Oligomerisation products of 2,2'-[(1-methylethylidene)bis(4,1-phenyleneoxymethylene)]bisoxirane with acrylic acid and fatty acids, C18-unsatd., dimers and nonanoic acid

Endpoint summary

Description of key information

Based on the EC3 value, the substance is considered to be a skin sensitiser with moderate sensitisation potency.

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:

From September 28, 2011 to October 10, 2011

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

Type of study:

mouse local lymph node assay (LLNA)

Specific details on test material used for the study:

Specific Gravity / Density: 1.14 g/cm3; pH (1% in water, indicative range): 5.5 – 5.5 (determined at WIL Research Europe); Stability at higher temperatures: yes, maximum temperature 40°C.

Species:

mouse

Strain:

CBA:J

Remarks:

inbred, SPF-Quality

Sex:

female

Details on test animals and environmental conditions:

Animals:
Species: mouse, CBA/J strain, inbred, SPF-Quality. Recognized by the international guidelines as the recommended test system (e.g. OECD, EC, EPA). Source: Janvier, Le Genest-Saint-Isle, France. Number of animals: 20 females (nulliparous and non-pregnant), five females per group. Age and body weight: young adult animals (approx. 9 weeks old) were selected. Body weight variation was within +/- 20% of the sex mean. Identification: tail mark with marker pen. Health inspection: a health inspection was performed prior to treatment, to ensure that the animals were in a good state of health. Special attention was paid to the ears, which were intact and free from any abnormality. Reliability check: reliability test with three concentrations of Hexylcinnamaldehyde in Acetone/Olive oil (4:1 v/v), performed not more than 6 months previously and using the same materials, animal supplier, animal strain and essential procedures. For both scientific and animal welfare reasons, no concurrent positive control group was added to the study. An extensive data base is available with reliability checks performed each half year during at least the recent 9 years showing reproducible and consistent positive results.
Conditions:
Animals were housed in a controlled environment, in which optimal conditions were considered to be approximately 15 air changes per hour, a temperature of 21.0 ± 3.0ºC (actual range: 20.1 – 23.4ºC), a relative humidity of 40-70% (actual range: 41 - 70%) and 12 hours artificial fluorescent light and 12 hours darkness per day. Accommodation: animals were group housed in labeled Makrolon cages (MIII type; height 18 cm) containing sterilised sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France). Paper (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) and shelters (disposable paper corner home, MCORN 404, Datesand Ltd, USA) were supplied as cage-enrichment. The acclimatization period was at least 5 days before the start of treatment under laboratory conditions. On Day 6, the animals were group housed in Makrolon MII type cages with a sheet of paper instead of sawdust and cage enrichment. Certificates of analysis were examined and then retained in the NOTOX archives. Diet: free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany). Water: free access to tap water. Results of analysis for diet (nutrients and contaminants), sawdust, paper, shelters 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.

Vehicle:

dimethylformamide

Concentration:

0 (vehicle alone), 10, 25 and 50%

No. of animals per dose:

5

Details on study design:

- Allocation: 5 animals/group.
- Induction - Days 1, 2, and 3: The dorsal surface of both ears was topically treated (25 μL/ear) with the test substance concentration, at approximately the same time on each day. The concentrations were stirred with a magnetic stirrer immediately prior to dosing. The control animals were treated in the same way 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 (0.2 mL/animal) 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) and 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).

Positive control results:

The six-month reliability check with Alpha-hexylcinnamicaldehyde indicates that the Local Lymph Node Assay as performed at NOTOX is an appropriate model for testing for contact hypersensitivity.

Key result

Parameter:

SI

Value:

4.2

Test group / Remarks:

10%

Key result

Parameter:

SI

Value:

7.2

Test group / Remarks:

25%

Key result

Parameter:

SI

Value:

15

Test group / Remarks:

50%

Key result

Parameter:

EC3

Value:

ca. 6.9

Pre-screen test:

At 25% and 50% test substance concentrations no signs of systemic toxicity were noted and no severe irritation was observed. Variations in ear thickness during the observation period were less than 25% from Day 1 pre-dose values. Based on these results, the highest test substance concentration selected for the main study was a 50% concentration.

Main study:

-Skin reactions / Irritation. The very slight erythema of the ears as shown by animals at 25% on Days 3-4 and animals at 50% on Days 2-4 was considered not to have a toxicologically significant effect on the activity of the nodes. -Systemic toxicity (body weights). No mortality occurred and no clinical signs of systemic toxicity were observed in the animals of the main study. Body weights and body weight gain of experimental animals remained in the same range as controls over the study period.

