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EC number: 237-377-8 | CAS number: 13767-32-3
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
- Nanomaterial porosity
- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
Skin sensitisation
Administrative data
- Endpoint:
- skin sensitisation: in vivo (LLNA)
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 14 November 2018 - 19 November 2018
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 019
- Report date:
- 2019
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 442B (Skin Sensitization: Local Lymph Node Assay: BrdU-ELISA)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: EU Method B.51 (Skin sensitisation. local lymph node assay: BrdU-ELISA)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of study:
- mouse local lymph node assay (LLNA): BrdU-ELISA
Test material
- Reference substance name:
- Molybdenum zinc tetraoxide
- EC Number:
- 237-377-8
- EC Name:
- Molybdenum zinc tetraoxide
- Cas Number:
- 13767-32-3
- Molecular formula:
- MoO4Zn
- IUPAC Name:
- molybdenum(6+) zinc(2+) tetraoxidandiide
- Test material form:
- solid: particulate/powder
Constituent 1
In vivo test system
Test animals
- Species:
- mouse
- Strain:
- CBA:J
- Remarks:
- CBA/JRj
- Sex:
- female
- Details on test animals and environmental conditions:
- TEST ANIMALS
- Source: Elevage Janvier Labs (F-53941 Le Genest Saint Isle).
- Females (if applicable) nulliparous and non-pregnant: yes.
- Age at study initiation: 8 weeks old.
- Weight at study initiation: average weight 19.5-24.4 g (treated groups and control group)
- Housing: Individually (to avoid any test item absorption by oral route) in a suspended solid-floor polypropylene cages furnished with softwood woodflakes.
- Diet: Ad libitum (Enviogo 2016)
- Water: Ad libitum (tap water)
- Acclimation period: at least 5 days
- Indication of any skin lesions: no
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19ºC-25ºC.
- Humidity (%): 30%-70%.
- Air changes (per hr): 10 changes/hour
- Photoperiod (hrs dark / hrs light): 12 h light (7.00 to 19.00) / 12 h darkness
Study design: in vivo (LLNA)
- Vehicle:
- acetone/olive oil (4:1 v/v)
- Concentration:
- 80%, 50% and 25%.
- No. of animals per dose:
- 4
- Details on study design:
- PRE-SCREEN TESTS: A preliminary screening test was performed using one mouse. The mouse was treated by daily application of 25 μL of the test item diluted at 90% in Acetone/olive oil (4:1 v/v) (AOO) to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The mouse was observed daily from day 1 to day 6. The bodyweight of the mice were recorded on Day 1 (prior to dosing) and on Day 6.
- Compound solubility: A preliminary solubility test was performed and Acetone/olive oil (4:1 v/v) was chosen as vehicle since it produced the most suitable formulation at the required concentration.
- Irritation: No sign of excessive irritation was noted at the tested concentration of 80%.
- Systemic toxicity: No mortality and no signs of systemic toxicity were noted.
- Ear thickness measurements: values were within the acceptable range.
- Erythema scores: no signs of erithema were observed.
MAIN STUDY
Groups of four mice were treated with the test item diluted at 80%, 50% and 25% in Acetone/olive oil (4:1, v/v) based on the results of the pre-screen test. The mice were treated by daily application of 25 µL of the appropriate concentration of the test item to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3).
- Clinical observations: all animals were observed daily on Days 1, 2, 3 4, 5 and 6. Any signs of toxicity or signs of ill health during the test were recorded.
- Body weight: the bodyweight of each mouse was recorded on Day 1 (prior to dosing) and Day 6 (prior to termination).
- Ear thickness: On day 1 and on day 3 (before application) as well as on day 6 (after sacrifice) of each experiment, the thickness of the right ear of each animal of the vehicle control and treated groups was measured by a micrometer. Furthermore, on day 6, punch biopsies of 8 mm in diameter of the apical area of both ears were prepared and weighted in order to assess the irritation potential of the test item and the two lymph nodes per mouse were weighed.
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Skin sensitisation. Local Lymph Node Assay:BrdU-ELISA
- Criteria used to consider a positive response: BrdU was measured by ELISA using a commercial kit (Roche Applied Science, Mannheim, Germany, Catalogue Number 11 647 229 001 - Batch No. 29134900). 100 µL of the suspension of lymph node cells (LNC) was added to the wells of a flat-bottom microplate in triplicate. After fixation and denaturation of the LNC, anti-BrdU antibody was added to each well and allowed to react. Subsequently the anti-BrdU antibody was removed by washing and the substrate solution was then added and allowed to produce chromogen. After 5 to 30 min, 30 µL of 1 M H2SO4 was added in each well, then shaken for one minute. Absorbance at 450 nm with a reference wavelength of 690 nm was then measured.
