estr-4-ene-3,11,17-trione cyclic 3-(1,2-ethanediylmercaptole)

Endpoint summary

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed at a registered GLP site and reported a high standard andis in compliance with recognised OECD standards with no deviations.

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Principles of method if other than guideline:

No analysis was carried out to determine the homogeneity, concentration or stability of the test item formulation.. The test item was formulated within two hours of it being applied to the test system; it is assumed that the formulation was stable for this duration. This exception is considered not to affect the purpose or integrity of the study.

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Species: Mouse
- Strain: CBA/CaOlaHsd
- Sex: female
- Source: Harlan Laboratories UK Ltd., Oxon, UK.
- Age at study initiation: 8-12 weeks
- Weight at study initiation: 15-23g
- Housing: individually housed in suspended solid floor polypropylene cages furnished with softwood woodflakes
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least five days

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

IN-LIFE DATES: From: day 1 To: day 6

Vehicle:

propylene glycol

Concentration:

10%, 5% or 2.5% w/w

No. of animals per dose:

5

Details on study design:

RANGE FINDING TESTS:
- Compound solubility:
For the purpose of the study, the test item was freshly prepared as a suspension in propylene glycol. This vehicle was chosen as it produced the highest concentration that was suitable for dosing. (10%, 2 g of 25% dilution made up to 5 g)
The test item was formulated within two hours of being applied to the test system. It is assumed that the formulation was stable for this duration.
No analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation.

- Irritation:
One mouse was treated by daily application of 25 µL of the test item at a concentration of 10% w/w in propylene glycol, 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. 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. 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.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay in the Mouse
- Criteria used to consider a positive response: 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 (Stimulation Index).
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."

TREATMENT PREPARATION AND ADMINISTRATION:
Test Item Administration
Groups of five mice were treated with the test item at concentrations of 10%, 5% or 2.5% w/w in propylene glycol. 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 ofthe 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, ARC UK Ltd) 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
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 labeled with the project 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 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 ofTCA and transferred to 10 mL of scintillation fluid (Opt~hase 'Trisafe'). ³HTdR incorporation was measured by B-scintillation counting. The "Poly Q™" vials containing the samples and scintillation fluid were placed in the sample changer ofthe 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: Phenylacetaldehyde (CAS No 122-78-1)

Statistics:

Statistical Analysis
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:

The Stimulation Index expressed as the mean radioactive incorporation for the treatment group divided by the mean radioactive incorporation of the vehicle control group is as follows:
Concentration (% v/v) in propylene glycol : 2.5
Stimulation Index : 6.32
Result : Positive

Parameter:

SI

Remarks on result:

other: see table below

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: see table below

Estimation of the Proliferative Response of Lymph Node Cells

The radioactive disintegrations per minute per lymph node and the stimulation index are given in the table below.

The Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group are as follows:

Individual Disintegrations per Minute and Stimulation Indices

Concentration (% w/w) in propylene glycol	Animal Number	dpm/ Animal^a	Mean dpm/Animal (Standard Deviation)	Stimulation Index^b	Result
Vehicle	1-1	5766.43	3290.73 (±1410.87)	n/a	n/a
	1-2	2477.42
	1-3	2473.95
	1-4	3139.07
	1-5	2596.78
2.5	2-1	2568.76	1985.23(±813.53)	0.60	Negative
	2-2	1923.10
	2-3	883.67
	2-4	1596.63
	2-5	2954.00
5	3-1	3029.33	2725.12(±848.88)	0.83	Negative
	3-2	4033.32
	3-3	1841.62
	3-4	2233.70
	3-5	2487.65
10	4-1	6081.50	3844.89(±1732.95)	1.17	Negative
	4-2	5309.17
	4-3	2635.11
	4-4	2212.37
	4-5	2986.28

where:

dpm = Disintegrations per minute

a = Total number of lymph nodes per animal is 2

b = Stimulation Index of 3.0 or greater indicates a positive result

n/a = Not applicable.

Clinical Observations, Body Weight 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 change of the test animals between Day 1 and Day 6 was comparable to those observed in the corresponding control group animals over the same period.

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

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

Executive summary:

Introduction

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.

Methods

Following a preliminary screening test in which no clinical signs of toxicity were noted at a concentration of 10% 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 suspension in propylene glycol at concentrations of 10%, 5% or 2.5% w/w. A further group of five animals was treated with propylene glycol alone.

Results

The Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group are as follows:

Concentration (% w/w) in propylene glycol	Stimulation Index	Result
2.5	0.60	Negative
5	0.83	Negative
10	1.17	Negative

Conclusion

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

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

Introduction

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.

Methods

Following a preliminary screening test in which no clinical signs of toxicity were noted at a concentration of 10% 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 suspension in propylene glycol at concentrations of 10%, 5% or 2.5% w/w. A further group of five animals was treated with propylene glycol alone.

Results

The Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group are as follows:

Concentration (% w/w) in propylene glycol	Stimulation Index	Result
2.5	0.60	Negative
5	0.83	Negative
10	1.17	Negative

Conclusion

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

Migrated from Short description of key information:
At the highest tested concentration of 10% v/v, the SI was observed to be 1.17, therefore the test item was considered to be a non-sensitizer under the conditions of the test.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

In the LLNA, the endpoint of interest is the proliferative response in the draining lymph nodes of mice exposed topically to the test material. Substances that elicit a 3-fold or greater proliferative activity compared to the controls (induce a stimulation index (SI) of 3 or more) are considered to be skin sensitizers.

In this case, at the highest tested concentration of 10%, the SI was observed to be 1.17, therefore the material should not be classified as a skin sensitiser.