Methyl trans-3-oxo-2-pentylcyclopentanecarboxylate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 November - 6 December 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted by GLP accredited laboratory. Method according to OECD guideline.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

Temporary deviations from the minimal level of temperature and relative humidity. Laboratory historical data do not indicate an effect of the deviations and the study integrity was not adversely affected.

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

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:

methyl trans-3-oxo-2-pentylcyclopentanecarboxylate

EC Number:

700-527-2

Cas Number:

1271488-66-4

Molecular formula:

C12H20O3

IUPAC Name:

methyl trans-3-oxo-2-pentylcyclopentanecarboxylate

Details on test material:

- Name of test material (as cited in study report): Yasmolys
- Substance type: pure active substance
- Physical state: liquid

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

The animals were CBA/J strain mice (inbred, SPF quality) from Janvier, Le Genest-Saint-Isle, France. Animals ±9 weeks old were selected having body weights +/-20% of the sex mean. They were identified at the tail with a marker pen. Special attention during health inspection was paid to the ears, whcih were intact and free from any abnormality.
A controlled environment was maintained in the room with optimal conditions of approximately 15/h air changes, a temperature of 17.9-22.2ºC, a relative humidity of 21-61% and a 12 hour artificial fluorescent light/12 hour dark cycle per day.
3 animals were present per cage in labeled Macrolon cages containing sterilised sawdust as bedding material and paper as cage-enrichment. Animals were acclimitised for a period of 5 days prior to exposure to the test substance. The animals had free access to tap water and pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest , Germany.

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

0, 25, 50, 100% test substance in vehicle

No. of animals per dose:

5 animals per dose group (4 dose groups)

Details on study design:

Pre-screen test
50% and 100% test substance in the vehicle were daily applied on a mouse (2 mice per concentration) for 3 consecutive days. The highest concentration should cause no systemic toxicity and have a maximum irritation score of 2.

Main study
Three groups of five animals were daily topically treated at the dorsal of both ears with one dose level per group for three consecutive days (Day 1, 2 and 3). The negative control animals were treated in the same way with the vehicle only.

On Day 6, the animals were injected at their tail veins with 0.25 ml sterile phosphate buffer (PBS) containing 20 µCi 3H-methyl thymidine. After 5 hours, the animals were sacrificed and the draining auricular lymph node was excised. The relative sizes of the nodes were visually examined and abnormalities recorded. The nodes were pooled for each animal.

A single cell suspension of lymph node cells (LNC) was prepared by mechanical disaggregation through a 125 µm in diameter stainless steel gauze. Cells are washed with PBS and centrifuged at 200g for 10 minutes at 4°C. The DNA was precipitated by exposing the LNC to 5% trichloro acetic acid (TCA) until the next day.

Precipitates were recovered by centrifugation and resuspended in 1ml TCA.
Radio activity was measured by a scintillation counter as expressed in disintegration per minute (dpm).

The stimulation index (SI) is the ratio of dpm of each dose group to the dpm of the vehicle control group. It was calculated for each dose group.

Positive control substance(s):

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

Results and discussion

Positive control results:

Hexyl cinnamaldehyde (CAS 101-86-0) was used as a positive control. The positive control test was performed in September 2010 on female mice of the CBA/J strain. The hexyl cinnamaldehyde concentrations were 0, 5, 10 and 25%. The disintergrations per minute increase over that range from 678 to 2502. the corresponding stimulation indici increased from 1 to 3.7. On this basis, an EC3 of 18% was calculated and fitted into the historical range of 2-20%. The results of the last six-months reliability checks were 13.8, 13.9, 16.0, 11.9, 16.9 and 10.7%. It was concluded that the results were reproducible and accurate.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: see Remark

Remarks:

The stimulation index for each test substance concentration, was the ratio of the mean radio activity for that concentration divided by the mean radio activity level of the vehicle control. The stimulation index was coorelated with the concentration of the test substance and increased from 0.9 to 1.9 when the concentration was increased from 25 to 100%. In case of application of the undiluted test substance to the dorsal ear surface of the mouse, the stimulation index remained below 3.

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: see Remark

Remarks:

The nodes were pooled for each animal and the mean radio-activity value measured (Table 3). The values were expressed in disintegrations per minute and the mean radio activity levels were averaged for each test substance concentration. From a test substance concentration of 25% to 100% (undiluted), the radio activity increased from 464 to 975 dpm. The vehicle control was 523 dpm.

Any other information on results incl. tables

Main test

The body weights in Table 1 are in the same range as the historical controls over the study period. The slight body weight loss noted in some animals was considered not toxicological significant.