The BrdU labelling index was defined as: BrdU labelling index = (ABSem - ABS blankem) - (ABSref - ABS blankref)
The test item will be regarded as a sensitiser if at least one concentration of the test item results is greater than 1.6 compared to control values.
However, the strength of the dose-response relationship, the statistical significance and the consistency of the solvent/vehicle and positive control responses may also be used when determining whether a borderline result (SI value between 1.6 and 1.9) is declared positive. Any test item failing to produce a SI>1.6 will be classified as a "non-sensitiser".
The EC1.6 value (theoretical concentration resulting in a SI value of 1.6) was detemined by linear interpolation of points on the dose-response curve, immediately above and below the 1.6 -fold threshold. The equation used for calculation of EC1.6 was:
EC1.6 = c + [(1.6 - d) / (b - d)] x (a - c)
Legend: a = the lowest concentration giving stimulation index > 1.6
b = the actual stimulation index caused by a
c = the highest concentration failing to produce a stimulation index of 1.6
d = the actual stimulation index caused by c
According to Regulation (EC) No. 286/2011, the positive test item will be classified in subcategory 1A or 1B in accordance with:
If the EC value ≤ 2, the test item will be classified in "sub-category 1A".
If the EC value > 2, the test item will be classified in "sub-category 1B".
TREATMENT PREPARATION AND ADMINISTRATION:
The test item was freshly prepared in Acetone/olive oil (4:1, v/v). Groups of four mice were treated with the test item diluted at 80%, 50% and 25% in Acetone/olive oil (4:1, v/v). The mice were treated by daily application of 25 µL of the appropiate concentration of the test item to the dorsal surface of each ear for three consecutive days (Days 1,2,3). The test item formulation was administered using an automatic micropipette and spread over the dorsal surface of the ear using the tip of the pipette. A further group of four mice received the vehicle alone in the same manner.
On day 5 (5 mg/mouse) of BrdU (10 mg/mL) solution was injected by intra-peritoneal route.
The Brdu solution was prepared by weighing 206.90 mg of 5-bromo-2'-deoxyuridine (SIGMA – Batch No. HMBF7815V) in a glass sample bottle adding 20.69 mL of physiological saline. The preparations were magnetically stirred during 10 min.
On day 6 (end of the test), the animals were euthanized with sodium pentobarbital (Dolethal®). The draining auricular lymph nodes from the four mice were excised.
From each mouse, a single-cell suspension through of lymph node cells (LNC) excised bilaterally was prepared by gentle mechanical disaggregation through a disposable plastic pestle to crush the lymph nodes followed by passage through a #70 nylon mesh in 15 mL of PBS (Ca2 +/Mg2+ - free) into a well of a multi-well 6. The optimised volume was based on achieving a mean absorbance of the negative control group within 0.1 -0.2. - Positive control substance(s):
- hexyl cinnamic aldehyde (CAS No 101-86-0)
Results and discussion
- Positive control results:
- EC1.6= 24.66%. The substance has to be classified in category 1 "Skin sensitisation".
In vivo (LLNA)
Resultsopen allclose all
- Key result
- Parameter:
- SI
- Value:
- 0.92
- Test group / Remarks:
- 25%
- Key result
- Parameter:
- SI
- Value:
- 0.88
- Test group / Remarks:
- 50%
- Key result
- Parameter:
- SI
- Value:
- 0.82
- Test group / Remarks:
- 80%
- Cellular proliferation data / Observations:
- CELLULAR PROLIFERATION DATA
No increase in ear thickness and in ear weight was noted in animals treated at 25%, 50% and 80%.
DETAILS ON STIMULATION INDEX CALCULATION
No stimulation index of more than 1.6 was recorded whatever the tested concentration. The Stimulation Index (SI) calculated by individual approach was 0.92, 0.88 and 0.82 for the treated groups at 25%, 50% and 80%, respectively.
EC1.6 CALCULATION
The EC1.6 cannot be determined in this study.
CLINICAL OBSERVATIONS:
No mortality and no signs of systemic toxicity were noted in the test and control animals during the test.
Residual test item was noted in all animals treated at 50% between Days 2 and 5.
