Reaction products of tetrazotized 5-amino-2-[(E)-2-(4-amino-2-sulfophenyl)ethenyl]benzenesulfonic acid with 6-amino-4-hydroxynaphthalene-2-sulfonic acid and 4-amino-5-hydroxynaphthalene-2,7-disulfonic acid in 2-[bis(2-hydroxyethyl)amino]ethanol

Endpoint summary

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

Reference

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 25C 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).

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 (±93.0)	NA
		403	359
		404	245
		405	234
		406	353
10	2	407	2914	2687 (±1063.5)	10.12
		408	4304
		409	2506
		410	2323
		411	1386
25	3	412	2325	3742 (±1102.0)	14.10
		413	3736
		414	4635
		415	3030
		416	4985
50	4	417	1917	2309 (± 403.1)	8.70
		418	2504
		419	2100
		420	2106
		421	2920
Positive control	5	422	3412	4174 (±1893.4)	15.73
		423	6933
		424	4335
		425	1718
		426	4473
Vehicle	6	487	388	421 (±140.4)	NA
		488	654
		489	393
		490	272
		491	398
0.2	7	492	580	404 (±129.6)	0.96
		493	488
		494	273
		495	297
		496	380
2	8	497	376	572 (± 117.0)	1.36
		498	582
		499	643
		500	584
		501	677
Positive control	9	502	3477	3316 (±726.0)	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

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.

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.