Butyl anthranilate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 August 2016 to 11 July 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline-conform study under GLP without deviations

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Butyl anthranilate

EC Number:

231-816-7

EC Name:

Butyl anthranilate

Cas Number:

7756-96-9

Molecular formula:

C11H15NO2

IUPAC Name:

butyl 2-aminobenzoate

Test material form:

liquid

Details on test material:

- Name of test material (as cited in study report): Anthranilsäurebutylester

Specific details on test material used for the study:

Identification: Anthranilsäurebutylester
Batch: 1/12
CAS No.: 7756-96-9
EINECS: 231-816-7
Purity: 99.9%
Appearance: Bright yellow clear liquid
Expiry Date: 02 May 2018
Storage Conditions:
(provided by the Sponsor) At room temperature
Stability in Solvent: Not indicated by the Sponsor
Certificate of Analysis: AZ 1014/Toxd1
Dose calculation not adjusted to purity.

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS: mice, CBA/CaOlaHsd
- Source: Harlan Laboratories B.V., Postbus 6174, 5960 AD Horst / The Netherlands
- Age at study initiation: 8 - 10 weeks
- Weight at study initiation: 17.2 - 22.4 g
- Housing: group caging
- Diet: pelleted standard diet (Harlan Laboratories B.V., 5960 AD Horst, The Netherlands), ad libidum
- Water: tap water, (Gemeindewerke, 64380 Rossdorf, Germany), ad libitum
- Acclimation period: At least 5 days prior to the start of dosing under test conditions after health examination. Only animals without any visible signs of illness were used for the study

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 + 2°C
- Humidity (%): 45-65 %
- Photoperiod (hrs dark / hrs light): artificial light 6:00 a.m. - 6:00 p.m.
- Bedding: granulated soft wood bedding (Rettenmaier & Söhne GmbH + Co. KG, 73494 Rosenberg, Germany)

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

2.5, 5, and 10%

No. of animals per dose:

4

Details on study design:

Vehicle and Dose Selection
A solubility experiment was performed according to the recommendations given by OECD 429. The highest test item concentration, which could be technically used was 100% of the undiluted test item. Test item solution at different concentrations was prepared using acetone/olive oil (4+1, v/v) as vehicle. Vortexing was used to formulate the test item.
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 and stated in raw data and report. Two mice were treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 50 and 100% once daily each on three consecutive days. Prior to the first application of the test item and before sacrifice the body weight was determined. Clinical signs were recorded at least once daily. Eventual signs of local irritation were documented and a score was used to grade a possible erythema of the ear skin. Furthermore, prior to the first application of the test item (day 1), on day 3 and before sacrifice (day 6) the ear thickness was determined using a micrometer. Additionally, for both animals, the ears were punched after sacrifice (day 6) at the apical area using a biopsy punch (Ø 8 mm corresponding to 0.5 cm2) and were immediately pooled per animal and weighed using an analytical balance. Eventual 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 (for detailed results see Appendix 1).
At the tested concentrations the animals did not show any signs of systemic toxicity. From day 1 to 6, the animals showed an erythema of the ear skin (Score 1 to 2, see Appendix 1 for details). Additionally, excitement, scaly ears, slight eschar formation, slight erythema of scalp, and visible swelling of ears was observed, as well as an increase of ear weight above the threshold of 25% (see Appendix 1 for details).
Therefore, a second pre-test was performed using test item concentrations of 10 and 25%. From day 2 to 4, the animals showed an erythema of the ear skin (Score 1, see Appendix 1 for details). Additionally, scaly ears (animal treated with 10%) and slight eschar formation (animal treated with 25%) was observed on day 6.
Thus, the test item in the main study was assayed at 2.5, 5, and 10%. The highest concentration tested was the highest level that could be achieved whilst avoiding systemic toxicity and excessive local skin irritation as confirmed in the pre-experiment.

Test Item Preparation
The test item was placed into a volumetric flask on a tared balance and acetone/olive oil (4+1, v/v) was quantitatively added (weight per volume).
The different test item concentrations were prepared individually.
The preparations were made freshly before each dosing occasion.

