2-Methoxy-4-methylphenyl methyl carbonate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06-02-2012 to 28-02-2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study performed under GLP. All relevant validity criteria were met.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected: July 2011; signature: August 2011

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

2-methoxy-4-methylphenyl methyl carbonate

EC Number:

700-673-7

Cas Number:

132638-45-0

Molecular formula:

C10H12O4

IUPAC Name:

2-methoxy-4-methylphenyl methyl carbonate

Test material form:

solid

Details on test material:

- Physical state: Extremely pale yellow crystalline solid
- Storage condition of test material: Approximately 4°C in the dark, under nitrogen

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Remarks:

CBA/CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Recognised Supplier
- Age at study initiation: 8 to 12 weeks
- Weight at study initiation: 15 to 23g
- Housing: individually housed in suspended solid-floor polypropylene cages furnished with softwood woodflakes.
- Diet (ad libitum): 2014C Teklad Global Rodent diet (Recognised Supplier); ad libitum
- Water (ad libitum): mains tap water; ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25
- Humidity (%): 30 to 70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 hours light / 12 hours dark

IN-LIFE DATES: Fro: 06-02-2012 To: 28-02-2012

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

- Preliminary test: 50% w/w
- Main test: 50%, 25% and 10% w/w

No. of animals per dose:

Preliminary test: 1
Main test: 5 per dose group

Details on study design:

RANGE FINDING TESTS:
Using available information regarding the systemic toxicity/irritancy potential of the test substance, a preliminary screening test was performed using one mouse per test item concentration. The mouse was treated by daily application of 25 µL of the undiluted test item or the test item at a concentration of 50% w/w in acetone/olive oil 4:1, to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The mice were observed twice daily on Days 1, 2 and 3 and once daily on Days 4, 5 and 6. Local skin irritation was scored daily according to the scale included in the full study report. Any clinical signs of toxicity, if present, were also recorded. The body weight of each mouse was recorded on Day 1 (prior to dosing) and on Day 6. The thickness of each ear was measured using a gauge, pre dose and post dose on Day 1, post dose on Days 2 and 3 and on Days 6. Any changes in the ear thickness were noted. Mean ear thickness changes were calculated between time periods Days 1 and 3 and Days 1 and 6. A mean ear thickness increase of equal to or greater than 25% was considered to indicate excessive irritation and limited biological relevance to the endpoint of sensitization.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response: The test item will be regarded as a sensitiser 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-sensitiser".

TREATMENT PREPARATION AND ADMINISTRATION:
Groups of five mice were treated with the test item at concentrations of 50%, 25% or 10% w/w in acetone/olive oil 4:1. A further group of five mice received the vehicle alone in the same manner. The preliminary screening test suggested that the test item 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 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.

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:80μCi/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.
- Bodyweights: The bodyweight of each mouse was recorded on Day 1 (prior to dosing) and Day 6 (prior to termination).
- Ear thickness measurements: The thickness of each ear was measured using a gauge, pre-dose on Day 1, post dose on Days 2 and 3 and on Days 4, 5 and 6. Any changes in the ear thickness were noted. Daily mean ear thickness changes were calculated. A mean ear thickness increase of equal to or greater than 25% was considered to indicate excessive irritation and limited biological relevance to the endpoint of sensitisation. Due to technician error, the Day 1 post-dose ear thickness measurements were not performed. This deviation from the study plan was considered not to affect the purpose or integrity of the study.

Preparation of Single Cell Suspension:
A single cell suspension of the lymph node cells for 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 labelled with the study number and dose concentration. The lymph node cells 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 lymph node cells were pelleted at 1400 rpm (approximately 190 g) for 10 minutes. The pellet was re suspended in 10 mL of PBS and re pelleted. To precipitate out the radioactive material, the pellet was re suspended in 3 mL of 5% Trichloroacetic acid (TCA).

Determination of 3HTdR Incorporation:
After approximately 18 hours incubation at approximately 4 degrees Celsius, the precipitates were recovered by centrifugation at 2100 rpm (approximately 450 g) for 10 minutes, resuspended in 1 mL of TCA and transferred to 10 mL of scintillation fluid. 3HTdR incorporation was measured by beta-scintillation counting. The vials containing the samples and scintillation fluid were placed in the sample changer of the scintillator and left to stand in darkness for approximately 20 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 20 minutes, the vials were shaken vigorously. The number of radioactive disintegrations per minute was then measured using a recognised scintillation system.

Positive control substance(s):

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

other: phenylacetaldehyde (Historical Control Data)

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.

A dpm value considered to be an outlier was identified for animal 1-2 (control group), possibly due to unsuccessful 3HTdR injection, and therefore this animal was removed from the calculation of the mean dpm/animal. This was considered not to affect the purpose or integrity of the study and the results were considered to be satisfactory/valid.

