Barium bis(dihydrogenorthophosphate)

Endpoint summary

Description of key information

Skin sensitisation (OECD 429): not skin sensitising

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:

26 October 2016 - 13 December 2016

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:

2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Hess. Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

other: CBA/CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS B.V., Inc, Postbus 6174, 5960 AD Horst / The Netherlands
- Females nulliparous and non-pregnant: yes
- Microbiological status of animals, when known: not specified
- Age at study initiation: 8 - 9 weeks
- Weight at study initiation: 17.1 - 20.9 g
- Housing: in a group in Makrolon Type II (pre-test) / III (main study) cages with wire mesh top
- Diet: 2018C Teklad Global 18% protein rodent diet (certified), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least five days
- Indication of any skin lesions: no

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 45 - 65%
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): 12/12

Vehicle:

propylene glycol

Concentration:

2.5, 5, 10, and 25%

No. of animals per dose:

5

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: The highest test item concentration, which can be technically used, was a 25% suspension in propylene glycol. Grinding of the test item in a mortar was used to formulate the test item. At higher concentrations, an applicable formulation of the test item was not achieved, neither by the use of other vehicles (acetone/olive oil (4+1, v/v), DMF, MEK, DMSO, ethanol/water, 1% pluronic) nor by using additional methods to formulate the test item (e.g. vortexing, sonicating, warming to 37°C).
- Irritation: Two mice were treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 10 or 25% once daily each on three consecutive days. Both animals showed a very slight erythema of the ear skin (score 1). Increased spontaneous activity was observed in both animals transiently after the 1st and 2nd application. Furthermore, animal 2 showed scaly ears on day 3 to 6 as well as very slight eschar formation on the day of preparation. Nevertheless, since this slight eschar formation was not supported by any additional indicators of excessive skin irritation, it was decided to use this test item concentration of 25% as highest possible in the main study. A fourth concentration was, however, included in the main study to achieve at least 3 evaluable concentrations in case the irritation at the 25% group would be more intense in the main study as observed in the pre-test leaving this group unevaluable.
- Systemic toxicity: both animals did not show any signs of systemic toxicity
- Ear thickness measurements: prior to first application: 10%: mean 212.5 µm, 25%: mean 225.0 µm; prior to 3rd application: 10%: mean 212.5 µm, 25%: mean 225.0 µm; prior to necropsy: 10%: mean 227.5 µm, 25%: mean 242.5 µm
- Erythema scores: maximum of score 1

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: radioacitve method
- Criteria used to consider a positive response: A test item is regarded as a sensitiser in the LLNA if the following criteria are fulfilled:
• First, that exposure to at least one concentration of the test item resulted in an incorporation of 3H-methyl thymidine (3HTdR) at least 3-fold or greater than that recorded in control mice, as indicated by the Stimulation Index.
• Second, that the data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.

TREATMENT PREPARATION AND ADMINISTRATION: The test item was placed into an appropriate container on a tared balance and propylene glycol (PG) was added (weight per weight). Grinding of the test item in a mortar was used to formulate the test item. The different test item concentrations were prepared individually. Homogeneity of the test item in vehicle was maintained during treatment using a magnetic stirrer. The preparations were made freshly before each dosing occasion.
Topical application: Each test group of mice was treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 2.5, 5, 10, and 25% in PG. The application volume, 25 μL/ear/day, was spread over the entire dorsal surface (diameter about 8 mm) of each ear once daily for three consecutive days. A further group of mice (control animals) was treated with an equivalent volume of the relevant vehicle alone (control animals). Five days after the first topical application (day 6) 250 μL of phosphate-buffered saline containing 20.5 μCi of 3HTdR (equivalent to 81.8 μCi/mL 3HTdR) were injected into each test and control mouse via the tail vein. Approximately five hours after treatment with 3HTdR all mice were euthanized by using CO2 and lymph nodes are harvested and analysed. Ear weights were determined.

