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Skin sensitisation

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skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2022-01-25 to 2022-03-08 (Only draft interim report available. Data will be updated upon availability of the final report.)
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Only draft interim report available. Data will be updated upon availability of the final report.

Data source

Reference Type:
study report

Materials and methods

Test guideline
according to guideline
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
GLP compliance:
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Reference substance name:
Reference substance 001
Cas Number:
Molecular formula:
Test material form:
solid: particulate/powder
Details on test material:
- State of aggregation: white crystalline powder
Specific details on test material used for the study:
- Storage condition of test material: stored at 2 to 8 °C, protected from light, under nitrogen and with desiccant

In vivo test system

Test animals

other: CBA/CaCrl
Details on test animals and environmental conditions:
- Source: Charles River (UK) Ltd., Margate
- Females nulliparous and non-pregnant: yes
- Age at study initiation (main study): approximately 8 to 9 weeks old
- Weight at study initiation (main study): 17 to 21 g

- Housing:
acclimatisation period: housed in groups of up to five in cages
main study (from day 1): singly housed

Cages 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 material: clean European softwood bedding (Datesand Ltd., Manchester, UK); porivded on a weekly basis

Environmental enrichment: wooden aspen chew blocks and nesting materials (nesting materials were removed from the cages prior to dosing on Day 1)

- Diet (ad libitum): 5LF2 EU Rodent Diet 14 %
- Water (ad libitum): mains water

- Acclimation period: 15 days

All animals were given a clinical inspection for ill health on arrival and weighed. The condition of the animals was assessed daily throughout the acclimatisation period. 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.

- Temperature: 19 °C to 25 °C
- Humidity: 40 % to 70%
- Air changes: minimum of 15 air changes/hour
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

other: water incorporating 1 % Pluronic® L92
10, 25 and 50 % of the test substance
No. of animals per dose:
5 female mice
Details on study design:
The murine local lymph node assay (LLNA) is the first-choice method for in vivo testing of skin sensitisation. A previous OCED 429 study was submitted to ECHA for review, however the following requirements of the guideline were not fulfilled and therefore a new in vivo study on the endpoint skin sensitisation was required:
- No dose level selection rationale was provided for selecting the highest dose (e.g. 50% in vehicle aqua ad iniectabilia)
- A wholly aqueous vehicle was used without incorporation of an appropriate solubiliser (e.g. 1% Pluronic® L92).

The in vitro test systems for skin sensitisation are not relevant for inorganic substances forming anionic species in aquatic media and all three assays (OECD 442 C, D and E) have their limitations as in vitro methods for sodium dithionite, primarily because this substance is a reducing agent which will not modify cysteine residues, thereby yielding by default negative responses.

The vehicle for the test article was water incorporating 1% Pluronic® L92. The vehicle was chosen following a solubility trial because it produced an homogenous suspension incorporating 50% w/v of the test article.

Test item formulations were freshly prepared as required using water incorporating 1% Pluronic® L92 on Days 1, 2 and 3. The formulations were stored at room temperature, in sealed, air-tight containers prior to dosing. Formulations were allowed to equilibrise for 3 hours prior to dosing (formulation were stirred for the first 30 minutes and then allowed to stand for the remainder of the 3 hours). After equilibration, formulations were used within two hours of preparation.

The formulations were mixed by multiple inversion of the containers prior to administration to ensure homogeneity.

Based on information suggesting that irritation and/or toxicity is possible, an initial 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 water incorporating 1% Pluronic® L92) 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 prior to administration of the test item on Day 1 and prior to termination on Day 6. Both ears were observed for erythema and scored using the Draize scale.

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 concentration for the main study was selected so that the highest concentration maximised exposure whilst avoiding systemic toxicity and excessive local irriation.

Death or signs of systemic toxicity were not noted.

No erythema was observed at the application sites. Furthermore, no increase in ear thickness of ≥ 25% was noted on any day of measurement. Therefore, no excessive local irritation was observed during the preliminary screening test.

Based on this information the dose levels selected for the main test were 10%, 25% and 50% w/v in water incorporating 1% Pluronic® L92.

Please also refer to the section "Overall remarks, attachements".

Doses were selected from the concentration series 75%, 50%, 25%, 10%, 5%, 2.5%, 1.0%, 0.5% etc. Three consecutive concentrations were selected on the basis of the preliminary screening test. As the vehicle for the test article was different to that used for the positive control formulation, a positive control vehicle group (acetone / olive oil in a ratio of 4:1 v/v) was also included in the study.

Treatment regimen:
Groups of five female mice were subjected to application (0.025 mL/pinna) of the vehicle control, positive control vehicle, positive control or one of the test formulations to the outer aspect of both auditory pinnae, once daily on Days 1, 2 and 3.

