Boronic acid, (4-ethylphenyl)-

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2007-07-02 to 2007-09-26

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2002-04-24

Deviations:

no

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:

(4-ethylphenyl)boronic acid

EC Number:

613-145-5

Cas Number:

63139-21-9

Molecular formula:

C8 H11 B O2

IUPAC Name:

(4-ethylphenyl)boronic acid

Test material form:

solid

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Remarks:

CBA/CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Netherlands B.V., Postbus 6174
- Females nulliparous and non-pregnant: Yes
- Age at study initiation: 7 - 8 weeks (beginning of acclimatisation)
- Weight at study initiation: 17.9 - 23 g
- Housing: single, Makrolon Type I cages, with wire mesh top and granulated soft wood bedding
- Diet: Ad libitum
- Water: Ad libitum
- Indication of any skin lesions: Only animals without any visible signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 + 3°C
- Humidity (%): 30-85
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Concentration:

5, 10, and 25% (w/v) of the test item in dimethyl formamide;

25% = highest test item concentration, which could be technically used

No. of animals per dose:

5 females/dose

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: The highest test item concentration, which could be technically used was a 25 % solution in dimethyl formamide
- Irritation: To determine the highest non-irritant test concentration or the highest technically applicable concentration, a pretest was performed in two mice on three consecutive days. At concentrations of 2.5, 5, 10, and 25%, the treated mice did not show any signs of irritation.

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay (LLNA)
- Criteria used to consider a positive response:
1) The exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the stimulation index.
2) 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 a volumetric flask on a tared balance and the vehicle was quantitatively added. The test item concentrations were prepared serially. Homogeneity of the test item in the vehicle was maintained during treatment with the magnetic stirrer.

Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear lobe (left and right) with different test item concentrations of 5, 10, and 25% (w/v) in dimethyl formamide. The application volume, 25 µL, was spread over the entire dorsal surface (0 ~ 8 mm) of each ear lobe once daily for three consecutive days. A further group of mice was treated with an equivalent volume of the relevant vehicle alone (control animals).

3HTdR (specific activity, 2 Ci/mmol; concentration, 1 mCi/mL) was administered five days after the first topical application. All mice were administered with 250 µL of 79.9 µCi/mL^3 HTdR (corresponds to 20.0 µCi 3HTdR per mouse) by intravenous injection via a tail vein.

Positive control substance(s):

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

Statistics:

The ANOVA (Dunnett-test) was conducted to assess whether the difference between test item groups and negative control (vehicle) group is statistically significant. Statistical significance was at the five per cent level (p < 0.05). However, both biological and statistical significance were considered together.

Results and discussion

Positive control results:

The validation- / positive control experiment was performed with hexyl cinnamic aldehyde in acetone:olive oil (4+1) using CBA/CaOlaHsd mice in May 2007. The stimulation index was 4.88 for the highest concentration of the postive control (25% w/v).

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

2.54

Variability:

SD= 0.88

Test group / Remarks:

25% (w/v) of the test item

Results 2

Key result

Parameter:

SI

Value:

2.65

Variability:

SD= 1.09

Test group / Remarks:

10% (w/v) of the test item

Results 3

Key result

Parameter:

SI

Value:

1.54

Variability:

SD= 0.52

Test group / Remarks:

5% (w/v) of the test item

Results 4

Key result

Parameter:

SI

Value:

1

Variability:

SI normalized (1), calculated from number of radioactive disintegrations per minute (DPM) per lymph node: 538.5 ± 176.6

Test group / Remarks:

control group (vehicle dimethyl formamide)

Cellular proliferation data / Observations:

DETAILS ON STIMULATION INDEX CALCULATION
The proliferative response of lymph node cells is expressed as the number of radioactive disintegrations per minute per lymph node (DPM/node) and as the ratio of 3HTdR incorporated into lymph node cells of test lymph nodes relative to that recorded for control lymph nodes (stimulation index). Before DPM/node 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 none of the tested concentrations induced an S.I. greater than 3.

CLINICAL OBSERVATIONS
No symptoms of local toxicity at the ears of the animals and no systemic findings were observed during the study period.

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.

