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EC number: 916-899-6 | CAS number: -
- 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
Endpoint summary
Administrative data
Description of key information
Based on a Local Lymph Node Assay (LLNA) following OECD TG 429, Bayscript Blaukomponente TEA should be considered as moderate skin sensitiser (Skin Sens 1B).
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:
- Experimental Start Date (animal arrival): 12 May 2020 Experimental Completion Date: 09 September 2020
- 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)
- Version / remarks:
- adopted 22 July 2010
- 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)
- Specific details on test material used for the study:
- dark blue to brown crystalline powder
- Species:
- mouse
- Strain:
- CBA/Ca
- Sex:
- female
- Details on test animals and environmental conditions:
- Animal Specifications and Acclimatisation
Nulliparous, non-pregnant female CBA/CaCrl strain mice were obtained from Charles River (UK) Ltd., Margate. All animals were given a clinical inspection for ill health on arrival and a sample was weighed.
Mice were arbitrarily selected from the delivery boxes and allocated to the appropriate number of cages. The condition of the animals was assessed daily throughout the acclimatisation period of 8 to 30 days. A second inspection was performed prior to study commencement to ensure the animals were suitable for the study. Overtly healthy animals were arbitrarily allocated to the study groups on the day prior to commencement of treatment.
Animals in the initial main study were in a body weight range of 19 to 23 g on the day before dosing commenced. Animals in the second main experiment were in a body weight range of 19 to 25 g on the day before dosing commenced. Individual body weights were within ±20% of the mean body weight for mice on the study. Based on information from the supplier the mice were approximately 11 to 13 weeks old on Day 1.
Environmental Conditions, Diet and Water
The animals were kept in the following conditions except for short periods of time where experimental procedures dictated otherwise.
Housing
The animals were housed in groups of up to five during acclimatisation in cages that conformed to the 'Code of Practice for the Housing and Care of Animals Bred, Supplied or Used for Scientific Purposes’ (Home Office, London, 2014). From Day 1, the preliminary study animal was individually housed and the main study animals were housed in groups of up to three.
Bedding was provided on a weekly basis to each cage by use of clean European softwood bedding (Datesand Ltd., Manchester, UK). The bedding had been analysed for specific contaminants and the results retained on file at Covance.
No contaminants were present in bedding at levels which might have interfered with achieving the objective of the study.
Water
Mains water was provided, ad libitum, via cage-mounted water bottles. The water had been periodically analysed for specific contaminants.
No contaminants were present in water at levels which might have interfered with achieving the objective of the study. Results are retained on file at Covance.
Diet
5LF2 EU Rodent Diet 14%, was freely available to the animals at all times. Each batch of diet had been analysed for specific constituents and contaminants by the manufacturer.
No contaminants were present in diet at levels which might have interfered with achieving the objective of the study. Results are retained on file at Covance.
Environment
The animal rooms were designed to permit a minimum of 15 air changes per hour. The temperature and humidity ranges were 19 to 25C and 40 to 70% respectively. Daily recordings of maximum and minimum temperature and humidity were made.
Fluorescent lighting was controlled automatically to give a cycle of 12 hours light and 12 hours dark.
Environmental Enrichment
In order to enrich both the environment and the welfare of the animals, they were provided with wooden Aspen chew blocks and nesting materials. The nesting materials were removed from the cages the day before dosing.
Animal Identification and Assignment to Study
A unique number inscribed onto the tail with indelible ink on the day prior to dosing individually identified the mice. A colour-coded card on each cage gave information including study number, animal number and sex.
Mice were arbitrarily selected from the delivery boxes and allocated to the appropriate number of cages. Overtly healthy animals were arbitrarily allocated to the study groups on the day prior to commencement of treatment. - Vehicle:
- dimethylformamide
- Concentration:
- Preliminary study: 50% w/v in DMF
Main study: 10, 25, 50 % w/v - No. of animals per dose:
- Five
- Details on study design:
- Test Article Formulation
Formulations were freshly prepared as required using DMF on Days 1, 2 and 3. The formulations were stored at room temperature, in sealed, air-tight containers prior to dosing and were used within two hours of preparation.
