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EC number: 915-371-2 | CAS number: -
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Administrative data
Description of key information
OECD 429 (LLNA): Not a skin sensitiser
Key value for chemical safety assessment
Skin sensitisation
Link to relevant study records
- Endpoint:
- skin sensitisation: in vivo (LLNA)
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2019-03-06 to 2019-03-12
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
- Deviations:
- yes
- Remarks:
- Deviations were not considered to adversely affect the results or integrity of the study
- GLP compliance:
- yes (incl. QA statement)
- Type of study:
- mouse local lymph node assay (LLNA)
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Emerald Kalama Chemical Ltd. (United Kingdom); Batch no: A170421E
- Expiration date of the lot/batch: 2019-06-06
- Purity test date: 2018-02-07
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Controlled room temperature (15-25oC, <70% relative humidity)
- Stability under test conditions: Analytical determination of the test item concentration, stability and homogeneity was not performed because of the character and the short period of study.
- Solubility and stability of the test substance in the solvent/vehicle: The solubility of the test item was examined in a short Preliminary Compatibility Test. The following standard OECD 429 vehicle was assessed: Acetone: Olive oil 4:1 (v/v) mixture (AOO). The best vehicle taking into account the test item characteristics, and the requirements of the relevant OECD guideline was considered to be AOO. 100% (undiluted) was the highest concentration which was suitable for the test. The formulations appeared to be solutions by visual examination.
FORM AS APPLIED IN THE TEST (if different from that of starting material) : Clear, pale yellow liquid - Species:
- mouse
- Strain:
- CBA/Ca
- Remarks:
- CBA/CaOlaHsd
- Sex:
- female
- Details on test animals and environmental conditions:
- TEST ANIMALS
- Source: Envigo (San Pietro al Natisone (UD) Zona Industriale Azzida, 57, 33049 Italy)
- Females (if applicable) nulliparous and non-pregnant: yes, nulliparous, non-pregnant
- Microbiological status of animals, when known: SPF at arrival
- Age at study initiation: 11 weeks old (age-matched, within one week)
- Weight at study initiation: 20.2 – 21.9 grams (The weight variation in animals in the study did not exceed ± 20% of the mean weight.)
- Housing: Group caging (Type II. polypropylene / polycarbonate) / mice were provided with glass tunnel-tubes
- Diet (e.g. ad libitum): ssniff® SM Rat/Mouse – Breeding and Maintenance, 15 mm, autoclavable "Complete feed for Rats and Mice – Breeding and Maintenance" (Batch number: 639 38520, Expiry date: 30 April 2019, produced by ssniff Spezialdiäten GmbH (Ferdinand-Gabriel-Weg 16, D-59494 Soest, Germany), and Gel diet Transport (Batch Number: 60182980010101, Expiry date: 25 October 2019 and Batch Number: 60190070110101, Expiry date: 08 January 2020, Scientific Animal Food & Engineering, Route de Saint Bris, 89290 Augy, France) ad libitum.
- Water (e.g. ad libitum): tap water from the municipal supply from 500 mL bottles, ad libitum
- Acclimation period: at least 14 days
- Indication of any skin lesions: Not specified
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.6 – 25.9°C
- Humidity (%): 22 - 74%
- Air changes (per hr): 15-20 air exchanges/hour
- Photoperiod (hrs dark / hrs light): 12 hours daily, from 6.00 a.m. to 6.00 p.m
- IN-LIFE DATES: From: 2019-02-06 To: 2019-03-12 - Vehicle:
- acetone/olive oil (4:1 v/v)
- Remarks:
- Acetone: Olive oil 4:1 (v/v) mixture (AOO)
- Concentration:
- Preliminary Irritation / Toxicity Test: Test material concentrations of 100% (undiluted), 50, 25, and 10% (w/v) in AOO
Main Test: Test material concentrations of 10, 5, and 2.5% (w/v) in AOO - No. of animals per dose:
- 4 females per concentration
- Details on study design:
- PRE-SCREEN TESTS:
- Compound solubility: The solubility of the test item was examined in a short Preliminary Compatibility Test. The following standard OECD 429 vehicle was assessed: Acetone: Olive oil 4:1 (v/v) mixture (AOO). The best vehicle taking into account the test item characteristics, and the requirements of the relevant OECD guideline was considered to be AOO. 100% (undiluted) was the highest concentration which was suitable for the test. The formulations appeared to be solutions by visual examination.
