4-amino-N-[4-(aminocarbonyl)phenyl]benzamide

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: well performed GLP an OECD guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

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:

4-amino-N-[4-(aminocarbonyl)phenyl]benzamide

EC Number:

277-874-7

EC Name:

4-amino-N-[4-(aminocarbonyl)phenyl]benzamide

Cas Number:

74441-06-8

Molecular formula:

C14H13N3O2

IUPAC Name:

4-amino-N-(4-carbamoylphenyl)benzamide

Details on test material:

- Name of test material (as cited in study report): p-Aminobenzoylaminobenzamid TF

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories B.V., Postbus 6174, 5960 AD Horst / The Netherlands
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: 17.8 - 24.7 g
- Housing: single caging
- Diet: pelleted standard diet, ad libidum
- Water: tap water, 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 (%): 45-70 %
- Photoperiod (hrs dark / hrs light): artificial light 6:00 a.m. - 6:00 p.m.
- Bedding: granulated soft wood bedding

Study design: in vivo (LLNA)

Vehicle:

methyl ethyl ketone

Concentration:

The highest test item concentration which can be used was a 25 % (w/v) suspension in methyl ethyl ketone after warming to 37 °C and vortexing.
In the pre-test, two mice were treated with test item concentrations of 10 or 25 %.
In the main test test item concentrations of 5, 10, and 25% were used.

No. of animals per dose:

4

Details on study design:

RANGE FINDING TESTS:

- Compound solubility: The highest test item concentration which can be technically used was a 25 % (w/v) suspension in methyl ethyl ketone after warming at 37 °C and vortexing.
In the pre-test, two mice were treated with test item concentrations of 10 or 25 %. At the tested concentrations, the animals did not show any signs of local skin irritation or systemic toxicity.
The test item in the main study was assayed at 5, 10 and 25 %.

MAIN STUDY:

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

ADMINISTRATION OF 3H-METHYL THYMIDINE AND DETERMINATION OF INCORPORATED 3H-METHYL THYMIDINE

Five days after the first topical application, all mice were intraveneously injected into a tail vein with radio-labelled thymidine (3HTdR). Approximately five hours after treatment with 3HTdR all mice were sacrificed and the draining lymph notes were excised and pooled per group. Single cell suspensions lymph node cells were prepared from pooled lymph nodes, which were subsequently washed with phosphate buffered saline and incubated with trichloroacetíc acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of 3H-methyl thymidine measured in a ß-scintillation counter.

INTERPRETATION OF RAW DATA

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. A test item is regarded as a sensitiliser 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.

OBSERVATIONS

In addition to the sensitising reactions the following observations and data were recorded during the test and observation period:
-Mortality / Viability: Once daily (week day) from experimental start to necropsy.
-Body weights: Prior to the first application and prior to sacrifice.
-Ear weights: After sacrifice biopsy punches were taken from each ear.
-Clinical signs (local / systemic): In the main experiment, clinical signs were recorded within 1 hour after each application, and 24 +/- 4 hours after the first and the second application as well as on the day of preparation. 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 for body weight.

Results and discussion

Positive control results:

Experiment performed in June 2009 using concentrations of 5, 10, and 25 % alpha-Hexylcinnamaldehyde in acetone:olive oil (4+1). These concentrations yielded S.I.´s of 1.79, 2.09, and 6.84, respectively.
The EC3 value calculated was 12.9 % (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:

SI

Remarks on result:

other: see Remark

Remarks:

In this study Stimulation Indices of 1.49, 1.31 and 1.45 were determined with the test item at concentrations of 5, 10 and 25 % (w/v) in methyl ethyl ketone, respectively. The EC3 value could not be calculated, since none of the tested concentrations induced an S.I. greater than 3.

Any other information on results incl. tables

Calculation and results of individual data; Vehicle: methyl ethyl ketone

Test item concentration % (w/v)	Group	Measurement DPM	Calculation			Result
			DPM-BG^a)	number of lymph nodes	DPM per lymph node^b^)	S.I.
---	BG I	20	---	---	---	---
---	BG II	21	---	---	---	---
---	1	2023	2003	8	250.3	---
5	2	3002	2982	8	372.7	1.49
10	3	2641	2621	8	327.6	1.31
25	4	2921	2901	8	362.6	1.45

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

1     =  Control Group

2-4 =   Test Group

S.I. =   Stimulation Index

^a)   =   The mean value was taken from the figures BG I and BG II

^b)   =   Since the lymph nodes of the animals of a dose group were pooled, DPM/node was determined by dividing the measured value by the number of lymph nodes pooled

The EC3 value could not be calculated, since all S.I.´s are below 3.

VIABILITY / MORTALITY

No deaths occurred during the study period.

CLINICAL SIGNS

No symtoms 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.

EAR WEIGHTS

The measured ear weights of all animals treated were recorded after necropsy. A relevant increase in ear weights was not observed.

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information

Conclusions:

The test item p-Aminobenzoylaminobenzamid TF was not a skin sensitiser under the described conditions.

Executive summary:

In the study the test item p-Aminobenzoylaminobenzamid TF suspended in methyl ethyl ketone 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 % (w/v).

The animals did not show any signs of toxicity during the course of the study and no cases of mortality were observed. An increase in the ear weights of the treated animals was also not observed.

In this study Stimulation Indices (S.I.) of 1.49, 1.31, and 1.45 were determined with the test item at concentrations of 5, 10, and 25 % in methyl ethyl ketone, respectively. The EC3 value could not be calculated, since none of the tested concentrations induced a S.I. greater than 3.

In conclusion, the test item p-Aminobenzoylaminobenzamid TF was not a skin sensitiser.