Multi constituent substance

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: Study performed according to OECD guideline 429 in compliance to GLP.

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Reference 2

Reference Type:

other: Published secondary source

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Multi constituent substance

EC Number:

916-604-0

IUPAC Name:

Multi constituent substance

Details on test material:

Test substance : Basic Brown 17 (COLIPA number B007)
Batch number : 64960101
Methylsulphate anion : 11.6%
Chloride ion : 3.3%
Water : 6.5%
Basic Red 118 : 4.5%
Purity : 94.2% (HPLC)

In vivo test system

Study design: in vivo (LLNA)

Vehicle:

other: ethanol : water (7 : 3 v/v)

Concentration:

The test item was prepared in the vehicle at the following concentration : 0.2, 0.5, 1.0, 3.0, 6.0 % (w/v)

No. of animals per dose:

Four females per dose

Details on study design:

Five groups of four female mice were treated daily with the test item at concentrations of 0.2, 0.5, 1.0, 3.0, 6.0 % (w/v) in ethanol : water (7:3 v/v). Each test group of mice was treated by topical application to the dorsal surface of each ear lobe with the different test item concentrations. A volume of 25uL was spread over the entire dorsal surface of each ear lobe once daily for 3 consecutive days. The control group was treated with the vehicle alone. Five days after the first topical administration all mice were treated with radio labelled thymidine by intravenous injection via the tail vien. Approximately 5 hours after administration with radio labelled thymidine all mice were euthanised. The draining lymph nodes were excised and pooled for each experimental group. After preparation the level of radio labelled incorporation was then measured by scintillation counting. The proliferative response of lymph node cells is expressed as the ratio of incorporation into lymph node cells of treated animals relative to that recorded in the control animals (stimulation index). A test item is regarded as a sensitiser if the exposure to at least one concentration resulted in at least a 3 fold increase in the incorporation of radio labelled thymidine compared with concurrent controls as indicated by the stimulation index together with consideration of dose response.

Positive control substance(s):

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

Results and discussion

In vivo (LLNA)

Results

Parameter:

SI

Remarks on result:

other: The stimulation index was 1.0, 1.0, 1.3, 0.9, 1.3 for test item concentrations of 0.2, 0.5, 1.0, 3.0, 6.0% (w/v) respectively. The stimulation index was 2.4, 3.6, 11.2 for positive control concentrations of 5.0, 10.0, 25.0% (w/v) respectively.

Cellular proliferation data / Observations:

No clinical signs were observed in any animals of the control groups, the 0.2% dose group, the 0.5% dose group or the 1.0% dose group. On the third application day, a slight erythema was observed at both dosing sites in all mice of the 3.0% dose group. On the second application day, a moderate or slight erythema was observed at both dosing sites in all mice of the 6.0% dose group, persisting for the remainder of the in life phase of the study. No deaths occurred during the study period. The body weight of the animals, recorded prior to the first application and prior to necropsy, was within the range commonly recorded for animals of the strain and age.

Any other information on results incl. tables

Test 1

Concentration %(w/v)		Measurement DPM	DPM-BG(a)	Number of lymph nodes	DPM per lymph node(b)	SI
-	BG1	9	-	-	-	-
-	BG2	7	-	-	-	-
-	CG1	2087	2079	7	297	-
0.2	TG2	2370	2362	8	295	1.0
0.5	TG3	2465	2457	8	307	1.0
1.0	TG4	3095	3087	8	386	1.3

Test 2

Concentration % (w/v)		Measurement DPM	DPM-BG(a)	Number of lymph nodes	DPM per lymph node(b)	SI
-	BG1	7	-	-	-	-
-	BG2	1	-	-	-	-
-	CG5	2199	2195	8	274	-
3.0	TG6	1995	1991	8	249	0.9
6.0	TG7	2882	2878	8	360	1.3

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

CG= Control group

TG= Test group

SI= Stimulation index

a)= The mean BG was calculated from the BG1 and BG2 values

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

Applicant's summary and conclusion

Interpretation of results:

other: a conclusion on sensitisation cannot be established

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

The test item is not a skin sensitiser when tested up to a concentration of 6% (w/v). The maximum test concentration is too low. A conclusion on sensitisation cannot be established.

Executive summary:

In order to study a possible allergic potential of Basic Brown 17, five groups each of four female mice were treated daily with the test item at concentrations of 0.2%, 0.5%, 1.0%, 3.0%, 6.0% in ethanol/water (7/3 v/v) by topical application to the dorsum of each ear lobe for three consecutive days. A loading of 6.0% was the highest technically applicable concentration in the vehicle. Two control groups each of four mice were treated with the vehicle (ethanol/water (7/3 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 group. 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 of 3H-methyl thymidine measured in a beta-scintillation counter. All treated animals survived the scheduled study period. No clinical signs were observed in any animals of the control groups, animals dosed at 0.2%, 0.5%, 1.0%. On the third application day, a slight erythema was observed at both dosing sites in all mice of the 3.0% dose group. Since the second application day, a moderate or slight erythema was observed at both dosing sites in all mice of the 6.0% dose group, persisting for the remainder of the in-life phase of the study. The stimulation index was 1.0, 1.0, 1.3, 0.9, 1.3 for test item concentrations of 0.2%, 0.5%, 1.0%, 3.0%, 6.0% respectively. No constant dose - dpm/LN level (indicated by stimulation index values) relationship was observed in the study. As no test item related findings, such as significant body weight loss or local/systemic findings, were observed up to the concentration of 1%, this indicated that the test item does not show an allergic potential at lower concentrations. At the higher concentrations tested, (3.0% and 6.0%), some test item related signs, such as slight to moderate ear erythema, was observed at the local dosing sites but no clear change of dpm/LN was caused by this local irritant effect. This demonstrates that the test item caused skin irritation but does not show an allergic potential at lower test concentrations.