Experimental Design and Study Conduct
The main experiment had to be repeated, since in the first experiment eschar formation was observed in all animals of the high dose. The raw data of the first experiment will be archived in this study file.

Test Item Administration
Each test group of mice was treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 2.5, 5, and 10% in acetone/olive oil (4+1, v/v). 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).

Administration of 3H-methyl-thymidine
Five days after the first topical application (day 6) 250 µL of phosphate-buffered saline containing 20.2 µCi of 3H-methyl thymidine (equivalent to 80.9 µCi/mL 3HTdR) were injected into each test and control mouse via the tail vein.

Terminal Procedure
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.

Preparation of Single Cell Suspensions
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 approximately 18 hours for precipitation of macromolecules.

Determination of cellular proliferation (incorporation of 3HTdR)
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 -scintillation counter. Similarly, background 3HTdR levels were also measured in two 1 mL-aliquots of 5 % trichloroacetic acid. The -scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute.

Observations
Clinical Observations
All animals were observed on a daily basis, including pre- and post-dose observations on days 1, 2 and 3. Any clinical signs of systemic toxicity, local skin irritation or signs of ill health during the study were recorded.

Determination of Ear Thickness
In the pre-test, the ear thickness was determined prior to the first application of the test item (day 1), on day 3, and on day 6 prior to sacrifice using a micrometer.

Determination of ear weights
In the pre-test and main experiment, after the lymph nodes have been excised, both ears of mice were punched at the apical area using a biopsy punch (Ø 8 mm corresponding to 0.5 cm2). For each animal both punches were immediately weighed per animal using an analytical balance. The values obtained were taken down manually. The results are described in the report.

Determination of Body Weights
The body weights was recorded on day 1 (prior to dosing) and prior to sacrifice (pre-test) or prior to treatment with 3HTdR (main experiment)

Data Evaluation
Interpretation of raw data
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 3HTdR 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.
A test item is regarded as a sensitiser in the LLNA if the following criteria are fulfilled:
• First, that 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.
• 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.

General Calculations
The mean values and standard deviations were calculated in the body weight tables.
Where appropriate, the EC3 value were calculated according to the equation
EC3 = (a-c) [(3-d)/(b-d)] + c
where EC3 is the estimated concentration of the test item required to produce a 3-fold increase in draining lymph node cell proliferative activity; (a, b) and (c, d) are respectively the co-ordinates of the two pair of data lying immediately above and below the S.I. value of 3 on the local lymph node assay dose response plot.
All calculations conducted on the DPM values and the ear weights were performed with a validated test script of “R”, a language and environment for statistical computing and graphics.
Within the program a statistical analysis conducted on the DPM values and the ear weights to assess 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. An outlier (DPM value determined for animal 17) was detected in both statistical outlier tests, but was not excluded from any calculations since exclusion of the outlier would not have any influence on the outcome of the study.
Both biological and statistical significance were considered together.

Positive control substance(s):

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

Statistics:

The mean values and standard deviations were calculated in the body weight tables.

Results and discussion

Positive control results:

Experiment performed in April 2017 (Harlan study number 1830700) using concentrations of 5, 10, and 25 % alpha-hexyl cinnamic aldehyde in acetone:olive oil (4:1). These concentrations yielded S.I.´s of 1.78, 3.43, and 10.06, respectively.
The EC3 value calculated was 8.7 % (w/v).
The positive control substance alpha-hexyl cinnamic aldehyde was found to be a skin sensitizer under the described conditions, demonstrating the validity of the study.

In vivo (LLNA)

Results

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: see depicted table below

Any other information on results incl. tables

 Calculation and Results of Individual Data

Vehicle: acetone/olive oil (4+1, v/v)

