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Administrative data

Description of key information

LLNA (OECD429, GLP): sensitising

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
08 February 2010 - 29 March 2010
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study has been performed according to OECD and EC guidlines and according to GLP principles. A reliability of 2 is assigned in accordance with the ECHA Practical guide #6 on the reporting of read-across in IUCLID, due to the read-across purpose.
Reason / purpose:
reference to same study
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:
other: CBA/J
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River France, L’Arbresle Cedex, France.
- Age at study initiation: Young adult animals (approx. 10 weeks old) were selected
- Weight at study initiation: Body weight variation was within +/- 20% of the sex mean.
- Housing: Individual housing in labeled Macrolon cages (MI type; height 12.5 cm) containing sterilized sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France). Paper (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) was supplied as cage-enrichment. The paper was removed on Day 1 prior to dosing and was supplied again after scoring of the ears on Day 3. During the acclimatization period the accommodation was as described above except that the animals were group housed in Macrolon cages (MIII type; height 18 cm).
- Diet (e.g. ad libitum): Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water (e.g. ad libitum): Free access to tap water.
- Acclimation period: The acclimatization period was at least 5 days before the start of treatment under laboratory conditions.
- Health inspection: A health inspection was performed prior to treatment, to ensure that the animals are in a good state of health. Special attention was paid to the ears, which were intact and free from any abnormality.

Results of analysis for diet (nutrients and contaminants), sawdust, paper and water were assessed and did not reveal any findings that were considered to have affected the study integrity. All certificates and results of analysis are retained in the NOTOX archives.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.1 – 23.3ºC
- Humidity (%): 39 – 65%
Temporary deviations from the minimum level of relative humidity occurred. Laboratory historical data do not indicate an effect of the deviations.
- Air changes (per hr): approximately 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours artificial fluorescent light and 12 hours darkness per day.

IN-LIFE DATES: From: 08 February 2010 To: 29 March 2010
Vehicle:
methyl ethyl ketone
Concentration:
0-25-50-100%
No. of animals per dose:
5
Details on study design:
The vehicle was selected based on trial formulations performed at NOTOX and on test substance data supplied by the sponsor.

RANGE FINDING TESTS:
- Irritation: 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 (maximally grade 2) at the highest concentration.
Two test substance concentrations were tested; a 25% and 50% concentration. The highest concentration was the maximum concentration as required in the test guidelines (undiluted for liquids, 50% for solids).
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 (8-14 weeks old. Each animal was treated with one concentration on three consecutive days. Approximately 3-4 hours after the last exposure, the irritation of the ears was assessed. Bodyweights were determined on Day 3. The animals were sacrificed after the final observation and no necropsy was performed.
- Lymph node proliferation response: Not determined in the preliminary irritation study.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay
- Criteria used to consider a positive response: DPM values are presented for each animal and for each dose group. A Stimulation Index (SI) is calculated for each group. The SI is the ratio of the DPM/group compared to DPM/vehicle control group. If the results indicate a SI ≥ 3, the test substance may be regarded as a skin sensitizer, based on the test guideline and recommendations done by ICCVAM (Reference 1). The results were evaluated according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations (2007) and the Regulation (EC) No 1272/2008 of the European Parliament and of the Council of 16 December 2008 on classification, labeling and packaging of substances and mixtures. The EC3 value (the estimated test substance concentration that will give a SI =3) was determined, using linear interpolation (Reference 2)..

Ref 1. National Institute of Environmental Health Sciences, The Murine Lymph Node Assay: A test method for assessing the allergic contact dermatitis potential of chemicals/compounds. Independent peer review by the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) and the National Toxicology Program Center for the Evaluation of Alternative Toxicological Methods (NICEATM), NIH publication; No 99-4494, February 1999.
Ref 2. Basketter DA, Lea LJ, Dickens A, Briggs, D, Pate I, Dearman RJ and Kimber I. A comparison of statistical approaches to the derivation of EC3 values from local lymph node assay dose responses. J Appl Toxicol 1999;19:261-266.

ANIMAL ASSIGNMENT
Three groups of five animals were treated with one test substance concentration per group. One group of five animals was treated with vehicle.

TREATMENT PREPARATION AND ADMINISTRATION:
Test substance preparation: The test substance formulations (w/w) were prepared within 4 hours prior to each treatment. Homogeneity was obtained to visually acceptable levels.
Rationale for vehicle: The vehicle was selected based on trial formulations performed at NOTOX and on test substance data supplied by the sponsor.

Induction - Days 1, 2 and 3:
The dorsal surface of both ears was epidermally treated (25 μL/ear) with the test substance concentration, at approximately the same time per day. The concentrations were mixed thoroughly using a vortex mixer immediately prior to dosing. The control animals were treated the same as the experimental animals, except that the vehicle was administered instead of the test substance.

