Boric acid (H3BO3), solid soln. with strontium oxide, europium-doped

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Boric acid (H3BO3), solid soln. with strontium oxide, europium-doped

EC Number:

310-028-8

EC Name:

Boric acid (H3BO3), solid soln. with strontium oxide, europium-doped

Cas Number:

102110-29-2

Molecular formula:

SrB4O7:Eu

IUPAC Name:

Boric acid (H3BO3), solid soln. with strontium oxide, europium-doped

Test material form:

solid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Zhejiang Jingneng Phosphor Material Co., Ltd; 170825
- Expiration date of the lot/batch: 24 August 2019
- Purity: 99%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature, protected from light

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Envigo (formed partially from Harlan in September 2015), 5800 AN Venray, The Netherlands
- Females (if applicable) nulliparous and non-pregnant: yes
- Microbiological status of animals, when known: The animals were derived from a controlled full-barrier maintained breeding system (SPF).
- Age at study initiation: 10 - 11 weeks
- Weight at study initiation: 18.0 - 21.9 g
- Housing: The animals were kept in groups of 5 animals in IVC cages, type II L, polysulphone cages on Altromin saw fibre bedding
- Diet: Altromin 1324 maintenance diet for rats and mice ad libitum
- Water: Free access to tap water, sulphur acidified to a pH value of approx. 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals)
- Acclimation period: 5 days
- Indication of any skin lesions: No

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 55 ± 10%
- Air changes (per hr): at least 10 x / hour
- Photoperiod (hrs dark / hrs light): Artificial light, sequence being 12 hours light, 12 hours dark

Study design: in vivo (LLNA)

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

Pre-screen: 50 and 100% in AOO
Main test: 25, 50 and 100% in AOO

No. of animals per dose:

5 females per dose in the pre-screen test
5 females per dose in the main test

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: Before the initiation of the pre-screen test, a solubility test was performed to define the vehicle and the maximum concentration which is technically applicable to the animals. The maximum technically applicable concentration of the test item was found to be 100% (w/v).

- Irritation: The mice were observed daily for any local irritation at the application site. Excessive local irritation was indicated by an erythema score ≥ 3 and/or ear swelling of ≥ 25%.
- Systemic toxicity: The mice were observed daily for any clinical signs of systemic toxicity.
- Ear thickness measurements: Ear thickness measurements were performed on day 1 (pre-dose), day 3 (approximately 48 hours after the first dose) and day 6.
- Erythema scores: Both ears were observed for erythema and scored.
Body weights were also recorded pre-test and prior to termination.

Four animals were treated by topical application with the test item on three consecutive days at the following concentrations to the entire dorsal surface of each ear: (i) animals no. 51 and no. 52 were treated with a test item concentration of 50% (suspended in AOO), (ii) animals no. 53 and no. 54 were treated with a test item concentration of 100% (suspended in AOO). (iii) one further animal was treated with 100% AOO and served as negative control. Immediately before the first application, approximately 48 hours after the first application and shortly before sacrificing the thickness of both ears of all animals was measured (see Table 2 in the appendix). During this period also all clinical signs were recorded. Neither signs of systemic toxicity nor signs of excessive irritation at any application site could be detected in any animal. For details see Table 4 in the appendix. All animals showed the expected weight development, which allows for a weight loss of up to 2 g throughout the duration of the pre-screen test (see Table 3 in the appendix).

MAIN STUDY
Three test groups (25, 50 and 100%) and 1 negative control group (AOO) were tested. The animals were weighed prior to the application and at the end of the test period (prior to the treatment with 3HTdR). Prior to the application and once a day thereafter all animals were observed in order to detect signs of toxicity, including dermal irritation at site of application.

Topical Application
Each mouse was treated by topical application of 25 µL of the selected solution to the entire dorsal surface of each ear. Topical applications were performed once daily over three consecutive days. The first treatment day is defined as study day 1.

