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Administrative data

Description of key information

An assessment of the contact hypersensitivity to EA-3098 was assessed by a LLNA study.

The Stimulation index values calculated for the substance concentrations 1, 10 and 25%  were 1.3, 1.0 and 0.9 respectively.

If results indicate a SI ≥3 the test substance may be regarded as a sensitiser. Therefore, the test substance is not considered to be a skin sensitiser based on the LLNA study.

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:
September 2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of relevant results.
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River France, L'Arbresle Cedex, France.
- Age at study initiation: approx. 12 weeks
- Weight at study initiation: body weight variation was within +/- 20% of the sex mean
- Housing: Individually housed in labeled Macrolon cages (MI type; height 12.5 cm) containing sterilized sawdust as bedding material
- Diet: ad libitum access to standard pelleted laboratory animal diet
- Water: ad libitum access to tap water
- Acclimation period: at least five days before the start of treatment under laboratory conditions.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.0 +/- 3.0°C (actual range: 20.8 - 22.4°C)
- Humidity (%): 30 - 70 % (actual range: 46-92%)
- Air changes (per hr): approximately 15 per hour
- Photoperiod (hrs dark / hrs light): 12 hours artificial fluorescent light and 12 hours darkness per day

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
Preliminary study 100% (undiluted), 50%, 25%, 10%, 5%, 2.5% and 1%

Main study: 1%, 10% and 25%
No. of animals per dose:
Preliminary study: 2

Main study: 5 animals per dose level
Details on study design:
TEST SUBSTANCE PREPARATION:
Vehicle: Acetone/Olive oil (4:! v/v)

RANGE FINDING TESTS:
A preliminary irritation study was conducted in order to select the highest test substance concentration to be used In the main study. In principle, this concentration should be well tolerated systemically by the animals and may give moderate irritation (grade 2) at the highest.

A series of two test substance concentrations was tested, selected from the series: 100% (undiluted), 50%, 25%, 10%, 5%, 2.5%, 1% and if needed further lower concentrations using the same steps. The highest concentration, selected from this series, was the maximum concentration that could technically be applied.
The test system, procedures and techniques were identical to those used during days 1 to 3 of the main study unless otherwise specified. Two young adult animals were selected (5-14 weeks old). Each animal was treated with one concentration on three consecutive days. Approximately 4 hours after the last exposure, the ear was cleaned of residual test substance with tap water and the irritation was assessed. Bodyweights were determined on day 3. The animals were sacrificed after the final observation and no necropsy was performed.


MAIN STUDY
TREATMENT PREPARATION AND ADMINISTRATION:
Three groups of five animals were treated with one test substance concentration per group. One group of five animals was treated with vehicle.

Allocation:
Group Induction
1: vehicle control Vehicle
2: experimental 1% test substance
3: experimental 10% test substance
4: experimental 25% test substance

Induction - Days 1, 2 and 3:
Experimental animals:
The dorsal surface of both ears was epidermally treated (25 µl/ear) with the test substance concentration, at approximately the same time per day.

Vehicle control animals:
The control animals were treated the same as the experimental animals, except that instead of the test substance, the vehicle alone was administered.

Treatment - Day 6:
All animals:
Each animal was injected via the tail vein with 0.25 ml of sterile phosphate buffered saline (PBS) containing 20 µCi of 3H-methyl thymidine.

After approximately five hours, all animals were killed by intra peritoneal injection with pentobarbital. The draining (auricular) lymph node of each ear was excised.
The relative size of the nodes (as compared to normal) was estimated by visual examination and abnormalities of the nodes and surrounding area were recorded. The nodes were pooled for each animal in approximately 3 ml PBS.

Tissue processing for radioactivity - Day 6:
A single cell suspension of lymph node cells (LNC) was prepared in PBS by gentle separation through stainless steel gauze (diameter 125 µm). LNC was washed twice with an excess of PBS by centrifugation at 200 g for 10 minutes at 4°C. To precipiate the DNA, the LNC were exposed to 5% trichloroacetic acid at 4°C during the night.

