N-(1,1,3,3-tetramethylbutyl)acrylamide

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:

Between 20 February 2012 and 06 March 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

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)

Remarks:

No analysis was carried out to determine the homogeneity, concentration or stability of the test item formulation. This exception is considered not to affect the purpose or integrity of the study.

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

other: CBA/Ca (CBA/CaOlaHsd)

Sex:

female

Details on test animals and environmental conditions:

Female CBA/Ca (CBA/CaOlaHsd) strain mice were supplied by Harlan Laboratories UK Ltd., Oxon, UK. On receipt the animals were randomly allocated to cages. The animals were nulliparous and non pregnant. After an acclimatisation period of at least five 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 eight to twelve weeks old.
The animals were individually housed in suspended solid floor polypropylene cages furnished with softwood woodflakes. Free access to mains tap water and food (2014C Teklad Global Rodent diet supplied by Harlan Laboratories UK Ltd., Oxon, UK) was allowed throughout the study.
The temperature and relative humidity were controlled to remain within target ranges of 19 to 25°C and 30 to 70%, respectively. Any occasional deviations from these targets were considered not to have affected the purpose or integrity of the study. The rate of air exchange was approximately fifteen changes per hour and the lighting was controlled by a time switch to give twelve hours continuous light (06.00 to 18.00) and twelve hours darkness.
The animals were provided with environmental enrichment items which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.

Vehicle:

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

Concentration:

The test item at concentrations of 50%, 25% or 10% w/w in acetone/olive oil 4:1

No. of animals per dose:

Groups of four mice were treated

Details on study design:

Preliminary Screening Test
As no toxicological information was available regarding the systemic toxicity/irritancy potential of the test item, a preliminary screening test was performed using one mouse. The mouse was treated by daily application of 25 µl of the test item at a concentration of 50% w/w in acetone/olive oil 4:1, to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The mouse was observed twice daily on Days 1, 2 and 3 and once daily on Days 4, 5 and 6. Local irritation was scored daily according to the scale included as Appendix 4. Any clinical signs of toxicity, if present, were also recorded. The bodyweight was recorded on Day 1 (prior to dosing) and on Day 6.
The thickness of each ear was measured using an Oditest micrometer (Dyer, PA), 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 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 sensitisation.
Main Test
Test Item Administration
Groups of four mice were treated with the test item at concentrations of 50%, 25% or 10% w/w in acetone/olive oil 4:1. The preliminary screening test suggested that the test item would not produce systemic toxicity or excessive local 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.
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.
Bodyweights: The bodyweight 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. 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 labelled with the project 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 ten minutes. 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).
Determination of 3HTdR Incorporation: After approximately eighteen hours incubation at approximately 4°C, the precipitates were recovered by centrifugation at 2100 rpm (approximately 450 g) for ten minutes, resuspended in 1 ml of TCA and transferred to 10 ml of scintillation fluid (Optiphase 'Trisafe'). 3HTdR incorporation was measured by ß scintillation counting. The "Poly QTM" vials containing the samples and scintillation fluid were placed in the sample changer of the scintillator and left for approximately twenty 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 twenty 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)

Positive control results:

Current Positive Control Study for the Local Lymph Node Assay
Introduction. A study was performed to assess the sensitivity of the strain of mouse used at these laboratories to a known sensitiser. The methodology for the LLNA is detailed in the OECD Guideline for the Testing of Chemicals No. 429 and Method B.42 of Commission Regulation (EC) No. 440/2008. The study described in this document is based on these test methods but has been refined in order to reduce the number of animals required. The reduced LLNA (rLLNA) has been endorsed by the non Commission members of the European Centre for the Validation of Alternative Methods (ECVAM) Scientific Advisory Committee (ESAC) at its 26th meeting held on 26 27 April 2007 at ECVAM, Ispra, Italy.
Test Item : α Hexylcinnamaldehyde, tech., 85%
Project number: 41201832
Study dates : 06 April 2012 to 12 April 2012
Methods. A group of five animals was treated with 50 µl (25 µl per ear) of α Hexylcinnamaldehyde, tech., 85% as a solution in acetone/olive oil 4:1 at a concentration of 25% v/v. A further control group of five animals was treated with acetone/olive oil 4:1 alone.
Results. The Stimulation Index expressed as the mean radioactive incorporation for the treatment group divided by the mean radioactive incorporation of the vehicle control group is as follows:
Concentration (% v/v) in
acetone/olive oil 4:1 Stimulation Index Result
25 5.76 Positive
Conclusion. α Hexylcinnamaldehyde, tech., 85% was considered to be a sensitiser under the conditions of the test.