The test item was weighed and formulations prepared daily on a weight:volume basis (as% (w/v)) in the Pharmacy of Citoxlab Hungary Ltd.
Analytical determination of the test item concentration, stability and homogeneity was not performed because of the character and the short period of study.
- Irritation: A Preliminary Irritation/Toxicity Test was started according to the Study Plan on CBA/CaOlaHsd mice using four doses (2 animals/dose) at test material concentrations of 100% (undiluted), 50, 25, and 10% (w/v) in AOO. The preliminary experiment was conducted in a similar experimental manner to the main study, but it was terminated on Day 6 and the radioactive proliferation assay was not performed.
The maximum concentration of test item in an acceptable solvent was established according to OECD guideline 429. Based on the observation of the solubility test, the maximum available concentration was 100% (undiluted).
- Systemic toxicity: In the Preliminary Irritation / Toxicity Tests, all mice were observed daily for any clinical signs of systemic toxicity.
During the main study Clinical observations were performed twice a day (before and after treatments) on Days 1, 2 and 3 and once daily on Days 4, 5 and 6. Individual body weights were recorded on Day 1 (beginning of the test) and on Day 6 (prior to 3HTdR injection) with a precision of ± 0.1 g.
- Ear thickness measurements: In the Preliminary Irritation / Toxicity Tests, all mice were observed daily for local irritation at the application site. Both ears of each mouse were observed for erythema and scored using the scoring system presented below. Ear thickness was also measured using a thickness gauge on Day 1 (pre-dose), Day 3 (approximately 48 hours after the first dose) and Day 6. Additional quantification of the ear thickness was performed by ear punch weight
determination after the euthanasia of the experimental animals.
- Erythema scores: Erythema Scoring
Observation Score
No erythema 0
Very slight erythema (barely perceptible) 1
Well-defined erythema 2
Moderate to severe erythema 3
Severe erythema (beet redness) to eschar formation 4
preventing grading of erythema
Note: Excessive local skin irritation is indicated by an erythema score ≥ 3 and/or an increase in ear thickness of ≥ 25% on any day of measurement.
MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay (LLNA)
- Criteria used to consider a positive response: The test material is regarded as a sensitizer if both of the following criteria are fulfilled:
- That exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than recorded in control mice, as indicated by the stimulation index.
- The data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.
TREATMENT PREPARATION AND ADMINISTRATION:
During the study, animals were topically dosed with 25 µL of the appropriate formulation using a pipette on the dorsal surface of each ear. Each animal was dosed once a day for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6.
The Preliminary Irritation/Toxicity Tests were started according to the Study Plan on CBA/CaOlaHsd mice using four doses (2 animals/dose) at test item concentrations of 100% (undiluted), 50, 25, and 10% (w/v) in AOO. The preliminary experiment was conducted in a similar experimental manner to the main study, but it was terminated on Day 6 and the radioactive proliferation assay was not performed.
Based on these observations in the preliminary test, 10% (w/v) dose was selected as the top dose for the main test. - Positive control substance(s):
- hexyl cinnamic aldehyde (CAS No 101-86-0)
- Statistics:
- The number of radioactive disintegrations per minute (DPM) was measured for each pooled group of nodes. The measured DPM values were corrected with the background DPM value ("DPM"). The average of the two measured DPM values of 5% (w/v) TCA solutions was used as background DPM value. The results were expressed as "DPN" (DPM divided by the number of lymph nodes) following the industry standard for data presentation.