On Day 6 the mice were injected intravenously 0.25 mL phosphate buffered saline incorporating 20 µCi of 3HTdR (0.74 MBq) into a tail vein of each mouse. After this treatment, the mice were returned to their cages.

Five hours after intravenous injection of the 3HTdR, all mice were killed by exanguination under a deep plane of inhalation anasthesia (isoflurane).

Terminal procedure:
Each mouse was incised in order to remove the auricular lymph nodes. 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.

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 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 syringe and passing it through a stainless steel gauze containing a fabric filter, cut to size. Any visible sediment remaining prior to filtering was left in the conical tube. The liquor was centrifuged and the supernatant was discarded and the pellet was resuspended in 5 mL phosphate buffered saline. This was centrifuged and 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 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 (10 mL) was added.

Once prepared the scintillation vials were placed in the appropriate carrier racks. Two background vials were prepared, one containing 10 mL of scintillation fluid and the other containing 1 mL of 5% w/v aqueous trichloroacetic acid and 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 was 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.

Data evaluation:
The scintillation counter provided data including the DPM value (disintegrations per minute during a ten minute period) for each individual animal. The DPM value was transformed into a mean DPM value for each group. The mean DPM value for each test group was divided by the mean DPM for the control group to provide the Stimulation Index (SI) value for each test group.

The test result is not valid for those groups producing an SI value of 3.0 or more when the sites of application have shown excessive irritation and for those groups that have shown indications of systemic toxicosis.

The test article is regarded as a sensitiser when the maximum value of the SI is 3.0 or above.

The test article is classified as a non-sensitiser when the maximum value of the SI is less than 3.0. (This result is unchanged by observations of irritation at sites of application of the test formulation).

- clinical signs: twice daily on Days 1 to 5 and once on Day 6
- observations of irritation or other changes at treatment site: twice daily on Days 1 to 5 and once on Day 6
- routine health checks: at the beginning and end of the working day throughout the acclimatisation and study periods
- body weights: Day 1 (the first day of dosing, prior to administration) and on Day 6 prior to intravenous administration of 3HTdR
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
As a Stimulation Index value of 3 or more was not obtained for any group in the main study, the DPM values were not statistically analysed.

Results and discussion

Positive control results:
The positive control vehicle group (acetone/olive oil (4:1 v/v)) and the positive control group animals were noted to have greay fur to the back of ears and neck on Days 1 to 6.

The positive control article produced a stimulation Index of 7.65, demonstrating adequate performance of the assay.

In vivo (LLNA)

Resultsopen allclose all
Key result
Test group / Remarks:
10 % test item concentration
Remarks on result:
other: These are interim results only. The LLNA test needs to be repeated in May 2022 and the results will be updated upon the availability of the final study report.
Key result
Test group / Remarks:
25 % test item concentration
Remarks on result:
other: These are interim results only. The LLNA test needs to be repeated in May 2022 and the results will be updated upon the availability of the final study report.
Key result
Test group / Remarks:
50 % test item concentration
Remarks on result:
other: These are interim results only. The LLNA test needs to be repeated in May 2022 and the results will be updated upon the availability of the final study report.
Cellular proliferation data / Observations:
- mortality: all animals survived treatment with sodium dithionite.
- clinical sings: there were no clinical signs indicative of a systemic effect of treatment among mice treated with the test item vehicle (water incorporating Pluronic® L92) or with 10, 25 or 50% w/v formulations of the test article.

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

Please also refer to the section "Overall remarks, attachments".

Applicant's summary and conclusion

During the conduct of the LLNA study, a dosing error occurred, whereby the mid dose animals (25 % (w/v) concentration of test item) received the incorrect dose (50 % (w/v) concentration of the test item) on Day 2 of the study. Therefore, the mid dose results were deemed to be compromised and as such the EC3 value could not be calculated. Therefore, a repeat of the study was conducted. Adequate performance of the assay was demonstrated in the initial study therefore positive control and corresponding vehicle control group were not required.

In the repeated experiment, the SI values were 1.52, 1.55 and 2.03 at the concentrations of 10 %, 25 % and 50 %, respectively. This result indicates the lack of sensitisation potential of sodium dithionite.

Nonetheless, due to the failure of the first test it was decided to conduct a third LLNA test to ascertain that the results obtained in the second study are reliable. Therefore, no statement regarding the classification or non-classification of sodium dithionite according to Regulation (EC) No 1272/2008 and subsequent adaptations can be given at this point. The repetition of the LLNA test is scheduled for May 2022. Upon the availability of the final report of the LLNA study this endpoint will be updated.