Parameter:

SI

Value:

1.3

Test group / Remarks:

10% w/w in acetone/olive oil 4:1

Parameter:

SI

Value:

1.03

Test group / Remarks:

25% w/w in acetone/olive oil 4:1

Parameter:

SI

Value:

1.11

Test group / Remarks:

50% w/w in acetone/olive oil 4:1

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: See Table 4

Interpreation of Results

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 sensitiser 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‑sensitiser".

Preliminary Screening Test

Clinical observations, bodyweight and mortality data are given in Table 1 and local skin irritation is given in Table 2. The ear thickness measurements and mean ear thickness changes are given in Table 3.

No signs of systemic toxicity, visual local skin irritation or excessive irritation indicated by an equal to or greater than 25% increase in mean ear thicknesswere noted.

Based on this information the dose levels selected for the main test were 50%, 25% and 10% w/w in acetone/olive oil 4:1.

Main Test

Estimation of the Proliferative Response of Lymph Node Assay

The radioactive disintegrations per minute per lymph node and the stimulation index are given in Table 4.

The Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group are as follows:

Concentration (% w/w) in acetone/olive oil 4:1	Stimulation Index	Result
10	1.30	Negative
25	1.03	Negative
50	1.11	Negative

Clinical Observations and Mortality Data

Individual clinical observations and mortality data for test and control animals are given in Table 5.

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

Bodyweight

Individual bodyweights and bodyweight changes for test and control animals are given in Table 6.

Bodyweight changes of the test animals between Day 1 and Day 6 were comparable to those observed in the corresponding control group animals over the same period, except for one animal treated with the test item at a concentration of 50% w/w in acetone/olive oil 4:1 which showed a slightly greater than expected bodyweight loss.

Table 1              Clinical Observations, Bodyweight and Mortality Data – Preliminary Screening Test

Concentration (% w/w) in acetone/olive oil 4:1	Animal Number	Bodyweight (g)		Day
				1		2		3		4	5	6
		Day 1	Day 6	Pre-Dose	Post Dose	Pre-Dose	Post Dose	Pre-Dose	Post Dose
50	S-1	19	19	0	0	0	0	0	0	0	0	0

0=      No signs of systemic toxicity

Table 2              Local Skin Irritation – Preliminary Screening Test

Concentration (% w/w) in acetone/olive oil 4:1

Animal Number

Local Skin Irritation

Day 1

Day 2

Day 3

Day 4

Day 5

Day 6

left

right

left

right

left

right

left

right

left

right

left

right

50

S-1

0

0

0

0

0

0

0

0

0

0

0

0

Table 3              Measurement of Ear Thicknessand Mean Ear Thickness Changes – Preliminary Screening Test

Concentration (%w/w) in acetone/olive oil 4:1	Animal Number	Ear Thickness Measurement (mm)
		Day 1		Day 3		Day 6
		pre‑dose		post dose
		left	right	left	right	left	right
50	S-1	0.225	0.220	0.260	0.250	0.235	0.240
overall mean (mm)		0.223		0.255		0.238
overall mean ear thickness change (%)		na		14.607		6.742

na=     Not applicable

Table 4              Disintegrations per Minute, Disintegrations per Minute/Node and Stimulation Index

Concentration (% w/w) in acetone/olive oil 4:1	dpm	dpm/Node^a	Stimulation Index^b	Result
Vehicle	9347.79	1168.47	na	na
10	12110.02	1513.75	1.30	Negative
25	9638.90	1204.86	1.03	Negative
50	10416.39	1302.05	1.11	Negative

dpm= Disintegrations per minut

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

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

na =    Not applicable

Table 5              Individual Clinical Observations and Mortality Data

Concentration (% w/w) in acetone/olive oil 4:1	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
10	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
25	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
50	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

