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EC number: 602-927-1 | CAS number: 123312-89-0
- 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
Dermal absorption
Administrative data
- Endpoint:
- dermal absorption in vitro / ex vivo
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 18 Jul 2011 to 28 Jul 2011
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 012
- Report date:
- 2012
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 428 (Skin Absorption: In Vitro Method)
- Version / remarks:
- 2004
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: OECD (Guidance Document No. 28). The Conduct of Skin Absorption studies
- Version / remarks:
- 2004
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: European Commission Guidance Document on Dermal Absorption
- Version / remarks:
- 2004
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Dermal Technology Laboratory Ltd. Med IC4, Keele University Science and Business Park, Keele, Staffordshire, ST5 5NL, United Kingdom
Test material
- Reference substance name:
- 6-methyl-4-[(E)-[(pyridin-3-yl)methylidene]amino]-2,3,4,5-tetrahydro-1,2,4-triazin-3-one
- EC Number:
- 602-927-1
- Cas Number:
- 123312-89-0
- Molecular formula:
- C10H11N5O
- IUPAC Name:
- 6-methyl-4-[(E)-[(pyridin-3-yl)methylidene]amino]-2,3,4,5-tetrahydro-1,2,4-triazin-3-one
Constituent 1
- Radiolabelling:
- yes
Administration / exposure
- Type of coverage:
- open
- Vehicle:
- water
- Duration of exposure:
- 24 hours
- Doses:
- - Test solutions: 1/2000 w/w aqueous spray strength dilution containing a nominal 0.25 g test substance/kg, a 1/500 w/w aqueous spray strength dilution containing a nominal 1 g test substance/kg and a formulation concentrate containing a nominal 500 g test substance/kg
- Nominal doses: 2.59, 10.5 and 1626 µg test substance per cm2
- Dose volume: 25.4 µL - Details on in vitro test system (if applicable):
- SKIN PREPARATION
- Type of skin: Human skin, abdomen, female, age 51 to 91 years
- Preparative technique: The skin samples were immersed in water at 60ºC for 40-45 seconds and the epidermis teased away from the dermis. Each membrane was given an identifying number and stored frozen, at approximately -20ºC, on aluminium foil until required for use.
- Membrane integrity check: yes, range 10.19 to 24.94 kΩ
- Storage conditions: stored for 50 to 56 weeks and one sample 192 weeks
PRINCIPLES OF ASSAY
Cells were selected such that each application was represented by six intact membranes from at least four different donors. The receptor chambers of the cells containing small magnetic stirrer bars were filled with a recorded volume of receptor fluid (50% v/v ethanol/water) and placed in a water bath maintained at a temperature of 32ºC ± 1ºC.
A pre-treatment sample (0.1 mL (aqueous dilutions) or 0.5 mL (slurry)) was taken from each receptor chamber for analysis by LSC. The volume reduction in the receptor chamber was immediately compensated by fresh receptor fluid.
The test substance formulations were applied to the skin surface by volume using a suitable positive displacement pipette. The dose was applied drop-wise over the skin to maximise coverage. The weight of dose applied was recorded.
Skin absorption for each application was followed for 24 hours. The exposed skin area was left unoccluded.
Samples (0.1 mL (aqueous dilutions) or 0.5 mL (slurry)) of receptor fluid were taken from the receptor chambers of this static cell system 1, 2, 3, 4, 6, 8, 10, 12, 16, 20 and 24 hours after application using an autosampler. The receptor fluid in the chambers was stirred continuously and the receptor volume was maintained by the replacement of a volume of fresh receptor fluid, equal to the sample volume, after each sample had been taken. The samples of receptor fluid were analysed by LSC.
After the final receptor fluid sample had been taken at the end of the exposure period, the remaining fluid in the receptor chamber was discarded. The receptor chamber was again refilled with fresh receptor fluid (approximately 5 mL), which was, afterwards, discarded.
The donor chamber was carefully removed and the underside (surface in contact with the membrane) wiped with one natural sponge pre-wetted with a dilute soap solution (3% Teepol in water) which was added to the wash sponges (below). The donor chambers were washed with methanol and the sample of the washing taken for analysis by LSC.
The epidermal surface of the skin was decontaminated by gently swabbing the application site with natural sponges pre-wetted with 3% Teepol in water, decontamination was assessed to be complete using a Geiger counter to measure the radioactivity on the skin/sponges, and with a further two sponges pre-wetted with water (one cell received three sponges) .The sponges were digested in a solubilising agent (Soluene 350) and a sample taken for analysis by LSC.