Stimulation index (SI = DPN value of a treated group divided by the DPN value of the negative control group) for each treatment group was also calculated. A stimulation index of 3 or greater is an indication of a positive result. - Positive control results:
- Larger than normal lymph nodes were observed in the positive control group. No mortality, cutaneous reactions or signs of toxicity were observed for the positive control
substance in the study. A lymphoproliferative response in line with historical positive control data (stimulation index value of 4.4) was noted for HCA in the main experiment. This value was considered to confirm the appropriate performance of the assay. Furthermore, the DPN values observed for the positive control substance in this
experiment were in within the historical control range. - Key result
- Parameter:
- SI
- Value:
- 1
- Variability:
- DPN: 594.3
- Test group / Remarks:
- Negative control (AOO)
- Key result
- Parameter:
- SI
- Value:
- 1.2
- Variability:
- DPN = 719.8
- Test group / Remarks:
- Test Material - 10% (w/v) in AOO
- Key result
- Parameter:
- SI
- Value:
- 1.1
- Variability:
- DPN = 639.6
- Test group / Remarks:
- Test Material - 5% (w/v) in AOO
- Key result
- Parameter:
- SI
- Value:
- 0.9
- Variability:
- DPN = 556.8
- Test group / Remarks:
- Test Material - 2.5% (w/v) in AOO
- Key result
- Parameter:
- SI
- Value:
- 4.4
- Variability:
- DPN = 2618.8
- Test group / Remarks:
- Positive control (25% (w/v) HCA in AOO
- Cellular proliferation data / Observations:
- CELLULAR PROLIFERATION DATA
The appearance of the lymph nodes was normal in the negative control group and in the test item treated dose groups. Larger than normal lymph nodes were observed in the positive control group.
DETAILS ON STIMULATION INDEX CALCULATION
The number of radioactive disintegrations per minute (DPM) was measured for each pooled group of nodes. The measured DPM values were corrected with the background DPM value ("DPM"). The average of the two measured DPM values of 5% (w/v) TCA solutions was used as background DPM value. The results were expressed as "DPN" (DPM divided by the number of lymph nodes) following the industry standard for data presentation.
Stimulation index (SI = DPN value of a treated group divided by the DPN value of the negative control group) for each treatment group was also calculated. A stimulation index of 3 or greater is an indication of a positive result.
The stimulation index values were 1.2, 1.1, and 0.9 at concentrations of 10, 5, and 2.5% (w/v), respectively.
EC3 CALCULATION
Not specified
CLINICAL OBSERVATIONS:
No mortality or signs of systemic toxicity were observed during the study. No test material residue was present on the ears of the animals.
BODY WEIGHTS
Decreases were noted in body weight, however were considered incidental and not related to treatment. Two out of 4 animals in the negative control, 3 out of 4 animals in the 10% (w/v) dose group, and 1 out of 4 animals in the positive control group showed marked body weight loss (>5%). The mean body weight loss in the 10% (w/v) dose group was -6.2% (mean value). Based on the unequivocal result for this dose group in the preliminary assays, the lack of systematic toxicity in any of the dose groups, and marked body weight losses in both the negative and positive control groups, these changes were considered to be due to individual variety, and not a factor to effect the results of the proliferation assay for this dose group. - Interpretation of results:
- other: Not a skin sensitiser
- Remarks:
- Based on EU CLP and GHS
- Conclusions:
- Under the conditions of the present assay, the test material (Reaction mass of 3-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile and 4-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile), tested in AOO, did not show a sensitisation potential (non-sensitiser) in the local lymph node assay.
No classification/labelling is triggered according to Regulation (EC) No 1272/2008 (CLP) / GHS (rev. 7) 2017. - Executive summary:
A Key OECD Guideline 429 study was conducted to determine the skin sensitisation potential of the test material (Reaction mass of 3-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile and 4-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile) following dermal exposure in mice.
The solubility of the test material was examined in a short Preliminary Compatibility Test. Acetone: Olive oil 4:1 (v/v) mixture (AOO) was used as the vehicle and 100% (undiluted) was the highest concentration found to be suitable for the test. A preliminary irritation/toxicity test was performed in CBA/CaOlaHsd mice using four doses (2 animals/dose): 100% (undiluted), 50, 25, and 10% (w/v) in AOO. Based on the results, 10% (w/v) was selected as top dose for the main test.
In the main assay, twenty female CBA/CaOlaHsd mice were allocated to five groups, each group comprised four animals:
- three groups of animals received the test material (formulated in AOO) at either 10, 5 or 2.5% (w/v),
- a negative control group received the vehicle (AOO) only,
- a positive control group received 25% (w/v) α-Hexylcinnamaldehyde (HCA) dissolved in AOO.