No increase in ear thickness and in ear weight was noted in animals treated at 25%, 50% and 90%.
Therefore, the test item has to be considered as not excessively irritant at these concentrations.
BODY WEIGHTS
Bodyweight changes of the test animals between Day 1 and Day 6 were comparable to those observed in the corresponding control group animals over the same period.
Any other information on results incl. tables
Table 1. Preliminary screening test: Clinical observation, bodyweight and mortality data
Concentration % |
Animal |
Bodyweight (g) |
DAY |
||||||
Day 1 |
Day 6 |
1 |
2 |
3 |
4 |
5 |
6 |
||
80% |
Sf1218 |
21.2 |
22.3 |
0 |
0 |
0 |
0 |
0 |
0 |
0: No sign of systemic toxicity and no sign of erythema
|
Ear thickness (mm) on day 1 |
Ear thickness (mm) on day 3 |
Ear thickness (mm) on day 6 |
Ear weight (mg) on day 6 |
Weight Lymph nodes (mg) |
Sf1218 |
0.21 |
0.21 |
0.21 |
28.4 |
5.3 |
The concentration of 80% was chosen as the highest concentration for the main study.
Table 2. Main study: Individual clinical observation and mortality data
Groups |
Test item |
Amimals |
Day 1 |
Day 2 |
Day 3 |
Day 4 |
Day 5 |
Day 6 |
1 |
AOO |
Nº Sf 1284 |
0 |
0 |
0 |
0 |
0 |
0 |
Nº Sf 1285 |
0 |
0 |
0 |
0 |
0 |
0 |
||
Nº Sf 1286 |
0 |
0 |
0 |
0 |
0 |
0 |
||
Nº Sf 1287 |
0 |
0 |
0 |
0 |
0 |
0 |
||
2 |
10% |
Nº Sf 1289 |
0 |
0 |
0 |
0 |
0 |
0 |
Nº Sf 1290 |
0 |
0 |
0 |
0 |
0 |
0 |
||
Nº Sf 1291 |
0 |
0 |
0 |
0 |
0 |
0 |
||
Nº Sf 1292 |
0 |
0 |
0 |
0 |
0 |
0 |
||
3 |
25% |
Nº Sf 1294 |
0 |
0* |
0* |
0* |
0* |
0 |
Nº Sf 1295 |
0 |
0* |
0* |
0* |
0* |
0 |
||
Nº Sf 1296 |
0 |
0* |
0* |
0* |
0* |
0 |
||
Nº Sf 1297 |
0 |
0* |
0* |
0* |
0* |
0 |
||
4 |
50% |
Nº Sf 1299 |
0 |
0 |
0 |
0 |
0 |
0 |
Nº Sf 1300 |
0 |
0 |
0 |
0 |
0 |
0 |
||
Nº Sf 1301 |
0 |
0 |
0 |
0 |
0 |
0 |
||
Nº Sf 1303 |
0 |
0 |
0 |
0 |
0 |
0 |
0: No sign of systemic toxicity
*: Residual test item
AOO: Acetone/olive oil (4:1 v/v)
Table 3. Individual body weight and body weight gain
Groups |
Test item |
Animals No. |
Body weight (g) |
Body weight gain (g) |
|
Day 1 |
Day 6 |
||||
1 |
AOO |
Sf1284 |
20.8 |
21.4 |
0.6 |
Sf1285 |
20.4 |
20.2 |
-0.2 |
||
Sf1286 |
20.6 |
20.5 |
-0.1 |
||
Sf1287 |
20.6 |
21.3 |
0.7 |
||
MEAN |
20.6 |
20.9 |
0.2 |
||
Standard deviation |
0.2 |
0.6 |
0.5 |
||
2 |
25% |
Sf1289 |
20.8 |
21.6 |
0.8 |
Sf1290 |
21.8 |
22.8 |
1.0 |
||
Sf1291 |
21.2 |
22.0 |
0.8 |
||
Sf1292 |
20.6 |
20.6 |
0.0 |
||
MEAN |
21.1 |
21.8 |
0.7 |
||
Standard-deviation |
0.5 |
0.9 |
0.4 |
||
3 |
50% |
Sf1294 |
21.2 |
21.5 |
0.3 |
Sf1295 |
21.7 |
21.6 |
-0.1 |
||
Sf1296 |
24.4 |
22.4 |
-2.0 |
||
Sf1297 |
22.8 |
23.3 |
0.5 |
||
MEAN |
22.5 |
22.2 |
-0.3 |
||
Standard-deviation |
1.4 |
0.8 |
1.1 |
||
4 |
80% |
Sf1299 |
20.2 |
20.4 |
0.2 |
Sf1300 |
22.1 |
22.0 |
-0.1 |
||
Sf1301 |
19.8 |
19.9 |
0.1 |
||
Sf1303 |
19.5 |
19.6 |
0.1 |
||
MEAN |
20.4 |
20.5 |
0.1 |
||
Standard-deviation |
1.2 |
1.1 |
0.1 |
AOO: Acetone/olive oil
Table 4. BrdU index & Stimulation index per group and calculation of EC1.6
Groups |
Test item |
BrdU-index (mean*) |
Stimulation Index SI (mean + standard deviation) |
Result |
EC1.6 value |
1 |
AOO |
0.589 |
n.a. |
n.a. |
n.a. |
2 |
25% |
0.540 |
0.92±0.10 |
negative |
n.a |
3 |
50% |
0.520 |
0.88±0.04 |
negative |
|
4 |
80% |
0.482 |
0.82±0.03 |
negative |
*: mean:Σindividual value / 4
AOO: Acetone/olive oil
Table 5. Individual Ear thickness and irritation level.
