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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010-06-29 -to 2010-11-05
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP, Guideline study available as unpublished report, no restrictions.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010

Materials and methods

Test guidelineopen allclose all
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)

Test material

Constituent 1
Chemical structure
Reference substance name:
Methyl 3-mercaptopropionate
EC Number:
220-912-4
EC Name:
Methyl 3-mercaptopropionate
Cas Number:
2935-90-2
Molecular formula:
C4H8O2S
IUPAC Name:
methyl 3-sulfanylpropanoate
Constituent 2
Reference substance name:
Methyl 3-mercaptoproprionate
IUPAC Name:
Methyl 3-mercaptoproprionate
Details on test material:
- Name of test material (as cited in study report): 3-Mercaptopropanoic acid methyl ester
- Physical state: clear, colourless liquid
- Analytical purity: no data
- Lot/batch No.: 2010-06-1996
- Storage condition of test material: room temperature 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: Harlan Laboratories U.K. Ltd., Oxon, UK
- Age at study initiation: 8-12 weeks old
- Weight at study initiation: 15 to 23 g.
- Housing: Individually housed in suspended solid-floor polypropylene cages furnished with softwood woodflakes.
- Diet: 2014 Teklad Global Rodent (Harlan Laboratories U.K., Ltd., Oxon, UK) ad libitum
- Water: Tap water ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19 to 25oC
- Humidity: 30 to 70%
- Air changes: 15/hr
- Photoperiod: 12 hr light / 12 hr dark

Study design: in vivo (non-LLNA)

Induction
Vehicle:
other:
Challenge
Vehicle:
other:

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
5, 10 and 25%
No. of animals per dose:
five (individual animals used)
Details on study design:
RANGE FINDING TESTS:
Three mice (one/dose) were given a topical application of 25 uL of undiluted test substance or concentrations of 10, 25 or 50% v/v in acetone/olive oil (4:1) to the dorsal surface of each ear for one day (Day 1). For the animals given 10 or 25% test substance, a daily topical application was also given on the following two consecutive days (Days 2 and 3). The mice were observed twice on Day 1. The surviving mice were observed twice daily on Days 2 and 3, and once daily on Days 4-6.

The animal treated with the undiluted test substance was humanely killed, post dose on Day 1, due to the occurrence of clinical signs of toxicity that exceeded the moderate severity limit set forth in the UK Home Office Project Licence. Signs of systemic toxicity noted in this animal were clonic convulsions and increased respiratory rate. The animal treated with the test substance at a concentration of 50% v/v in acetone/olive oil (4:1) was found dead post dose on Day 1. No signs of systemic toxicity were noted in the animals treated with dilutions of 10 or 25% test substance in acetone/olive oil (4:1).

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local lymph node assay using tritiated-methyl thymidine (3HTdR).
- Criteria used to consider a positive response: The proliferation response of lymph node cells was expressed as the number of radioactive disintegrations per minute (dpm) per lymph nodes from each individual animal 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 material was regarded as a sensitiser if at least one concentration of the test substance results in a three-fold or greater increase in 3HTdR incorporation compared to control values.

TREATMENT PREPARATION AND ADMINISTRATION:
Mice (five/group) were given daily topical applications of 25 uL of 5, 10 or 25% test substance v/v in acetone/olive oil (4:1) to the dorsal surface of each ear (50 uL per animal) for three consecutive days. An additional group of five mice were given topical applications of acetone/olive oil (4:1).

Five days following the first topical application of the test substance or vehicle (Day 6), all mice were injected via the tail vein with 250 uL of phosphate buffered saline (PBS) containing 3HTdR (80 uCi/ml, specific activity 2.0 Ci/mmol), giving a total of 20 uCi to each mouse. Five hours following the administration of 3HTdR, the mice were killed by carbon dioxide asphyxiation followed by cervical separation, and the draining auricular lymph nodes were excised and processed from each individual animal to which one mL of PBS was added. A single cell suspension of the lymph node cells from each individual animal 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, and the lymph node cell suspension was transferred to a centrifuge tube. The petri dish was washed with an additional 5 mL of PBS, which was then added to the centrifuge tube. The lymph node cells were pelleted at 1,400 rpm for ten minutes. The pellet was resuspended in 10 mL of PBS and re-pelleted. The pellet was then resuspended in 3 mL of 5% trichloroacetic acid (TCA). After approximately 18 hours incubation at approximately 4oC, the precipitates were recovered by centrifugation at 2,100 rpm for ten minutes, resuspended in 1 mL of TCA and transferred to 10 mL of scintillation fluid. 3HTdR incorporation was measured as disintegrations per minute (dpm) by beta-scintillation counting.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Data was processed to give group mean values for disintegrations per minute and standard deviations where appropriate. Individual and group mean disintegrations per minute values were assessed for dose response relationships by analysis of homogeneity of variance followed by one way analysis of variance (ANOVA). In the event of a significant result from the ANOVA, pairwise comparisons were performed between control and treated groups. For homogenous datasets, Dunnett's Multiple Comparison test was used and for non-homogenous datasets, Dunnett's T3 Multiple Comparison Method was used. Probability values (p) of less than 0.05 were considered significant.

Results and discussion

Positive control results:
Hexyl cinnamic aldehyde was considered to be a sensitiser under the conditions of the test (SI = 7.25).

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: The Stimulation Indices (SI) were 2.87, 2.14 and 2.44 for the 5, 10 and 25% concentration groups, respectively.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see Remark
Remarks:
The mean +/- standard deviations for the vehicle control, 5%, 10% and 25% concentration groups were 1549.65 +/- 557.57, 4447.43 +/- 1172.53, 3313.95 +/- 845.48 and 3780.08 +/- 1669. 38, respectively. The values for all three treatment groups were significantly different from controls (p<0.05).

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Methyl 3-mercaptoproprionate was considered to be a non-sensitiser in the mouse local lymph node assay.
Executive summary:

Methyl 3-mercaptoproprionate was topically applied to the dorsal surface of the ear of CBA/Ca female mice in a local lymph node assay (LLNA). A preliminary screening test was conducted in which no clinical signs of toxicity were noted at a concentration of 25% v/v in acetone/olive oil (4:1). Thus, mice (five/group) were treated with 50 uL (25 uL per ear) with concentrations of 5, 10 or 25% methyl 3 -mercaptoproprionate v/v in acetone/olive oil (4:1). An additional group of mice was treated with acetone/olive oil (4:1). The Stimulation Index (SI) expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control was less than three for all of the treatment groups. Thus, methyl 3 -mercaptoproprionate was considered to be a non-sensitiser under the conditions of the test.

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