Test material solutions were applied to the dorsal surface of the ears of the experimental animals (25 µL/ear) for three consecutive days (Days 1, 2 and 3) and then maintained on study for an additional 3 days. Cell proliferation in the (local) lymph nodes was assessed by measuring disintegrations per minute after the incorporation of tritiated methyl thymidine (3HTdR) into the lymph nodes and the values obtained were used to calculate stimulation indices (SI) in comparison with the control group.
No mortality or signs of systemic toxicity were observed during the study.Decreases were noted in body weight, however were considered incidental and not related to treatment. Two out of 4 animals in the negative control, 3 out of 4 animals in the 10% (w/v) dose group, and 1 out of 4 animals in the positive control group showed marked body weight loss (>5%). The mean body weight loss in the 10% (w/v) dose group was -6.2%.
The SI values were 1.2, 1.1, and 0.9 at test material concentrations of 10, 5, and 2.5% (w/v), respectively.
The SI value for the positive control substance α-Hexylcinnamaldehyde (HCA) dissolved in AOO was 4.4 therefore demonstrating the appropriate performance of the assay. The lymphoproliferative response of the HCA was in line with historical control data for the positive control, therefore confirming the validity of the assay.
Under the conditions of this assay, the test material (Reaction mass of 3-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile and 4-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile), tested in AOO, did not show a sensitisation potential (non-sensitizer) in the Local Lymph Node Assay.
No classification/labelling is triggered according to EU Regulation (EC) No 1272/2008 (CLP) or GHS (rev. 7) 2017.
Reference
Table 2. Summary of Preliminary Study Data |
||||||
Preliminary Concentrations |
Physical Formulation |
Clinical Observations |
Body Weight |
Erythema |
Ear Thickness |
Ear Biopsy Weight |
100% (undiluted) |
A |
U |
E |
E |
A |
A |
50% (w/v) |
A |
U |
E |
E |
A |
A |
25% (w/v) |
A |
U |
E |
A |
A |
A |
10%(w/v) |
A |
A |
E |
A |
A |
A |
U = Unacceptable; A = Acceptable; E = Equivocal
Table 3. Individual Body Weights for all Animals with Group Means |
||||
Animal Number |
Test Group Name |
Initial Body Weight (g) |
Terminal Body Weight* (g) |
Change# (%) |
7585 |
Negative (vehicle) control in AOO |
21.6 |
19.9 |
-7.9 |
7598 |
21.2 |
21.0 |
-0.9 |
|
7595 |
20.6 |
20.4 |
-1.0 |
|
7597 |
20.7 |
19.6 |
-5.3 |
|
|
Mean |
21.0 |
20.2 |
-3.8 |
|
||||
7586 |
Test material 10% (w/v) in AOO |
21.5 |
19.7 |
-8.4 |
7592 |
21.0 |
20.6 |
-1.9 |
|
7596 |
20.8 |
19.3 |
-7.2 |
|
7593 |
20.3 |
18.8 |
-7.4 |
|
|
Mean |
20.9 |
19.6 |
-6.2 |
|
||||
7599 |
Test material 5% (w/v) in AOO |
21.9 |
21.1 |
-3.7 |
7584 |
20.7 |
20.8 |
0.5 |
|
7588 |
20.6 |
21.3 |
3.4 |
|
7600 |
20.4 |
21.7 |
6.4 |
|
|
Mean |
20.9 |
21.2 |
1.7 |
|
||||
7587 |
Test material 2.5% (w/v) in AOO |
21.5 |
22.5 |
4.7 |
7602 |
21.7 |
22.4 |
3.2 |
|
7594 |
20.3 |
20.5 |
1.0 |
|
7590 |
20.2 |
19.6 |
-3.0 |
|
|
Mean |
20.9 |
21.3 |
1.5 |
|
||||
7603 |
Positive control 25% (w/v) HCA in AOO |
21.5 |
21.2 |
-1.4 |
7591 |
21.9 |
20.6 |
-5.9 |
|
7601 |
20.3 |
19.5 |
-3.9 |
|
7589 |
20.2 |
21.5 |
7.4 |
|
|
Mean |
21.0 |
20.8 |
-1.0 |
Notes:
*: Terminal body weights were measured on Day 6.