Any other information on results incl. tables

Table 1: Calculation and results of individual data

 

Test item concentration		Measurement	Calculation	Result	Calculation	DPM per lymph node b)	Result
% (w/v)	Group	DPM	DPM-BG per anima (2 lymph nodes) a)	S.I.	number of lymph nodes		S.I.
—	BG I	16.00	—	—	—	—	—
—	BG II	18.00	—	—	—	—	—
—	1_1	822.00	805.0	—	—	—	—
—	1_2	1636.00	1619.0	—	—	—	—
—	1_3	1094.00	1077.0	—	—	—	—
—	1_4	741.00	724.0	—	—	—	—
—	1_5	1177.00	1160.0	—	10	538.5	1.00
5	2_1	1183.00	1166.0	1.1	—	—	—
5	2_2	2032.00	2015.0	1.9	—	—	—
5	2_3	1030.00	1013.0	0.9	—	—	—
5	2_4	2237.00	2220.0	2.1	—	—	—
5	2_5	1877.00	1860.0	1.7	10	827.4	1.54
10	3_1	4752.00	4735.0	4.4	—	—	—
10	3_2	2789.00	2772.0	2.6	—	—	—
10	3_3	2699.00	2682.0	2.5	—	—	—
10	3_4	1574.00	1557.0	1.4	—	—	—
10	3_5	2536.00	2519.0	2.3	10	1426.5	2.65
25	4_1	2015.00	1998.0	1.9	—	—	—
25	4_2	2271.00	2254.0	2.1	—	—	—
25	4_3	3537.00	3520.0	3.3	—	—	—
25	4_4	3979.00	3962.0	3.7	—	—	—
25	4_5	1973.00	1956.0	1.8	10	1369.0	2.54

 

Vehicle: dimethyl formamide

BG  =  Background (1 mL 5% trichloroacetic acid) in duplicate

1     =  Control Group

2-4  =  Test Group

S.I.  =  Stimulation Index

 

= The mean of the background value (BGI and BGII) was subtracted from the value. = DPM/node was determined by dividing the sum of the measured values from all lymph nodes within a group by the number of lymph nodes taken from that group

 

 

Table 2: Results for ear thickness

  

Animal No.	Dose Group	Ear thickness before the 1^st application (Dm)				Ear thickness prior to treatment with ^3HTdR (Dm)				Ear thickness gain (Dm)
		right	left	mean	Mean dose group ± SD	right	left	mean	Mean dose group ± SD	Thick­ness gain	Mean dose group ± SD
1		220	240	230		290	290	290		60
2		250	250	250		260	290	275		25
3		240	240	240		240	270	255		15
4		250	260	255		250	280	265		10
5		230	240	235	242 ± 11	240	240	240	265 ± 22	5	23 ± 22
6	2	250	250	250		260	270	265		15
7	2	250	250	250		250	250	250		0
8	2	265	250	258		280	270	275		18
9	2	240	250	245		270	280	275		30
10	2	240	250	245	250 ± 7	260	270	265	266 ± 11	20	17 ± 11
11	3	260	270	265		270	280	275		10
12	3	240	240	240		250	250	250		10
13	3	230	240	235		280	260	270		35
14	3	250	260	255		260	260	260		5
15	3	250	240	245	248 ± 12	250	250	250	261 ± 12	5	13 ± 13
16	4	250	260	255		250	280	265		10
17	4	250	240	245		260	260	260		15
18	4	230	240	235		250	270	260		25
19	4	250	250	250		280	270	275		25
20	4	250	240	245	246 ± 8	270	260	265	265 ± 11	20	19 ± 7

 

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test item was found to be not a skin sensitiser in a LLNA according to OECD guideline 429.

Executive summary:

In the study according to OECD guideline 429, the test item dissolved in dimethyl formamide was assessed for its possible contact allergenic potential. For this purpose a local lymph node assay was performed using test item concentrations of 5, 10, and 25%. All treated animals survived the scheduled study period and no signs of toxicity were observed. A relevant increase in ear thickness gain could not be observed after treatment with the test item. In this study Stimulation Indices (S.I.) of 1.54, 2.65, and 2.54 were determined with the test item at concentrations of 5, 10, and 25% in dimethyl formamide, respectively. The EC3 value could not be calculated, since none of the tested concentrations induced an S.I. greater than 3. The test item was not a skin sensitiser in this assay.