Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 May 2012 to 20 June 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Chemical structure
Reference substance name:
(4-chloro-2-fluoro-3-methoxyphenyl)boronic acid
EC Number:
696-001-4
Cas Number:
944129-07-1
Molecular formula:
C7H7BClFO3
IUPAC Name:
(4-chloro-2-fluoro-3-methoxyphenyl)boronic acid
Test material form:
other: solid (unspecified)
Details on test material:
- Appearance: White Solid
- Storage conditions of the test material: Ambient

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/Ca
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: approximately 9 to 11 weeks
- Weight at study initiation: 20.15 to 22.46 g at the start of dermal sensitisation.
- Housing: Animals were housed individually in solid floor standard polysulfone cages (Size: approximately 360 mm long x 205 mm deep x 140 mm high),with stainless steel top grills that provided access to pelleted feed and drinking water. Steam sterilised corn cob was used as bedding and changed along with the cage twice a week. Cages were placed on 5 tier racks. Mice were provided with huts as environmental enrichment in each cage during the experimental period.
- Diet (e.g. ad libitum): Standard mouse pelleted maintenance diet was provided ad libitum
- Water (e.g. ad libitum): Deep bore-well water passed through activated charcoal filter and exposed to UV rays in a water filter-cum-purifier was provided to animals ad libitum in polycarbonate bottles with stainless steel sipper tubes.
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 23 °C
- Humidity (%): 64 to 66 % (relative)
- Air changes (per hr): 13.3 air changes/hour
- Photoperiod (hrs dark / hrs light): 12 hour light / 12 hour dark cycle (light from approximately 06:00 to 18:00)

IN-LIFE DATES: From: 15 May 2012 To: 20 June 2012

Study design: in vivo (LLNA)

Vehicle:
dimethyl sulphoxide
Remarks:
Selected on the basis of miscibility and the fact that at concentrations of ≤50 %, the test material was pipettable and suitable for application to the dorsal surface of the mouse ear.
Concentration:
5, 15 and 50 % of the test material
No. of animals per dose:
5 female animals per dose
Details on study design:
RANGE-FINDING TEST
Prior to the main LLNA study, concentrations of 1, 5, 10, 25 and 50 % w/v in DMSO and the vehicle control were evaluated to determine the highest achievable concentration that avoids overt systemic toxicity and excessive local irritation.
Both ears of six female mice (one mouse / concentration) were topically treated for three consecutive days (Days 1, 2 and 3). No treatment was made on Days 4 and 5.
The test material was administered using an adjustable micropipette with a disposable tip. All mice received 25 μL of one concentration of the test material, spread over the dorsal surface of each ear in a manner to prevent test material loss (50 μL total / mouse). Similarly, the vehicle alone was applied to the ears of one animal. Prior to each application, both the ears were evaluated for erythema. Ears were also evaluated on Day 6. All mice were weighed on Days 1 and 6.
Animals were observed daily for clinical signs of toxicity. Ear thickness was measured using a micrometer prior to dosing on Days 1 and 3 and prior to euthanasia on Day 6. Additionally, on Day 6, ear thickness was determined by ear punch weight, after animals were euthanised.
Erythema scores, ear thickness and body weight data following test material applications were compared to the response of the animals treated with vehicle alone. The final determination of the maximum dose for the main LLNA involved consideration of dose-response and biological variability and plausibility.

MAIN STUDY
The application of the test material (25 μL/ear) was made on the dorsum of both ears as described above. Five female mice/group received the appropriate treatment once daily for three consecutive days (Days 1, 2 and 3). No treatment was made on Days 4 and 5.

- Preparation of 3H-Methyl Thymidine (3H -TdR)
A solution of 3H-TdR (80 μCi/mL, specific activity 6.7 Ci/mmol in sterile phosphate buffer saline (PBS) was freshly prepared and analysed for radioactivity.

- Injection of of 3H-Methyl Thymidine (3H -TdR)
On Day 6, a volume of 250 μL (20 μCi) of 3H-TdR in PBS was administered to each mouse via the lateral tail vein using a 1.0 mL disposable syringe fitted with a 26 SWG, 1/2 inch needle.

- Collection of auricular lymph nodes
Approximately five hours post injection of 3H-TdR, animals were euthanised by an overdose of Isoflurane anaesthesia. The auricular lymph nodes (bilateral) were excised and placed in PBS, processed and the radioactivity was counted.

- Processing of auricular lymph nodes
A single cell suspension of the lymph node cells (LNC) from each mouse was prepared by gentle mechanical disaggregation using a tissue homogeniser for 30 seconds at medium speed using PBS (approx. 10 mL).
After all the nodes had been processed, the tubes containing the suspensions of LNC were centrifuged at 200 x gravity for 10 minutes at 4 °C. The supernatant was poured into a container for collection of radiolabelled waste. 10 mL of PBS was added to each tube and inverted to re-suspend the pellet. The tubes were centrifuged again and supernatant was poured off. After the final (second) wash, the cell pellet was suspended in 3.0 mL of 5 % trichloroacetic acid (TCA) and stored overnight at 4 °C for approximately 18 hours. Clumping of LNC was avoided by ensuring that the pellet was completely re-suspended in a small volume of TCA before making up to the final volume. The suspended precipitates were centrifuged at 200 x gravity for 10 minutes at 4 °C and the supernatant poured off into a container for collection of radiolabelled waste. The pellet from each tube was reconstituted in 1 mL of 5 % TCA and subsequently transferred to a scintillation vial containing 10 mL of scintillation cocktail. Two additional 2 mL aliquots of distilled water were used to rinse the tubes and the rinses were added to the scintillation vial containing the pellet in TCA and cocktail. The samples were mixed using a snapping wrist action.
The radioactivity in each precipitate was measured for 5 minutes using a β-scintillation counter as disintegrations per minute (dpm) per mouse. Any test material that produces an SI of ≥3 in the LLNA should be considered “positive” for contact sensitisation.

