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EC number: 208-341-9 | CAS number: 523-24-0
- 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
Skin sensitisation
Administrative data
- Endpoint:
- skin sensitisation: in vivo (LLNA)
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- The study was conducted between 05 April 2017 and 25 April 2017
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 017
- Report date:
- 2017
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.42 (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:
- Diammonium phthalate
- EC Number:
- 208-341-9
- EC Name:
- Diammonium phthalate
- Cas Number:
- 523-24-0
- Molecular formula:
- C8H6O4.2H3N
- IUPAC Name:
- diammonium phthalate
- Test material form:
- solid: crystalline
Constituent 1
- Specific details on test material used for the study:
- Identification: Diammonium phthalate
Batch: 7172002
Purity: 99.3%
Physical state, appearance: White crystalline powder
Expiry Date: August 2017
Storage Conditions: At room temperature, protected from moisture
In vivo test system
Test animals
- Species:
- mouse
- Strain:
- CBA
- Remarks:
- CBA/CaOlaHsd
- Sex:
- female
- Details on test animals and environmental conditions:
- TEST ANIMALS:
Source: Envigo RMS B.V., / The Netherlands
Number of animals for the pre-test: 2 females
Number of animals for the main study: 16 females
Number of animals per group: 4 females (nulliparous and non-pregnant)
Number of test groups: 3
Number of control (vehicle) groups: 1
Age (beginning of treatment): Pre-test: 10 - 11 weeks; Main study: 8 - 9 weeks
Body weight (at start of experiment): Main study: 16.7 to 20.7 g
Identification: The animals were distributed into the test groups at random. All animals belonging to the same experimental group were kept in one cage. In the main experiment, the animals were identified by tail tags. In the pre-experiment, animals were identified by cage number.
Acclimation: 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:
The animals were kept conventionally. The experiment was conducted under standard laboratory conditions.
Housing: group
Cage Type: Makrolon Type II (pre-test) / III (main study), with wire mesh top
Bedding: granulated soft wood bedding
Feed: 2018C Teklad Global 18% protein rodent diet (certified), ad libitum
Water: tap water, ad libitum
Environment: temperature 22 ± 2°C
relative humidity approx. 45-65% (except for deviation)
artificial light 6.00 a.m. - 6.00 p.m.
Study design: in vivo (LLNA)
- Vehicle:
- other: ethanol/water (3+7, v/v)
- Concentration:
- 0, 5, 10, and 25% (w/v).
- No. of animals per dose:
- 4 females per dose
- Details on study design:
- TEST ITEM PREPARATION:
VEHICLE AND DOSE SELECTION:
A solubility experiment was performed according to the recommendations given by OECD 429. The highest test item concentration which could be technically used was a 25% solution in ethanol/water (3+7, v/v). Vortexing was used to formulate the test item. At higher concentrations, an applicable formulation of the test item was not achieved by the use of other vehicles.
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 . Two mice were treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 10 and 25% 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 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. Additionally, for both animals, the ears were punched after sacrifice (day 6) at the apical area using a biopsy punch (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.
At the tested concentrations the animals did not show any signs of systemic toxicity. On days 4 and 5, the animals showed an erythema of the ear skin (Score 1).
Thus, the test item in the main study was assayed at 5, 10, and 25%. The highest concentration tested was the highest level that could be achieved whilst avoiding systemic toxicity and excessive local skin irritation as confirmed in the pre-experiment.
TEST ITEM PREPARATION:
The test item was placed into a volumetric flask on a tared balance and ethanol/water (3+7, v/v) was quantitatively added (w/v).
The different test item concentrations were prepared serially.
The preparations were made freshly before each dosing occasion.
EXPERIMENTAL DESIGN AND STUDY CONDUCT
Test Item Administration
Each test group of mice was treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 5, 10, and 25% in ethanol/water (3+7, v/v). The application volume, 25 μL/ear/day, was spread over the entire dorsal surface (Ø ~ 8 mm) of each ear once daily for three consecutive days. A further group of mice (control animals) was treated with an equivalent volume of the relevant vehicle alone (control animals).
Administration of 3H-methyl-thymidine:
Five days after the first topical application (day 6) 250 μL of phosphate-buffered saline containing 20.4 μCi of 3H-methyl thymidine (equivalent to 81.4 μCi/mL 3HTdR) were injected into each test and control mouse via the tail vein.
Terminal Procedure:
Approximately five hours after treatment with 3HTdR all mice were euthanized by using CO2, which was, after harvesting of the lymph nodes, followed by cervical dislocation to ensure death.
Preparation of Single Cell Suspensions:
The draining lymph nodes were rapidly excised and pooled for each experimental group (8 nodes per group). Single cell suspensions (in phosphate buffered saline) of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 μm mesh size). After washing two times with phosphate buffered saline (approx. 10 mL) the lymph node cells were resuspended in 5 % trichloroacetic acid (approx. 3 mL) and incubated at approximately +4 °C for at least 18 hours for precipitation of macromolecules.
