Barium 4-dodecylphenolate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020-09-15 to 2020-10-20

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2010-07-22

Deviations:

no

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Barium 4-dodecylphenolate

EC Number:

300-141-0

EC Name:

Barium 4-dodecylphenolate

Cas Number:

93922-04-4

Molecular formula:

C18H30O.1/2Ba

IUPAC Name:

barium 4-dodecylphenolate

Test material form:

solid

Details on test material:

- State of aggregation: brownish, inhomogeneous solid with a chewy texture

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: stored at 15 - 25 °C, dry and protected from light in the original container

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/CaCrl

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Ltd., Margate
- Females nulliparous and non-pregnant: yes
- Age at study initiation (main study): approximately 11 to 12 weeks
- Weight at study initiation (main study): 18 to 24 g
- Housing:
acclimatisation period: housed in groups of up to five in cages

preliminary study (from Day 1): individually housed

main study (from day 1): housed in groups of two or three until immediately after dosing on Day 2 when they were 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)

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

- Diet (ad libitum): 5LF2 EU Rodent Diet 14%
- Water (ad libitum): mains water
- Acclimation period: 8 to 29 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 Day 1, prior to the commencement of treatment.

ENVIRONMENTAL CONDITIONS
- 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)

Vehicle:

methyl ethyl ketone

Concentration:

25 %, 50 % and 75 % of the test substance

No. of animals per dose:

5 female mice

Details on study design:

RATIONALE FOR USE OF AN IN VIVO MODEL
Barium-4-dodecylphenolate is a UVCB, therefore it is not possible to define the molar ratio between substance and peptides for OECD 442 C: In Chemico Skin Sensitisation and so OECD 442 C is not applicable in this situation.

The in vitro methods described in OECD 442 D and E were found to be unsuitable for use with barium-4-dodecylphenolate as the compound was insoluble in all suitable vehicles at sufficient concentrations to meet the OECD 442 E and D Test Guidelines (Dreher, 2020 and Dreher, 2020)*.

Since it is not possible to conduct the specific OECD in vitro methods, an in vivo evaluation is required.

TEST ARTICLE FORMULATION
Formulations were freshly prepared as required using the vehicle on Days 1, 2 and 3. The vehicle was chosen because it was the first of the vehicles listed in the protocol that produced an overtly stable solution, emulsion or dispersion incorporating 75% w/v of the test article. Furthermore, due to the physical-chemical properties of the test item, the test item was stirred and heated to 40-60°C prior to the incorporation of the vehicle in order to facilitate homogenous sampling of the test material. Then a formulation sample for application was allowed to cool down to room temperature prior to use.

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.

PRE-SCREEN TESTS:
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 (75% w/v in vehicle) 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 prior to dosing 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.

Due to the clinical signs and erythema noted in the initial preliminary test, a second preliminary screening test was conducted as described above with one animal, treated by daily application of 25 μL of the test article at a concentration of 50% w/v in vehicle.

Results:
Both animals survived treatment with barium -4-dodecylphenolate.

In the initial preliminary test (75% concentration of test item) clinical signs of hair loss and slight sores/scabs were observed from Day 2 to Day 6. A well-defined erythema (Draize score: 2) was also noted from Day 2 to Day 6.

In the second preliminary test (50% concentration of test item) clinical signs included hair loss from Day 2 to Day 6, dry skin from Days 3 to 5 and greasy fur to the back of the ears and neck on Day 3 to Day 5. A very slight erythema ( Draize score: 1) was noted on Day 2, a well-defined erythema (Draize score: 2) was noted on Days 3 and 4 and no erythema was noted on Days 5 and 6.
These signs were not indicative of systemic toxicity or excessive irritation.

Based on this information the dose levels selected for the main test were 25%, 50% and 75% w/v in the vehicle.

MAIN STUDY
Doses were selected from the concentration series 75%, 50%, 25%, 10%, 5%, 2.5%, 1.0%, 0.5% etc. Due to the physical properties of the substance (32.4% water content), the concentration of 75% was included in the concentration series even though this concentration is not included in the OECD guideline. 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. 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 placed in a thermacage in order to dilate the peripheral blood vasculature and thus facilitate intravenous dosing of 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 ± 15 minutes after intravenous injection of the 3HTdR, all mice were sacrificed by exsanguination under a deep plane of inhalation anaesthesia.

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 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 (10 mL) was added. In addition, 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. Then all vials were placed into a 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.