OBSERVATIONS
Cage-side examination was conducted twice daily and the following parameters were evaluated: decreased / increased activity, repetitive behaviour, vocalisation, incoordination / limping, injury, neuromuscular function (convulsion, fasciculation, tremor and twitches), altered respiration, blue / pale skin and mucous membranes, severe eye injury (rupture), alterations in faecal consistency, and faecal/urinary quantity.
In addition, all animals were observed for morbidity and mortality, twice daily and for the availability of feed and water.
Individual body weights of the animals were recorded on Day 1 (initial) shortly before the test material administration and prior to sacrifice on Day 6.
The ears were graded for erythema prior to test material application on days 1, 2, 3 and on day 6 as follows:
- No erythema: 0
- Very slight erythema (barely perceptible): 1
- Well defined erythema: 2
- Moderate to severe erythema: 3
- Severe erythema (beet redness) to eschar formation preventing grading of erythema: 4
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
A mean dpm value ± SD (standard deviation) was calculated for each group and stimulation index (SI) was calculated using the absolute dpm value for each mouse as the numerator, and the mean dpm value from the vehicle-treated mice as the denominator.

The % increase in ear thickness was calculated for each ear using the following equation:
% Ear swelling = [(B - A) / A] x 100
where:
A = ear thickness measurement on Day 1 (μm)
B = ear thickness measurement on Day 3 or 6 (μm)

The SI was calculated for each mouse using the following equation:
SI = Disintegrations per minute (dpm) of individual mouse / Average dpm of the vehicle control mice

Means and SD were generated for body weight data (absolute and gain) and LLNA response (dpm values). The body weight and dpm data were analysed by one-way analysis of variance. When the differences are indicated by the ANOVA, a comparison of treated vs. control groups was done using a Dunnett’s t-test (p ≤ 0.05).

Results and discussion

Positive control results:
A positive response was elicited from the positive control, achieving a stimulation index (SI) of 5.28 in comparison to vehicle-treated mice. See Table 1.

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
1
Test group / Remarks:
0 % (vehicle)
Remarks on result:
other: See Table 1.
Key result
Parameter:
SI
Value:
1.54
Test group / Remarks:
5%
Remarks on result:
other: See Table 1.
Key result
Parameter:
SI
Value:
1.93
Test group / Remarks:
15%
Remarks on result:
other: see Table 1
Key result
Parameter:
SI
Value:
2.3
Test group / Remarks:
50%
Remarks on result:
other: see table 1
Key result
Parameter:
SI
Value:
5.28
Test group / Remarks:
HCA (positive control)
Remarks on result:
other: see table 1

Any other information on results incl. tables

Table 1: Summary of Disintegrations Per Minute (dpm) and Stimulation Indices (SI)

Dose

(%)

Number of Mice

dpm / mouse

SI

0

(Vehicle)

5

1571.60

325.05

1.00

5

5

2423.80

784.11

1.54

15

5

3031.80*

637.57

1.93

50

5

3614.80*

1293.47

2.30

25 HCA

(Positive Control)

5

8304.80*

568.43

5.28

*Significantly higher than the vehicle control group

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Under the conditions of this study, topical application of the test material at 5, 15 and 50 % in DMSO did not elicit a stimulation index (SI) that met the 3-fold threshold, thus indicating a lack of dermal sensitisation potential in the mouse LLNA. As such the test material requires no classification in accordance with EU criteria.
Executive summary:

The potential of the test material to cause contact sensitisation was investigated in a mouse LLNA study conducted in accordance with the standardised guidelines OECD 429, EU Method B.42 and USA EPA OPPTS 870.2600 under GLP conditions.

A screening study was conducted in which three daily topical applications of 0, 1, 5, 10, 25 or 50 % of the test material in dimethyl sulphoxide were given to one animal at each dose level. Erythema was absent and body weights and ear thickness were unaffected. Therefore the results from this study were used to determine the dosing concentrations in the definitive LLNA test.

In the main study, on Days 1 to 3 five female mice/group received 5, 15 or 50 % of the test material in dimethyl sulphoxide or the vehicle alone (DMSO). One group of five mice received 25 % α-hexylcinnamaldehyde in DMSO and served as the positive control. On day 6, uptake of 3H-thymidine into the auricular lymph nodes draining the site of chemical application was measured five hours post administration.

Erythema was absent and body weights were unaffected in all dose groups. The test material at concentrations of 5, 15 and 50 % elicited proliferative responses with stimulation indices (SI) of 1.54, 1.93, and 2.30, respectively, in comparison to vehicle-treated mice.

Proper conduct of the LLNA was confirmed via a positive response using 25 % α-hexylcinnamaldehyde (a moderate contact sensitiser) which elicited proliferation with a stimulation index of 5.28 in comparison to vehicle-treated mice.

Under the conditions of this study, topical application of the test material at 5, 15 and 50 % in DMSO did not elicit a stimulation index (SI) that met the 3-fold threshold, thus indicating a lack of dermal sensitisation potential in the mouse LLNA. As such the test material requires no classification in accordance with EU criteria.