To assess penetration through human stratum corneum, successive layers of the stratum corneum were removed by the repeated application of adhesive tape to a maximum of 5 strips.
The surface of the skin was allowed to dry naturally. Strips of adhesive tape were sequentially pressed onto the skin surface and then carefully peeled off to remove layers of the stratum corneum. The adhesive strips were immersed individually in methanol to extract any test material. The extracts were sequentially numbered and analysed by LSC. The total number of tape strips was recorded.
The remaining epidermis was removed from the receptor chamber, digested in Soluene 350 and the whole digest analysed by LSC.
Results and discussion
- Absorption in different matrices:
- The mean data for distribution in the test system are presented in Table 1 to 3 in terms of amount and percentage of the applied dose.
The absorption rates for each individual cell are given in Table 4.
Key result in 'percutaneous absorption', based on 1/2000 dilution of the formulation concentrate. Absorption = absorption in stratum corneum (tape strips 3-5) + remaining epidermis + absorbed = 2.425%
Undiluted concentrate: amounts of test substance absorbed at 8, 12 and 24 hours were 0.160, 0.234 and 0.410 μg/cm2 respectively. These respective amounts expressed as percentages of the applied dose equated to 0.010, 0.014 and 0.025%. Absorption flow was 0.015 µg/cm2/h during the 24 hour exposure period.
1/500 dilution: amounts of the substance absorbed at 8, 12 and 24 hours were 0.017, 0.025 and 0.051 μg/cm2 respectively. These respective amounts expressed as percentages of the applied dose equated to 0.157, 0.236 and 0.484%. Absorption flow was 0.002 µg/cm2/h during the 24 hour exposure period.
1/2000 dilution: amounts of the substance absorbed at 8, 12 and 24 hours were 0.012, 0.014 and 0.021 μg/cm2 respectively. These respective amounts expressed as percentages of the applied dose equated to 0.459, 0.557 and 0.801%. Absorption flow was 0.001 µg/cm2/h during the 24 hour exposure period. - Total recovery:
- - Total recovery: Table 1 to 3 in ‘any other information on results incl. tables’.
- Recovery of applied dose acceptable: Yes
- Limit of detection (LOD): Table 5 to 7 in ‘any other information on results incl. tables’.
- Undiluted concentrate: Total recovery 108%, of which 107% was washed off at 24 hours.
- 1/500 dilution: Total recovery was 105%, of which 103% was washed off at 24 hours.
- 1/2000 dilution: Total recovery was 106%, of which 103% was washed off at 24 hours.
Percutaneous absorptionopen allclose all
- Key result
- Time point:
- 24 h
- Dose:
- 1/2000 dilution of formulation concentrate: 2.59 µg/cm2
- Parameter:
- percentage
- Absorption:
- 2.451 %
- Time point:
- 24 h
- Dose:
- 1/500 dilution of formulation concentrate: 10.5 µg/cm2
- Parameter:
- percentage
- Absorption:
- 1.2 %
- Time point:
- 24 h
- Dose:
- 33.3% w/w concentrate: 1626 µg/cm2
- Parameter:
- percentage
- Absorption:
- 0.051 %
Any other information on results incl. tables
Table 1. 33.3% w/w aqueous slurry of the formulation concentrate: 1626 µg test substance per cm2
Test Compartment |
µg test substance per cm² |
% of applied dose |
||
n=6 |
Mean |
SEM |
Mean |
SEM |
Donor chamber |
0.185* |
0.081 |
0.011* |
0.005 |
Skin wash |
1748 |
7.08 |
107 |
0.435 |
Stratum corneum(tape strips 1 & 2) |
0.120* |
0.031 |
0.007* |
0.002 |
Stratum corneum(tape strips 3 – 5) |
0.096* |
0.046 |
0.006* |
0.003 |
Remaining epidermis |
0.321 |
0.074 |
0.020 |
0.005 |
Absorbed |
0.410 |
0.064 |
0.025 |
0.004 |
Total recovered |
1749 |
7.14 |
108 |
0.439 |
* < Limit of Quantitation Values
Table 2. 1/500 w/w aqueous dilution of the formulation concentrate: 10.5 µg test substance per cm2
Test Compartment |