Determination of cellular proliferation (incorporation of 3HTdR):
The precipitates were then resuspended in 5 % trichloroacetic acid (1 mL) and transferred to scintillation vials with 10 mL of scintillation liquid and thoroughly mixed. The level of 3HTdR incorporation was then measured in a β-scintillation counter. Similarly, background 3HTdR levels were also measured in two 1 mL-aliquots of 5 % trichloroacetic acid. The β-scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM).
OBSERVATIONS:
Clinical Observations:
All animals were observed on a daily basis, including pre- and post-dose observations on days 1, 2 and 3. Any clinical signs of systemic toxicity, local skin irritation or signs of ill health during the study were recorded.
Determination of Ear Thickness:
In the pre-test, the ear thickness was determined prior to the first application of the test item (day 1), on day 3, and on day 6 prior to sacrifice using a micrometer.
In the pre-test, after the lymph nodes had been excised, both ears of mice were punched at the apical area using a biopsy punch (Ø 8 mm corresponding to 0.5 cm2). For each animal both punches were immediately weighed per animal using an analytical balance.
Determination of Body Weights:
The body weights were recorded on day 1 (prior to dosing) and prior to sacrifice (pre-test) or prior to treatment with 3HTdR (main experiment)
DATA EVALUATION:
Interpretation of raw data:
The proliferative response of the lymph node cells is expressed as the number of radioactive disintegrations per minute per lymph node (DPM/lymph node) and as the ratio of 3HTdR incorporated into lymph node cells of test animals relative to that recorded for lymph nodes of control animals (Stimulation Index; S.I.). Before DPM/lymph node values were determined, mean scintillation-background DPM was subtracted from test and control raw data.
A test item is regarded as a sensitiser 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. - Positive control substance(s):
- other: α-Hexylcinnamaldehyde
Results and discussion
- Positive control results:
- The sensitivity and reliability of the experimental technique employed was assessed by use of α-hexyl cinnamaldehyde dissolved in acetone/olive oil (4+1 v/v) which is known to have skin sensitisation properties in mice. The periodic positive control experiment was performed using CBA/CaOlaHsd mice in October 2016.
Test item concentration %: 0
Stimulation Index (S.I.): 1.00
Test item concentration %: 5
Stimulation Index (S.I.): 1.50
Test item concentration %: 10
Stimulation Index (S.I.) 3.84
Test item concentration %: 25%
Stimulation Index (S.I.): 11.76
EC3 calculated as 8.2% (w/v)
The positive control substance was shown to be a skin sensitiser and was within historical control data values.
In vivo (LLNA)
Resultsopen allclose all
- Parameter:
- SI
- Value:
- 1
- Test group / Remarks:
- Control Group
- Remarks on result:
- other: non-sensitising
- Parameter:
- SI
- Value:
- 0.89
- Test group / Remarks:
- 5% test concentration
- Remarks on result:
- other: non-sensitising
- Parameter:
- SI
- Value:
- 0.98
- Test group / Remarks:
- 10% test concentration
- Remarks on result:
- other: non-sensitising
- Parameter:
- SI
- Value:
- 1.2
- Test group / Remarks:
- 25% test concentration
- Remarks on result:
- other: non-sensitising
- Cellular proliferation data / Observations:
- CELLULAR PROLIFERATION DATA:
See table in any other information on results incl. tables section.
EC3 CALCULATION:
The EC3 value could not be calculated, since all S.I.´s were below the threshold value of 3.
CLINICAL OBSERVATIONS:
No deaths occurred during the study period. No signs of systemic toxicity were observed during the study period. On day 3, the animals showed a transient 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.
Any other information on results incl. tables
Calculation and Results of Individual Data
Vehicle: ethanol/water (3+7, v/v)
Test item concentration % |
Group |
Measurement, DPM |
Calculation |
Result |
||
DPM-BGa) |
Number of lymph nodes |
DPM per lymph nodeb) |
S.I. |
|||
- |
BG I |
14 |
- |
- |
- |
- |
- |
BG II |
16 |
- |
- |
- |
- |
0 |
1 |
5307 |
5292 |
8 |
661.5 |
1.00 |
5 |
2 |
4748 |
4733 |
8 |
591.6 |
0.89 |
10 |
3 |
5207 |
5192 |
8 |
649.0 |
0.98 |
25 |
4 |
6386 |
6371 |
8 |
796.4 |
1.20 |
1 = Control Group
2-4 = Test Group
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
Applicant's summary and conclusion
- Interpretation of results:
- GHS criteria not met
- Conclusions:
- The test item Diammonium phthalate was not a skin sensitiser under the test conditions of this study.
- Executive summary:
In the study the test item Diammonium phthalate formulated in ethanol/water (3+7, v/v) was assessed for its possible skin sensitising potential.
For this purpose a local lymph node assay was performed using test item concentrations of 5, 10, and 25% (w/v). The highest concentration tested was the highest concentration that could technically be achieved.
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, the animals showed a transient erythema of the ear skin (Score 1).
I
n this study Stimulation Indices (S.I.) of 0.89, 0.98, and 1.20 were determined with the test item at concentrations of 5, 10, and 25% (w/v) in ethanol/water (3+7, v/v), respectively.
The test item Diammonium phthalate was not a skin sensitiser under the test conditions of this study.
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