1,1'-{(phenylmethanediyl)bis[(3-substituted benzene-4,1-diyl)diazene-2,1-diyl{1-[3-(dimethylamino)propyl]-2-hydroxy-4-methyl-6-oxo-1,6-dihydropyridine-5,3-diyl}]}dipyridinium chloride lactate salts

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

January 07 - February 02, 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well documented and reported study, conducted according to internationally accepted technical guidelines and in compliance with GLP in recognized contract research organization.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

of 2002

Deviations:

yes

Remarks:

During the range-finding study animal housing at 16-22ºC and 20-65% relative humidity. These deviations did not affect the choice of dose range for the main study. Study integrity was not adversely affected by these deviations.

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

of 2008

Deviations:

yes

Remarks:

During the range-finding study animal housing at 16-22ºC and 20-65% relative humidity. These deviations did not affect the choice of dose range for the main study. Study integrity was not adversely affected by these deviations.

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

of 2003

Deviations:

yes

Remarks:

During the range-finding study animal housing at 16-22ºC and 20-65% relative humidity. These deviations did not affect the choice of dose range for the main study. Study integrity was not adversely affected by these deviations.

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Reference

Name:

Unnamed

Type:

Constituent

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Mouse (females only), strain: CBA/J (inbred, SPF-Quality), with appropriate range of bodyweight at study start.
- Source: Charles River France, L’Arbresle Cedex, France.
- Age at treatment start (1st induction): Approx. 10 weeks.
- Weight at treatment start (1st induction): Minimum 20 g, maximum 24 g.
- Housing: Individual housing in M I type cages. (During acclimatization group housing in M III type cages).
- Bedding material: Commercially available sterilized saw dust (Litalabo, S.P.P.S., Argenteuil, France).
- Cage enrichment: Commercially available paper (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, UK).
This was removed from Day 1 (prior to dosing) until ear scoring on Day 3. - Diet (ad libitum): Commercially available pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest,
Germany).
- Water (ad libitum): Tap water
- Acclimation period: At least 5 days before treatment start under laboratory conditions.
- Health inspection: A health inspection was performed prior to treatment, to ensure that the animals are in a good state of health. Special attention was paid to the ears, which were intact and free from any abnormality.


ENVIRONMENTAL CONDITIONS

During the preliminary range-finding study the lower limits of temperature and relative humidity in the animal rooms were lower than recommended in the technical guidelines: 16-22ºC and 20-65% relative humidity. These deviations did not affect the choice of dose range for the main study. Study integrity was not adversely affected by these deviations.

During the main study animal housing and environmental conditions were appropriate for sensitization testing by the LLNA test in the mouse: Controlled environment with approximately 15 air changes per hour, 12 hours artificial fluorescent light and 12 hours darkness per day and 20 – 24ºC. The relative humidity during the main study was 38 – 63%.

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Concentration:

Range-Finding Test:
Induction on Days 1, 2 and 3 at the following concentrations:
10% (1 female), 25% (3 females), 50% (2 females).
The concentrations of test substance are expressed as percentage of water- and minor impurity-free test substance in the vehicle (w/w). One animal of the 25% group was treated only on Days 1 and 2, as it was found dead on Day 3 prior to dosing.

Main Study:
Induction on Days 1, 2 and 3 at the following concentrations:
0% (vehicle control, 5 females), 10% (5 females), 25% (5 females), 50% (5 females).
The concentrations of test substance are expressed as percentage of water- and minor impurity-free test substance in the vehicle (w/w).

No. of animals per dose:

Range-Finding Test:
1 to 3 female animals per dose (for details see Table 2 in section "Remarks on results including tables and figures").

Main Study:
5 female animals per dose (for details see Table 3 in section "Remarks on results including tables and figures").

Positive control substance(s):

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

Results and discussion

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: see Remark

Remarks:

Mean Stimulation Index (SI) values for the experimental groups treated with 10, 25 and 50% test substance dilutions* were 3.0, 7.9 and 3.9, respectively. Deatailed SI results are presented in Table 5 in section "Remarks on results including tables and figures". Table 4 of this section indicates which individual DPM values were included in the respective group mean DPM values and which were considered to be outliers. Group mean DPM values were used for calculation the SI values. ------------------------------------------------------------------------------------------------------------------------------------- * Expressed as percentage of water- and minor impurity-free test substance in the vehicle (w/w).

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: see Remark

Remarks:

Mean DPM/animal values for the experimental groups treated with 10, 25 and 50% test substance dilutions* were 1043, 2749 and 1361 DPM/animal, respectively. For the vehicle control group, on average 350 DPM/animal were recorded. Deatailed DPM results are presented in Tables 4 and 5 in section "Remarks on results including tables and figures". ------------------------------------------------------------------------------------------------------------------------------------- * Expressed as percentage of water- and minor impurity-free test substance in the vehicle (w/w).

Any other information on results incl. tables

 

 

Table 2: Range-finding (Preliminary Irritation) Study. Skin Reactions after Epidermal Exposure and Body Weights
		Day 1	Day 3
			Skin Reactions at Dorsal Ear Surface^B
			Left Ear		Right Ear
Animal No.	% Test Substance^A	Bodyweight (g)	Erythema	Edema	Erythema	Edema	Bodyweight (g)
5	10	21	G	0	G	0	20
1	25	24	G	0	G	0	^C
3	25	23	G	0	G	0	22^D
6	25	22	G	0	G	0	20
2	50	24	G	0	G	0	22^E
4	50	22	G	0	G	0	21^E

^A.Vehicle: Dimethylformamide. The concentrations of test substance in the vehicle are expressed as percentage of water- and minor impurity-free test substance in the vehicle (w/w).