µg test substance per cm² |
% of applied dose |
||
n=6 |
Mean |
SEM |
Mean |
SEM |
Donor chamber |
0.045 |
0.017 |
0.432 |
0.157 |
Skin wash |
10.9 |
0.479 |
103 |
4.56 |
Stratum corneum(tape strips 1 & 2) |
0.008 |
0.003 |
0.074 |
0.025 |
Stratum corneum(tape strips 3 – 5) |
0.008 |
0.002 |
0.079 |
0.017 |
Remaining epidermis |
0.066 |
0.013 |
0.630 |
0.126 |
Absorbed |
0.051 |
0.019 |
0.484 |
0.177 |
Total recovered |
11.1 |
0.463 |
105 |
4.41 |
* < Limit of Quantitation Values
Table 3. 1/2000 w/w aqueous dilution of the formulation concentrate: 2.59 µg test substance per cm2
Test Compartment |
µg test substance per cm² |
% of applied dose |
||
n=6 |
Mean |
SEM |
Mean |
SEM |
Donor chamber |
0.014 |
0.013 |
0.540 |
0.491 |
Skin wash |
2.67 |
0.057 |
103 |
2.19 |
Stratum corneum(tape strips 1 & 2) |
0.008 |
0.004 |
0.301 |
0.140 |
Stratum corneum(tape strips 3 – 5) |
0.012 |
0.007 |
0.454 |
0.266 |
Remaining epidermis |
0.030 |
0.022 |
1.17 |
0.861 |
Absorbed |
0.021 |
0.006 |
0.801 |
0.218 |
Total recovered |
2.75 |
0.043 |
106 |
1.65 |
* < Limit of Quantitation Values
Table 4. Mean absorption rates
Application of Test Materials and Actual Concentration of Dose Preparation |
Mean Absorption Rates |
Mean Amount and Percentage of Dose Absorbed |
|||
Time period (h) |
Absorption rate (µg/cm2/h±SEM) |
Time (h) |
Amount (µg/cm2) |
Percentage Absorbed (%) |
|
33.3% w/w aqueous slurry |
|
|
|
|
|
(163 g/kg) |
0-8 |
0.016 ± 0.004 |
6 |
0.136 |
0.008 |
10 µLcm2(1626 µg/cm2) |
8-12 |
*0.024 ± 0.008 |
8 |
0.160 |
0.010 |
Unoccluded |
12-24 |
0.014 ±0.004 |
12 |
0.234 |
0.014 |
Duration of exposure: 24h |
0-24 |
0.015 ± 0.003 |
24 |
0.410 |
0.025 |
n = 6 |
|
|
|
|
|
1/500 w/w aqueous dilution |
|
|
|
|
|
(1.05 g/kg) |
0-8 |
0.002 ± 0.001 |
6 |
0.013 |
0.125 |
10 µL/cm2(10.5 µg/cm2) |
8-12 |
0.002 ± 0.001 |
8 |
0.017 |
0.157 |
Unoccluded |
12-24 |
0.002 ± 0.001 |
12 |
0.025 |
0.236 |
Duration of exposure: 24h |
0-24 |
0.002 ± 0.001 |
24 |
0.051 |
0.484 |
n = 6 |
|
|
|
|
|
1/2000 w/w aqueous dilution |
|
|
|
|
|
(0.259 g/kg) |
0-8 |
0.001 ± 0.0003 |
6 |
0.010 |
0.403 |
10 µL/cm2(2.59 µg/cm2) |
8-12 |
0.001 ± 0.0001 |
8 |
0.012 |
0.459 |
Unoccluded |
12-24 |
0.001 ± 0.0001 |
12 |
0.014 |
0.557 |
Duration of exposure: 24h |
0-24 |
0.001 ± 0.0001 |
24 |
0.021 |
0.801 |
n = 5 |
|
|
|
|
|
Table 6. Mean limit of quantitation values for 33.3% w/w Aqueous slurry of the formulation concentrate: 1626 µg test substance per cm2
Study compartment |
DPM Value |
µg/cm2/h |
µg/cm2 |
% of applied dose |
Absorption rate (0-8 hours) |
- |
0.015 |
- |
0.001 |
Absorption rate (8-12 hours) |
- |
0.031 |
- |
0.002 |
Absorption rate (12-24 hours) |
- |
0.010 |
- |
0.0006 |
Absorption rate (0-24 hours) |
- |
0.005 |
- |
0.0003 |
Receptor fluid |
39.6 |
- |
0.124 |
0.008 |
Donor chamber |
16 |
- |
0.204 |
0.013 |
Skin wash |
17 |
- |
0.542 |
0.033 |
Stratum corneum |
18 |
- |
0.574 |
0.035 |
Remaining epidermal membranes |
24 |
- |
0.008 |
0.0005 |
Table 6. Mean limit of quantitation values for 1/500 w/w Aqueous dilution of the formulation concentrate: 10.5 µg test substance per cm2
Study compartment |
DPM Value |
µg/cm2/h |
µg/cm2 |
% of applied dose |
Absorption rate (0-8 hours) |
- |
0.0007 |
- |
0.006 |
Absorption rate (8-12 hours) |
- |
0.001 |
- |
0.012 |
Absorption rate (12-24 hours) |
- |
0.0004 |
- |
0.004 |
Absorption rate (0-24 hours) |
- |
0.0002 |
- |
0.002 |
Receptor fluid |
33.3 |
- |
0.005 |
0.050 |
Donor chamber |
28 |
- |
0.004 |
0.037 |
Skin wash |
20 |
- |
0.007 |
0.066 |
Stratum corneum |
25 |
- |
0.009 |
0.083 |
Remaining epidermal membranes |
19 |
- |
0.001 |
0.010 |
Table 7. Mean limit of quantitation values for 1/2000 w/w Aqueous dilution of the formulation concentrate: 2.59 µg test substance per cm2
Study compartment |
DPM Value |
µg/cm2/h |