The formulations were mixed by multiple inversion of the containers prior to administration to ensure homogeneity.
Concentrations of test article were expressed gravimetrically and in terms of test article received (without regard to purity or active content).
Formulations Analysis
Triplicate samples (3 x 1 mL) were taken from each Bayscript Blaukomponente TEA formulation used in the main studies. A single sample (1 mL) of the vehicle control was also retained. All samples were analysed for achieved concentration and homogeneity.
Duplicate samples (2 x 1 mL) were taken from each stratum of one low and one high concentration (spanning the range of concentrations to be used in the main studies) and analysed for achieved concentration. The remaining bulk formulations were sampled at 6 hours (3 x 1 mL) and analysed for achieved concentration following 6 hours storage at room temperature, protected from light, in order to determine stability. The analytical methods and results are presented in the Formulations Analysis Report. Any remaining unanalysed samples were retained until report finalisation and were then discarded.
Preliminary Screening Test
In the absence of toxicological information regarding irritation and/or systemic toxicity, a preliminary screening test was conducted with one animal.
The mouse was treated by daily application of 25 µL of the test article at the maximum suitable concentration (50% w/v in DMF) to the dorsal surface of each ear for three consecutive days (Days 1, 2 and 3). The mouse was observed daily for any signs of toxicity or irritation at the application site. The body weight was recorded on Day -1 and prior to termination on Day 6. Both ears were observed for erythema and scored
Ear thickness measurements were taken using a thickness gauge on Day 1 (pre-dose), Day 3 (approximately 48 hours after the first dose) and Day 6.
Excessive local irritation is indicated by an erythema score ≥3 and/or an increase in ear thickness of ≥25% on any day of measurement.
The animal was killed by an intraperitoneal injection of an overdose of sodium pentobarbitone at the end of the observation period. Death was confirmed by cervical dislocation.
Main Study
Groups of five female mice were assigned to study according to the table given below. Doses were selected from the concentration series 50%, 25%, 10%, 5%, 2.5%, 1.0%, 0.5% etc. In the initial main study, three consecutive concentrations were selected on the basis of the preliminary screening test so that the highest concentration maximised exposure whilst avoiding systemic toxicity and excessive local irritation (Groups 1 to 5).
A low concentration of 10% was incorrectly used in the initial main study. A second main experiment was therefore conducted using a concentration of 2%, with an additional dose group using a concentration of 0.2% included due to the high SI values noted in the initial main study, to ensure that an EC3 concentration could be calculated (Groups 6 to 9).
Inlife Procedures
Test Article Administration
Each mouse was manually restrained with both auditory pinnae left free. The outer aspect of both pinnae of each mouse was treated by direct application of the appropriate test or control formulation (0.025 mL/pinna) dispensed from an automatic micro pipette.
Treatment Regimen
The nine groups of five female mice were subjected to application of the vehicle control, positive control or one of the test formulations to the outer aspect of the auditory pinnae, once daily on Days 1, 2 and 3.
On Day 6 the mice were placed in a thermacage in order to dilate the peripheral blood vasculature and thus facilitate intravenous dosing. Each mouse was transferred to a cylindrical restrainer. A plastic syringe and fine gauge hypodermic needle were used to administer 0.25 mL phosphate buffered saline incorporating 20 μCi of 3HTdR into a tail vein of each mouse by slow bolus injection.
After this treatment, the mice were returned to their cages.
Approximately five hours after intravenous injection of the 3HTdR, all mice were killed by exsanguination under a deep plane of inhalation anasthesia. Killing was organised to minimise the interval between death and the recovery of the auricular lymph nodes to no more than fifteen minutes.