Radioactivity measurements - Day 7:
Precipitates were recovered by centrifugation, resuspended in 1 ml TCA and transferred to 10 ml of Ultima gold cocktail as the scintillation fluid. Radioactive measurements were performed using a Packadrd scintillation counter (1900TR). Counting time was to a statistical precision of +/- 0.2% or a maximum of 5 minutes , whichever comes first. The Packard 1900TR was programmed to automatically substract background and convert Counts per minute (CPM) to Disintegration per minute (DPM).

Obsevation:
Mortality/Viability: Twice daily
Toxicity: At least once daily
Body weights: On days 1 (pre-treatment) and 6
Irritation: On day 3 (3-4 hours after treatment), the skin reactions were assessed. Descriptions of all other (local) effects were recorded.



Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Positive control results:
The SI values calculated for the substance (alpha-hexylcinnamicaldehyde, tech. 85%) concentrations 5, 10 and 25% were 2.1, 3.6 and 7.5 respectively. An EC3 value of 8.0% was calculated using linear interpolation.
The calculated EC3 value was found to be in the acceptable range of 2 and 20%.
Parameter:
SI
Value:
1.3
Test group / Remarks:
1% test substance
Remarks on result:
other: non-sensitising
Parameter:
SI
Value:
1
Test group / Remarks:
10% test substance
Remarks on result:
other: non-sensitising
Parameter:
SI
Value:
0.9
Test group / Remarks:
25% test substance
Remarks on result:
other: non-sensitising
Cellular proliferation data / Observations:
Radioactivity measurements:
Mean DPM/animals values for the experimental groups treated with test substance concentrations 1, 10 and 25% were 61, 207 and 170 respectively.
The mean DPM/animal value for the vehicle control group was 199.

The SI values calculated for the substance concentrations 1, 10 and 25% were 1.3, 1.0 and 0.9 respectively.

Results:

Preliminary irritation study:

The results of the epidermal exposures for the selection of the highest test substance concentration to be tested in the main study are described in Table 1 (attached background material).

Based on the results, the highest test substance concentration selected for the main study was a 25% concentration.

Main study:

Induction phase (Table 2 - attached background material):

The skin effects seen after the third epidermal exposure are presented in Table 2. No irritation was observed in any of the animals examined.

Macroscopy of the nodes and surrounding area (Table 2 - attached background material):

All nodes of the experimental and control groups were considered normal in size. No macroscopic abnormalities of the surrounding area were noted.

Body weights (Table2 - attached background material):

Body weights and body weight gain of experimental animals remained in the same range as controls over the study period. The slight body weight loss, noted in some animals, was considered not toxicologically significant.

Toxicity and Mortality:

No mortality occurred and no symptoms of systemic toxicity were observed in the animals of the main study.

Calculation of stimulation index (SI)

Group

Treatment

Induction

Mean DPM

SI

2

Experimental

1% test substance

261

1.3

3

Experimental

10% test substance

207

1.0

4

Experimental

24% test substance

170

0.9

1

Vehicle control

Acetone/Olive oil (4:1 v/v)

199

1

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The SI values calculated for the substance concentrations 1, 10 and 25% were 1.3, 1.0 and 0.9 respectively.
There was no indication that the test substance could elicit an SI ≥3. It was established that the EC3 value (if any) exceeds 25%.
The test substance was considered to be a non-sensitiser under the conditions of the test.
Executive summary:

Assessment of the contact hypersensitivity to EA-3098 in a LLNA study was assessed using the following guidelines:

- OECD No. 429 (2002)

- EC Council Directive 67/548/EEC, Annex V, B.42 (2004)

- EPA, OPPTS 870.2600 (2003) "Skin Sensitisation"

Test substance concentrations selected for the main study were based on the results of a preliminary study.