µg/cm2 |
% of applied dose |
Absorption rate (0-8 hours) |
- |
0.0007 |
- |
0.026 |
Absorption rate (8-12 hours) |
- |
0.001 |
- |
0.051 |
Absorption rate (12-24 hours) |
- |
0.0004 |
- |
0.017 |
Absorption rate (0-24 hours) |
- |
0.0002 |
- |
0.009 |
Receptor fluid |
34.8 |
- |
0.005 |
0.206 |
Donor chamber |
26 |
- |
0.004 |
0.140 |
Skin wash |
17 |
- |
0.006 |
0.229 |
Stratum corneum |
32 |
- |
0.011 |
0.432 |
Remaining epidermal membranes |
18 |
- |
0.001 |
0.039 |
Applicant's summary and conclusion
- Conclusions:
- In this GLP compliant OECD 428 study, the penetration of the test substance through the human epidermis is generally very slow and the vast majority of the applied dose could be removed by gentle skin washing after 24 hours. The dermal absorption value obtained from the undiluted formulation concentrate (33.3% w/w), 0.051%. For 1/2000 aqueous dilution of the concentrate formulation, a dermal absoption of 2.425% was derived. This value is used as reference value for risk assessment, since it is considered to be conservative.
- Executive summary:
The in vitro dermal absorption of the test substance concentrate formulation through human epidermal membranes was measured in an OECD 428 study under GLP. [14C]-test substance doses were applied as a nominal 33.3% w/w aqueous slurry of the formulation concentrate (500 g/kg) and as 1/500 w/w (nominally, 1 g/kg) and 1/2000 w/w (nominally, 0.25 g/kg) aqueous dilutions of the formulation concentrate. The doses were applied at 10 μL/cm2
and left unoccluded for an exposure period of 24 hours.The absorption process was followed by taking samples of the receptor fluid (50% v/v ethanol/water) at recorded intervals throughout the exposure period. The distribution of the test substance within the test system and a 24 hour absorption profile were determined, using liquid scintillation counting (LSC).
For the 33.3% w/w aqueous slurry of the formulation concentrate the mean recovery was 108% of the dose applied. The vast majority of the applied test substance (mean 107%) was washed off the skin at 24 hours. The mean absorption rate through the human epidermis was 0.015 µg/cm2/h, accounting for a 24-hour accumulating dose of 0.827 µg/cm2 (0.051% of the dose applied) in the stratum corneum, remaining epidermis and systemic available fraction. For the 1/500 w/w aqueous dilution of the formulation concentrate the mean recovery was 105% of the dose applied.The vast majority of the applied test substance (mean 103%) was washed off the skin at 24 hours. The mean absorption rate through the human epidermis was 0.002 µg/cm2/h, accounting for a 24-hour accumulating dose of 0.125 µg/cm2 (1.193% of the dose applied) in the stratum corneum, remaining epidermis and systemic available fraction. For the 1/2000 w/w aqueous dilution of the formulation concentrate the mean recovery was 106% of the dose applied.The vast majority of the applied test substance (mean 103%) was washed off the skin at 24 hours. The mean absorption rate through the human epidermis was 0.001 µg/cm2/h, accounting for a 24-hour accumulating dose of 0.063 µg/cm2 (2.425% of the dose applied) in the stratum corneum, remaining epidermis and systemic available fraction.
In conclusion, the penetration of the test substance through the human epidermis is generally very slow and the vast majority of the applied dose could be removed by gentle skin washing after 24 hours. The dermal absorption value obtained from the undiluted formulation concentrate (33.3% w/w), 0.051%. For 1/2000 aqueous dilution of the concentrate formulation, a dermal absoption of 2.425% was derived. The latter value is used as reference value for risk assessment, since it is considered to be conservative.
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