4-Nitrophenyl 2-(methylsulphonyl)ethanoate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2014-06-03 to 2014-06-17

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

22 July 2010

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:

4-nitrophenyl 2-methanesulfonylacetate

EC Number:

808-288-7

Cas Number:

1158363-70-2

Molecular formula:

C9H9NO6S

IUPAC Name:

4-nitrophenyl 2-methanesulfonylacetate

Specific details on test material used for the study:

Batch No.: PNP-0020313
Purity: 99.5%

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: supplied by Harlan Laboratories UK Ltd.
- Females nulliparous and non-pregnant: yes
- Age at study initiation: eight to twelve weeks
- Weight at study initiation: 15 to 23 g
- Housing: individually housed in suspended solid floor polypropylene cages furnished with softwood woodflakes.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25 °C
- Humidity (%): 30 to 70%
- Air changes (per hr): approximately fifteen changes per hour
- Photoperiod (hrs dark / hrs light): twelve hours continuous light (06.00 to 18.00) and twelve hours darkness.

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Concentration:

25%, 10% or 5% w/w in dimethyl formamide

No. of animals per dose:

5

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: Dimethyl formamide was chosen as it produced the highest concentration that was suitable for dosing.
- Result:: One mouse was treated by daily application of 25 μL of test item at a concentration of 25% w/w in dimethyl formamide to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). No signs of systemic toxicity, visual local skin irritation or irritation indicated by an equal to or greater than 25% increase in mean ear thickness were noted.
Based on this information the dose levels selected for the main test were 25%, 10% and 5% w/w in dimethyl formamide.

MAIN STUDY
- Test Item Formulation:
The test item was formulated within two hours of being applied to the test system.
- Test Item Administration:
Groups of five mice were treated with thee test item at concentrations of 25%, 10% or 5% w/w in dimethyl formamide. 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 five 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) 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.
Body Weights: The body weight of each mouse was recorded on Day 1 (prior to dosing) and Day 6 (prior to termination).
- Terminal Procedures: Five hours following the administration of 3HTdR all mice were killed by carbon dioxide asphyxiation followed by cervical separation.
- Criteria used to consider a positive response:
The test item will be regarded as a sensitizer 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-sensitizer."

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.

Results and discussion

Positive control results:

α-Hexylcinnamaldehyde, tech., 85% was considered to be a sensitizer (SI=6.13 at a concentration of 15% v/v in dimethyl formamide) under the conditions of the test.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

5.88

Test group / Remarks:

5% w/w in dimethyl formamide

Results 2

Parameter:

SI

Value:

7.31

Test group / Remarks:

10% w/w in dimethyl formamide

Results 3

Parameter:

SI

Value:

7.24

Test group / Remarks:

25% w/w in dimethyl formamide

Cellular proliferation data / Observations:

- 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.
- Body Weight:
Body weight changes of the test animals between Day 1 and Day 6 were comparable to that observed in the corresponding control group animals over the same period.

Applicant's summary and conclusion

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

The test item was considered to be a sensitizer under the conditions of the test.

Executive summary:

A study was performed to assess the skin sensitization potential of the test item in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear according to OECD 429.

Following a preliminary screening test in which no clinical signs of toxicity were noted at a concentration of 25% 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 five animals, were treated with 50 μL (25 μL per ear) of the test item as a solution in dimethyl formamide at concentrations of 25%, 10% or 5% w/w. A further group of five animals was treated with dimethyl formamide alone.

The Stimulation Index (SI) was determined to be 5.88, 7.31, 7.24 for concentration of 5 %, 10% or 25% w/w, respectively.

The test item was considered to be a sensitizer under the conditions of the test.