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EC number: 801-656-8 | CAS number: 1416808-92-8
- 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
Genetic toxicity: in vivo
Administrative data
- Endpoint:
- genetic toxicity in vivo, other
- Remarks:
- Combined Micronucleus and Alkaline Comet Test in the Rat
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Justification for type of information:
- testing proposal decision TPE-D-2114322496-49-01/F
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 017
- Report date:
- 2017
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- other: OECD474 combined with OECD489
- Version / remarks:
- Reviewed and agreed by the Laboratory Animal Welfare Officer and the Ethical Committee (DEC 14-45) as required by the Dutch Act on Animal Experimentation (February 1997).
- GLP compliance:
- yes
- Type of assay:
- other: combined in vivo micronucleus and in vivo COMET assay
Test material
- Reference substance name:
- (3E)-2-chloro-3-(hydroxymethylidene)cyclohex-1-ene-1-carbaldehyde
- EC Number:
- 801-656-8
- Cas Number:
- 1416808-92-8
- Molecular formula:
- C8H9ClO2
- IUPAC Name:
- (3E)-2-chloro-3-(hydroxymethylidene)cyclohex-1-ene-1-carbaldehyde
- Test material form:
- solid: particulate/powder
Constituent 1
Test animals
- Species:
- rat
- Strain:
- Wistar
- Details on species / strain selection:
- recommended by international guidelines
- Sex:
- male
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Strain:Crl:WI(Han)
- Age at study initiation: 6-9 weeks
- Weight at study initiation: 150-155 g
- Assigned to test groups randomly: yes
- Fasting period before study: limited supply of food the night before dosing (7 g/rat)
- Housing: 5 animals per sex in Macrolon cages (type MIV) with sawdust bedding and paper as enrichment
- Diet: pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) ad libitum
- Water: tap water ad libitum
- Acclimation period: at least 5 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.6 – 21.2°C
- Humidity (%): 40-70%
- Air changes (per hr): ca 10/hr
- Photoperiod (hrs dark / hrs light): 12/12
Administration / exposure
- Route of administration:
- oral: gavage
- Vehicle:
- - Vehicle(s)/solvent(s) used: propylene glycol
- Justification for choice of solvent/vehicle: no data
- Concentration of test material in vehicle: no data
- Amount of vehicle (if gavage or dermal): no data - Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
The substance was suspended in propylene glycol, specific gravity 1.036. The test formulation was kept on ice directly after weighing. The test item was sonicated (started at a low temperature and thereafter slowly increasing; range 3.0ºC- 24.5ºC for 16-64 minutes in the main assay) and a blender was (very shortly) used to get a homogeneous suspension.The formulation was clear yellow with small particles.
Treatment:
The first dose of the test item and vehicle was administered at t=0 h. The second and third dose were administered at approximately t=24 h and t=45 h, respectively. The positive control CP was administered once at t = 0 h and EMS was administered at t=24 (± 2 h) and t=45 (± 2 h). The animals were sacrificed by abdominal aorta bleeding under isoflurane anaesthesia at approximately t = 48 h. - Duration of treatment / exposure:
- 3 days
- Frequency of treatment:
- once daily
- Post exposure period:
- none
Doses / concentrationsopen allclose all
- Dose / conc.:
- 250 mg/kg bw/day (nominal)
- Dose / conc.:
- 500 mg/kg bw/day (nominal)
- Dose / conc.:
- 1 000 mg/kg bw/day (nominal)
- Remarks:
- MTD as derived in pre-test
- No. of animals per sex per dose:
- 5 males/dose (+ 3 additional animals dosed at 1000 mg/kg bw as replacement in case of mortality in the main group)
Males were selected based on a toxicity test at 500, 1000 and 2000 mg/kg bw where no substantial differences in toxicity between sexes were found. - Control animals:
- yes, concurrent vehicle
- Positive control(s):
- For micronucleus part: cyclophosphamide
For Comet part: Ethyl Methanesulfonate
Examinations
- Tissues and cell types examined:
- Micronucleus test: bonemarrow
Comet Assay: liver, stomach and duodenum - Details of tissue and slide preparation:
- Micronucleus test: bone marrow was isolated within 3 hours after third treatment from one femur, suspended in fetal calf serum and cetrifuged. Smears were prepared from the pellet that was resuspended in fetal calf serum, drops of this suspension were placed on the slid, air dreid and stained based on Giemsa (2 slides per animal). Micronuclei were scored in 4000 polychromatic erythrocytes and the ratio of polychromatic to normochromatic erythrocytes was determined by counting and differentiating at least the first 1000 erythrocytes
Comet assay: liver, stomach and duodenum were isolated within 3 hours after third treatment, washed (stomach and duodenum), transferred in mincing buffer (stomach and duodenum) and minced on ice. For stomach and duodenum surface epithelia was gently scraped one time softly.