In the main study, three groups of five experimental animals were epidermally exposed to test substance concentrations of 1%, 10% or 25% on three consecutive days. Five vehicle control animals were similarly treated, but with vehicle alone (acetone/olive oil (4:1 v/v)). Three days after the last exposure, all animals were injected with 3H-methyl thymidine and after five hours the draining (auricular) lymph nodes were excised. After precipitating the DNA of the lymph node cells, radioactivity measurements were performed. The activity was expressed as the number of Disintegrations Per Minute (DPM) and a simulation index calculated for each group.

No irritation was observed in any of the animals examined.

All nodes of the experimental and control groups were considered normal in size. No macroscopic abnormalities of the surrounding area were noted.

Mean DPM/animal values for the experimental groups with test substance concentrations 1, 10 and 25% were 261, 207 and 170 respectively. The mean DPM/animal value for the vehicle control group was 199.

The SI values calculated for the substance concentrations 1, 10 and 25% were 1.3, 1.0 and 0.9 respectively.

There was no indication that the test substance could elicit an SI >3. It was established that the EC3 value (if any) exceeds 25%.

The six month reliability check with hexylcinnamic aldehyde indicates that the LLNA as performed is an appropriate method for testing contact hypersensitivity.

Based on these results EA-3098 is considered to be a non-sensitiser.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

Local Lymph Node Assay

Introduction

Assessment of the contact hypersensitivity to EA-3098 in a LLNA study was assessed using the following guidelines:

- OECD No. 429 (2002)

- EC Council Directive 67/548/EEC, Annex V, B.42 (2004)

- EPA, OPPTS 870.2600 (2003) "Skin Sensitisation"

Method

Test substance concentrations selected for the main study were based on the results of a preliminary study.

In the main study, three groups of five experimental animals were epidermally exposed to test substance concentrations of 1%, 10% or 25% on three consecutive days. Five vehicle control animals were similarly treated, but with vehicle alone (acetone/olive oil (4:1 v/v)). Three days after the last exposure, all animals were injected with 3H-methyl thymidine and after five hours the draining (auricular) lymph nodes were excised. After precipitating the DNA of the lymph node cells, radioactivity measurements were performed. The activity was expressed as the number of Disintegrations Per Minute (DPM) and a simulation index calculated for each group.

Results

No irritation was observed in any of the animals examined.

All nodes of the experimental and control groups were considered normal in size. No macroscopic abnormalities of the surrounding area were noted.

Mean DPM/animal values for the experimental groups with test substance concentrations 1, 10 and 25% were 261, 207 and 170 respectively. The mean DPM/animal value for the vehicle control group was 199.

The SI values calculated for the substance concentrations 1, 10 and 25% were 1.3, 1.0 and 0.9 respectively.

There was no indication that the test substance could elicit an SI >3. It was established that the EC3 value (if any) exceeds 25%.

The six month reliability check with hexylcinnamic aldehyde indicates that the LLNA as performed is an appropriate method for testing contact hypersensitivity.

Conclusion

Based on these results EA-3098 is considered to be a non-sensitiser.

The LLNA study on the test substance was negative and this study is considered valid for the assessment of the substance.

Justification for selection of skin sensitisation endpoint:

The study has been conducted according to OECD Guideline 429 and GLP and is adequately reported. The study has been assigned a reliability 1.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available
Additional information:

Migrated from Short description of key information:

No data available. However, as inhalation is not expected to be a significant route of exposure and aerosols of the substance are not anticipated to be respirable, sensitisation of the respiratory system is not considered to be of concern, also taking into account the negative result for sensitization in the LLNA study.  

Justification for classification or non-classification

Skin sensitisation (Local Lymph Node Assay):

The SI values calculated for the substance concentrations 1, 10 and 25% were 1.3, 1.0 and 0.9 respectively.

There was no indication that the test substance could elicit an SI >3. It was established that the EC3 value (if any) exceeds 25%.

If results indicate a SI ≥3 the test substance may be regarded as a sensitiser. Therefore, based on the results of the LLNA study, the test substance does not meet the criteria for a positive result for skin sensitisation and therefore does not meet the criteria for classification as a skin sensitiser.

The conducted LLNA study on the substance is considered valid for assessment of the substance and for classification purposes.