Registration Dossier

Administrative data

Description of key information

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study was conducted by a GLP accredited laboratory using OECD Testing Guideline 429. The study was conducted on the substance 6-[(p-tosyl)amino]hexanoic acid, compound with 2,2’,2’’- nitrilotriethanol (1:1) (tosyl salt) which is structurally similar to the registered substance.
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymphnode assay (LLNA)
Test material information:
Composition 1
Species:
mouse
Strain:
other: CBA/Ca
Sex:
female
Details on test animals and environmental conditions:
Species Selection

The sex and strain of mouse has been selected for this study in conformance with the design of the validated test method (OECD Guidelines for Testing of Chemicals Method 429 adopted 22 July 2010).

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 15 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 main study were in a body weight range of 17 to 20 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 9 to 10 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 group housed during acclimatisation and individually housed from Day –1 in cages that conformed to the 'Code of Practice for the Housing and Care of Animals Used in Scientific Procedures' (Home Office, London, 1989).
Bedding was provided on a weekly basis to each cage by use of clean European softwood bedding (Datesand Ltd, Manchester, UK).
Each batch of bedding was analysed for specific contaminants. No contaminants were present in bedding at levels which might have interfered with achieving the objective of the study. Results are retained on file at Covance.

Diet

Throughout the study the animals had access ad libitum to SQC Rat and Mouse Maintenance Diet No 1, Expanded, (Special Diets Services Ltd, Witham, UK). Each batch of diet was analysed for specific constituents and contaminants.
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.

Water

Mains water was provided ad libitum via water bottles. The water is 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.

Environment

Rooms were air conditioned to provide a minimum 15 to 20 air changes/hour. The temperature and relative humidity ranges were maintained in the specified ranges of 20 to 24°C and 45 to 65%, respectively.
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

The animals were arbitrarily assigned to treatment groups one day before treatment commenced.
A number written in indelible ink on the tail individually identified the mice. A colour coded card on each cage gave information including study number, animal number and sex.
Vehicle:
dimethylformamide
Concentration:
Following a preliminary screening test using 50% w/v formulation, the test substance was prepared for administration at 10, 25 and 50% w/v in dimethylformamide (DMF)
No. of animals per dose:
4 females
Details on study design:
Preliminary screening test

As no toxicological information was available regarding the systemic toxicity/irritancy potential of the test substance, a preliminary screening test was performed with one mouse. The mouse was treated by daily application of 25 µl of the test substance 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 six days from the initiation of treatment. Any signs of toxicity or irritation during this period were recorded. Ear thickness measurements were made using a thickness gauge on Day 1 (pre-treatment), Day 3 and Day 6.

Both ears were also observed for erythema and scored using the following scale:

No erythema: Score = 0
Very slight erythema (barely perceptible): Score = 1
Well-defined eryhthema: Score = 2
Moderate to severe erythema: Score = 3
Severe erythema (beet redness) to eschar formation preventing grading of erythema: Score = 4

Body weight was recorded on Day 1 and on Day 6. The animal was killed by intraperitoneal injection of sodium pentobarbitone. Death was confirmed by cervical dislocation.

Constitution and function of groups

Groups of four 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. 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.

Group number Group description Concentration (% w/v) Number of animals in group
1 Vehicle control - 4
2 Test – low concentration 10 4
3 Test – intermediate concentration 25 4
4 Test – high concentration 50 4


Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
mercaptobenzothiazole (CAS No 149-30-4)
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
Test results were expressed in terms of Stimulation Indices, the ratios of the scintillation count per group obtained from the test groups relative to the corresponding scintillation count 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. Test substance (10% w/v): 1.3 Test susbtance (25% w/v): 1.3 Test substance (50% w/v): 2.0
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Vehicle control: 696 DPM Test substance (10% w/v): 886 DPM Test substance (25% w/v): 937 DPM Test substance (50% w/v): 1376 DPM Scintillation fluid with 5% w/v trichloroacetic acid: 43 DPM

Preliminary screening test

Death or signs of systemic toxicty/excessive irritation were not noted. Based on this information the dose levels selected for the main test were 10%, 25% and 50% w/v in dimethylformamide.

Main test

Mortality

All animals survived treatment.

Clinical signs

There were no clinical signs indicative of a systemic effect. The vehicle and test formulation application sites remained free of irritation. Greasy fur to the back of the head and neck was noted in all Group 4 animals from Day 3 to Day 6.