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).

OBSERVATIONS
- 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

*References:
- Dreher, D (2020): Barium-4-dodecylphenolate: In Vitro Skin Sensitisation (ARE-Nrf2 Luciferase Test Method). Covance Study Number 8415390
- Dreher, D (2020): Barium-4-dodecylphenolate: In Vitro Skin Sensitisation (human Cell Line Activation Test). Covance Study Number 8415391

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

As a stimulation Index value of 3 or more was obtained in all groups, DPM values were analysed using the methods detailed below:

Tests were performed using a one-sided risk for increased response with increasing dose.

The positive control vehicle group and the positive control were excluded from statistical analyses.

The vehicle control group was taken as the baseline group with which the treated groups were compared.

Significant results are reported as P<0.05, P<0.01 or P<0.001.

Procedure I (ANOVA)
One-way analysis of variance (ANOVA) will be performed. Levene's test will be used to test for equality of variances among groups. Where Levene's test is non-significant (p≥0.01), comparisons between each treated group and control will be made using Dunnett's test. A linear contrast will be used to test for dose response.

Where the data prove unsuitable for this procedure, non-parametric methods (Procedure II) will be used.

Where data for only two groups are available, a two-sample t-test will be used.

Procedure II (non-parametric):
The Kruskal-Wallis non-parametric ANOVA will be performed, together with the Wilcoxon Rank Sum test for each treated group against control. Where the ANOVA is not significant, the Wilcoxon tests will not be reported. The Terpstra-Jonckheere test for dose response will also be performed.

Where data for only two groups are available, the Wilcoxon Rank Sum test will be used.

Results and discussion

Positive control results:

The positive control vehicle group and the positive control group animals were noted to have greasy fur to back of ears and neck on Days 1 to 6.

The positive control article produced a stimulation Index of 7.61, demonstrating adequate performance of the assay.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

20.97

Test group / Remarks:

25 % concentration of the test item

Results 2

Key result

Parameter:

SI

Value:

16.55

Test group / Remarks:

50 % concentration of the test item

Results 3

Key result

Parameter:

SI

Value:

14.5

Test group / Remarks:

75 % concentration of the test item

Cellular proliferation data / Observations:

STATISTICAL ANALYSIS
DPM/animal data were analysed using one-way analysis of variance (ANOVA). Levene’s test was used to test for equality of variances among groups and this showed no evidence of heterogeneity (P≥0.01). Comparisons between each treated group and vehicle control were made using Dunnett’s test. The test was performed using a one-sided risk for increased response with increasing dose. In addition, a linear contrast was used to test for an increasing dose response.

All dose groups showed a statistically significant increase in mean DPM/animal data when compared to the vehicle control group.

Please also refer to the field "Attached background material" below.

CLINICAL OBSERVATIONS:
- mortality: with the exception of a single animal receiving 75 % of the test item, all animals survived treatment with barium-4-dodecylphenolate. The animal of the 75 % concentration group was sacrificed in extremis due to the severity of clinical signs.
- clinical signs: test article vehicle control application sites remained free of irritation and clinical signs. However, signs of excessive irritation were noted on Day 3 for one animal of 75% concentration group, which was sacrificed in extremis. The animal had a moderate to severe erythema (Draize score: 3), scabbing/sores and discharge from the sores. Other findings were greasy fur to back of ears and neck (Days 1 and 2), hair loss (Days 2 and 3) and dry skin (Day 3).

The remaining animals of all concentration levels (25 %, 50 % and 75% of the test item) were noted to have greasy fur to back of ears and neck (Days 1 and 2), hair loss (Days 2 to 6) (Days 3 to 6 for 25 % concentration group only), dry skin (Days 3 to 6), slight discharge (Day 4, 75 % concentration group only) and scabbing (Day 6, 75 % concentration group only). Furthermore, very slight erythema (Draize score: 1) was also noted in the group receiving 25 % of the test item on Days 3 to 6, with very slight to well-defined erythema (Draize scores of 1 and 2, respectively) noted for the animals receiving 50 % and 75 % of the test item from Days 2 to 6.

Please also refer to the field "Attached background material" below.

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

Applicant's summary and conclusion

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

The Local Lymph Node Assay demonstrated that barium-4-dodecylphenolate has the potential to cause skin sensitisation and therefore should be classified and labelled for skin sensitisation (Category 1; H317) according to Regulation (EC) No. 1272/2008 and its subsequent regulations.