Excision of the nodes - Day 6:
Each animal was injected via the tail vein with 0.25 mL of sterile phosphate buffered saline (PBS) (Merck, Darmstadt, Germany) containing 20 μCi of 3H-methyl thymidine (PerkinElmer Life and Analytical Sciences, Boston, MA, US). After approximately five hours, all animals were killed by intraperitoneal injection with Euthasol® 20% (AST Farma BV, Oudewater, The Netherlands). 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 were washed twice with an excess of PBS by centrifugation at 200g for 10 minutes at 4ºC. To precipitate the DNA, the LNC were exposed to 5% trichloroacetic acid (TCA) (Merck, Darmstadt, Germany) stored in the refrigerator until the next day.

Radioactivity measurements - Day 7
Precipitates were recovered by centrifugation, resuspended in 1 mL TCA and transferred to 10 mL of Ultima Gold cocktail (PerkinElmer Life and Analytical Sciences, Boston, MA, US) as the scintillation fluid. Radioactive measurements were performed using a Packard scintillation counter (2800TR). Counting time was to a statistical precision of ± 0.2% or a maximum of 5 minutes whichever came first. The scintillation counter was programmed to automatically subtract background and convert Counts Per Minute (CPM) to Disintegrations Per Minute (DPM).

Observations:
Mortality/Viability: Twice daily.
Toxicity: At least once daily.
Body weights: On Days 1 (pre-treatment) and 6.
Necropsy: No necropsy was performed according to protocol.
Irritation: On Day 3 (3-4 hours after treatment), the skin reactions were assessed. Skin reactions were graded according to the following numerical scoring system. Furthermore descriptions of all other (local) effects were recorded.

Grading Irritation Reactions:
Erythema and eschar formation:
0: No erythema
1: Slight erythema (barely perceptible)
2: Well-defined erythema
3: Severe erythema (beef redness) to slight eschar formation (injuries in depth)

Oedema formation:
0: No oedema
1: Slight oedema (barely perceptible)
2: Moderate oedema
3: Severe oedema
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Not performed.
Positive control results:
The six monthly reliability check with Hexylcinnamaldehyde, indicates that the Local Lymph Node Assay as performed at NOTOX is an appropriate model for testing for contact hypersensitivity. See attached document "492991 Reliability check".


The calculated EC3 value was found to be in the acceptable range of 2 and 20%. The results of the 6 monthly HCA reliability checks of the recent years were 13.1, 15.6, 14.1, 13.8, 13.9, 16.0 and 11.9%.

Based on the results, it was concluded that the Local Lymph Node Assay as performed at NOTOX is an appropriate model for testing for contact hypersensitivity. The raw data, protocol and report from this study are kept in the NOTOX archives. The test described above was performed in accordance with NOTOX Standard Operating Procedures and the report was audited by the QA-unit

See attached document "Reliability check".
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Mean DPM/animal values for the experimental groups treated with test substance concentrations 25, 50 and 100% were 774, 1299 and 2310 DPM respectively. The mean DPM/animal value for the vehicle control group was 289 DPM.
Key result
Parameter:
SI
Value:
2.7
Test group / Remarks:
25%
Key result
Parameter:
SI
Value:
4.5
Test group / Remarks:
50%
Key result
Parameter:
SI
Value:
8
Test group / Remarks:
100%

Tables and figures have been included in the attached document "LLNA tables and figures".

 

Preliminary irritation study:

The results of the epidermal exposures for the selection of highest test substance concentration to be tested in the main study are described in the table. Based on these results, the highest test substance concentration selected for the main study was a 100% concentration. See also Table 1 in the attached document LLNA tables and figures

 

Main study:

The slight irritation of the ears as shown by all animals treated at 50 and 100% was considered not to have a toxicologically significant effect on the activity of the nodes. No oedema was observed in any of the animals examined.

All auricular lymph nodes of the animals of the control groups and animals at 25% were

considered normal in size. A few auricular lymph nodes at 50% and the majority of nodes at 100% were enlarged in size. No macroscopic abnormalities of the surrounding area were noted in any of the animals.

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.

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

Interpretation of results:
other: Sensitising (skin sensitizer, Category 1)
Remarks:
Based on CLP criteria
Conclusions:
The results indicate that the test substance could elicit an SI ≥ 3. The data showed a doseresponse and an EC3 value (the estimated test substance concentration that will give a SI =3) of 29.2% was calculated.

Based on these results:
- according to the recommendations made in the test guidelines, Standolized linseed oil would be regarded as skin sensitizer.
- according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations (2007), Standolized linseed oil should be classified as skin sensitizer (Category 1).
- according to the Regulation (EC) No 1272/2008 on classification, labeling and packaging of substances and mixtures, Standolized linseed oil should be classified as skin sensitizer (Category 1) and labeled as H317: May cause an allergic skin reaction.
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
See attached justification
Reason / purpose:
read-across source
Positive control results:
The six monthly reliability check with Hexylcinnamaldehyde, indicates that the Local Lymph Node Assay as performed at NOTOX is an appropriate model for testing for contact hypersensitivity. See attached document "492991 Reliability check".