The minced liver tissue was added to 10 mL of collagenase (20 Units/mL; Sigma Aldrich, Zwijndrecht, The Netherlands) dissolved in HBSS (Ca2+- and Mg2+-free) and incubated in a shaking waterbath at 37 °C for 21 minutes. Thereafter, a low centrifugation force was applied two times to remove large undigested liver debris (40 g for 5 min). The supernatant was collected and centrifuged to precipitate the cells (359 g for 10 min).
The minced stomach and duodenum tissues were added to 10 mL of mincing buffer and incubated in a shaking waterbath at 37 °C for 15 minutes. Thereafter, a low centrifugation force was applied two times to remove undigested debris (40 g for 5 min). The supernatant was collected and filtered through a 100 µm Cell Strainer to purify the cell suspension. The purified cell suspension is centrifuged to precipitate the cells (359 g for 10 min).
Cell pellets of all tissues were suspended in ice cold HBSS (Ca2+- and Mg2+-free) and kept on ice. Three slides (pre-coated Comet slides) per animal were prepared in fresh alkaline solution and electrophoresis was performed at 4°C during 30 minutes.(1 Volt/cm for liver and 0.7 Volt/cm for stomach and duodenum). The slides were examined with a fluorescence microscope connected to a Comet Assay IV image analysis system. One hundred fifty Comets per slide were examined.
-Only horizontal orientated Comets were scored, with the head on the left and the tail on the right.
-Cells that showed overlap or were not sharp were not scored. - Evaluation criteria:
- Micronucleus assay:
A test item is considered positive in the micronucleus test if:
a)At least one of the treatment groups exhibits a statistically significant (Dunnett’s test, one-sided, p < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes compared with the concurrent negative control.
b)Any of the results are outside the 95% control limits of the historical control data range.
A test item is considered negative in the micronucleus test if:
a)None of the treatment groups exhibits a statistically significant (Dunnett’s test, one-sided, p < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes compared with the concurrent negative control.
b)All results are within the 95% control limits of the negative historical control data range.
Comet assay:
A test compound is considered positive in the Comet assay (in a tissue) if the following criteria are met:
It induces a toxicologically as well as a statistically significant (Dunnett’s test, one-sided, p < 0.05) dose-dependent increase in percentage Tail Intensity. In case of other non-dose-dependent significant increases the data interpretation will be on a case by case base.
A test compound is considered as negative in the Comet assay (in a tissue) if the following criteria are met:
None of the tested concentrations show a statistically significant (Dunnett’s test, one-sided, p < 0.05) dose-dependent increase in percentage Tail Intensity. - Statistics:
- Student's t-test, Dunnett test (ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany))
Results and discussion
Test resultsopen allclose all
- Key result
- Sex:
- male
- Genotoxicity:
- negative
- Remarks:
- micronucleus test
- Toxicity:
- yes
- Remarks:
- at 500 mg/kg bw and above
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: abnormalities (large dark spots in the plasma of polychromatic erythrocytes) were observed in bone marrow slides at all doses
- Key result
- Sex:
- male
- Genotoxicity:
- positive
- Remarks:
- dose dependent significant increase of tail intensity in liver, stomach and duodenum
- Toxicity:
- yes
- Remarks:
- at and above 500 mg/kg bw
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- 1000 mg/kg bw: lethargy, dark yellow colored urine, ventral recumbency, ataxia, hairless skin was yellow colored, rough coat, hunched posture. One animal had a low body temperature and showed slow breathing. One animal from the main group died just prior isolation of the cells/tissues and was replaced by one of the additional animals. One of the additional animals also died approximately 3 hours after the third dose.
