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EC number: 284-716-0 | CAS number: 84962-20-9
- 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. Mar - 27. Mar 2012
- 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 guideline
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of study:
- mouse local lymph node assay (LLNA)
Test material
- Reference substance name:
- Phosphoric acid, mixed esters with Bu alc. and ethylene glycol
- EC Number:
- 284-716-0
- EC Name:
- Phosphoric acid, mixed esters with Bu alc. and ethylene glycol
- Cas Number:
- 84962-20-9
- IUPAC Name:
- Reaction mass of Phosphoric acid mono-(2-hydroxy-ethyl) ester and Phosphoric acid monobutyl ester and Phosphoric acid bis-(2-hydroxy-ethyl) ester
- Reference substance name:
- Phosphoric acid, mixed with Ethylene glycol and Butanol
- IUPAC Name:
- Phosphoric acid, mixed with Ethylene glycol and Butanol
- Test material form:
- other: liquid
Constituent 1
Constituent 2
In vivo test system
Test animals
- Species:
- mouse
- Strain:
- CBA
- Sex:
- female
- Details on test animals and environmental conditions:
- TEST ANIMALS: mice, CBA/CaOlaHsd
- Source: Harlan Laboratories B.V., Postbus 6174, 5960 AD Horst / The Netherlands
- Age at study initiation: 8 - 9 weeks
- Weight at study initiation: 17.3 - 23.4 g
- Housing: group caging
- Diet: pelleted standard diet (Harlan Laboratories B.V., 5960 AD Horst, The Netherlands), ad libidum
- Water: tap water, (Gemeindewerke, 64380 Rossdorf, Germany), ad libitum
- Acclimation period: At least 5 days prior to the start of dosing under test conditions after health examination. Only animals without any visible signs of illness were used for the study
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 + 2°C
- Humidity (%): 45-65 %
- Photoperiod (hrs dark / hrs light): artificial light 6:00 a.m. - 6:00 p.m.
- Bedding: granulated soft wood bedding (Rettenmaier & Söhne GmbH + Co. KG, 73494 Rosenberg, Germany)
Study design: in vivo (LLNA)
- Vehicle:
- dimethylformamide
- Concentration:
- 10, 25, and 50% (w/w)
- No. of animals per dose:
- 4
- Details on study design:
- RANGE FINDING TESTS:
A solubility experiment was performed according to the recommendations given by OECD 429. The highest test item concentration, which can be technically used, was 100% of the undiluted test item. Test item solution at different concentrations was prepared using dimethylformamide as vehicle. Vortexing was used to formulate the test item.
To determine the highest non-irritant test concentration that at the same time did not induce signs of systemic toxicity, a pre-test was performed in two animals and stated in raw data and report. Two mice were treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 50 and 100% once daily each on three consecutive days. Prior to the first application of the test item and before sacrifice the body weight was determined. Clinical signs were recorded at least once daily. Eventual signs of local skin irritation were documented and a score was used to grade a possible erythema of the ear skin. Furthermore, prior to the first application of the test item (day 1), on day 3 and before sacrifice (day 6) the ear thickness was determined using a micrometer (S0247 Kroeplin, 36381 Schlüchtern, Germany). Additionally, for both animals, the ears were punched after sacrifice (day 6) at the apical area using a biopsy punch (Stiefel, Ø 8 mm corresponding to 0.5 cm2) and were immediately pooled per animal and weighed using an analytical balance. Eventual ear irritation was considered to be excessive if an erythema of the ear skin of a score value ≥3 was observed at any observation time and/or if an increase in ear thickness of ≥25% was recorded on day 3 or day 6 (for detailed results see Annex 1).
Due to the strong local skin irritation and toxic reaction of the animal treated with 100% test item concentration, the third application was omitted, but the observation was continued until day 6. From day 2 to day 6, the animals showed an erythema of the ear skin (animal treated with 50%: from day 3 to day 6 Score 1, animal treated with 100%: day 2, day 5, and day 6 Score 1, day 3 and day 4 Score 2). Swelling of the ears was observed on day 3 (both animals) and on day 4 (only the animal treated with 100%). The animal treated with 100% appeared to be exited was observed on day 3, day 4, and day 5. Furthermore, at the 100% test item concentration, an open wound was observed in the neck region from day 3 to 6. For detailed results of the observations and measurements, see Annex 1.
Based on the above mentioned results, 100% could not be used as highest test item concentrationin the main study. Thus, the test item in the main study was assayed at 10, 25, and 50% (w/w).
MAIN STUDY:
TOPICAL APPLICATION:
Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear (left and right) with different test item concentrations of 10, 25, and 50% (w/w). in dimethylformamide. The application volume, 25 µL was spread over the entire dorsal surface (~ 8 mm) of each ear once daily for three consecutive days. A futher group of mice was treated with an equivalent volume of the relevant vehicle alone (control animals).
ADMINISTRATION OF 3H-METHYL THYMIDINE AND DETERMINATION OF INCORPORATED 3H-METHYL THYMIDINE
Five days after the first topical application, all mice were intraveneously injected into a tail vein with radio-labelled thymidine (3HTdR). Approximately five hours after treatment with 3HTdR all mice were sacrificed and the draining lymph nodes were excised and pooled per group. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes, which were subsequently washed with phosphate buffered saline and incubated with trichloroacetíc acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of 3H-methyl thymidine measured in a ?-scintillation counter.