Table 6              Individual Bodyweights and Bodyweight Changes

Concentration (% w/w) in acetone/olive oil 4:1	Animal Number	Bodyweight (g)		Bodyweight Change (g)
		Day 1	Day 6
Vehicle	1-1	19	19	0
	1-2	21	19	-2
	1-3	22	20	-2
	1-4	20	21	1
10	2-1	22	20	-2
	2-2	23	21	-2
	2-3	21	20	-1
	2-4	21	20	-1
25	3-1	19	20	1
	3-2	19	21	2
	3-3	17	18	1
	3-4	21	20	-1
50	4-1	21	19	-2
	4-2	20	20	0
	4-3	19	19	0
	4-4	23	20	-3

Interpretation of results:

not sensitising

Remarks:

Migrated information

Conclusions:

The test item was considered to be a non-sensitiser under the conditions of the test.

Executive summary:

Introduction. A study was performed to assess the skin sensitisation potential of the test item in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear. The method was designed to be compatible withthe following:

OECD Guideline for the Testing of Chemicals No. 429 "Skin Sensitisation: Local Lymph Node Assay" (adopted 22 July 2010)

Method B42 Skin Sensitisation (Local Lymph Node Assay) of Commission Regulation (EC) No. 440/2008

Methods. Following a preliminary screening test in which no clinical signs of toxicity were noted at a concentration of 50% 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 solution in acetone/olive oil 4:1at concentrations of 50%, 25% or 10% w/w. A further group of four animals was treated with acetone/olive oil 4:1alone.

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 are as follows:

Concentration (%w/w) in acetone/olive oil 4:1	Stimulation Index	Result
10	1.30	Negative
25	1.03	Negative
50	1.11	Negative

Conclusion. The test item was considered to be a non-sensitiser under the conditions of the test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

In the Local Lymph node assay, N-(1,1,3,3-tetramethylbutyl)acrylamide was shown to not be a skin sensitizer up to the maximum exposure for the assay of 50%, with a lack of any indication of a dose related increase in the stimulation index.

Migrated from Short description of key information:

The is a recent full GLP Klimisch 1 validity Local Lymph node assay on N-(1,1,3,3-tetramethylbutyl)acrylamide CAS 4223-03-4, there was no indication of potential for skin sensitization.

Justification for selection of skin sensitisation endpoint:

This study on N-(1,1,3,3-tetramethylbutyl)acrylamide CAS 4223-03-4 is done to GLP and is Klimisch 1 validity

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

The is a recent full GLP Klimisch 1 validity Local Lymph node assay on N-(1,1,3,3-tetramethylbutyl)acrylamide CAS 4223-03-4, there was no indication of potential for skin sensitization even at the highest concentration recommended for this assay of 50%. This was a well conducted GLP study with a well characterised test substance. Available information indicates that respiratory sensitisers are positive in the local lymph node assay, therefore it is expected that N-(1,1,3,3-tetramethylbutyl)acrylamide also has no potential as a respiratory sensitier.

Migrated from Short description of key information:

There is good evidence, that respiratory sensitisers are positive in the Local Lymph Node Assay in mice.  The absence of any indication of skin sensitization potential for N-(1,1,3,3-tetramethylbutyl)acrylamide in this study is a good indication that it also has no potential to induce respiratory sensitsation.  

Justification for selection of respiratory sensitisation endpoint:

There is good evidence, that respiratory sensitisers are positive in the Local Lymph Node Assay in mice.  The absence of any indication of skin sensitization potential for N-(1,1,3,3-tetramethylbutyl)acrylamide in this study is a good indication that it also has no potential to induce respiratory sensitsation.  

Justification for classification or non-classification

The clearly negative finding in the local lymph node assay and the information that this also indicates a lack of potential for respiratory sensitization, supports a conclusion that N-(1,1,3,3-tetramethylbutyl)acrylamide CAS 4223-03-4 is not classified as a skin or respiratory sensitizer according to the EU CLP (GHS) criteria