Amides, tall-oil fatty, N,N-di-Me

Endpoint summary

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 December 2012 to 23 January 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study conducted according to relevant guidelines, with no deviations.

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:

The animals were nulliparous and non-pregnant female CBA/Ca (CBA/CaOlaHsd) mice, supplied by Harlan Laboratories UK Ltd. The animals were acclimatised for at least 5 days, and randomly allocated to treatment groups. At the start of the study the mice weighed 15 to 23 g and were 8 to 12 weeks old. They were individually housed in suspended solid floor polypropylene cages furnished with softwood woodflakes. Mains tap water and food (2014C Teklad Global Rodent diet) were provided ad libitum. Temperature was maintained as 19 to 25°C and relative humidity at 30 to 70%. Lighting was provided on a 12 hour cycle, and there were approximately 15 air changes per hour.

Vehicle:

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

Concentration:

Preliminary test: 25%, 50% or 100% v/v in acetone/olive oil 4:1
Main test: 0%, 5%, 10% or 25% v/v in acetone/olive oil 4:1

No. of animals per dose:

Preliminary test: 1 mouse/dose
Main test: 4 mice/dose

Details on study design:

The test material was freshly prepared as a solution in acetone/olive oil 4:1. The test material was formulated within two hours of being applied to the test system, it was assumed that the formulation was stable for this duration.

A preliminary screening test was performed using three mice (one mouse per concentration), The mice were treated by daily application of 25 µL of either undiluted test material, or concentrations of 50% or 25% v/v in acetone/olive oil 4:1 to the dorsal surface of each ear for three consecutive days (Days 1, 2 3). The mice were observed twice daily on Days 1, 2 and 3 and once daily on Days 4, 5 and 6. Local skin irritation was scored daily. Any clinical signs of toxicity were recorded. Bodyweights were recorded on Day 1 (prior to dosing) and on Day 6. The thickness of each ear was measured using an Oditest micrometer, pre-dose on Day 1, post-dose on Day 3 and on Day 6. Any changes in ear thickness were noted. Mean ear thickness changes were calculated between time periods Day 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.

In the main test, groups of four mice were treated with the test material at concentrations of 5%, 10% or 25% v/v in acetone/olive oil 4:1. The preliminary screening test suggested that there would be no systemic toxicity or excessive local irritation at a concentration of 25%. The mice were treated by daily application of 25 µL of the appropriate concentration to the dorsal surface of the ear for three consecutive days (Days 1, 2, 3). The controls received the vehicle alone in the same manner. Five days following the first topical application (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) giving a total of 20 µCi to each mouse.

Clinical observations were made twice daily on Days 1, 2 and 3 and on a daily basis on Days 4, 5 and 6. Bodyweights were recorded on Day 1 (prior to dosing) and Day 6 (prior to termination).

Five hours following the administration of 3HTdR all mice were killed. 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. A single cell suspension of pooled lymph node cells was prepared by gentle mechanical disaggregation through a 200-mesh stainless steel gauze. The cells were rinsed through the gauze with 4 mL of PBS into a petri dish. The cell suspension was transferred to a centrifuge tube, the petri dish was washed with an additional 5 mL of PBS and the washings added to the tube. The cells were pelleted at 1400 rpm for 10 minutes. The pellet was resuspended in 10 mL of PBS and re-pelleted. To precipitate out the radioactive material, the pellet was resuspended in 10 mL of PBS and re-pelleted. To precipitate out the radioactive material, the pellet was resuspended in 3 mL of 5% trichloroacetic acid (TCA). After approximately 18 hours incubation at ca. 4°C, the precipitates were removed by centrifugation at 2100 rpm for 10 minutes, resuspended in 1 mL of TCA and transferred to 10 mL of scintillation fluid. 3HTdR incorporation was measured by scintillation counting. The vials were placed in the sample changer and left for approximately 20 minutes in the dark, to reduce the risk of luminescence. The vials were shaken vigorously afterwards. The number of radioactive disintegrations per minute was then measured.

The proliferation response of lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph node (disintegrations/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). Test materials causing a threefold or greater increase in 3HTdR incorporation in at least once concentration compared to control values are regarded as sensitisers. The EC3 value was also calculated using the following equation: EC3 = c + [[(3-d)/(b-d)] x (a-c)] where a is the lowest concentration giving a stimulation index > 3, b is the actual stimulation index caused by 'a', c is the highest concentration failing to produce a stimulation index of 3 and d is the actual stimulation index caused by 'c'.

