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Diss Factsheets

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to OECD and GLP guidelines, and was considered to be relevant, adequate and reliable.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report date:
2015

Materials and methods

Test guidelineopen allclose all
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 (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Reference substance name:
Reaction mass of (1,1’ oxybis(ethylbenzene) and styrene, oligomers
IUPAC Name:
Reaction mass of (1,1’ oxybis(ethylbenzene) and styrene, oligomers
Constituent 2
Reference substance name:
SMPO Heavy Ends
IUPAC Name:
SMPO Heavy Ends
Test material form:
liquid: viscous
Details on test material:
- Name of test material (as cited in study report): SMPO Heavy Ends; Reaction mass of (1,1’ oxybis(ethylbenzene) and styrene, oligomers
- Substance type: UVCB
- Physical state: Brown liquid
- Analytical purity: 100% (UVCB)
- Purity test date: No data
- Lot/batch No.: BC604 17-10-2014
- Expiration date of the lot/batch: No data
- Stability under test conditions: Not indicated
- Storage condition of test material: At room temperature protected from light
- Other: pH (1% in water, indicative range) = 6.2 – 6.7 (determined by WIL Research Europe)

In vivo test system

Test animals

Species:
mouse
Strain:
other: Mouse, CBA/J strain, inbred, SPF-Quality
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, 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.
- Fasting period before study: No data
- Housing: Animals were group housed in labeled Makrolon cages (MIII type; height 18 cm) containing sterilized sawdust as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany). 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.
- 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.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24°C
- Humidity (%):40 to 70%,
- Air changes (per hr): At least 10 air changes/hour
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 14 January 2015 To: 02 February 2015

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
0 (vehicle: acetone/olive oil 4:1 v/v%), 25, 50 and 100 % w/w
No. of animals per dose:
5 females per group (main study only)
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: The vehicle was selected on the basis of maximizing the solubility using the test substance data provided by the sponsor and trial formulation results performed at WIL Research Europe. The vehicle was chosen from the vehicles specified in the test guideline: Acetone/Olive oil (4:1 v/v), N,N-dimethylformamide, methylethylketone, propylene glycol and dimethylsulfoxide.
- Irritation: No irritation and no signs of systemic toxicity were observed in any of the animals examined. Brown staining of the dorsal surface of the ears by test substance remnants was noted all animals between Days 1 and 3. The staining did not hamper the scoring of the ears.
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 100% concentration.
- Lymph node proliferation response: Assessment of lymph node proliferation and necropsy were not performed in pre-screen test.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Test method: Three groups of five animals were treated with one test substance concentration per group. The highest test substance concentration was selected from the pre-screen test. One group of five animals was treated with vehicle.
- Criteria used to consider a positive response: DPM (Desintegrations Per Minute) values are presented for each animal and for each dose group. A Stimulation Index (SI) is calculated for each group using the individual SI values. The individual SI is the ratio of the DPM/animal 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 (2011) (including all amendments) 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, including all amendments.
Consideration was given to the EC3 value (the estimated test substance concentration that will give a SI =3).

TREATMENT PREPARATION AND ADMINISTRATION:
The test substance formulations (w/w) were prepared within 4 hours prior to each dosing. No adjustment was made for specific gravity of the vehicle. Homogeneity was obtained to visually acceptable levels.
Correction of the purity/composition of the test substance is not applicable, since the test method requires a logical concentration range rather than specific dose levels to be dosed.
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.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

