N-isopropylmethacrylamide

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was perfonned between 12 November 2007 and 05 December 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

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)

Test material

Test material information

Constituent 1

Reference substance name:

N-isopropylmethacrylamide

EC Number:

237-331-7

EC Name:

N-isopropylmethacrylamide

Cas Number:

13749-61-6

Molecular formula:

C7H13NO

IUPAC Name:

N-isopropylmethacrylamide

Test material form:

other: solid

Details on test material:

Sponsor's identification: N-Isopropyl methacrylamide (CAS number 13749-61-6) technical grade
Description: white crystalline solid
Batch number: 58838
Date received: 01 October 2007
Storage conditions: approximately 4°C in the dark

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Female CBA/Ca (CBA/CaOlaHsd) strain mice were supplied by Harlan UK Limited
- Age at study initiation: Eight to twelve weeks old.
- Weight at study initiation: Weight range of 15 to 23 g.
- Housing: The animals were individually housed in suspended solid-floor polypropylene cages furnished with softwood wood flakes.
- Diet (e.g. ad libitum): Free access to mains tap water was allowed throughout the study.
- Water (e.g. ad libitum): Free access to food (Certified Rat and Mouse Diet) was allowed throughout the study.
- Acclimation period: At least five days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Controlled to remain within target range of 19 to 25°C.
- Humidity (%): Controlled to remain within target range of 30 to 70%.
- Air changes (per hr): Approximately fifteen changes per hour.
- Photoperiod (hrs dark / hrs light): Controlled by a time switch to give twelve hours continous light (06.00 to 18.00) andd twelve hours darkness.

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Remarks:

For the purpose of the study, the test material was freshly prepared in dimethyl formamide. This vehicle was chosen as it produced the highest concentration that was suitable for dosing.

Concentration:

Main test: test material at concentrations of 5%, 10% or 25% v/v in dimethyl formamide.

No. of animals per dose:

Groups of four mice per dose.

Details on study design:

PRELIMINARY SCREENING TEST:
As no toxicological information was available regarding the systemic toxicity/irritancy potential of the test material, a preliminary screening test was performed using one mouse. The mouse was treated by daily application of 25 µl of the test material at a concentration of 25% v/v in dimethyl formamide, 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. Any signs of toxicity or excessive local irritation noted during this period were recorded. The bodyweight of the mouse was recorded on Day 1 (prior to dosing) and on Day 6.

MAIN TEST:
TEST MATERIAL ADMINISTRATION:
Groups of four mice were treated with the test material at concentrations of 5%, 10% or 25% v/v in dimethyl formamide. The preliminary screening test suggested that the test material 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 material to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The test material 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 material (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.

OBSERVATIONS:
CLINICAL OBSERVATIONS:
All animals were observed twice daily on Days I, 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, re-suspended 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 Q" 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.

Positive control substance(s):

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

Results and discussion

Positive control results:

Three groups, each of five animals, were treated with 50 µl (25 µl per ear) of α-Hexylcinnamaldehyde, Tech, 85% as a solution in dimethyl formamide at concentrations of 5%, 10% and 25% v/v. A further control group of five animals was treated with dimethyl formamide alone.

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 (% v/v) in demethyl formamide: 5
Stimulation Index: 2.11
Result: Negative

Concentration (% v/v) in demethyl formamide: 10
Stimulation Index: 2.82
Result: Negative

Concentration (% v/v) in demethyl formamide: 25
Stimulation Index: 6.37
Result: Positive

α-Hexylcinnamaldehyde, Tech, 85% was considered positive under the conditions of the test.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

1.23

Test group / Remarks:

5 %, 5 animals

Results 2

Parameter:

SI

Value:

1

Test group / Remarks:

25 %, 5 animals

Results 3

Parameter:

SI

Value:

1.77

Test group / Remarks:

10 %, 5 animals

Any other information on results incl. tables

Preliminary Screening Test:

Clinical observations, bodyweight and mortality data are given in Table 1.

No signs of systemic toxicity were noted.

Based on this information the dose levels selected for the main test wer 5%, 10% and 25% v/v in dimethyl formamide.

Clinical Observations and Mortality Data:

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

Bodyweight:

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.

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

 

Concentration (% v/v) in dimethyl formamide	Animal number	Bodyweight (g)		Day
		Day 1	Day 6	1		2		3		4	5	6
				Pre-dose	Post dose	Pre-dose	Post dose	Pre-dose	Post dose
25	S-1	19	19	0	0	0	0	0	0	0	0	0

0 = No signs of systemic toxicity

Table 2: Disintegrations per Minute, Disintegrations per Minute/Node and Stimulation Index

 

Concentration (% v/v) in dimethyl formamide	Dpm	Dpm/Node^a	Stimulation Index^b	Result
Vehicle	3596.38	449.55	na	na
5	4410.37	551.30	1.23	Negative
10	6348.28	793.54	1.77	Negative
25	3610.40	451.30	1.00	Negative

 

Dpm = disintegrations per minute

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

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test material 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 material in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear. The method was designed to meet the requirements of the following:

• OECD Guideline for the Testing of Chemicals No. 429 "Skin Sensitisation: Local Lymph Node Assay" (adopted 24 April 2002)

• Method B42 Skin Sensitisation (Local Lymph Node Assay) of Commission Directive 2004173/EC

Methods.

Following a preliminary screening test, three groups, each of four animals, were treated with 50 µl (25 µl per ear) of the test material as a solution in dimethyl formamide at concentrations of 5%, 10% or 25% v/v. A further group of four animals was treated with dimethyl formamide alone.

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 (% v/v) in dimethyl formamide	Stimulation Index	Result
5	1.23	Negative
10	1.77	Negative
25	1.00	Negative

Conclusion.

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