Clinical Signs
Treated mice were observed twice daily on Days 1 to 5 and once on Day 6 for clinical signs of reaction to treatment or for irritation or other changes at the sites of application of the test article.
Routine Health Checks
All animals were examined at the beginning and end of the working day throughout the acclimatisation and study periods to ensure they were in good health.
Body Weights
Mice were weighed on Day -1 (the day before dosing) and on Day 6 prior to intravenous administration of 3HTdR.
Terminal Procedures
Recovery of Lymph Nodes
Once death had been confirmed each mouse was placed on a bench lined with paper with its ventral aspect uppermost. A mid-line incision from the lower jaw to the sternum was made and the cervical structures were exposed by reflecting the skin. The auricular lymph nodes were located and removed using curved end forceps. Any connective tissue was removed from the capsule of the nodes. The auricular lymph nodes of each mouse were placed into individual petri dishes containing 5 mL phosphate buffered saline.
Preparation for Scintillation Count
The lymph nodes collected into each petri dish were cut open and disaggregated by squashing the fragments with a sharp blade. The resultant liquor was transferred into code-identified conical tubes. The petri dishes were rinsed with an additional 5 mL phosphate buffered saline and the second liquor was added to the first liquor. At each transfer, debris such as fragments of capsule, were retained in the petri dish wherever possible.
After 5 minutes the pooled liquor was filtered into a second conical tube by transferring the liquor into a 10 mL syringe and passing it through a stainless steel gauze containing a fabric filter, cut to size (Clarcor UK, Lockertex Filtration Products, Warrington, UK). Any visible sediment remaining prior to filtering was left in the conical tube. The liquor was centrifuged at 200 g for 10 minutes.
Following centrifugation, the supernatant was discarded and the pellet was resuspended in 5 mL phosphate buffered saline. This was centrifuged at 200 g for 10 minutes. The supernatant was discarded and the pellet resuspended in 3 mL of 5% w/v aqueous trichloroacetic acid. The suspension was stored for 18 hours at 2 to 8°C (nominal 4°C).
On the following day the suspension was re-centrifuged at 200 g for 10 minutes and the supernatant was drawn off and discarded. The pellet was resuspended in 1 mL 5% w/v aqueous trichloroacetic acid then subjected to ultrasonic dispersion for 25 minutes to ensure a homogenous suspension. The suspension (1 mL) was transferred to a scintillation vial and scintillation fluid (ca 10 mL) was added.
Scintillation Counting
Once prepared the scintillation vials were placed in the appropriate carrier racks. Two background vials were prepared, one containing ca 10 mL of scintillation fluid and the other containing 1 mL of 5% w/v aqueous trichloroacetic acid and ca 10 mL scintillation fluid. The carrier rack was passed into the scintillation counter. All vials, including the background samples, were submitted for liquid scintillation counting for 10 minutes, using a 3H quench curve.
Incorporation of 3HTdR is measured by ß-scintillation counting as disintegrations per minute (DPM) over a ten-minute period. This value was corrected to account for the background containing 5% w/v aqueous trichloroacetic acid and scintillation fluid. - Positive control substance(s):
- hexyl cinnamic aldehyde (CAS No 101-86-0)
- Positive control results:
- The positive control article produced a Stimulation Index of 15.73 (initial main study) and 7.88 (second main study), demonstrating adequate performance of the assay.
- Key result
- Parameter:
- SI
- Value:
- 0.96
- Test group / Remarks:
- 0.2% w/v in applied formulation
- Key result
- Parameter:
- SI
- Value:
- 1.36
- Test group / Remarks:
- 2% w/v in applied formulation
- Key result
- Parameter:
- SI
- Value:
- 10.12
- Test group / Remarks:
- 10% w/v in applied formulation
- Key result
- Parameter:
- SI
- Value:
- 14.1
- Test group / Remarks:
- 25% w/v in applied formulation
- Key result
- Parameter:
- SI
- Value:
- 8.7
- Test group / Remarks:
- 50% w/v in applied formulation
- Key result
- Parameter:
- EC3
- Value:
- 3.5
- Test group / Remarks:
- All groups
- Cellular proliferation data / Observations:
- Preliminary Screening Test
Death or signs of systemic toxicity/excessive irritation were not noted.