Groups |
Test item |
Animals No. |
Day 1 |
Day 3 |
Day 6 |
Ear thickness increase D3/D1 |
Ear thickness increase D6/D1 |
|
1 |
AOO |
Sf1284 |
0.21 |
0.21 |
0.21 |
0.0 |
0.0 |
|
Sf1285 |
0.21 |
0.21 |
0.21 |
0.0 |
0.0 |
|||
Sf1286 |
0.21 |
0.21 |
0.21 |
0.0 |
0.0 |
|||
Sf1287 |
0.21 |
0.21 |
0.21 |
0.0 |
0.0 |
|||
MEAN |
0.21 |
0.21 |
0.21 |
0.0 |
0.0 |
|||
Standard-deviation |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
|||
2 |
25% |
Sf1289 |
0.20 |
0.21 |
0.20 |
5.0 |
0.0 |
|
Sf1290 |
0.20 |
0.21 |
0.21 |
5.0 |
5.0 |
|||
Sf1291 |
0.21 |
0.21 |
0.20 |
0.0 |
-4.8 |
|||
Sf1292 |
0.21 |
0.21 |
0.20 |
0.0 |
-4.8 |
|||
MEAN |
0.21 |
0.21 |
0.20 |
2.5 |
-1.1 |
|||
Standard-deviation |
0.01 |
0.00 |
0.00 |
2.9 |
4.7 |
|||
3 |
50% |
Sf1294 |
0.21 |
0.21 |
0.19 |
0.0 |
-9.5 |
|
Sf1295 |
0.20 |
0.21 |
0.20 |
5.0 |
0.0 |
|||
Sf1296 |
0.21 |
0.21 |
0.21 |
0.0 |
-4.8 |
|||
Sf1297 |
0.21 |
0.21 |
0.21 |
0.0 |
0.0 |
|||
MEAN |
0.21 |
0.21 |
0.20 |
1.0 |
-3.6 |
|||
Standard-deviation |
0.00 |
0.00 |
0.01 |
2.5 |
4.6 |
|||
4 |
80% |
Sf1299 |
0.21 |
0.21 |
0.20 |
0.0 |
-4.8 |
|
Sf1300 |
0.20 |
0.21 |
0.21 |
5.0 |
5.0 |
|||
Sf1301 |
0.21 |
0.21 |
0.21 |
0.0 |
0.0 |
|||
Sf1303 |
0.21 |
0.21 |
0.19 |
0.0 |
-9.5 |
|||
MEAN |
0.21 |
0.21 |
0.20 |
1.3 |
-2.3 |
|||
Standard-deviation |
0.00 |
0.00 |
0.01 |
2.5 |
6.2 |
AOO: Acetone/olive oil
Table 6. Individual Ear biopsy weight and lymph node weight.
