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Diss Factsheets

Administrative data

skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25.05 - 11.07.2011
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was performed according to EC and OECD guidelines and according to GLP principles.

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guidelineopen allclose all
according to guideline
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
according to guideline
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
according to guideline
EPA OPPTS 870.2600 (Skin Sensitisation)
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Chemical structure
Reference substance name:
1,5,2,4-dioxadithiane 2,2,4,4-tetraoxide
EC Number:
Cas Number:
Molecular formula:
1,5,2,4-dioxadithiane 2,2,4,4-tetraoxide
Details on test material:
- Name of test material (as cited in study report): MMDS
- Substance type: organic
- Physical state: White powder
- Analytical purity: Not indicated by the sponsor; treated as 100% pure
- Purity test date: no data
- Lot/batch No.: 100910
- Expiration date of the lot/batch: 20 April 2012
- Stability under test conditions: hydrolyses rapidly, handle in nitrogen environment in glovebox
- Storage condition of test material: In refrigerator (2-8°C) in the dark under nitrogen

In vivo test system

Test animals

Details on test animals and environmental conditions:
- Source: Janvier, Le Genest-Saint-Isle, France
- Age at study initiation: approx. 11 weeks old
- Weight at study initiation: 22-25 g
- Housing: Animals were group housed in labeled Makrolon cages (MIII type; height 18 cm) containing sterilised sawdust as bedding material. Paper and shelters (disposable paper corner home) 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
- Water: Free access to tap water
- Acclimation period: at least 5 days before the start of treatment under laboratory conditions

- Temperature (°C): 21.1 – 23.6ºC
- Humidity (%): 41 - 70%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 hours artificial fluorescent light and 12 hours darkness per day

Study design: in vivo (LLNA)

other: dewatered DMF
0; 0.5; 1.0; 2.5 ( % w/w)
No. of animals per dose:
Details on study design:
A pre-screen test was conducted in order to select the highest test substance concentration to be used in the main study.
Initially, two test substance concentrations were tested: a 50% and 25% concentration. The test system, procedures and techniques were identical to those used in the main study except that assessment of lymph node proliferation and necropsy were not performed, and oedema was scored for all animals at 25 and 50% in the pre-screen test. Two young adult animals per concentration were selected (in the range of 8 to14 weeks old). Each animal was treated with one concentration on three consecutive days. Ear thickness measurements were conducted using a digital thickness gaugeprior to dosing on Days 1 and 3, and on Day 6. Animals were sacrificed after the final observation.
Based on the results of the initially treated animals, two additional animals were treated in a similar manner with three lower concentrations (2.5%, 5% and 10%) at a later stage.

- Name of test method: LLNA
- Criteria used to consider a positive response: If the results indicate a SI ≥ 3, the test substance may be regarded as a skin sensitizer.

Dehydrated N,N-dimethylformamide was used as a suitable vehicle on the basis of maximizing the solubility using the test substance data provided by the sponsor and trial formulation results. The test substance and formulation were handled under nitrogen as much as possible, using a glove box. The test substance formulations (w/w) were prepared within 4 hours prior to each treatment. No adjustment was made for specific gravity of the vehicle. Homogeneity was obtained to visually acceptable levels.

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
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 (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).

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.

Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

Positive control results:
Positive control - reliability check is carried out at regular intervals to check the sensitivity of the test system and the reliability of the experimental techniques. In this study, performed in March 2011, 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 was fabricated under lot no.04012JE (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 1.7, 1.5 and 6.6 respectively. An EC3 value of 14.4% 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 reliability checks of the recent years were13.8, 13.9, 16.0, 11.9, 16.9 and 10.7%. Based on these results it was concluded that the Local Lymph Node Assay as performed at NOTOX is suitable for testing for contact hypersensitivity.

In vivo (LLNA)

Resultsopen allclose all
Remarks on result:
other: The SI values calculated for the substance concentrations 0.5, 1.0 and 2.5% were 3.2, 8.6 and 14.8 respectively.
other: disintegrations per minute (DPM)
Remarks on result:
other: Mean DPM/animal values for the experimental groups treated with test substance concentrations 0.5, 1.0 and 2.5% were 911, 2473 and 4246 DPM respectively. The mean DPM/animal value for the vehicle control group was 288 DPM.

Any other information on results incl. tables

Pre-screen test

Both animals showed up to moderate to severe erythema at 50% and up to well-defined erythema at 25% between Days 2 and 6. White test substance remnants were present on both ears of both animals at 50% between Days 1 and 6, which did not hamper scoring of the skin reactions. No signs of systemic toxicity were observed in any of the animals examined. An increase in ear thickness was recorded up to 63% and 69% from Day 1 pre-dose values at a 25% and 50% concentration, respectively. Since none of the tested concentrations complied with the criteria for selection of the highest test substance concentration to be used in the main study, an additional pre-screen test was initiated at 2.5, 5 and 10%.

In the additional pre-screen test, very slight erythema was observed at 10% for both animals on Days 2 and 3. No irritation was observed at 2.5 and 10%, and no signs of systemic toxicity were noted at any concentration. An increase in ear thickness was recorded up to 41% and 53% from Day 1 pre-dose values at a 5% and 10% concentration, respectively. Variations in ear thickness at 2.5% were less than 20% from Day 1 pre-dose values.

Based on these results, the highest test substance concentration selected for the main study was a 2.5% concentration. Main test Skin reactions / Irritation:

No irritation of the ears was observed in any of the animals examined.

Body weights:

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 several animals across the dose groups was considered not toxicologically significant since the changes were slight in nature and no concentration-related incidence was apparent.

Toxicity and mortality:

No mortality occurred and no symptoms of systemic toxicity were observed in the animals of the main study. Macroscopy of the auricular lymph nodes and surrounding area: Both auricular lymph nodes of all animals at 0.5, 1.0 and 2.5% appeared larger in size when compared to the control group. The largest auricular lymph nodes were found at 2.5%. No macroscopic abnormalities of the surrounding area were noted in any of the animals.

Applicant's summary and conclusion

Interpretation of results:
Migrated information Criteria used for interpretation of results: EU
The SI values calculated for the substance concentrations 0.5, 1.0 and 2.5% were 3.2, 8.6 and 14.8 respectively.
These results indicate that the test substance could elicit an SI ≥ 3. The data showed a dose-response and an EC3 value (the estimated test substance concentration that will give a SI =3) was established to be between 0 and 0.5%
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, the test substance is considered to be skin sensitizer.