500 mg/kg bw: lethargy, dark yellow colored urine, ataxia
250 mg/kg bw: yellow colored urine
Any other information on results incl. tables
Micronucleus test
Treatment |
Dose (mg/kg bw |
Number of micronucleated polychromatic erythrocytes (mean ± S.D.)(1,2) |
Ratio polychromatic/normochromatic erythrocytes (mean ± S.D.)(1,3) |
|
|
|
|
Vehicle control |
0 |
3.8±1.6 |
0.95±0.04 |
substance |
1000 |
3.2±1.3 |
0.85±0.22 |
substance |
500 |
4.2±0.8 |
0.91±0.09 |
substance |
250 |
4.2±0.8 |
1.00±0.10 |
CP |
20 |
31.2±4.8(4) |
0.43±0.17 |
Vehicle control = Propylene Glycol
CP = Cyclophosphamide.
(1) Five animals per treatment group.
(2) At least 4000 polychromatic erythrocytes were evaluated with a maximum deviation of 5%.
(3) The ratio was determined from at least the first 1000 erythrocytes counted.
(4) Significantly different from corresponding control group (Students t test, p < 0.05).
Comet test
Liver Tail Intensity (%)(1) |
S.D. |
Stomach Tail Intensity (%)(1) |
S.D. |
Duodenum Tail Intensity (%)(1) |
S.D. |
|
Vehicle Control |
8.13 |
1.21 |
13.19 |
8.86 |
26.69 |
13.07 |
V171629 250 mg/kg |
30.48 |
10.20 |
46.62a |
5.53 |
69.10a |
15.21 |
V171629 500 mg/kg |
19.80 |
22.77 |
41.32a |
12.86 |
43.69 |
12.57 |
V171629 1000 mg/kg |
38.93a |
24.16 |
35.60a |
19.52 |
59.30a |
8.54 |
EMS 200 mg/kg |
94.78*** |
1.26 |
93.30*** |
2.45 |
92.12*** |
1.42 |
(1) Five animals per treatment group.
***=
p < 0.001(Student’sttest)
a= p < 0.05 Dunnett’s t test
Vehicle Control = Propylene glycol
EMS = Ethyl Methanesulfonate
Applicant's summary and conclusion
- Conclusions:
- It is concluded that the substance is not clastogenic or aneugenic in the bone marrow micronucleus test in male rats up to a dose of 1000 mg/kg (the maximum tolerated dose) under the experimental conditions described in this report. However, treatment with the substance caused bone marrow abnormalities (polychromatic erythrocytes with large dark spots in the plasma). Moreover, it is concluded that the substance shows a positive result (genotoxic effect) in the Comet assay in liver, stomach and duodenum post dosing of male rats up to a dose of 1000 mg/kg under the experimental conditions described in this summary.
- Executive summary:
In a combined micronucleus and Comet assay, rats (5 males/group) received three daily doses at 0, 250, 500 and 1000 mg/kg bw of the substance in propylene glycol. Cyclophosphamide (MN) and ethylmethane sulphonate (Comet) were includeed as positive controls. At 500 and 1000 mg/kg bw a dose related increase of clinical signs was noted. At 1000 mg/kg bw one of the animals died on day 3. Preparation of bone marrow smears and concomitant scoring of mictonuclei showed that the substance is not clastogenic or aneugenic in the bone marrow.
Pre-coated Comet slides with stomach, duodenum and liver cells were subjected to electrophoresis and the tail intensity was dose related significantly increased in all three tissues. Therefore it was concluded that the substance shows a genotoxic effect.
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