3-Fluorphenylboric acid

Endpoint summary

Description of key information

The test item was determined to be no skin sensitiser (reference 7.4.1 -1).

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:

16 Nov - 21 Dec 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:

22 July 2010

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

06 July 2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Netherlands B.V. Postbus 6174 NL - 5960 AD Horst
- Age at study initiation: 8-9 weeks
- Weight at study initiation: 17.5-19.9 g
- Housing: single
- Diet: pelleted standard diet (Harlan Winkelmann GmbH, Borchen, Germany), ad libitum
- Water: tap water, ad libitum
- Acclimation period: yes, at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 + 2
- Humidity (%): 45-65
- Photoperiod (hrs dark / hrs light): 12/12

HUSBANDRY
Cage type: Makrolon, Type II/III with wire mesh top
Bedding: granulated soft wood bedding (Harlan Winkelmann GmbH, Borchen, Germany)

Vehicle:

dimethylformamide

Concentration:

0.25, 0.5, and 1 % (w/w)

No. of animals per dose:

5

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: A solubility experiment was performed according to the recommendations given by OECD 429. The highest test item concentration, which can be technically used, was a 50 % solution in DMF.
To determine the highest non-irritant test concentration that at the same time did not induce signs of systemic toxicity, a pre-test was performed in two animals. Ear irritation was considered to be excessive if an erythema of the ear skin of a score value >3 was observed at any observation time and/or if an increase in ear thickness of >25 % was recorded on day 3 or day 6. In a first pre-test 2 animals were treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 25 and 50 % once daily each on three consecutive days. The animal treated with 50 % had to be sacrificed 1 h after the first application and the 25 % treated animals 1 h after the third application. A second pre-test was performed using concentrations of 5 and 10 %. Both animals had to be sacrificed after the third application. Therefore, a third pre-test was performed using concentrations of 1 and 2.5%.
- Irritation:
Following effects were observed in treated animals:
50 %: within 1h after the first application erythema of the ear skin (sore 1)
25 %: within 1h after the first application erythema of the ear skin (sore 1), within 1h after third application open wound at the base of the ear
10 %: within 1h after application on day 1-3 erythema of the ear skin (sore 1) and also 24h after the second application, after the third application open wounds and eschar on the ear
5 %: 24h after the second application erythema of the ear skin (sore 1), after the third application open wounds and eschar on the ear
2.5 %: day 3 to 6 erythema of the ear skin (Score 1), scaly ear, slight eschar formation and visible swelling of the ear
1 %: day 3 to 6 erythema of the ear skin (Score 1), scaly ear, slight eschar formation and visible swelling of the ear
- Systemic toxicity:
Following effects were observed in treated animals:
50 %: within 1h after the first application reduced spontaneous activity, swollen eyes, facial expression of pain and reduced movement
25 %: within 1h after the first application reduced spontaneous activity, within 1h after the second reduced spontaneous activity and eyelid closure, within 1h after third application reduced spontaneous activity, eyelid closure and tumbling
- Ear thickness measurements: 2.5 %: increase of ear thickness above the recommended threshold
- Erythema scores: see above

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
The animals were distributed into the test groups at random.
- Criteria used to consider a positive response: First, that exposure to at least one concentration of the test item resulted in an incorporation of HTdR 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:
- Test item preparation:
The test item was placed into a volumetric flask on a tared balance and DMF was quantitatively added (weight per volume). Warming to 37°C and vortexing was used to formulate the test item.
The different test item concentrations were prepared serially. 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 0.25, 0.5, and 1% in DMF. The application volume, 25 μL/ear/day, was spread over the entire dorsal surface (∅ ∼ 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).
- Determination of Ear Thickness:
Prior to the first and third application of the test item (study days 1 and 3) and prior to treatment with 3HTdR (study day 6), the ear thickness was determined using a micrometer.
- Administration of 3H-methyl-thymidine:
Five days after the first topical application (day 6) 250 μL of phosphate-buffered saline containing 20.3 μCi of 3H-methyl thymidine (equivalent to 81.3 μCi/mL 3HTdR) were injected into each test and control mouse via the tail vein.
- Determination of incorporated 3HTdR:
Approximately five hours after treatment with 3HTdR all mice were euthanized by using CO2, which was, after harvesting of the lymph nodes, followed by cervical dislocation to ensure death.
The draining lymph nodes were rapidly excised and pooled per animal (2 nodes per animal). Single cell suspensions (in phosphate buffered saline) of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 μm mesh size). After washing two times with phosphate buffered saline (approx. 10 mL) the lymph node cells were resuspended in 5 % trichloroacetic acid (approx. 3 mL) and incubated at approximately +4 °C for at least 18 hours for precipitation of macromolecules. The precipitates were then resuspended in 5 % trichloroacetic acid (1 mL) and transferred to scintillation vials with 10 mL of scintillation liquid and thoroughly mixed. The level of 3HTdR incorporation was then measured in a β-scintillation counter. Similarly, background 3HTdR levels were also measured in two 1 mL-aliquots of 5 % trichloroacetic acid. The β-scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute.
- Observations:
In addition to the sensitising reactions the following observations and data were recorded during the test and observation period:
Mortality / Viability: At least once daily from experimental start to necropsy.
Body weights: In the pre-test: prior to the first application and prior to sacrifice. In the main experiment: prior to the first application and prior to treatment with 3HTdR.
Ear thickness: In the pre-test prior to the first application of the test item (day 1), on day 3 and before sacrifice (day 6). In the main experiment prior to the first application (day 1), on study day 3 and prior to treatment with 3HTdR (day 6).
Ear weights: In the pre-test after sacrifice; biopsy punches were taken from each ear.
Clinical signs (local / systemic): Clinical signs (local irritation at the application site or systemic toxicity) were recorded at least once daily. Especially the treatment sites were observed carefully.

