Ethyl 2-methyl-1,3-dioxolane-2-acetate

Endpoint summary

Description of key information

Short description of key information:

Not sensitising, mouse (LLNA), OECD 429, EU Method B.42, EPA OPPTS 870.2600, Hanzell (2013)

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 May 2013 to 4 June 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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

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)

Species:

mouse

Strain:

other: CBA/Ca

Sex:

not specified

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories UK Ltd., Oxon, UK
- Age at study initiation: 8 - 12 weeks old
- Weight at study initiation: 15 - 23 g
- Housing: individually in suspended solid floor polypropylene cages furnished with softwood woodflakes
- Diet: 2014C Teklad Global Rodent diet, ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days

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

Vehicle:

other: ethanol/diethyl phthalate 1:3

Concentration:

0 (vehicle control), 25, 50 and 100% v/v in vehicle

The test material was formulated within 2 hours of being applied to the test system. It was assumed that the formulation was stable for this duration.

No. of animals per dose:

5 animals per group (main test)

Details on study design:

RANGE FINDING TESTS
A single mouse was treated by daily application of 25 μL of the undiluted test item, 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 body weight was recorded on Day 1 (prior to dosing) and on Day 6.
The thickness of each ear was measured using a Mitutoyo 547-300S gauge (Mitutoyo Corporation), pre-dose on Day 1, post dose on Day 3 and on Day 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.

MAIN STUDY
- Test material administration:
The mice were 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 days (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 five mice received the vehicle alone in the same manner.
- ³H-methyl thymidine administration:
Five days following the first topical application of the test material 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, ARC UK Ltd) giving a total of 20 μCi to each mouse.
- Observations and bodyweights:
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 body weight of each mouse was recorded on Day 1 (prior to dosing) and Day 6 (prior to termination).
- Terminal procedures:
Termination: Five hours following the administration of ³HTdR all mice were killed by carbon dioxide asphyxiation followed by cervical separation. 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.
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. 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 ten minutes. The pellet was resuspended in 10 mL of PBS and re-pelleted. To precipitate out the radioactive material, the pellet was resuspended in 3 mL of 5% Trichloroacetic acid (TCA).
Determination of ³HTdR Incorporation: After approximately eighteen hours incubation at approximately 4 °C, the precipitates were recovered by centrifugation at 2100 rpm (approximately 450 g) for ten minutes, resuspended in 1 mL of TCA and transferred to 10 mL of scintillation fluid (Optiphase 'Trisafe'). ³HTdR incorporation was measured by β-scintillation counting. The "Poly Q™" vials containing the samples and scintillation fluid were placed in the sample changer of the scintillator and left for approximately twenty 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 twenty minutes, the vials were shaken vigorously. The number of radioactive disintegrations per minute was then measured using the Beckman LS6500 scintillation system (Beckman Instruments Inc, Fullerton, CA, USA).

Positive control substance(s):

other: α-hexylcinnamaldehyde, tech., 85%

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.

Positive control results:

At positive control concentrations of 5, 10 and 25% v/v in ethanol/diethyl phthalate 1:3, the stimulation index (expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group) was calculated to be 1.85, 3.37 and 8.22, respectively. Under the conditions of the study, α-hexylcinnamaldehyde, tech., 85% was considered to be a sensitiser.

Key result

Parameter:

SI

Value:

1.6

Test group / Remarks:

25%

Key result

Parameter:

SI

Value:

0.83

Test group / Remarks:

50%

Key result

Parameter:

SI

Value:

0.85

Test group / Remarks:

100%

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: See Table 1.

Preliminary Screening Test Results

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 undiluted test material and the test material at concentrations of 50% or 25% v/v in ethanol/diethyl phthalate 1:3 were selected for the main test.

Main Test - Observations and Bodyweight Data

There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test.

Body weight change of the test animals between Day 1 and Day 6 were comparable to those observed in the corresponding control group animals over the same period.

Table 1: Individual Disintegrations per Minute and Stimulation Indices (Main Test)

Concentration (% v/v) in vehicle	dpm/ animal^*	Mean dpm/animal (± SD)	Stimulation Index^#	Result
Vehicle	486.63	722.25 (± 234.79)	na	na
	1028.37
	540.38
	651.44
	904.45
25	837.77	1158.62 (± 645.38)	1.60	Negative
	525.69
	2119.26
	1501.33
	809.03
50	485.49	598.61 (± 131.71)	0.83	Negative
	788.43
	493.66
	680.36
	545.13
100	389.59	615.58 (± 304.60)	0.85	Negative
	365.97
	428.28
	908.54
	985.50

^* Total number of lymph nodes per animal is 2

^# Stimulation Index of 3.0 or greater indicates a positive result

na Not Applicable

Interpretation of results:

GHS criteria not met

Conclusions:

Under the conditions of the study the test material was considered to be a non-sensitiser.

Executive summary:

The skin sensitisation potential of the test material was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guidelines OECD 429, EU Method B.42 and EPA OPPTS 870.2600.

Following a preliminary screening test in which no clinical signs of toxicity were noted at a concentration of 100% v/v, 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 undiluted test material or the test material as a solution in ethanol/diethyl phthalate 1:3 at concentrations of 50% or 25% v/v. A further group of five animals was treated with ethanol/diethyl phthalate 1:3 alone.

At test material concentrations of 25, 50 and 100% v/v in ethanol/diethyl phthalate 1:3, the stimulation index (expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group) was calculated to be 1.60, 0.83 and 0.85, respectively.

Under the conditions of the study the test material was considered to be a non-sensitiser.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

In the key study the skin sensitisation potential of the test material was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guidelines OECD 429, EU Method B.42 and EPA OPPTS 870.2600.

Following a preliminary screening test in which no clinical signs of toxicity were noted at a concentration of 100% v/v, 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 undiluted test material or the test material as a solution in ethanol/diethyl phthalate 1:3 at concentrations of 50% or 25% v/v. A further group of five animals was treated with ethanol/diethyl phthalate 1:3 alone.

At test material concentrations of 25, 50 and 100% v/v in vehicle, the stimulation index (expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group) was calculated to be 1.60, 0.83 and 0.85, respectively.

Under the conditions of the study the test material was considered to be a non-sensitiser.

Since the key study was conducted under GLP conditions and in accordance with standardised guidelines it was assigned a reliability score of 1 in line with the criteria of Klimisch.

Short description of key information:

Not sensitising, mouse (LLNA), OECD 429, EU Method B.42, EPA OPPTS 870.2600, Hanzell (2013)

Justification for selection of skin sensitisation endpoint:

The study was conducted under GLP conditions and in accordance with standardised guidelines.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation 1272/2008, the test material does not require classification as a skin sensitiser.