Oligomerisation products of 2,2'-[(1-methylethylidene)bis(4,1-phenyleneoxymethylene)]bisoxirane with acrylic acid and lauric acid

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

Study period:

June 13, 2012 to June 25, 2012

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)

Species:

mouse

Strain:

other: CBA/J

Sex:

female

Details on test animals and environmental conditions:

- Source: Janvier, Le Genest-Saint-Isle, France
- Age at study initiation: Young adult animals (approx. 9 weeks old)
- Weight at study initiation: Body weight variation was within +/- 20% of the sex mean (19-23 grams)
- Housing: Animals were group housed in labeld makrolon cages.
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap water.
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
Environmental controls for the animal room were set to maintain 18 to 24°C, a relative humidity of 40 to 70%, approximately 15 room air changes/hour, and a 12-hour light/12-hour dark cycle. Any variations to these conditions were maintained in the raw data and had no effect on the outcome of the study.

IN-LIFE DATES: From: 13 to 25 June 2012

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

0, 10, 25 , 50%

No. of animals per dose:

5

Details on study design:

The vehicle was selected based on trial formulations performed at WIL Research Europe and on test substance data supplied by the sponsor.

RANGE FINDING TESTS:
In the interest of animal welfare and to minimize any testing likely to produce severe responses in animals, a weight of evidence analysis was performed prior to start of this study. All available information was evaluated (e.g. existing human and animal data, literature, substance data supplied by the sponsor, analysis of structure activity relationships (SAR), physicochemical properties and reactivity (pH, buffering capacity).

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. The results were evaluated according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (20011) and the Regulation (EC) No 1272/2008 of the European Parliament and of the Council of 16 December 2008 on Classification, Labelling and Packaging of substances and mixtures.

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 WIL Research Europe and on test substance data supplied by the sponsor.

Induction - Days 1, 2 and 3; Excision of nodes - Day 6; Tissue processing for radioacitivity - Day 6; Radioactivity measurements - Day 7; Performed according to test guidelines.

Observations:
Mortality/Viability: Twice daily.
Body weights: On Day 1 (pre-dose) and Day 6 (prior to necropsy).
Clinical signs: Once daily on Days 1-6 (on Days 1-3 between 3 and 4 hours after dosing).
Irritation: Once daily on Days 1-6 (on Days 1 - 3 immediately after dosing) according to the following numerical scoring system. Furthermore, a description of all other (local) effects was recorded according to guidelines.

Necropsy: 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.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

Not performed.

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. See attached document 'Reliability check'.

Key result

Parameter:

SI

Value:

ca. 34

Remarks on result:

other: For the substance concentrations of 50%

Key result

Parameter:

SI

Value:

ca. 18.9

Remarks on result:

other: For the substance concentrations of 25 %

Key result

Parameter:

SI

Value:

ca. 17.5

Remarks on result:

other: For the substance concentrations of 10 %

Results Pre-screen test:

A maximum of moderate to severe erythema was noted in animals treated at 70%. The animals at 50% showed very slight up to well-defined erythema. Variations in ear thickness, in animals treated at 50%, 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

Other results - main study:

Skin reactions / Irritation:

No irritation of the ears was observed in the animals at 10%. Very slight erythema was noted at 25% and very slight up to well-defined erythema was noted at 50%. The very slight up to well-defined irritation of the ears as shown by animals treated at 25% and 50% 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. The body weight loss noted for some animals across the dose groups was considered not toxicologically significant since the changes were slight in nature and no concentration-related incidence was apparent.

Macroscopy of the auricular lymph nodes and surrounding area:

All auricular lymph nodes of the test substance treated animals were considered larger in size compared to normal. No macroscopic abnormalities of the surrounding area were noted in any of the animals.

Conclusions:

Under the study conditions, the test substance was a strong skin sensitiser in mouse.

Executive summary:

An in vivo LLNA study was carried out in accordance with OECD Guideline 429, EU method B.42, EPA OPPTS 870.2600, under GLP conditions in female mice. Three experimental groups of five female CBA/J mice were treated with test substance concentrations of 10, 25 or 50% w/w on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated. 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 and pooled for each animal. After precipitating the DNA of the lymph node cells, radioactivity measurements were performed. No irritation of the ears was observed in the animals at 10%. Very slight erythema was noted at 25% and very slight up to well-defined erythema was noted at 50%. The very slight up to well-defined irritation of the ears as shown by animals treated at 25% and 50% was considered not to have a toxicologically significant effect on the activity of the nodes. All auricular lymph nodes of the test substance treated animals were considered larger in size compared to normal. No macroscopic abnormalities of the surrounding area were noted in any of the animals. Stimulation Index (SI) values calculated for the substance concentrations 10, 25 and 50% were 17.5, 18.9 and 34.0 respectively. These results show that the test substance elicits an SI ≥ 3. The EC3 value (the estimated test substance concentration that will give a SI =3) was established to be between 0 and 10%. Although it was possible to strengthen the outcome of the study by adding lower concentrations. Since the SI values clearly exceeded 3 and since extension of the study would not alter the classification, this was considered not appropriate for ethical reasons. The results of a reliability test with three concentrations of hexylcinnamaldehyde (a positive control substance) in acetone/olive oil (4:1 v/v), performed under the same conditions not more than 6 months previously, indicated that the assay performed at the testing facility was an appropriate model. Under the study conditions, the test substance was a strong skin sensitiser in mouse (Wil, 2012).

 

 

(Wil,2012).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

An in vivo LLNA study was carried out in accordance with OECD Guideline 429, EU method B.42, EPA OPPTS 870.2600, under GLP conditions in female mice. Three experimental groups of five female CBA/J mice were treated with test substance concentrations of 10, 25 or 50% w/w on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated. 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 and pooled for each animal. After precipitating the DNA of the lymph node cells, radioactivity measurements were performed. No irritation of the ears was observed in the animals at 10%. Very slight erythema was noted at 25% and very slight up to well-defined erythema was noted at 50%. The very slight up to well-defined irritation of the ears as shown by animals treated at 25% and 50% was considered not to have a toxicologically significant effect on the activity of the nodes. All auricular lymph nodes of the test substance treated animals were considered larger in size compared to normal. No macroscopic abnormalities of the surrounding area were noted in any of the animals. Stimulation Index (SI) values calculated for the substance concentrations 10, 25 and 50% were 17.5, 18.9 and 34.0 respectively. These results show that the test substance elicits an SI ≥ 3. The EC3 value (the estimated test substance concentration that will give a SI =3) was established to be between 0 and 10%. Although it was possible to strengthen the outcome of the study by adding lower concentrations. Since the SI values clearly exceeded 3 and since extension of the study would not alter the classification, this was considered not appropriate for ethical reasons. The results of a reliability test with three concentrations of hexylcinnamaldehyde (a positive control substance) in acetone/olive oil (4:1 v/v), performed under the same conditions not more than 6 months previously, indicated that the assay performed at the testing facility was an appropriate model. Under the study conditions, the test substance was a strong skin sensitiser in mouse (Wil, 2012).

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the results of the mouse LLNA assay with the substance, the substance warrants classification as skin sensitizer (Category 1-H317: May cause an allergic skin reaction) according to the EU CLP Regulation (EC) No 1272/2008.