Positive control substance(s):

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

Statistics:

Mean values and standard deviations were calculated.
The DPM values were assessed 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.

Parameter:

SI

Value:

0.8

Test group / Remarks:

Test Group 0.25 %

Parameter:

SI

Value:

1.1

Test group / Remarks:

Test Group 0.5 %

Key result

Parameter:

SI

Value:

1.3

Test group / Remarks:

Test Group: 1 %

Parameter:

SI

Value:

1

Test group / Remarks:

Control Group

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
For the 1 % test substance group the mean DPM count/animal was 1961.7. For the 0.5 % test substance group the mean DPM count/animal was 1602.3. For the 0.25 % test substance group the mean DPM count/animal was 1277.5.
No outlier value was detected in both statistical tests for DPM values.

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 HTdR 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.
No significant lymphoproliferative response (SI ≥ 3) compared to the relevant control (DMF) was noted for the test item at the applied test concentrations.

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. From day 2 (within 1h after the second application) to day 4, the animals treated with a test item concentration of 0.5 and 1 % showed an erythema of the ear skin (Score 1). Furthermore, animals treated with 1 % of the test item showed scaly ears on test days 4 and 5. Animals treated with 0.25 % test item concentration showed an erythema of the ear skin (Score 1) on day 3 (within 1h after the third application).
The measured ear thickness of all animals treated was recorded prior to the 1st application, on study day 3 and prior to necropsy (day 6). A relevant increase in ear thickness was not observed.

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.

Interpretation of results:

not sensitising

Conclusions:

The test item was not a skin sensitiser under the test conditions of this study.

Executive summary:

In the study according to OECD test guideline 429 the test item formulated in dimethylformamide (DMF) was assessed for its possible skin sensitising potential.

For this purpose a local lymph node assay was performed using test item concentrations of 0.25, 0.5, and 1 %. The test item was topically applied to the dorsum of each ear for three consecutive days of five female mice per test item concentration. The highest concentration tested was the highest concentration that could technically be achieved whilst avoiding systemic toxicity and excessive local skin irritation, as confirmed by three pre-experiments.

The animals did not show any signs of systemic toxicity during the course of the study and no cases of mortality were observed. From day 2 (within 1h after the second application) to day 4, the animals treated with a test item concentration of 0.5 and 1 % showed an erythema of the ear skin (Score 1). Furthermore, the animals treated with 1 % of the test item showed scaly ears on days 4 and 5. Animals treated with 0.25 % test item concentration showed an erythema of the ear skin (Score 1) on day 3 (within 1h after the third application). A relevant increase in ear thickness was not observed.

In this study Stimulation Indices (S.I.) of 0.8, 1.1, and 1.3 were determined with the test item at concentrations of 0.25, 0.5, and 1 % (w/v) in DMF, respectively.

The test item was not a skin sensitiser under the test conditions of this study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

In the study according to OECD test guideline 429 the test item formulated in dimethylformamide (DMF) was assessed for its possible skin sensitising potential (reference 7.4.1 -1).

For this purpose a local lymph node assay was performed using test item concentrations of 0.25, 0.5, and 1 %. The test item was topically applied to the dorsum of each ear for three consecutive days of five female mice per test item concentration. The highest concentration tested was the highest concentration that could technically be achieved whilst avoiding systemic toxicity and excessive local skin irritation, as confirmed by three pre-experiments.

The animals did not show any signs of systemic toxicity during the course of the study and no cases of mortality were observed. From day 2 (within 1h after the second application) to day 4, the animals treated with a test item concentration of 0.5 and 1 % showed an erythema of the ear skin (Score 1). Furthermore, the animals treated with 1 % of the test item showed scaly ears on days 4 and 5. Animals treated with 0.25 % test item concentration showed an erythema of the ear skin (Score 1) on day 3 (within 1h after the third application). A relevant increase in ear thickness was not observed.

In this study Stimulation Indices (S.I.) of 0.8, 1.1, and 1.3 were determined with the test item at concentrations of 0.25, 0.5, and 1 % (w/v) in DMF, respectively.

The test item was not a skin sensitiser under the test conditions of this study.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No 1272/2008

The available data for skin sensitization is reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on this data, the substance is considered to be not classified for skin sensitisation (UN GHS: No category) under Regulation (EC) No 1272/2008, as amended for the twelfth time in Regulation (EC) 2019/521.