Administration of 3H-Methyl Thymidine
Five days after the first topical application all mice were dosed with 20 µCi 3H-methyl thymidine by intravenous injection (tail vein) of 250µL of 3H-methyl thymidine, diluted with PBS to a working concentration of 80 µCi/mL
.
Preparation of Cell Suspension
Approximately 5 hours after the injection of 3H-methyl thymidine all mice were sacrificed by cervical dislocation. The draining auricular lymph nodes were excised, individually pooled for each animal (2 lymph nodes per animal) and collected in phosphate buffered saline (PBS). A single cell suspension of pooled lymph node cells was prepared by gentle mechanical disaggregation through polyamide gauze (200 mesh size). After washing the gauze with PBS the cell suspension was pelleted in a centrifuge. The supernatant was discarded and the pellets were resuspended with PBS. This washing procedure was repeated. After the final wash each pellet was resuspended in approx. 1 mL 5% TCA at approx. 4° C for approximately 18 hours for precipitation of macromolecules. Each precipitate was once washed again, resuspended in 1 mL 5% TCA and 7 mL scintillation fluid was added. Then this solution was transferred into scintillation vials and stored at room temperature overnight.

Determination of Incorporated 3H -Methyl Thymidine
The 3H-methyl thymidine – incorporation was measured in a ß-counter and expressed as the number of disintegrations per minute (DPM). Similarly, background 3H-methyl thymidine levels were also measured (5% TCA). Determination of radioactivity was performed individually for each animal.

Evaluation of Results
EC3 values, calculated concentrations which induce stimulation indices of three, are determined by linear interpolation, EC3=c+[(3-d)/(b-d)]x(a c), between two points of the stimulation indices axis, one above (a,b) and one below (c,d) the stimulation index of three. If all measured points are above or below the stimulation index of three, no EC3 value can be stated.

Positive control substance(s):

other: A shared positive control was performed concomitantly, using 5 animals. 1% phenylenediamine (Sigma-Aldrich, lot no.: WXBC5926V, expiry date 05/12/2020) in AOO was used as positive-control substance.

Statistics:

CPM data of all dose and control groups were screened for statistical outliers using Grubbs’, Nalimov’s and Dixon’s test, respectively. Values that failed at least one of the tests were labelled as outliers and excluded for SI calculation.

Results and discussion

Positive control results:

The positive-control substance (1% phenylenediamine in AOO) exceeded the stimulation index of 3 confirming the reliability of the test system (see Table 7).

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

0.7

Test group / Remarks:

25%

Results 2

Parameter:

SI

Value:

0.6

Test group / Remarks:

50%

Results 3

Parameter:

SI

Value:

1

Test group / Remarks:

100%

Cellular proliferation data / Observations:

DETAILS ON STIMULATION INDEX CALCULATION: The proliferative response of lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph node (DPM/NODE) and as the ratio of 3H-methyl thymidine - incorporation into lymph node cells of test group animals relative to that recorded for control group animals (STIMULATION INDEX). Before DPM/NODE values were determined, background values were subtracted.

EC3 CALCULATION: The EC3 value (derived by linear interpolation) could not be calculated as the stimulation indices of all concentrations were below 3 (Table 5).

CLINICAL OBSERVATIONS: All animals survived throughout the test period without showing any clinical signs (see Table 8 in the appendix).

BODY WEIGHTS: All animals showed the expected weight development, which allows for a weight loss of up to 2 g throughout the study. For individual data see Table 6 in the appendix.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In an LLNA assay in female CBA/CaOlaHsd mice, boric acid (H3BO3), solid soln. with strontium oxide, europium-doped is not a skin sensitiser.

Executive summary:

In a dermal sensitization study (187129) with boric acid (H3BO3), solid soln. with strontium oxide, europium-doped (in acetone/olive oil (4:1 v/v)), young adult CBA/CaOlaHsd mice (5 females) were tested in the Local Lymph Node Assay. The reliability of the test system was confirmed by a shared positive control performed concomitantly, using 5 animals (1% phenylenediamine in AOO).

The positive-control substance (1% phenylenediamine in AOO) exceeded the stimulation index of 3 confirming the reliability of the test system. All animals showed the expected weight development, which allows for a weight loss of up to 2 g throughout the study. All animals survived throughout the test period without showing any clinical signs. The stimulation indexes were 0.7 at 25%, 0.6 at 50% and 1.0 at 100%. The EC3 value (derived by linear interpolation) could not be calculated as the stimulation indices of all concentrations were below 3. Therefore, the substance is not a skin sensitiser.