#: = (Terminal Body Weight – Initial Body Weight) / Initial Body Weight x 100
Table 4. DPM, DPN and Stimulation Index Values for all Groups |
|||||
Test Group Name |
Measured DPM / group |
DPM |
Number of lymph nodes |
DPN |
Stimulation Index |
Background (5% (w/v) TCA) |
35 |
- |
- |
- |
- |
Negative control (AOO) |
4789 |
4754.0 |
8 |
594.3 |
1.0 |
Test Material 10% (w/v) in AOO |
5793 |
5758.0 |
8 |
719.8 |
1.2 |
Test Material 5% (w/v) in AOO |
5152 |
5117.0 |
8 |
639.6 |
1.1 |
Test Material 2.5% (w/v) in AOO |
4489 |
4454.0 |
8 |
556.8 |
0.9 |
Positive control (25% (w/v) HCA in AOO) |
20985 |
20950.0 |
8 |
2618.8 |
4.4 |
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (not sensitising)
- Additional information:
A Key OECD Guideline 429 study (Citoxlab Hungary Ltd., 2019, K = 1) was conducted to determine the skin sensitisation potential of the test material (Reaction mass of 3-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile and 4-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile) following dermal exposure in mice.
The solubility of the test material was examined in a short Preliminary Compatibility Test. Acetone: Olive oil 4:1 (v/v) mixture (AOO) was used as the vehicle and 100% (undiluted) was the highest concentration found to be suitable for the test. A preliminary irritation/toxicity test was performed in CBA/CaOlaHsd mice using four doses (2 animals/dose): 100% (undiluted), 50, 25, and 10% (w/v) in AOO. Based on the results, 10% (w/v) was selected as top dose for the main test.
In the main assay, twenty female CBA/CaOlaHsd mice were allocated to five groups, each group comprised four animals:
- three groups of animals received the test material (formulated in AOO) at either 10, 5 or 2.5% (w/v),
- a negative control group received the vehicle (AOO) only,
- a positive control group received 25% (w/v) α-Hexylcinnamaldehyde (HCA) dissolved in AOO.
Test material solutions were applied to the dorsal surface of the ears of the experimental animals (25 µL/ear) for three consecutive days (Days 1, 2 and 3) and then maintained on study for an additional 3 days. Cell proliferation in the (local) lymph nodes was assessed by measuring disintegrations per minute after the incorporation of tritiated methyl thymidine (3HTdR) into the lymph nodes and the values obtained were used to calculate stimulation indices (SI) in comparison with the control group.
No mortality or signs of systemic toxicity were observed during the study.Decreases were noted in body weight, however were considered incidental and not related to treatment. Two out of 4 animals in the negative control, 3 out of 4 animals in the 10% (w/v) dose group, and 1 out of 4 animals in the positive control group showed marked body weight loss (>5%). The mean body weight loss in the 10% (w/v) dose group was -6.2%.
The SI values were 1.2, 1.1, and 0.9 at test material concentrations of 10, 5, and 2.5% (w/v), respectively.
The SI value for the positive control substance α-Hexylcinnamaldehyde (HCA) dissolved in AOO was 4.4 therefore demonstrating the appropriate performance of the assay. The lymphoproliferative response of the HCA was in line with historical control data for the positive control, therefore confirming the validity of the assay.
Under the conditions of this assay, the test material (Reaction mass of 3-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile and 4-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile), tested in AOO, did not show a sensitisation potential (non-sensitizer) in the Local Lymph Node Assay.
No classification/labelling is triggered according to EU Regulation (EC) No 1272/2008 (CLP) or GHS (rev. 7) 2017.
Respiratory sensitisation
Endpoint conclusion
- Endpoint conclusion:
- no study available
Justification for classification or non-classification
Based on the results of the LLNA assay, Reaction mass of 3-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile and 4-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile does not meet the criteria for classification as a skin sensitiser under EU Regulation (EC) No 1272/2008 (CLP) and GHS (rev. 7) 2017.
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