-Macroscopy of the auricular lymph nodes and surrounding area. The auricular lymph nodes of the animals at 25% and 50% appeared larger in size as compared to nodes of the control animals and animals at 10%. No macroscopic abnormalities of the surrounding area were noted in any of the animals.

-Radioactivity measurements. Mean DPM/animal values for the experimental groups treated with test substance concentrations 10, 25 and 50% were 1655, 2814 and 5850 DPM respectively. The mean DPM/animal value for the vehicle control group was 390 DPM.

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

Under the study conditions, the test substance was classified as following:
- according to the recommendations made in the test guidelines, Ebecryl® 3702 radiation curing resin would be regarded as skin sensitizer. - according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2007), Ebecryl® 3702 radiation curing resin should be classified as skin sensitizer (Category 1).
- according to the Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures, Ebecryl® 3702 radiation curing resin should be classified as skin sensitizer (Category 1) and labeled as H317: May cause an allergic skin reaction.

Executive summary:

A study was conducted to determine the skin sensitisation potential of the test substance according to OECD Guideline 429, EU Method B.42 and EPA OPPTS 870.2600 (Local Lymph Node Assay). Three groups of 5 female CBA/J mice were treated at concentrations of 10, 25 and 50% w/w on three consecutive days by open application on the ears. Five control animals were similarly treated, but with vehicle alone (dimethyl formamide). Three days after the last exposure, all animals were injected with 3H-methyl thymidine and, after 5 h, the draining (auricular) lymph nodes were excised and pooled for each animal. After precipitation of the DNA of the lymph node cells, radioactivity measurements were performed. The activity was expressed as Disintegrations Per Minute (DPM) and a Stimulation Index (SI) was subsequently calculated for each group. The very slight erythema of the ears - as shown by animals at 25% on Days 3-4 and animals at 50% on Days 2-4 - was considered not to have a toxicologically significant effect on the activity of the nodes. The auricular lymph nodes of the animals at 25 and 50% appeared larger in size as compared to controls and animals at 10%. No macroscopic abnormalities of the surrounding area were noted in any of the animals. No mortality occurred and no clinical signs of systemic toxicity were observed throughout the study. Body weights and body weight gain of experimental animals remained in the same range as controls over the study period. These test substance elicited an SI ≥ 3 (4.2, 7.2 and 15.0 at 10, 25 and 50%, respectively). The EC3 value (the estimated test substance concentration that will give a SI =3) was established to be <10%. Under the study conditions, the test substance was considered to be sensitizing to skin (Stitzinger, 2011).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

A study was conducted to determine the skin sensitisation potential of the test substance according to OECD Guideline 429, EU Method B.42 and EPA OPPTS 870.2600 (Local Lymph Node Assay). Three groups of 5 female CBA/J mice were treated at concentrations of 10, 25 and 50% w/w on three consecutive days by open application on the ears. Five control animals were similarly treated, but with vehicle alone (dimethyl formamide). Three days after the last exposure, all animals were injected with 3H-methyl thymidine and, after 5 h, the draining (auricular) lymph nodes were excised and pooled for each animal. After precipitation of the DNA of the lymph node cells, radioactivity measurements were performed. The activity was expressed as Disintegrations Per Minute (DPM) and a Stimulation Index (SI) was subsequently calculated for each group. The very slight erythema of the ears - as shown by animals at 25% on Days 3-4 and animals at 50% on Days 2-4 - was considered not to have a toxicologically significant effect on the activity of the nodes. The auricular lymph nodes of the animals at 25 and 50% appeared larger in size as compared to controls and animals at 10%. No macroscopic abnormalities of the surrounding area were noted in any of the animals. No mortality occurred and no clinical signs of systemic toxicity were observed throughout the study. Body weights and body weight gain of experimental animals remained in the same range as controls over the study period. This test substance elicited an SI ≥ 3 (4.2, 7.2 and 15.0 at 10, 25 and 50%, respectively). The EC3 value (the estimated test substance concentration that will give a SI =3) was established to be <10%. Under the study conditions, the test substance was considered to be sensitizing to skin (Stitzinger, 2011).

Considering that there was dose response relationship in the skin sensitisation response and the SI value was above 3 even at the lowest concentration, the EC3 value has been calculated using the two lowest concentrations in the below log-linear interpolation equation (Ryan et al., 2007):

EC3 = 2^(log2(c) + (3 - d) / (b - d) * (log2(a) - log2(c)))

Using this equation, the EC3 value can be calculated at 6.9%.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the results of a local lymph node assay and the EC3 value of 6.9%, the substance is considered to be moderately sensitizing, with a classification of Skin Sens. 1B - H317: May cause an allergic skin reaction as per CLP criteria (EC 1272/2008).