[No public or meaningful name is available]

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

12th March 2018 - 16th July 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Similar Substance 01

IUPAC Name:

Similar Substance 01

Test material form:

solid: particulate/powder

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/CaCrl

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 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 18 to 21 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 housed in groups of up to five during acclimatisation and housed in pairs from Day-1 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). 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.

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.

Environment
The animal rooms were designed to permit 15 to 20 air changes per hour. The temperature and humidity ranges were 20 to 24C and 45 to 65% 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.

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Concentration:

10%, 25%, 50%

No. of animals per dose:

4

Details on study design:

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 dimethylformamide (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.

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
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.

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 five groups of four female mice were subjected to application of the vehicle 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 suitable 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 exanguination 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.


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 lymphnodes were pooled by dose group into 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)

Results and discussion

Positive control results:

The positive control article produced a Stimulation Index of 8.5, demonstrating adequate performance of the assay

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

ca. 1.1

Remarks on result:

other: 10% concentration

Results 2

Key result

Parameter:

SI

Value:

ca. 1.1

Remarks on result:

other: 25% concentration

Results 3

Key result

Parameter:

SI

Value:

ca. 1.7

Remarks on result:

other: 50% concentration

Any other information on results incl. tables

Preliminary Screening Test

Death or signs of systemic toxicity/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 DMF.

Main Test

Mortality

All animals survived treatment with test item.

Clinical Signs

There were no clinical signs indicative of a systemic effect of treatment among mice treated with the vehicle or with 10, 25 or 50% w/v formulations of the test article.

The vehicle and test formulation application sites remained free of irritation.

Body Weights

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

Applicant's summary and conclusion

Interpretation of results:

other: non skin sensitizing

Conclusions:

The Local Lymph Node Assay demonstrated that the test item does not have the potential to cause skin sensitisation.
The test article did not meet the criteria for classification as a sensitiser according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS).

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 10, 25 and 50% w/v in dimethylformamide (DMF).

Groups of four female CBA / CaCrl mice were subjected to topical applications of 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 tritiated 3H-methyl thymidine was injected intravenously into each mouse. Approximately five hours later the auricular lymph nodes were recovered from each animal. The lymph nodes from each group 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 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.

The Local Lymph Node Assay demonstrated that the test item does not have the potential to cause skin sensitisation.

The positive control article produced a Stimulation Index of 8.5, demonstrating adequate performance of the assay