Main Test
Mortality
All animals survived treatment with Bayscript Blaukomponente TEA.
Clinical Signs
There were no clinical signs indicative of a systemic effect of treatment among mice treated with the vehicle or with 0.2, 2, 10, 25 or 50% w/v formulations of the test article.
The vehicle and test formulation application sites remained free of irritation.
Greasy fur behind the ears and on the back of the neck was noted in all Group 5 animals on Days 1 to 3, in all Group 2 to 4 animals on Days 2 and 3. Group 4 animals had thinning fur behind the ears on Day 6.
Greasy fur to the back of the neck and head was noted in all Group 9 animals on Days 1 to 4.
Body Weights
There was no indication of a treatment related effect on body weight (Table 10.5). - Interpretation of results:
- Category 1B (indication of skin sensitising potential) based on GHS criteria
- Conclusions:
- The positive control article produced a Stimulation Index of 15.73 (initial main study) and 7.88 (second main study), demonstrating adequate performance of the assay.
The Local Lymph Node Assay demonstrated that Bayscript Blaukomponente TEA has the potential to cause skin sensitisation. - Executive summary:
This study was conducted to assess the potential of the test article to cause skin sensitisation in the mouse.
Following a preliminary screening test using a 50% w/v formulation, the test article was prepared for administration at 0.2. 2, 10, 25 and 50% w/v in dimethylformamide (DMF).
Groups of five female CBA / CaCrl mice were subjected to topical applications of positive control, vehicle control or of one of the test formulations to the outer aspect of the auditory pinnae once daily on Days 1, 2 and 3. On Day 6 a 20 μCi dose of tritiated3H-methyl thymidine was injected intravenously into each mouse. Approximately five hours later the auricular lymph nodes were recovered from each animal. The pairs of nodes from each animal were pooled and suspensions of the cellular components of the lymph nodes were prepared in 5% w/v trichloroacetic acid and processed through a scintillation counter.
Test results (shown below) are expressed in terms of Stimulation Indices, the ratios of the mean scintillation counts obtained from the test groups relative to the corresponding mean scintillation count obtained from controls. The threshold level for the Stimulation Index to be considered a positive indicator of the potential to cause skin sensitisation is 3.0.
Concentration of Test Article in Applied Formulation (% w/v)
0.2%
2%
10%
25%
50%
StimulationIndex
0.96
1.36
10.12
14.10
8.70
The EC3 value was calculated to be 3.50.
The results of formulation analyses demonstrated homogeneity and stability of the samples (6 hours at room temperature) and achieved concentrations within 100±10% of the nominal test article concentrations, with a relative standard deviation (RSD)≤5%,and were therefore considered acceptable.No significant degradation (>10%) was observed over the storage period.No test article was detected in the vehicle samples.
The positive control article produced a Stimulation Index of 15.73 (initial main study) and 7.88 (second main study), demonstrating adequate performance of the assay.
The Local Lymph Node Assay demonstrated that Bayscript Blaukomponente TEA has the potential to cause skin sensitisation.
Reference
Formulations Analysis Results
The results of formulation analyses demonstrated homogeneity and stability of the samples (6 hours at room temperature) and achieved concentrations within 100±10% of the nominal test article concentrations, with a relative standard deviation (RSD)≤5%,and were therefore considered acceptable (Formulations Analysis Report).No significant degradation (>10%) was observed over the storage period.No test article was detected in the vehicle samples.