Positive control substance(s):

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

Statistics:

All calculations conducted on the DPM values and the ear weights were performed with a validated test script of “R”, a language and environment for statistical computing and graphics.
Within the program a statistical analysis was conducted on ear weights to assess whether the difference was statistically significant between the test item groups and negative control group. Statistical significance was set at the five per cent level (p < 0.05). Additionally, the Dean-Dixon-Test and Grubb’s Test were used for identification of possible outliers. Three outlier values (DPM values for animal 9, 12 and 16) were identified in one or both of the statistical test. However as exclusion of the values in question would not change the overall test result, the values were not excluded from calculation.

Positive control results:

The sensitivity and reliability of the experimental technique employed was assessed by use of α-hexyl cinnamaldehyde dissolved in acetone/olive oil (4+1 v/v) (compound listed in OECD 429 Guideline) which is known to have skin sensitisation properties in mice. The periodic positive control experiment was performed using CBA/CaOlaHsd mice in October 2016.
25%: SI = 11.8

Key result

Parameter:

SI

Value:

1.2

Variability:

0.5 - 2.4

Test group / Remarks:

2.5%

Key result

Parameter:

SI

Value:

0.9

Variability:

0.6 - 2.4

Test group / Remarks:

5%

Key result

Parameter:

SI

Value:

0.9

Variability:

0.7 - 1.1

Test group / Remarks:

10%

Key result

Parameter:

SI

Value:

1

Variability:

0.8 - 1.2

Test group / Remarks:

25%

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA: The lymph nodes of each individual animal were pooled and DPM values were measured from the pooled lymph node cell suspensions. Treatment with test substance concentrations of 2.5, 5, 10, and 25% in PG resulted in mean DPM values of 2270.2 ± 1397.9, 1713.6 ± 676.8, 1783.8 ± 299.4 and 1862.6 ± 343.8, respectively. The mean DPM value of the vehicle control was 1932.0 ± 1002.0.

DETAILS ON STIMULATION INDEX CALCULATION: The proliferative response of the lymph node cells is expressed as the number of radioactive disintegrations per minute per lymph nodes of each animal (DPM/animal) and as the ratio of 3HTdR incorporated into lymph node cells of test animals relative to that recorded for lymph nodes of control animals (Stimulation Index; S.I.). Before DPM/animal values were determined, mean scintillation-background DPM was subtracted from test and control raw data.

EC3 CALCULATION: The EC3 value could not be calculated, since all S.I.´s are below the threshold value of 3.

CLINICAL OBSERVATIONS: No deaths occurred during the study period. No signs of systemic toxicity were observed during the study period. On day 2, the animals treated with a test item concentration of 25% showed an erythema of the ear skin (score 1). Additionally, on day 3 (one hour after the third application), the animals treated with a test item concentration of 10 and 25% showed an erythema of the ear skin (score 1). Animals treated with 2.5 and 5% test item concentration did not show any signs of local skin irritation.

BODY WEIGHTS: The body weight of the animals, recorded prior to the first application and prior to treatment with 3HTdR, was within the range commonly recorded for animals of this strain and age.

EAR WEIGHTS: The measured ear weights of all animals treated was recorded on test day 6 (after necropsy). A relevant increase in ear weights was not observed.

Interpretation of results:

other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008

Conclusions:

The test substance Barium bis(dihydrogenorthophosphate) was not a skin sensitiser under the test conditions of this study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

Reliable data in order to evaluate the skin sensitising potential is available for the test substance.

The skin sensitising potential of the test substance was evaluated in a LLNA test according to OECD 429 and in compliance with GLP (Envigo, 2017). The study was conducted on five female mice per dose group (2.5%, 5%, 10% and 25%). Each animal received 25 µL of the substance to the dorsum of each ear. Animals were treated once daily for three days. After a two-day rest period, all animals were injected with tritiated methyl-thymidine in the tail vein. Approximately five hours later, animals were sacrificed, and the draining auricular lymph nodes removed and prepared for cell suspension and scintillation counting. A vehicle control (polypropylen glycol) group of five females each were run concurrently. A postive control in this laboratoy with alpha-hexylcinnamaldehyde is performed regularly, veryfing that the test system is reliable. The test substance produced a stimulation index smaller than 3 in all groups and is therefore not considered as skin sensitiser.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The available data on skin sensitisation do not meet the criteria for classification according to Regulation (EC) 1272/2008, and are therefore conclusive but not sufficient for classification.