Body weights

There was no indication of a treatment related effect on body weight.

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The Local Lymph Node Assay (LLNA) demonstrated that 6-[(p-tosyl)amino] hexanoic acid, compound with 2,2',2''-nitrilotriethanol (1:1) does not have the potential to cause skin sensitisation. The test substance did not meet the criteria for classification as a skin sensitiser according to EU CLP criteria.
Executive summary:

The skin sensitisation potential of the test substance was determined in accordance with the OECD Guideline for Testing of Chemicals 429. Following a preliminary screening test using 50% w/v of the test substance in dimethylformamide (DMF), the test substance was prepared for administration to groups of four female CBA/CaCrl mice. Volumes of 10% w/v, 25% w/v and 50% w/v of the test substance in DMF were applied to the ears of each mouse. A further group was exposed to the vehicle only (DMF). On Day 6 a 20 μCi dose of tritiated 3H-methyl thymidine was injected intravenously into each mouse. Approximately five hours later the auricular lymph nodes were recovered from each animal. The nodes from mice subjected to the same treatment 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 are expressed in terms of Stimulation Indices, the ratios of the mean scintillation count per group obtained from the test groups relative to the corresponding mean scintillation count 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. The Stimulation Indices were found to be 1.3 for the 10% w/v test substance group, 1.3 for the 25% w/v test substance group, and 2.0 for the 50% w/v test substance group. The Local Lymph Node Assay (LLNA) demonstrated that 6-[(p-tosyl)amino] hexanoic acid, compound with 2,2',2''-nitrilotriethanol (1:1) does not have the potential to cause skin sensitisation. The test substance did not meet the criteria for classification as a skin sensitiser according to EU CLP criteria. Read-across between the registered substance and the test substance (tosyl salt) is considered justified owing to the structural similarity of the two substances. Both are salts of a carboxylic acid and triethanolamine. The acid components are structural isomers and differ only in the position of a single methyl group in the N-methyl salt. This is not expected to change the behavior or properties of the substances, and both substances have shown similarity in the results of other physical endpoints such as water solubility.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

The skin irritation potential of the registered substance was determined via read-across to the results of a study on a structurally similar substance i.e. tosyl salt. The tosyl salt was tested in accordance with the OECD Guideline for Testing of Chemicals 429. Following a preliminary screening test using 50% w/v of the test substance in dimethylformamide (DMF), the test substance was prepared for administration to groups of four female CBA/CaCrl mice. Volumes of 10% w/v, 25% w/v and 50% w/v of the test substance in DMF were applied to the ears of each mouse. A further group was exposed to the vehicle only (DMF). 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 nodes from mice subjected to the same treatment 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 are expressed in terms of Stimulation Indices, the ratios of the mean scintillation count per group obtained from the test groups relative to the corresponding mean scintillation count 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. The Stimulation Indices were found to be 1.3 for the 10% w/v test substance group, 1.3 for the 25% w/v test substance group, and 2.0 for the 50% w/v test substance group. The Local Lymph Node Assay (LLNA) demonstrated that 6-[(p-tosyl)amino] hexanoic acid, compound with 2,2',2''-nitrilotriethanol (1:1) does not have the potential to cause skin sensitisation. The test substance did not meet the criteria for classification as a skin sensitiser according to EU CLP criteria. Read-across between the registered substance and the test substance (tosyl salt) is considered justified owing to the structural similarity of the two substances. Both are salts of a carboxylic acid and triethanolamine. The acid components are structural isomers and differ only in the position of a single methyl group in the N-methyl salt. This is not expected to change the behavior or properties of the substances, and both substances have shown similarity in the results of other physical endpoints such as water solubility.


Migrated from Short description of key information:
Study was conducted on the structurally similar substance tosyl salt using OECD 429 according to GLP requirements. The tosyl salt was found to be non-sensitising.

Justification for selection of skin sensitisation endpoint:
Study was conducted on the structurally similar substance tosyl salt using OECD 429 according to GLP requirements. The tosyl salt was found to be non-sensitising.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available
Additional information:

A skin sensitisation study was conducted on the structurally similar substance tosyl salt using OECD 429 according to GLP requirements. The tosyl salt was found to be non-sensitising.

Justification for classification or non-classification

Study was conducted on the structurally similar substance tosyl salt using OECD 429 according to GLP requirements. The tosyl salt was found to be non-sensitising.