Positive control substance(s):

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

Statistics:

Not required.

Positive control results:

In a separate study, a group of 5 mice were treated with alpha-Hexylcinnamaldehyde, tech., 85% at a concentration of 25% v/v in acetone/olive oil 4:1. Controls received the vehicle alone. The Stimulation Index was 6.29 therefore alpha-Hexylcinnamaldehyde, tech., 85% was considered to be a skin sensitiser.

Parameter:

SI

Remarks on result:

other: Stimulation indices of 1.09, 3.40 and 7.19 was obtained for test material concentrations of 5%, 10% and 25%, respectively.

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: DPM values were 10345.17, 11283.55, 35150.61 and 74432.01 for concentrations of 0%, 5%, 10% and 25%.

There were no deaths, and no signs of systemic toxicity were noted in the test or control animals during the main test. Bodyweight changes between Days 1 and 6 were comparable to those observed in the corresponding control group over the same period.

Table 1. Results obtained in the main LLNA study

Concentration (% v/v) in acetone/olive oil 4:1	dpm	dpm/Node^a	Stimulation Index^b	Result
Vehicle	10345.17	1293.15	1.00	Negative
5	11283.55	1410.44	1.09	negative
10	35150.61	4393.83	3.40	positive
25	74432.01	9304.00	7.19	positive

dpm = disintegrations per minute

a = disintegrations per minute/node

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

na = not applicable

Interpretation of results:

sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The EC3 value was calculated to be 9.13%. The test material was considered to be a skin sensitiser under the conditions of the test.

Executive summary:

The skin sensitisation potential of DMATO was evaluated in the Local Lymph Node Assay (LLNA) according to OECD Guideline 429 and GLP. Groups of four female CBA/Ca mice were treated with the test material at concentrations of 5%, 10% or 25% v/v in acetone/olive oil 4:1. Controls received the vehicle alone. The highest dose selected for the main study was chosen following a preliminary screening test in which no clinical signs of toxicity were noted at a concentration of 25%. The test material formulations were applied to the dorsum of each ear for 3 consecutive days. The draining auricular lymph nodes were harvested on Day 6, following administration of 3 -HTdR, and incorporation of 3 -HTdR was measured using scintillation counting. The Stimulation Index was expressed as the mean radioactive incorporation for each treatment group, divided by the mean radioactive incorporation of the vehicle controls group; values of greater than or equal to 3.0 indicate a positive result. The Stimulation Indices were 1.09, 3.40 and 7.19 for test concentrations 5%, 10% and 25%, respectively. The concentration expected to cause a 3 fold increase in 3HTdR incorporation (EC3 value) was calculated to be 9.13%. The test material was considered to be a skin sensitiser under the conditions of the test, and requires classification as a Category 1B Skin Sensitiser according to Regulation (EC) No 1272/2008.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

The skin sensitisation potential of DMATO was evaluated in the Local Lymph Node Assay (LLNA) according to OECD Guideline 429 and GLP. Groups of four female CBA/Ca mice were treated with the test material at concentrations of 5%, 10% or 25% v/v in acetone/olive oil 4:1. Controls received the vehicle alone. The highest dose selected for the main study was chosen following a preliminary screening test in which no clinical signs of toxicity were noted at a concentration of 25%. The test material formulations were applied to the dorsum of each ear for 3 consecutive days. The draining auricular lymph nodes were harvested on Day 6, following administration of 3 -HTdR, and incorporation of 3 -HTdR was measured using scintillation counting. The Stimulation Index was expressed as the mean radioactive incorporation for each treatment group, divided by the mean radioactive incorporation of the vehicle controls group; values of greater than or equal to 3.0 indicate a positive result. The Stimulation Indices were 1.09, 3.40 and 7.19 for test concentrations 5%, 10% and 25%, respectively. The concentration expected to cause a 3 fold increase in 3HTdR incorporation (EC3 value) was calculated to be 9.13%. The test material was considered to be a skin sensitiser under the conditions of the test, and requires classification as a Category 1B Skin Sensitiser according to Regulation (EC) No 1272/2008.

Migrated from Short description of key information:
A modern, guideline-compliant mouse LLNA is available for the submission substance.

Justification for selection of skin sensitisation endpoint:
Only study available for this endpoint

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the results of the available mouse LLNA (EC3 = 9.13%); DMATO requires classification as a Category 1B Skin Sensitiser according to Regulation (EC) No 1272/2008. Classification with (R43) 'May cause sensitisation by skin contact' is appropriate according to Directive 67/548/EEC.