Groups |
Test item |
Animals No. |
ear weight |
% of ear weight |
Lymph nodes (mg) |
|
1 |
AOO |
Sf1284 |
28.1 |
|
5.7 |
|
Sf1285 |
28.2 |
5.3 |
||||
Sf1286 |
29.2 |
4.9 |
||||
Sf1287 |
26.8 |
5.0 |
||||
MEAN |
28.1 |
5.2 |
||||
Standard-deviation |
1.0 |
0.4 |
||||
2 |
25% |
Sf1289 |
26.3 |
-0.6 |
6.1 |
|
Sf1290 |
30.2 |
5.2 |
||||
Sf1291 |
29.0 |
5.4 |
||||
Sf1292 |
26.1 |
6.7 |
||||
MEAN |
27.9 |
5.9 |
||||
Standard-deviation |
2.0 |
0.7 |
||||
3 |
50% |
Sf1294 |
28.4 |
1.2 |
7.6 |
|
Sf1295 |
30.1 |
5.2 |
||||
Sf1296 |
27.5 |
5.7 |
||||
Sf1297 |
27.6 |
5.8 |
||||
MEAN |
28.4 |
6.1 |
||||
Standard-deviation |
1.2 |
1.1 |
||||
4 |
800% |
Sf1299 |
28.2 |
-2.1 |
4.9 |
|
Sf1300 |
26.0 |
5.0 |
||||
Sf1301 |
27.9 |
4.9 |
||||
Sf1303 |
27.8 |
5.5 |
||||
MEAN |
27.5 |
5.1 |
||||
Standard-deviation |
1.0 |
0.3 |
AOO: Acetone/olive oil
Table 7. Summary of result – skin irritation
Groups |
Test item |
Ear thickness increase D6/D1 (%) |
Biopsy ear weight Increase (%) |
Excessive irritation# |
1 |
AOO |
0.0 |
n.a |
No |
2 |
25% |
-1.1 |
-0.6 |
No |
3 |
50% |
-3.6 |
1.2 |
No |
4 |
80% |
-2.3 |
-2.1 |
No |
#: O.E.C.D. criteria: (% increase in ear thickness higher than 25%, score of erythema higher than 3)
AOO: Acetone/olive oil
Table 8. BrdU index & Stimulation index per group and calculation of EC1.6 of the positive control (study performed 04 July 2018 – 11 July 2018)
Groups |
Test item |
BrdU-index (mean*) |
Stimulation Index SI (mean + standard deviation) |
Result |
EC1.6 value |
1 |
AOO |
0.637 |
n.a. |
n.a. |
n.a. |
2 |
5% |
0.614 |
0.96±0.10 |
negative |
24.66% |
3 |
10% |
0.744 |
1.17±0.17 |
negative |
|
4 |
25% |
1.023 |
1.61±0.20 |
positive |
AOO: Acetone/olive oil
Applicant's summary and conclusion
- Interpretation of results:
- GHS criteria not met
- Conclusions:
- The test item does not have to be classified as a skin sensitizer, under the tested conditions in the LLNA assay (OECD 442B). The Stimulation Index (SI) calculated by individual approach were 0.92, 0.88 and 0.82 at concentrations of test item 25%, 50% and 80%, respectively.
- Executive summary:
The skin sensitisation potential of the test item was tested in the LLNA assay according to OECD 442B and E.U. B.51 method, following GLP. Firstly, a preliminary screening test was performed using one mouse treated with 25 μL of the test item diluted at 90% in Acetone/olive oil (4:1 v/v) for three consecutive days. No mortality or signs of systemic toxicity and no signs of excessive irritation were noted in the preliminary study. In the main test, three groups of four mouse CBA/J were treated for three consecutive days with 50 µL (25 µL per ear) of the test item diluted at concentrations of 25%, 50% and 80% in Acetone/olive oil (4:1, v/v). A control group was treated with Acetone/olive oil (4:1, v/v). On day 5, 0.5 mL of BrdU solution (10mg/mL) was injected by intraperitoneal route. On day 6, the proliferation of lymphocytes in the draining auricular lymph nodes was deremined by measurement of BrdU content in DNA of lymphocyte using an ELISA kit. No mortality and no signs of systemic toxicity were noted in the test and control animals during the test. No increase in ear thickness and in ear weight was noted in animals treated at 25%, 50% and 80%. Therefore, the test item has to be considered as not excessively irritant at these concentrations.The Stimulation Index (SI) calculated by individual approach was 0.92, 0.88 and 0.82 for the treated groups at 25%, 50% and 80% respectively. Therefore, the EC1.6 cannot be determined due to the absence of SI value higher than 1.6. Under these experimental conditions, the test item does not have to be classified as a skin sensitizer in accordance with the CLP Regulation (EC) no. 1272/2008.
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