Boron

Endpoint summary

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:

2012-11-06 to 2012-11-29

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:

adopted 2010-07-22

Deviations:

yes

Remarks:

modified OECD 429, method according to Ehlings et al. 2005

Principles of method if other than guideline:

The test was performed in accordance with the method according to Ehling et al (2005): An European inter-laboratory validation of alternative endpoints of the murine local lymph node assay: first round, Toxicology 212 (2005) 60-68 and Ehling et al (2005): An European inter-laboratory validation of alternative endpoints of the murine local lymph node assay: 2nd round, Toxicology 212 (2005) 69-79.

Threshold values of the stimulation indices of lymph node cell count and ear weight were calculated by dividing the average values per group of the test item treated animals by the vehicle treated ones. Values above 1.4 (cell count) or 1.1 (ear weight) are considered positive
(these values were fixed empirically during the inter-laboratory validation of this method). In addition, the lymph node weights were determined for concentration related properties.

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2009-11-12

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

NMRI

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Age at study initiation: 62 days
- Weight at study initiation: 27 - 34 g
- Housing: before application the animals were housed in groups in MAKROLON cages (type III) with a basal surface of approx. 39 cm x 23 cm and a height of approx. 15 cm. After application the animals were housed singly in order to prevent their licking off the test item from the ears of the other animals. Granulated textured wood (Granulat A2, J. Brandenburg, 49424 Goldenstedt, Germany) was used as bedding material for the cages.
- Diet (ad libitum): commercial diet ssniff® R/M-H V1534 (ssniff Spezialdiäten GmbH, 59494 Soest, Germany)
- Water (ad libitum): tap water
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22°C ± 3°C (maximum range)
- Relative humidity: 55% ± 15% (maximum range)
- Air changes: 12 - 18 times per hour
- Photoperiod (hrs dark / hrs light): 12/12

Vehicle:

other: Acetone/olive oil (3+1, v/v)

Concentration:

10%, 25% and 50% (w/w) of boron, amorphous

No. of animals per dose:

6 female mice

Details on study design:

RANGE FINDING TESTS:
A preliminary experiment was carried out in 3 animals to examine the irritating potential and handling/application of the test item in order to select the appropriate concentrations. Three concentrations of 10, 25 and 50% of boron, amorphous in acetone/olive oil (3+1, v/v) were examined. Possible clinical signs would have been recorded.
Boron, amorphous was a dark brown powder. Hence, a 50% suspension was the highest feasible concentration of boron, amorphous in acetone/olive oil (3+1, v/v).
Results:
No pronounced irritating properties were observed in this preliminary experiment at concentrations of 10%, 25% or 50%, no differences in ear weight and ear thickness were noted. No clinical signs were recorded.

MAIN STUDY
The experimental schedule of the assay was as follows:
Day 1:
The weight of each animal and possible clinical signs were individually identified and recorded. In addition, ear swelling measurements were carried out at the helical edge of both ears using an Oditest micrometer.
Open application of 25 µL of the appropriate dilution of the test item, the vehicle alone or the positive control (as appropriate) were administered to the dorsum of each ear.
Days 2 and 3:
The application procedure carried out on day 1 was repeated.
Day 4 (24 hours after the last application the animals were sacrificed under ether anaesthesia by cutting the aorta abdominalis):
Ear swelling measurements (immediately before sacrificing the mice) were carried out at the helical edge of both ears using an Oditest micrometer.
Punch biopsies of 8 mm in diameter of the apical area of both ears were prepared and immediately weighed on an analytical balance.
Lateral pairs of auricular lymph nodes draining the ear tissue were excised, carefully separated from remaining fatty tissue and weighed on an analytical balance immediately following preparation. The lymph nodes were then stored on ice in PBS/0.5% BSA and subjected to the preparation of single cell suspensions by mechanical tissue disaggregation. The cells were counted automatically in a cell counter.

OBSERVATIONS
The following observations were made during the course of the study:
- Clinical signs: animals were observed once daily for any clinical signs of local systemic irritation at the application site or of systemic toxicity. Observations were recorded for each individual animal. Cageside observations included skin/fur, eyes, mucous membranes, respiratory and circulatory systems, somatomotor activity and behaviour patterns. The onset, intensity and duration of any signs observed were recorded.
In addition, animals were checked regularly throughout the working day from 7:30 a.m. to 4:30 p.m. On Saturdays and Sundays animals were checked regularly from 8:00 a.m. to 12:00 noon with a final check performed at approximately 4:00 p.m., if applicable.
- Body weight: the weight of each mouse was recorded at the time of allocation of animals to groups (test day 1) and at the time of necropsy (test day 4).

ANALYSIS OF RESULTS
The so-called stimulation (or LLN-) indices to determine the sensitising potential were calculated by dividing the average absolute lymph node weight or lymph node cell counts per group of the test item treated animals by the vehicle treated ones.
Thus, in case of no stimulating effect the index for the lymph node cell count is always below 1.4 (cut-off value). An index above 1.4 is considered positive.
For lymph node weight significance at p ≤ 0.01 is considered positive (U-test according to MANN and WHITNEY). A possible concentration-response-relationship for the lymph node weight in order to determine a possible sensitising potential was examined by linear regression analysis employing PEARSON's correlation coefficient. Outliers would have been determined according to the Nalimov test.
In addition, the acute inflammatory skin reaction (irritating potential) was measured by ear weight determination of circular biopsies of the ears and ear thickness measurements on test day 1 and test day 4 to identify skin irritation properties of the test item employing the U-test according to MANN and WHITNEY by comparing the test groups to the vehicle control.
The stimulation indices were calculated by dividing the average ear weight and average ear thickness on test day 4 per group of the test item treated animals by the vehicle treated ones. The cut-off threshold value for ear weight was set at 1.1.

