Butanedioic acid, 1-[2-[(1-oxo-2-propen-1-yl)oxy]ethyl] ester

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 performed between 15 May 2006 and 30 May 2006.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

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)

Remarks:

Date of Inspection: 30th August 2005. Date of Signature: 21st November 2005

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

478-130-6

EC Name:

-

Cas Number:

50940-49-3

Molecular formula:

C9H12O6

IUPAC Name:

2-(acryloyloxy)ethyl hydrogen succinate

Details on test material:

- Name of test material (as cited in study report): MAES
- Substance type: Organic
- Physical state: Colourless turbid liquid
- Lot/batch No.: J417708-A
- Storage condition of test material: Room temperature in the dark

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/Ca (CBA/CaOlaHsd)

Sex:

female

Details on test animals and environmental conditions:

- Female CBA/Ca (CBA/Ca CruBR) strain mice were supplied by Charles River UK Limited, Margate, Kent, UK and (CBA/CaOlaHsd) strain mice were supplied by Harlan UK Limited, Bicester, axon, 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 (Certified Rat and Mouse Diet (Code 5LF2) supplied by BCM IPS Limited, London, 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.

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Concentration:

10%,25% and 50% v/v in dimethyl formamide.

No. of animals per dose:

Groups of four mice per dose level were treated

Details on study design:

RANGE FINDING TESTS:
As no toxicological information was available regarding the systemic toxicity/irritancy potential of the test material, a preliminary screening test was performed using two mice. The mice were treated by daily application of 25 ~l of the undiluted test material and the test material at a concentration of 50% v/v in dimethyl formamide, to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The mice were observed twice daily on Days 1 and 2 and the surviving mouse twice daily on Day 3 and once daily on Days 4, 5 and 6. Any signs of toxicity or excessive local irritation noted dur ng this period were recorded. The bodyweight of each mouse was recorded on Day 1 (prior to dosing) and of the surviving mouse on Day 6. Based on this information the dose levels selected for the main test were 10%, 25% or 50% v/v in dimethyl formamide.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT

- Name of test method:
Local Lymph Node Assay in the Mouse.
- Criteria used to consider a positive response:
The proliferation response of lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph node(dpm/node) and as the ratio of 3HTdR incorporation in lymph node cells of test nodes relative to that recorded for the control nodes (stimulation Index). The test material will be regarded as a sensitiser if at least one concentration of the test material results in a threefold or greater increase in 3HTdR incorporation compared to control values. Any test material failing to produce a threefold or greater increase in 3HTdR incorporation will be classified as a "non-sensitier".

TREATMENT PREPARATION AND ADMINISTRATION:
For the purpose of the study, the test material was used undiluted and also freshly prepared in dimethyl formamide. This vehicle was chosen as it pr duced the highest concentration that was suitable for dosing. The concentrations used are given above. Determination, by analysis, of the concentration, homogeneity and stability of the test material preparations was not appropriate because it was not specified in the Study Plan and is not a requirement of the Test Guidelines.
Groups of four mice were treated with the test material at concentrations of 10%, 25% or 50% 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 w re 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 day (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, GE Healthcare UK Ltd) giving a total of 20 µCi to each mouse.

Positive control substance(s):

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

Statistics:

None provided.

Results and discussion

Positive control results:

Three groups, each of five animals, were treated with 50 µl (25 µl per ear) of a-Hexylcinnamaldehyde, Tech, 85% as a solution in acetone/olive oil (4:1 v/v) at concentrations of 5%, 10% and 25% vivo A further control group of five animals was treated with acetone/olive oil (4: 1 v/v) alone.

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

Concentration % v/v in acetone/olive oil 4:1 Stimulation Index (SI) Result
5 2.50 Negative
10 4.03 Positive
25 9.13 Positive

α-Hexylcinnamaldehyde, Tech, 85% was considered to be a sensitiser under the
conditions of the test.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

9.59

Test group / Remarks:

10%

Results 2

Parameter:

SI

Value:

13.62

Test group / Remarks:

25%

Results 3

Parameter:

SI

Value:

10.79

Test group / Remarks:

50%

Any other information on results incl. tables

Table 1.   Disintegrations per Minute, Disintegrations per Minute/Node and Stimulation Index

Concentration (%v/v) in dimethylformamide	dpm	dpm/Node^a	Stimulation Index^b	Result
Vehicle	5340.94	667.62	N/A	N/A
10	51218.04	6402.26	9.59	Positive
25	72735.61	9091.95	13.62	Positive
50	43238.61	7206.44*	10.79	Positive

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

N/A =      Not applicable

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 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)

§        Method B42 Skin Sensitisation (Local Lymph Node Assay) of Commission Directive 2004/73/EC

Test Material:                                                  a-Hexylcinnamaldehyde, Tech, 85%

Safepharm Laboratories Project number:           0039/0821

Study dates:                                                      05 April 2006 to 11 April 2006

Methods. Three groups, each of five animals, were treated with 50 µl (25 µl per ear) of a-Hexylcinnamaldehyde, Tech, 85% as a solution in acetone/olive oil (4:1 v/v) at concentrations of 5%, 10% and 25% vivo A further control group of five animals was treated with acetone/olive oil (4: 1 v/v) 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 are as follows:

Concentration % v/v in acetone/olive oil 4:1		Stimulation Index (SI)		Result
5		2.50		Negative
10		4.03		Positive
25		9.13		Positive

Conclusion. a-Hexylcinnamaldehyde, Tech, 85% was considered to be a sensitiser under the conditions of the test.

Applicant's summary and conclusion

Interpretation of results:

sensitising

Remarks:

Migrated information

Conclusions:

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

Executive summary:

Introduction. A study was perfonned 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 2004/73/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 fonnamide at concentrations of 10%, 25% or 50% v/v. A further group of four animals was treated with dimethyl fonnamide alone.

Results. The Stimulation Index (SI) 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 dimethyl formamide	Stimulation Index	Result
10	9.59	Positive
25	13.62	Positive
50	10.79	Positive

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