INTERPRETATION OF RAW DATA
The proliferation response of lymph node cells is expressed as the number of radioactive disintegrations per minute per lymph node (DPM/node) and as the ratio of 3HTdR incorporated into lymph node cells of test lymph nodes relative to that recorded for control lymph nodes (stimulation index). Before DPM/node values were determined, mean scintillation-background DPM was subtracted from test and control raw data. A test item is regarded as a sensitiliser in the LLNA if the following criteria are fulfilled:
-First, that exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the stimulation index.
-Second, that 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.
OBSERVATIONS
In addition to the sensitising reactions the following observations and data were recorded during the test and observation period:
Mortality / Viability: At least once daily from experimental start to necropsy.
Body weights: In the pre-test prior to the first application and prior to sacrifice. In the main experiment: prior to the first application and prior to treatment with 3HTdR.
Ear thickness: In the pre-test prior to the first application of the test item (day 1), on day 3 and before sacrifice (day 6).
Ear weights: In the pre-test after sacrifice; biopsy punches were taken from each ear.
Clinical signs (local / systemic): Clinical signs (systemic toxicity or local skin irritation) were recorded at least once daily. Especially the treatment sites were observed carefully. - Positive control substance(s):
- hexyl cinnamic aldehyde (CAS No 101-86-0)
- Statistics:
- The mean values and standard deviations of body weights were calculated.
Results and discussion
- Positive control results:
- Experiment performed in April 2012 (Harlan study number 1486301) using concentrations of 5, 10, and 25 % alpha-hexyl cinnamic aldehyde in acetone:olive oil (4:1). These concentrations yielded S.I.´s of 1.70, 1.81, and 5.90, respectively. The EC3 value calculated was 14.4 % (w/v).
The positive control substance alpha-hexyl cinnamic aldehyde was found to be a skin sensitizer under the described conditions, demonstrating the validity of the study.
In vivo (LLNA)
Resultsopen allclose all
- Key result
- Parameter:
- SI
- Value:
- 1.52
- Test group / Remarks:
- 10% test in DMF
- Key result
- Parameter:
- SI
- Value:
- 1.21
- Test group / Remarks:
- 25% test in DMF
- Key result
- Parameter:
- SI
- Value:
- 1.32
- Test group / Remarks:
- 1.32% test in DMF
Any other information on results incl. tables
Vehicle: dimethylformamide
Test item concentration % (w/w) |
Group |
Measurement DPM |
Calculation |
Result |
||
DPM-BGa) |
number of lymph nodes |
DPM per lymph nodeb) |
S.I. |
|||
--- |
BG I |
27 |
--- |
--- |
--- |
--- |
--- |
BG II |
22 |
--- |
--- |
--- |
--- |
0 |
1 |
2569 |
2545 |
8 |
318.1 |
1.00 |
10 |
2 |
3904 |
3880 |
8 |
484.9 |
1.52 |
25 |
3 |
3112 |
3088 |
8 |
385.9 |
1.21 |
50 |
4 |
3382 |
3358 |
8 |
419.7 |
1.32 |
BG = Background (1 ml 5% trichloroacetic acid) in duplicate
1 = Control Group
2-4= Test Group
S.I. = Stimulation Index
a) = The mean value was taken from the figures BG I and BG II
b) = Since the lymph nodes of the animals of a dose group were pooled, DPM/node was determined by dividing the measured value by the number of lymph nodes pooled
The EC3 value could not be calculated, since all S.I.´s are below the threshold value of 3.
VIABILITY / MORTALITY
No deaths occurred during the study period.
CLINICAL SIGNS
The animals did not show any signs of systemic toxicity during the course of the study. On day 3 and day 4,all animals treated withthe test item concentrations of 10, 25, and 50% showed an erythema of the ear skin (Score 1).
BODY WEIGHTS
The body weight of the animals, recorded prior to the first application and prior to treatment with 3HTdR, was within the range commonly recorded for animals of this strain and age.
Applicant's summary and conclusion
- Interpretation of results:
- not sensitising
- Remarks:
- Migrated information Criteria used for interpretation of results: EU
- Conclusions:
- The test item was not a skin sensitiser under the test conditions of this study.
- Executive summary:
In order to study a possible skin sensitising potential of the test item, three groups each of four female mice were treated once daily with the test item at concentrations of 10, 25, and 50% (w/w) in dimethylformamide by topical application to the dorsum of each ear for three consecutive days. The highest concentration tested was the highest concentration that could be applied whilst avoiding systemic toxicity and excessive local skin irritation as confirmed by a pre-experiment. A control group of four mice was treated with the vehicle (dimethylformamide) only. Five days after the first topical application the mice were injected intravenously into a tail vein with radio-labelled thymidine (3H-methyl thymidine). Approximately five hours after intravenous injection, the mice were sacrificed, the draining auricular lymph nodes excised and pooled per group. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes, which were subsequently washed and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of3H-methyl thymidine measured in ab-scintillation counter.
The animals did not show any signs of systemic toxicity during the course of the study and no cases of mortality were observed. On day 3 and day 4,all animals treated withthe test item concentrations of 10, 25, and 50% showed an erythema of the ear skin (Score 1).
A test item is regarded as a sensitiser in the LLNA if the exposure to one or more test concentration resulted in a 3-fold or greater increase in incorporation of3HTdR compared with concurrent controls, as indicated by the Stimulation Index (S.I.). The estimated concentration of test item required to produce a S.I. of 3 is referred to as the EC3 value.
In this study Stimulation Indices of 1.52, 1.21, and 1.32 were determined with the test item at concentrations of 10, 25, and 50% (w/w) in dimethylformamide. A dose response was not observed. The EC3 value could not be calculated, since none of the tested concentrations induced a S.I. greater than the threshold value of 3.
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