Positive control substance(s):

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

Statistics:

Please refer to "details on study design"

Positive control results:

The positive control group caused the expected increases in lymph node cell count and lymph node weight (statistically significant at p ≤ 0.01). The values for the stimulation index of lymph node cell count and lymph node weight were 1.818 and 1.692, respectively. Therefore, the study can be regarded as valid.

Parameter:

SI

Remarks:

lymph node cell count

Value:

0.883

Test group / Remarks:

10 % w/w test item

Remarks on result:

other: SI: 1.019 (lymph node weight)

Parameter:

SI

Remarks:

ear weight

Value:

1

Test group / Remarks:

10 % w/w test item

Remarks on result:

other: SI: 1.059 (ear thickness)

Parameter:

SI

Remarks:

lymph node cell count

Value:

1.182

Test group / Remarks:

25 % w/w test item

Remarks on result:

other: SI: 1.231 (lymph node weight)

Parameter:

SI

Remarks:

ear weight

Value:

1

Test group / Remarks:

25 % w/w test item

Remarks on result:

other: SI: 1.102 (ear thickness)

Parameter:

SI

Remarks:

lymph node cell count

Value:

0.802

Test group / Remarks:

50 % w/w test item

Remarks on result:

other: SI: 1.096 (lymph node weight)

Parameter:

SI

Remarks:

ear weight

Value:

0.925

Test group / Remarks:

50 % w/w test item

Remarks on result:

other: SI: 1.059 (ear thickness)

Cellular proliferation data / Observations:

DETAILS ON STIMULATION INDEX CALCULATION
Threshold values of the stimulation indices of lymph node cell count and ear weight were calculated by dividing the average values per group of the test item treated animals by the vehicle treated ones. Values above 1.4 (cell count) or 1.1 (ear weight) are considered positive (these values were fixed empirically during the inter-laboratory validation of this method).

RESULTS ON SKIN SENSITISATION
In the main study treatment with boron, amorphous at concentrations of 10%, 25% or 50% did not reveal statistical significantly increased values for lymph node cell count. The stimulation indices of the lymph node cell count did not exceed the threshold level of 1.4. Hence, the test item is classified as not sensitising.
The threshold level for the ear weight of 1.1 was not exceeded and no increase of ear thickness was observed, i.e. no irritating properties were noted.

CLINICAL OBSERVATIONS:
No signs of local or systemic intolerance were recorded.

BODY WEIGHTS:
The animal body weight was not affected by the treatment.

Interpretation of results:

not sensitising

Conclusions:

Under the present test conditions, boron, amorphous at concentrations of 10%, 25% or 50% (w/w) in acetone/olive oil (3+1 v/v) did not reveal any sensitising properties in the local lymph node assay and therefore should not be classified and labelled according to Regulation (EC) No.: 1272/2008 and its subsequent regulations.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

One reliable animal study described in Haferkorn (2013) (modified OECD 429; method according to Ehlings et al. 2005; GLP compliant) is considered to be reliable without restrictions. The substance was determined not to be a skin sensitiser.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

According to the CLP regulation the hazard identification and subsequently a proposal for classification as “Respiratory sensitiser” will normally be based on human experience. In this context, hypersensitivity is normally seen as asthma, but other hypersensitivity reactions such as rhinitis/conjunctivitis and alveolitis are also considered. The condition will have the clinical character of an allergic reaction. However, immunological mechanisms do not have to be demonstrated.

The evidence could be:

a)           clinical history and data from appropriate lung function tests related to exposure to the substance, confirmed by other supportive evidence which may include:

i.             in vivo immunological test (e.g. skin prick test);

ii.            in vitro immunological test (e.g. serological analysis);

iii.           studies that indicate other specific hypersensitivity reactions where immunological mechanisms of action have not been proven, e.g. repeated low-level irritation, pharmacologically mediated effects;

iv.          a chemical structure related to substances known to cause respiratory hypersensitivity;

b)           data from one or more positive bronchial challenge tests with the substance conducted according to accepted guidelines for the determination of a specific hypersensitivity reaction.

In long-time industrial experience in the production and handling of substance boron, no cases of respiratory hypersensitivity have been observed.

Justification for classification or non-classification

Skin sensitisation

The reference Haferkorn (2013) is considered as the key study on skin sensitisation and will be used for classification. The overall sensitisation results are as follows:

Local lymph node assay in mice

SIs of less than 3.0 (0.802 - 1.182) were observed at all test concentrations of boron, amorphous. Thus, the classification criteria acc. to regulation (EC) 1272/2008 as skin sensitiser are not met and boron, amorphous does not have to be classified as such.

Respiratory sensitisation

In long-time industrial experience in the production and handling of substance boron, no cases of respiratory hypersensitivity have been observed. Classification as respiratory sensitiser is not applicable.