Positive control results:
A reliability check is carried out at regular intervals to check the sensitivity of the test system and the reliability of the experimental techniques as used by WIL Research Europe. In this study, performed in November 2014, females of the CBA/J mouse strain (Janvier, Le Genest-Saint-Isle, France) were checked for sensitivity to Hexylcinnamaldehyde. The females were approx. 10 weeks old at commencement of the study. The study was based on the OECD Guideline No. 429, EC No 440/2008, Part B.42 and EPA, OPPTS 870.2600 “Skin Sensitization”. Alpha- Hexylcinnamaldehyde, technical grade (CAS no. 101-86-0) was fabricated under lot no. MKBJ8846V (Sigma- Aldrich, Steinheim, Germany). Concentrations used for this study were 5, 10 and 25% in Acetone/Olive oil (4:1 v/v).
The SI values calculated for the substance concentrations 5, 10 and 25% were 1.1, 3.1 and 3.7 respectively. An EC3 value of 9.8% was calculated using linear interpolation.
The calculated EC3 value was found to be in the acceptable range of 4.8 and 19.5%. The results of the 6 monthly HCA reliability checks of the recent years were 14.4, 16.5, 14.5, 13.4, 14.1 and 17.3%.
Based on the results, it was concluded that the Local Lymph Node Assay as performed at WIL Research Europe is an appropriate model for testing for contact hypersensitivity.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: The SI values calculated for the substance concentrations 25, 50 and 100% were 9.1, 10.2 and 21.6, respectively.
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 4753, 5300 and 11214 DPM, respectively. The mean DPM/animal value for the vehicle control group was 520 DPM.

Any other information on results incl. tables

Pre-screen test

No irritation and no signs of systemic toxicity were observed in any of the animals examined. Brown staining of the dorsal surface of the ears by test substance remnants was noted all animals between Days 1 and 3. The staining did not hamper the scoring of the ears.

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 100% concentration.

Main study

No irritation of the ears was observed in any of the animals examined. Brown staining of the dorsal surface of the ears by test substance remnants was noted all experimental animals between Days 1 and 3. The staining did not hamper the scoring of the ears.

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.

All auricular lymph nodes of the animals of the control group were considered normal in size. The nodes of all experimental animals were considered enlarged. The largest auricular lymph nodes were found in the higher dose groups.

No macroscopic abnormalities of the surrounding area were noted for any of the animals.

Mean DPM/animal values for the experimental groups treated with test substance concentrations 25, 50 and 100% were 4753, 5300 and 11214 DPM, respectively. The mean DPM/animal value for the vehicle control group was 520 DPM. The SI values calculated for the substance concentrations 25, 50 and 100% were 9.1, 10.2 and 21.6, respectively.

Applicant's summary and conclusion

Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Remarks:
Migrated information: Category 1
Conclusions:
Based on the study results:
- according to the recommendations made in the test guidelines (including all amendments), SMPO Heavy ends would be regarded as skin sensitizer.
- according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2011) (including all amendments), SMPO Heavy ends 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 (including all amendments), SMPO Heavy ends should be classified as skin sensitizer (Category 1) and labelled as H317: May cause an allergic skin reaction.
Executive summary:

SMPO Heavy ends was tested in the Mouse Local Lymph Node Assay according to OECD No.429. Test substance concentrations selected for the main study were based on the results of a pre-screen test. In the main study, three experimental groups of five female CBA/J mice were treated with test substance concentrations of 25, 50 or 100% w/w on three consecutive days, by open application on the ears. 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 and pooled for each animal. 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 stimulation index (SI) was subsequently calculated for each group. No irritation of the ears was observed in any of the animals examined.

All auricular lymph nodes of the animals of the control group were considered normal in size. The nodes of all experimental animals were considered enlarged. The largest auricular lymph nodes were found in the higher dose groups. No macroscopic abnormalities of the surrounding area were noted for any of the animals.

Mean DPM/animal values for the experimental groups treated with test substance concentrations 25, 50 and 100% were 4753, 5300 and 11214 DPM, respectively. The mean DPM/animal value for the vehicle control group was 520 DPM. The SI values calculated for the substance concentrations 25, 50 and 100% were 9.1, 10.2 and 21.6, respectively. These results indicate that the test substance could elicit a SI = 3. The data showed a dose-response and the EC3 value (the estimated test substance concentration that will give a SI =3) was established to be between >0 and 25%.

Based on these results, according to the Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures (including all amendments), SMPO Heavy ends should be classified as skin sensitizer (Category 1) and labelled as H317: May cause an allergic skin reaction.

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