Test item concentration			DPM values measured	DPM-BG per animal (2 lymph nodes)^a)	S.I.^b)
%	Group no.	Animal no.
---	---	BG I	20	---	---
---	---	BG II	23	---	---
0	1	1	1616	1594.5	---
0	1	2	2229	2207.5	---
0	1	3	1233	1211.5	---
0	1	4	928	906.5	---
0	1	5	1033	1011.5	---
2.5	2	6	3717	3695.5	2.7
2.5	2	7	6487	6465.5	4.7
2.5	2	8	7134	7112.5	5.1
2.5	2	9	5974	5952.5	4.3
2.5	2	10	4195	4173.5	3.0
5	3	11	6666	6644.5	4.8
5	3	12	5304	5282.5	3.8
5	3	13	7372	7350.5	5.3
5	3	14	8213	8191.5	5.9
5	3	15	11322	11300.5	8.2
10	4	16	8585	8563.5	6.2
10	4	17	11508*	11486.5	8.3
10	4	18	8807	8785.5	6.3
10	4	19	7897	7875.5	5.7
10	4	20	8851	8829.5	6.4

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

1      =   Control Group for the test item and for the positive control item

2-4 =   Test Groups

S.I.  =   Stimulation Index

^a)     =   values corrected for mean background value (BGI and BGII).

^b)          =     Stimulation Indices relative to the mean of the control group (Group 1)

*statistical outlier value in both outlier tests

Calculation of Stimulation Indices per Dose Group

Test item concentration	Group Calculation
	Mean DPM per animal (2 lymph nodes)^a)	SD	S.I.
Vehicle Control Group (acetone/olive oil (4+1, v/v))	1386.3	528.9	1.0
2.5% Anthranilsäurebutylester	5479.9	1479.1	4.0^S
5% Anthranilsäurebutylester	7753.9	2251.2	5.6^S
10% Anthranilsäurebutylester	9108.1	1383.3	6.6^S

Calculation of the EC3 value

The EC3 value could not be calculated, since all S.I.´s are above the threshold value of 3.

Viability / Mortality

No deaths occurred during the study period.

Clinical Signs

No signs of systemic toxicity were observed during the study period.From day 2 to 4, the animals treated with a test item concentration of 10% showed an erythema of the ear skin (Score 1, see Appendix 2 for details). Animals treated with 2.5 and 5% test item concentration did not show any signs of local skin irritation.

Body Weights

The body weight of the animals, recordedprior to the first application and prior to treatment with³HTdR, was within the range commonly recorded for animals of this strain and age.

 Ear Weights

The measured ear weight of all animals treated was recorded on test day 6 (after necropsy). A statistically significant increase of ear weight was observed for the mid and high dose (group mean increase compared to vehicle control: 10 and 18%, respectively).

Applicant's summary and conclusion

Conclusions:

The test item Anthranilsäurebutylester was found to be a skin sensitiser under the test conditions of this study.

Executive summary:

In order to study a possible skin sensitising potential of Anthranilsäurebutylester, three groups each of five female mice were treated once daily with the test item at concentrations of 2.5, 5, and 10% in acetone/olive oil (4+1, v/v) by topical application to the dorsum of each ear for three consecutive days. The highest concentration tested was the highest concentration that could be achieved whilst avoiding systemic toxicity and excessive local skin irritation as confirmed by two pre-experiments. A control group of five mice was treated with the vehicle (acetone/olive oil (4+1, v/v)) only. Five days after the first topical application the mice were injected intravenously into a tail vein with radio-labelled thymidine (³H-methyl thymidine). Approximately five hours after intravenous injection, the mice were sacrificed, the draining auricular lymph nodes excised and pooled per animal. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes, which were subsequently washed and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of³H-methyl thymidine measured in ab-scintillation counter.

All treated animals survived the scheduled study period and no signs of systemic toxicity were observed. From day 2 to 4, the animals treated with a test item concentration of 10% showed an erythema of the ear skin (Score 1, see Appendix 2 for details). Animals treated with 2.5 and 5% test item concentration did not show any signs of local skin irritation. A statistically significant increase in ear weights was observed for the mid and high dose.

A test item is regarded as a sensitiser in the LLNA if the exposure to one or more test concentration resulted in a 3-fold or greater increase in incorporation of³HTdR compared with concurrent controls, as indicated by the Stimulation Index (S.I.). The estimated concentration of test item required to produce a S.I. of 3 is referred to as the EC3 value.

In this study Stimulation Indices of 4.0, 5.6, and 6.6 were determined with the test item at concentrations of 2.5, 5, and 10% in acetone/olive oil (4+1, v/v). A dose response was observed. The EC3 value could not be calculated, since all obtained SI´s were above the threshold value of 3.