820

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

Female CBA/Ca (CBA/CaOlaHsd) strain mice were supplied by Envigo RMS B.V., Inc., Horst, The Netherlands. On receipt the animals were randomly allocated to cages. The animals were nulliparous and non pregnant. After an acclimatization period of at least 5 days the animals were selected at random and given a number unique within the study by indelible ink marking on the tail and a number written on a cage card. At the start of the study the animals were in the weight range of 15 to 23 g, and were 8 to 12 weeks old.

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Concentration:

Test item at concentrations of 50%, 25% or 10% w/w in dimethyl formamide was applied.

No. of animals per dose:

4

Details on study design:

Preliminary Screening Test
Using available information regarding the systemic toxicity/irritancy potential of the test item, a preliminary screening test was performed using three mice, one mouse per test item concentration. The mice were treated by daily application of 25 µL of the undiluted test item or the test item at concentrations of 50% or 25% v/v in dimethyl formamide, to the dorsal surface of each ear for two consecutive days (Days 1 and 2). The surviving mice were similarly treated with the test item at concentrations of 50% or 25% v/v in dimethyl formamide on the following day (Day 3). The mice were observed twice daily on Days 1 and 2. The surviving mice were observed twice on Day 3 and once daily on Days 4, 5 and 6. Local skin irritation was scored daily according to the scale included as Annex 5. Any clinical signs of toxicity, if present, were also recorded. The body weight of each mouse was recorded on Day 1 (prior to dosing) and of the surviving mice on Day 6. The body weight of the mouse that was humanely killed was recorded immediately prior to termination.
Where necessary the thickness of each ear was measured using a Mitutoyo 547 300S gauge (Mitutoyo Corporation), pre dose on Day 1, post dose on Day 3 and on Day 6. Any changes in the ear thickness were noted. Mean ear thickness changes were calculated between time periods Days 1 and 3 and Days 1 and 6. A mean ear thickness increase of equal to or greater than 25% was considered to indicate excessive irritation and limited biological relevance to the endpoint of sensitization.
Main Test
Test Item Administration
Groups of four mice were treated with the test item at concentrations of 50%, 25% or 10% v/v in dimethyl formamide. 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.
Due to an apparent technician error, acetone/olive oil 4:1 was recorded as being used as the vehicle on Day 1 of the main test. However, given that in the vehicle determination trial formulation in acetone/olive oil 4:1 was not possible and that the description of the formulation on Day 1 of the main test (amber colored solution) matched that of the vehicle determination for dimethyl formamide, it is reasonable to assume that the correct vehicle, dimethyl formamide, was used as intended.

3H-Methyl Thymidine Administration
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.

Observations
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).

Terminal Procedures
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  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:

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).
The test item will be 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 computerized system used in the studywas Delta Controls – ORCAview .

Results and discussion

Positive control results:

The Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group was 6.08 for 25% solution of α Hexylcinnamaldehyde, (tech., 85%) in dimethyl formamide.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

1.41

Test group / Remarks:

Group treated with 10% solution of test substance in dimethyl formamide

Results 2

Parameter:

SI

Value:

3.56

Test group / Remarks:

Group treated with 25% solution of test substance in dimethyl formamide

Results 3

Parameter:

SI

Value:

5.36

Test group / Remarks:

Group treated with 50% solution of test substance in dimethyl formamide

Cellular proliferation data / Observations:

There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test.
Body weight change 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.

Applicant's summary and conclusion

Interpretation of results:

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

Conclusions:

Results of Local Lymph Node Assay  study with Boron (2-propanamine) trifluoro-, rxn prod with butyl glycidyl ether indicate that the test item could elicit a SI ≥ 3. The data showed a dose-response and an EC3 value (the estimated test item concentration that causes a 3 fold increase in 3HTdR incorporation) of 21% was calculated. Based on the results, test item is considered to be sensitizer.

Executive summary:

Local lymph node assay in the mouse (Pooled Method) was used to determine sensitization potetial of boron (2-propanamine) trifluoro-, rxn prod with butyl glycidyl ether. Three groups, each of four animals, were treated with 50 µL (25 µL per ear) of the test item as a solution in dimethyl formamideat concentrations of 50%, 25% or 10% w/w. A further group of four animals was treated with dimethyl formamide alone.

Results of Local Lymph Node Assay study with Boron (2-propanamine) trifluoro-, rxn prod with butyl glycidyl ether indicate that the test item could elicit a SI ≥ 3. The data showed a dose-response and an EC3 value (the estimated test item concentration that causes a 3 fold increase in 3HTdR incorporation) of 21% was calculated.

Test item was considered to be a sensitizer under the conditions of the test.