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Description of key information

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:
March - June 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study following OECD method without significant deviations
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
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:
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 (main study only).
Age: Young adult animals (approx. 10 weeks old) were selected.
Body weight: Body weight variation was within +/- 20% of the sex mean.
Identification: Tail mark with marker pen.
Health inspection: At least prior to dosing. It was ensured that the animals were healthy and that the ears were intact and free from any abnormality.
Animal husbandry
Conditions: Environmental controls for the animal room were set to maintain 18 to 24 °C, a relative humidity of 40 to 70%, at least 10 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.
Accommodation: Animals were group housed in labeled Makrolon cages (MIII type; height 18 cm) containing sterilised 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: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
Water: Free access to tap water.
Diet, water, bedding and cage enrichment evaluations for contaminants and/or nutrients were performed according to facility standard procedures. There were no findings that could interfere with the study.
Vehicle:
methyl ethyl ketone
Remarks:
(sourced from Merck, Darmstadt, Germany)
Concentration:
Two test substance concentrations were tested in the pre-screen; a 25% and 40% concentration. The highest concentration was the highest possible concentration that could technically be applied.
In the main study concentraions of 10%, 25% and 40% were tested accordingly.
No. of animals per dose:
Five females per group (main study only)
Details on study design:
A pre-screen test was conducted in order to select the highest test item concentration to be used in the main study. In principle, this highest concentration should cause no systemic toxicity, may give well-defined irritation as the most pronounced response (maximum grade 2 (see section 6.7) and/or an increase in ear thickness < 25%) and/or is the highest possible concentration that can technically be applied.
Two test substance concentrations were tested; a 25% and 40% concentration. The highest concentration was the highest possible concentration that could technically be applied.
The test system, procedures and techniques were identical as those used in the main study except that the animals were approximately 11 weeks (at initiation of treatment) and that the assessment of lymph node proliferation and necropsy were not performed. Two young adult animals per concentration were selected. Each animal was treated with one concentration on three consecutive days. Animals were group housed in labeled Makrolon cages (MII type, height 14 cm). Ear thickness measurements were conducted using a digital thickness gauge (Kroeplin C110T-K) prior to dosing on Days 1 and 3, and on Day 6.
Animals were sacrificed after the final observation.
Main study
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.
Allocation:
Group 1: 1 - 5 animals, 0 % w/w test substance in MEK
Group 2: 06 - 10 animals, 10 % w/w test substance in MEK
Group 3: 11 - 15 animals, 25 % w/w test substance in MEK
Group 4: 16 - 20 animals, 40 % w/w test substance in MEK
Induction - Days 1, 2 and 3
The dorsal surface of both ears was topically treated (25 μL/ear) with the test substance, 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 five hours, all animals were killed by intraperitoneal injection (0.2 mL/animal) of 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
Following excision of the nodes, 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 4 °C. To precipitate the DNA, the LNC were exposed to 5% trichloroacetic acid (TCA) (Merck, Darmstadt, Germany) and then 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. Radioactivity 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.
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 within 1 hour after dosing) according to the following numerical scoring system. Furthermore, a description of all other (local) effects was recorded.
Grading Irritation Reactions (Erythema and eschar formation):
No erythema 0
Very slight erythema (barely perceptible) 1
Well-defined erythema 2
Moderate to severe erythema (beet redness) to slight eschar formation (injuries in depth) 3
Severe erythema (beet redness) to eschar formation preventing grading of erythema 4
Necropsy: No necropsy for gross macroscopic examination was performed according to protocol.
Electronic data capture: Observations/measurements in the study were recorded electronically using the following programs: REES Centron Environmental Monitoring system version SQL 2.0 (REES scientific, Trenton, NJ, USA); Quantasmart 2.03 (PerkinElmer Life Sciences, Boston, MA, USA): LSC software.
Interpretation: DPM 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 the DPM/vehicle control group mean. 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).
List of protocol deviations: There were no deviations from the protocol.
List of standard operating procedures deviations: Any deviations from standard operating procedures were evaluated and filed in the study file. There were no deviations from standard operating procedures that affected the integrity of the study.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
None applied as no effects could be seen
Positive control results:
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.
Parameter:
SI
Remarks on result:
other: The SI values calculated for the substance concentrations 10, 25 and 40% were 1.3, 1.3 and 1.0, respectively.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Mean DPM/animal values for the experimental groups treated with test substance concentrations 10, 25 and 40% were 891, 872 and 691 DPM, respectively. The mean DPM/animal value for the vehicle control group was 677 DPM.

Pre-screen test: No irritation and no signs of systemic toxicity were observed in any of the animals examined. White staining of the dorsal surface of the ears by test substance remnants was noted for all animals on Days 1, 2 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 40% concentration.

Main study: No irritation of the ears was observed in any of the animals examined. White staining of the dorsal surface of the ears by test substance remnants was noted for all experimental animals on Days 1, 2 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.

Macroscopy of the auricular lymph nodes and surrounding area: All auricular lymph nodes of the animals of the experimental and control groups were considered normal in size. No macroscopic abnormalities of the surrounding area were noted for any of the animals.

Radioactivity measurements and SI values: Mean DPM/animal values for the experimental groups treated with test substance concentrations 10, 25 and 40% were 891, 872 and 691 DPM, respectively. The mean DPM/animal value for the vehicle control group was 677 DPM. The SI values calculated for the substance concentrations 10, 25 and 40% were 1.3, 1.3 and 1.0, respectively.

Interpretation of results:
GHS criteria not met
Remarks:
Migrated information
Conclusions:
The test substance was found not being sensitising to skin in the local lymph node assay (LLNA).
Executive summary:

In the main study, three experimental groups of five female CBA/J mice were treated with test substance concentrations of 10, 25 or 40% w/w on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with vehicle alone (Methyl ethyl ketone).

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. 

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 experimental and control groups were considered normal in size. 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 10, 25 and 40% were 891, 872 and 691 DPM, respectively. The mean DPM/animal value for the vehicle control group was 677 DPM. The SI values calculated for the substance concentrations 10, 25 and 40% were 1.3, 1.3 and 1.0, respectively.

Since there was no indication that the test substance elicits a SI ≥ 3 when tested up to 40%, D-C16 was not considered to be a skin sensitizer. It was established that the EC3 value (the estimated test substance concentration that will give a SI =3) (if any) exceeds 40%.

Based on these results, D-C16 would not be regarded as a skin sensitizer according to the recommendations made in the test guidelines. The test substance does not have to be classified and has no obligatory labelling requirement for sensitization by skin contact 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 on classification, labelling and packaging of substances and mixtures (including all amendments).

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

In a LLNA study (OECD 429), three experimental groups of five female CBA/J mice were treated with test substance concentrations of 10, 25 or 40% w/w on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with vehicle alone (Methyl ethyl ketone). 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, 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 and all auricular lymph nodes of the animals of the experimental and control groups were considered normal in size. 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 10, 25 and 40% were 891, 872 and 691 DPM, respectively and for for the vehicle control group was 677 DPM. The SI values calculated for the substance concentrations 10, 25 and 40% were 1.3, 1.3 and 1.0, respectively.

Since there was no indication that the test substance elicits a SI ≥ 3 when tested up to 40%, D-C16 was not considered to be a skin sensitizer.


Migrated from Short description of key information:
The test substance was found not being sensitising to skin.

Justification for selection of skin sensitisation endpoint:
Fully compliant GLP study following OECD method without significant deviations

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the negative outcome of a LLNA study, the test substance is not considered being sensitising to skin and thus requires no classification for skin sensitisation according to GHS or Regulation EU 1272/2008 (CLP).