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Administrative data

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:
April-May 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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:
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 and a relative humidity of 40-70% and 12 hours artificial fluorescent light and 12 hours darkness per day.
For the main study, the actual temperature range was 19.9 – 22.6ºC and the actual relative humidity range was 40 - 56%.

Accommodation
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.

Acclimatization period
The acclimatization period was at least 5 days before the start of treatment under laboratory conditions. Accommodation was as described above except that the animals were group housed in Macrolon cages (MIII type; height 18 cm).

Diet
Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).

Water
Free access to tap water.
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
0, 5, 25 and 100%
No. of animals per dose:
5
Details on study design:
In the range-finding test, 50 and 100% was tested each on one animal. Slight irritation of the ears was noted in the animal treated at 50%. Based on these results, the highest test substance concentration selected for the main study was a 100% concentration.

Induction - Days 1, 2 and 3
The dorsal surface of both ears was epidermally topically treated (25 μL/ear) with the test substance concentration, at approximately the same time on each per day. The concentrations were mixed thoroughly using a vortex mixer immediately prior to dosing.
The vehicle 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
All animals:
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 the controlnormal ) 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 substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Not needed
Parameter:
SI
Value:
1.3
Test group / Remarks:
5%
Parameter:
SI
Value:
1.5
Test group / Remarks:
25%
Parameter:
SI
Value:
1.7
Test group / Remarks:
100%
Interpretation of results:
GHS criteria not met
Conclusions:
The SI values calculated for the substance concentrations 5, 25 and 100% were 1.3, 1.5 and 1.7 respectively. Since there was no indication that the test substance elicits an SI ≥ 3 when tested up to 100%, dichloromethane was considered not to be a skin sensitizer.
Executive summary:

The study was carried out based on the guidelines described in:

OECD, Section 4, Health Effects, No.429 (2002),

EC, No 440/2008; B42: "Skin Sensitization: Local Lymph Node Assay"

EPA, OPPTS 870.2600 (2003) “Skin Sensitization”.

Test substance concentrations selected for the main study were based on the results of a preliminary study.

In the main study, three experimental groups of five female CBA/J mice were treated with test substance concentrations of 5, 25 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 with3H-methyl thymidine and after five hours the draining (auricular) lymph nodes were excised and pooled for each animal. After precipitating theof 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.

The slight irritation of the ears as shown by several animals treated at 25 and 100% was considered not to have a toxicologically significant effect on the activity of the nodes. All auricular lymph nodes of the animals of the experimental and control groups were considered normal in size. Body weights and body weight gain of experimental animals remained in the same range as controls over the study period. Mean DPM/animal values for the experimental groups treated with test substance concentrations 5, 25 and 100% were 534, 648 and 735 DPM respectively. The mean DPM/animal value for the vehicle control group was 423 DPM. The SI values calculated for the substance concentrations 5, 25 and 100% were 1.3, 1.5 and 1.7 respectively. Since there was no indication that the test substance elicits an SI3 when tested up to 100%, DICHLOROMETHANE was considered not to be a skin sensitizer.

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.

Based on these results DICHLOROMETHANE would not be regarded as a skin sensitizer according to the recommendations made in the test guidelines. It does not have to be classified and has no obligatory labeling requirement for sensitization by skin contact according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations (2007) and theRegulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures.

 

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

A LLNA study in mice showed that dichloromethane has no sensitising properties. In view of the solvent's extensive, widespread and long-standing use, and the scarcity of published evidence in the area of skin sensitization indicates that DCM does not possess any significant sensitising potential.

According to MAK (2016) As there are no positive findings for skin and respiratory sensitization in humans to date, and the results of a local lymph node assay in mice were negative, dichloromethane is not designated with “Sh” or “Sa”(for substances which cause sensitization of the skin or airways).

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available
Additional information:

A single case of asthma or reactive airways dysfunction syndrome was mentioned (Sallie et al., 1996; see section 7.10.3). However, respiratory sensitization is a term that is used to describe asthma irrespective of the mechanism – immunological or non-immunological – by which this is caused. The difficulty that this definition causes is that it serves to embrace both true respiratory allergens which, by definition, induce effects via immunological mechanisms, and non-allergenic asthmagens in which case adverse effects are caused by non-immunological mechanisms. In a recent GLP LLNA study (2010), the SI values calculated for the substance concentrations of 5, 25 and 100% were 1.3, 1.5 and 1.7 respectively. Since there was no indication that the test substance elicits an SI ≥3 when tested up to 100%, DCM was considered not tobe a skin sensitiser. In addition, chemicals that test negative in the LLNA can be eliminated with confidence from further consideration as possible chemical respiratory allergens (Dearman et al., 2013). Moreover, in view of its long-standing and wide use no respiratory sensitisation has been reported.

According to MAK (2016): As there are no positive findings for skin and respiratory sensitization in humans to date, and the results of a local lymph node assay in mice were negative, dichloromethane is not designated with “Sh” or “Sa”(for substances which cause sensitization of the skin or airways).

Justification for classification or non-classification

Based on the available data, classification for skin and respiratory tract sensitisation is not warranted according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.