The calculated EC3 value was found to be in the acceptable range of 2 and 20%. The results of the 6 monthly HCA reliability checks of the recent years were 13.1, 15.6, 14.1, 13.8, 13.9, 16.0 and 11.9%.

Based on the results, it was concluded that the Local Lymph Node Assay as performed at NOTOX is an appropriate model for testing for contact hypersensitivity. The raw data, protocol and report from this study are kept in the NOTOX archives. The test described above was performed in accordance with NOTOX Standard Operating Procedures and the report was audited by the QA-unit

See attached document "Reliability check".
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Mean DPM/animal values for the experimental groups treated with test substance concentrations 25, 50 and 100% were 774, 1299 and 2310 DPM respectively. The mean DPM/animal value for the vehicle control group was 289 DPM.
Key result
Parameter:
SI
Value:
2.7
Test group / Remarks:
25%
Key result
Parameter:
SI
Value:
4.5
Test group / Remarks:
50%
Key result
Parameter:
SI
Value:
8
Test group / Remarks:
100%

Tables and figures have been included in the attached document "LLNA tables and figures".

 

Preliminary irritation study:

The results of the epidermal exposures for the selection of highest test substance concentration to be tested in the main study are described in the table. Based on these results, the highest test substance concentration selected for the main study was a 100% concentration. See also Table 1 in the attached document LLNA tables and figures

 

Main study:

The slight irritation of the ears as shown by all animals treated at 50 and 100% was considered not to have a toxicologically significant effect on the activity of the nodes. No oedema was observed in any of the animals examined.

All auricular lymph nodes of the animals of the control groups and animals at 25% were

considered normal in size. A few auricular lymph nodes at 50% and the majority of nodes at 100% were enlarged in size. No macroscopic abnormalities of the surrounding area were noted in any of the animals.

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.

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

Interpretation of results:
other: Sensitising (skin sensitizer, Category 1)
Remarks:
Based on CLP criteria
Conclusions:
The results indicate that the test substance could elicit an SI ≥ 3. The data showed a doseresponse and an EC3 value (the estimated test substance concentration that will give a SI =3) of 29.2% was calculated.

Based on these results:
- according to the recommendations made in the test guidelines, Standolized linseed oil would be regarded as skin sensitizer.
- according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations (2007), Standolized linseed oil should be classified as skin sensitizer (Category 1).
- according to the Regulation (EC) No 1272/2008 on classification, labeling and packaging of substances and mixtures, Standolized linseed oil should be classified as skin sensitizer (Category 1) and labeled as H317: May cause an allergic skin reaction.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

The results of Standolized Linseed Oil (SLO) can be read across to Standolized Soybean Oil (SSO) as SLO and SSO share similar structures and reactivity, based on the fatty acids composition of the raw material oils. Due to higher level of polyunsaturated fatty acid chains, SLO presents the worst case scenario in terms of toxicology. (see read across document for further details)

A Local Lymph Node Assay was performed with Standolized Linseed Oil in accordance with OECD 429 and under GLP-conditions. The results indicate that the test substance could elicit an SI ≥ 3. The data showed a dose response and an EC3 value (the estimated test substance concentration that will give a SI =3) of 29.2% was calculated.

After receiving the result of the LLNA study, the manufacturers of the substance contacted their company physician and asked their employees about any case of a skin reaction to the substance. Some company physicians even issued detailed questionnaires based on the LLNA result. The conclusion of these investigations is that during decades of manufacturing this substance, at different locations in the EU, not a single case of a skin reaction has been reported, as confirmed in writing by the company physicians.

It is evident that a careful and scientific consideration of the weight of all the evidence needs to be taken into account. Since the beginning of the 20thcentury, the substance is present in applications with a high potential for frequent skin exposure, like varnishes, coatings, paints and lacquers. Any indication of a skin sensitizing potential should have been noted for this substance. All the company physicians of the manufacturers confirm this assumption. Therefore, the outcome of the LLNA study is regarded as a false-positive result, supported by investigations reported in the literature, that unsaturated fatty acid functions present in the substance, give a false-positive result in the LLNA study (Kreiling et al (2008); Basketter et al (2009)).


Justification for selection of skin sensitisation endpoint:
One available study which was performed in accordance with OECD429 and under GLP-conditions

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available
Additional information:

No study is available to address the possible respiratory sensitisation of Standolized Soybean Oil. Based on the very low vapour pressure of the substance, no respiratory sensitisation is expected.

Justification for classification or non-classification

Based on the available information Standolized Soybean Oil does not need to be classified for skin or respiratory sensitisation in accordance with the criteria outlined in Annex VI of 67/548/EEC and Annex I of 1272/2002/EC.