Table 1 Body weight and relative size auricular lymph nodes of mice subjected to the test substance dissolved in acetone/olive oil in the main test

Concentration test substance		Day 1		size node
	Animal	bw (g)	bw (g)	left	right
0%	1	24	22	n	n
	2	21	21	n	n
	3	21	21	n	n
	4	23	22	n	n
	5	22	23	n	n
25%	1	23	23	n	n
	2	22	22	n	n
	3	22	22	n	n
	4	22	21	n	n
	5	22	22	n	n
50%	1	21	21	n	n
	2	21	21	n	n
	3	23	23	n	n
	4	21	21	n	n
	5	21	21	n	n
100%	1	22	22	n	n
	2	21	21	n	n
	3	21	22	n	n
	4	21	21	n	n
	5	22	22	n	n
bw: body weight;  n: normal

The skin reactions are tabulated in Table 2. Slight erythema of the ears was observed at a concentration level of 50% and 100% between days 2 and 3. It was considered not to have any toxicological significance on the activity of the nodes. No oedema was observed in any of the animals. All auricular lymph nodes of the animals were considered normal in size (Table 2). No macroscopic abnormalities of the surrounding area were noted in any of the animals.

Table2 Erythema (E) and Oedema (O) on the dorsal surface of the left and right ear of the mice exposed to the test substance in the main test

Concentration test substance		Day 1				Day 2				Day 3				Day 4				Day 5				Day 6
	Animal	left		right		left		right		left		right		left		right		left		right		left		right
		E	O	E	O	E	O	E	O	E	O	E	O	E	O	E	O	E	O	E	O	E	O	E	O
0%	1	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	2	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	3	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	4	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	5	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
25%	1	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	2	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	3	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	4	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	5	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
50%	1	0	0	0	0	1	0	1	0	1	0	1	0	0	0	0	0	0	0	0	0	0	0	0	0
	2	0	0	0	0	1	0	1	0	1	0	1	0	0	0	0	0	0	0	0	0	0	0	0	0
	3	0	0	0	0	1	0	1	0	1	0	1	0	0	0	0	0	0	0	0	0	0	0	0	0
	4	0	0	0	0	1	0	1	0	1	0	1	0	0	0	0	0	0	0	0	0	0	0	0	0
	5	0	0	0	0	1	0	1	0	1	0	1	0	0	0	0	0	0	0	0	0	0	0	0	0
100%	1	0	0	0	0	1	0	1	0	1	0	1	0	0	0	0	0	0	0	0	0	0	0	0	0
	2	0	0	0	0	1	0	1	0	1	0	1	0	0	0	0	0	0	0	0	0	0	0	0	0
	3	0	0	0	0	1	0	1	0	1	0	1	0	0	0	0	0	0	0	0	0	0	0	0	0
	4	0	0	0	0	1	0	1	0	1	0	1	0	0	0	0	0	0	0	0	0	0	0	0	0
	5	0	0	0	0	1	0	1	0	1	0	1	0	0	0	0	0	0	0	0	0	0	0	0	0

Erythema score: 0 (no erythema) - 4 (severe erythema to eschar formation)

Oedema score: 0 (no oedema) - 3 (severe oedema)

Table 3 Radio-activity measured in the individual animals

Concentration test substance		DPM per animal
	Animal
0%	1	518
	2	649
	3	350
	4	619
	5	480
25%	1	435
	2	496
	3	175
	4	604
	5	608
50%	1	340
	2	407
	3	533
	4	1042
	5	792
100%	1	700
	2	792
	3	958
	4	1328
	5	1098
DPM: disintegrations per minute

Table 4 Mean disintegrations per minute (DPM) and stimulation index (SI), including the standard error of the mean (SEM) calculated from the radio activity measurement in the animals subjected to the test concentration.

Concentration test substance	mean		mean
	DPM	SEM	DPM	SEM
0%	523	53	1	0.2
25%	464	79	0.9	0.3
50%	623	130	1.2	0.3
100%	975	112	1.9	0.1

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information

Conclusions:

The six-month reliability check with the positive control proved that this LLNA was appropiate for testing contact hypersensitivity.
Since there was no indication that the test substance applied undiluted at the dorsal surface of the mouse ear elicits a stimulation index SI≥3, the test substance is regarded as a non skin sensitiser.

Executive summary:

The contact hyper sensitivity of the test substance was assessed by the Local Lymph Node Assay conducted according to the OECD and EU test guidelines. Four experimental groups of five female CBA/J mice were treated with the test substance at concentrations of 0 (vehicle control), 25, 50 and 100%. The animals were visually scored for skin irritation reactions. Three days after the last exposure, all animals were injected with tritiated methyl thymidine. The auricular lymph nodes were isolated and their radio activity levels determined. The stimulation index was subsequently calculated. The positive control hexyl cinnamic aldehyde was included as a six-months reliability check, but not as a concurrent positive control group.

Slight erythema of the ears was observed at the animals treated with 50 and 100% of the test substance between days 2 and 3. No oedema was observed.

The stimulation index showed a clear dose-response relationship, but it remained below three for the undiluted test substance. The index was 1.9 and the test substance is therefore not regarded as a skin sensitiser.