Individual DPMs and Stimulation Index (SI)
Concentration (% w/v) in DMF |
Group Number |
Animal Number |
DPM / Animal |
Mean DPM / Animal (Standard Deviation) |
Stimulation Index (SI)a |
Vehicle |
1 |
402 |
136 |
265 |
NA |
403 |
359 |
||||
404 |
245 |
||||
405 |
234 |
||||
406 |
353 |
||||
10 |
2 |
407 |
2914 |
2687 |
10.12 |
408 |
4304 |
||||
409 |
2506 |
||||
410 |
2323 |
||||
411 |
1386 |
||||
25 |
3 |
412 |
2325 |
3742 |
14.10 |
413 |
3736 |
||||
414 |
4635 |
||||
415 |
3030 |
||||
416 |
4985 |
||||
50 |
4 |
417 |
1917 |
2309 |
8.70 |
418 |
2504 |
||||
419 |
2100 |
||||
420 |
2106 |
||||
421 |
2920 |
||||
Positive control |
5 |
422 |
3412 |
4174 |
15.73 |
423 |
6933 |
||||
424 |
4335 |
||||
425 |
1718 |
||||
426 |
4473 |
||||
Vehicle |
6 |
487 |
388 |
421 |
NA |
488 |
654 |
||||
489 |
393 |
||||
490 |
272 |
||||
491 |
398 |
||||
0.2 |
7 |
492 |
580 |
404 |
0.96 |
493 |
488 |
||||
494 |
273 |
||||
495 |
297 |
||||
496 |
380 |
||||
2 |
8 |
497 |
376 |
572 |
1.36 |
498 |
582 |
||||
499 |
643 |
||||
500 |
584 |
||||
501 |
677 |
||||
Positive control |
9 |
502 |
3477 |
3316 |
7.88 |
503 |
2780 |
||||
504 |
3646 |
||||
505 |
4256 |
||||
506 |
2420 |
a= Stimulation Index of 3.0 or greater indicates a positive result
NA= Not applicable
Endpoint conclusion
- Endpoint conclusion:
- adverse effect observed (sensitising)
- Additional information:
This study was conducted to assess the potential of the test article to cause skin sensitisation in the mouse (LLNA following OECD TG 429).
Following a preliminary screening test using a 50% w/v formulation, the test article was prepared for administration at 0.2. 2, 10, 25 and 50% w/v in dimethylformamide (DMF).
Groups of five female CBA / CaCrl mice were subjected to topical applications of positive control, vehicle control or of one of the test formulations to the outer aspect of the auditory pinnae once daily on Days 1, 2 and 3. On Day 6 a 20 μCi dose of tritiated3H-methyl thymidine was injected intravenously into each mouse. Approximately five hours later the auricular lymph nodes were recovered from each animal. The pairs of nodes from each animal were pooled and suspensions of the cellular components of the lymph nodes were prepared in 5% w/v trichloroacetic acid and processed through a scintillation counter.
Test results (shown below) are expressed in terms of Stimulation Indices, the ratios of the mean scintillation counts obtained from the test groups relative to the corresponding mean scintillation count obtained from controls. The threshold level for the Stimulation Index to be considered a positive indicator of the potential to cause skin sensitisation is 3.0.
Concentration of Test Article in Applied Formulation (% w/v)
0.2%
2%
10%
25%
50%
StimulationIndex
0.96
1.36
10.12
14.10
8.70
The EC3 value was calculated to be 3.50, and thus, shows a moderate skin sensitizing potential.
The positive control article produced a Stimulation Index of 15.73 (initial main study) and 7.88 (second main study), demonstrating adequate performance of the assay.
The Local Lymph Node Assay demonstrated that Bayscript Blaukomponente TEA has the potential to cause moderate skin sensitisation.
Respiratory sensitisation
Endpoint conclusion
- Endpoint conclusion:
- no study available
Justification for classification or non-classification
According to the available data the registered substance has to be classified for Skin Sensitization with Skin Sens 1B (H317) according to Regulation (EC) No 1272/2008, Annex I.
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