Results and discussion

Positive control results:

In a non-concurrent 'positive control study' performed according to OECD TG 429, the sensitivity of the strain of mouse used in this study was assessed using the known sensitiser, α-hexylcinnamaldehyde (85%) at 25% v/v in acetone/olive oil 4:1. The highest concentration tested showed a Stimulation Index (SI) of 4.05 and met the criteria for a 'positive' result.
The results of routine positive control testing (Historic Control Data) performed according to OECD TG 429, using α-hexylcinnamaldehyde (85%) and Phenylacetaldehyde (90%) indicated positive results using a variety of different vehicles. Information is documented in the full study report.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

<= 2.73

Test group / Remarks:

50% w/w in acetone/olive oil 4:1

Remarks on result:

other: The Stimulation Index for all 3 treated groups was less than three-fold higher than the Control group. (see table)

Results 2

Parameter:

other: disintegrations per minute (DPM)

Value:

<= 6 751.7

Variability:

±1453.57

Test group / Remarks:

50% w/w in acetone/olive oil 4:1

Remarks on result:

other: See table.

Results 3

Parameter:

SI

Value:

2.27

Test group / Remarks:

25% w/w in acetone/olive oil 4:1

Results 4

Parameter:

SI

Value:

2.37

Test group / Remarks:

10% w/w in acetone/olive oil 4:1

Any other information on results incl. tables

There were no clinical observations or evidence of skin irritation (visually or by ear thickness) and no effects of treatment on body weight.

Table 1: Stimulation Index (mean radioactive incorporation for each group divided by mean radioactive incorporation of vehicle control group).

Concentration (% w/w) in acetone/olive oil 4:1	Stimulation Index	Result
10	2.37	Negative
25	2.27	Negative
50	2.73	Negative

Table 2: Individual Disintegrations per Minute and Stimulation Indices

Concentration (% w/w) in acetone/olive oil 4:1	Animal Number	Dpm/Animal^1	Mean dpm/Animal (SD)	Stimulation Index^2	Result
Vehicle	1-1	2414.61	2405.58^4 (±123.36)	n/a	n/a
	1-2	898.23^3
	1-3	2571.57
	1-4	2281.32
	1-5	2354.82
10	2-1	5336.04	5704.63 (±2085.69)	2.37	negative
	2-2	8676.85
	2-3	3027.03
	2-4	4948.47
	2-5	6534.74
25	3-1	2472.24	5462.53 (±2998.21)	2.27	negative
	3-2	10495.82
	3-3	4850.04
	3-4	4371.17
	3-5	5123.40
50	4-1	7770.98	6571.70* (±1453.57)	2.73	negative
	4-2	4131.01
	4-3	6399.30
	4-4	7162.29
	4-5	7394.93

¹ n=2 lymph nodes per animal
² Stimulation index of 3.0 or greater indicates positive result
³ dpm/animal not included in total, considered an outlier
⁴ value based on n=4 animals due to excluded value
n/a not applicable
* p<0.05

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

Under the condition of this study, the test material is considered to be non-sensitising to skin.

Executive summary:

The study was performed to OECD TG 429, EU Method B.42 and US EPA OPPTS 870.2600, Local Lymph Node Assay under GLP to assess the skin sensitisation potential of the test material in the CBA/CaOlaHsd mouse following topical application to the dorsal surface of the ear. In a preliminary screening test single mice were treated by daily application of 25 μl of the solid test item diluted at 50% in Acetone/Olive Oil (4:1) 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. Local skin irritation was scored daily. Any clinical signs of toxicity, if present, were also recorded. The bodyweight was recorded on Day 1 (prior to dosing) and on Day 6. 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 the preliminary test, the concentrations selected for the main test were 0% (control), 10%, 25% and 50% w/w within Acetone/Olive Oil (4:1). The preliminary screening test suggested that the test item would not produce systemic toxicity or excessive local irritation at the highest suitable concentration (50% w/w).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. Any changes in the ear thickness were noted. Mean ear thickness changes were calculated daily between Days 1 to 3 and Days 1 to 6. A mean ear thickness increase of equal to or greater than 25% was considered to indicate excessive irritation and limited biological relevance to the endpoint of sensitisation. 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. The bodyweight of each mouse was recorded on Day 1 (prior to dosing) and Day 6 (prior to termination). At test termination, five hours after administration of 3HTdR, the test organisms were humanly euthanized. For each individual animal of each group the draining auricular lymph nodes were excised and processed. For each individual animal 1 mL of PBS was added to the lymph nodes. Following appropriate preparation, 3HTdR incorporation was measured by β-scintillation counting and the number of radioactive disintegrations per minute (dpm) was measured. The proliferation response of lymph node cells was expressed as the number of radioactive disintegrations per minute per animal and as the ratio of 3HTdR incorporation into lymph node cells of test nodes relative to that recorded for the control nodes. The substance was regarded as sensitising if at least one concentration of the substance resulted in a 3-fold or greater increase in3HTdR incorporation compared to control values. Any substance failing to produce a 3-fold or greater increase in 3HTdR incorporation was classified as non-sensitising. In the main test, there were no deaths or signs of systemic toxicity, and body weights were comparable to controls. A stimulation index (SI) was recorded for each concentration as follows: 10%w/w: SI = 2.37, 25%w/w: SI = 2.27 and 50%w/w: SI = 2.73. No concentrations resulted in an SI > 3.0. Accordingly, the test material was considered to be non-sensitising under the conditions of the test.