^B.  Attempt was made to clean the ears of residual test substance with tap water and vehicle.

^C. Animal was found dead prior to dosing on Day 3 (bodyweight 22 gram).

^D. Animal was found on Day 4.

^E.  Hard ears and bold spots behind the ears observed.

^G. No scoring possible due to brown discoloration of the ears by the test substance.

 

Table 3: Main Study Skin Reactions, Bodyweights and Relative Size of Auricular Lymph Nodes
			Day 1	Day 3				Day 6
				Skin Reactions at Dorsal Ear Surface					Size Lymph Nodes^D
				Left Ear		Right Ear			Left	Right
Group	% Test Substance^A	Animal No.^B	BW (g)^C	Ery-thema	Ede-ma	Ery-thema	Ede-ma	BW (g)^C
1	0%	1	24	0	0	0	0	23	n	n
	(vehicle)	2	22	0	0	0	0	21	n	n
		3	21	0	0	0	0	21	n	n
		4	22	0	0	0	0	21	n	n
		5	24	0	0	0	0	23	n	n
2	10%	6	22	G	0	G	0	21	n	n
		7	20	G	0	G	0	21	n	n
		8	21	G	0	G	0	22	n	n
		9	23	G	0	G	0	23	n	n
		10	23	G	0	G	0	21	n	n
3	25%	11	21	G	0	G	0	21	+++	n
		12	22	G	0	G	0	22	n	n
		13	24	G	0	G	0	22	+	n
		14	22	G	0	G	0	20	n	n
		15	23	G	0	G	0	21	n	n
4	50%^E	16	21	G	0	G	0	21	n	n
		17	20	G	0	G	0	20	n	n
		18	23	G	0	G	0	23	n	n
		19	22	G	0	G	0	21	n	+
		20	23	G	0	G	0	21	n	n

 

^A.Vehicle: Dimethylformamide. The concentrations of test substance in the vehicle are expressed

as percentage of water- and minor impurity-free test substance in the vehicle (w/w).

^B.  Animal (identification) number.

^C. BW = bodyweight (grams).

^D. Relative size of auricular lymph nodes (–, –– or –––: degree of reduction, +, ++ or +++: degree

of enlargement, n: considered to be normal).

^E.  Hard ears and bold spots behind the ears observed.

^G. No scoring possible due to brown discoloration of the ears by the test substance.

 

Table 4: Main Study Radioactivity Measurements (Individual Animals)
Group	% Test Substance^A	Animal No.	DPM^B/Animal
1	0%	1	635
	(vehicle)	2	120
		3	266
		4	1279^C
		5	378
2	10%	6	747
		7	952
		8	1324
		9	621
		10	1573
3	25%	11	8919^D
		12	2379
		13	2520
		14	3566
		15	2532
4	50%	16	804
		17	1273
		18	835
		19	3022
		20	870

 

^A.Vehicle: Dimethylformamide. The concentrations of test substance in the vehicle are
expressed as percentage of water- and minor impurity-free test substance in the vehicle (w/w).

^B.  DPM = Disintegrations per Minute

^C. Value rejected and not used for interpretation (outside historical range for vehicle).

^D. Value rejected and not used for interpretation (outlier response).

 

Table 5: Main Study Disintegrations Per Minute (DPM) and Stimulation Index (SI)
Group	% Test Substance^A	Mean DPM ± SEM^B			SI ± SEM
2	10%	1043	±	178	3.0	±	1.1
3	25%	2749	±	274	7.9	±	2.6
4	50%	1361	±	424	3.9	±	1.7
1	0% (vehicle)	350	±	109	1.0	±	0.4

 

^A.Vehicle: Dimethyl formamide. The concentrations of test substance in the vehicle are
expressed as percentage of water- and minor impurity-free test substance in the vehicle (w/w).

^B.  SEM = Standard Error of the Mean

 

Applicant's summary and conclusion

Interpretation of results:

sensitising

Remarks:

Migrated information

Conclusions:

In view of mean stimulation indices (SI) of 3.0, 7.9 and 3.9 attained in the 10%, 25% and 50% dose groups*, respectively, the substance was classified as "irritant (Xi)" and "May cause sensitisation by skin contact (R43)” according to EU-DSD classification rules [DIRECTIVE 67/548/EEC] and as "Category 1 (Warning: May cause an allergic skin reaction)" according to EU-GHS classification rules [REGULATION (EC) 1272/2008]. From the SI data attained, an EC3 value (the estimated test substance concentration that will give a SI of 3) of 10.1%* was determined by linear interpolation.
---------------------------------------------------------------------------------------------------------------------------
* Expressed as percentage of water- and minor impurity-free test substance in the vehicle (w/w).

Executive summary:

the test substance was tested for skin sensitisation in female CBA/J mice according to OECD Guideline 429 (Local Lymph Node Assay) and the corresponding EC and, EPA-OPPTS Technical Guidelines in compliance with GLP. Reliability grade 1 was assigned. The suitability of the mouse strain used and methods adopted by the testing laboratory were confirmed in previous reliability checks using hexyl cinnamic aldehyde (CAS No. 101-86-0) as a positive control agent.

 

From a preliminary range-finding (irritation) test, suitable concentrations of 10%, 25% and 50% the test substance in Dimethylformamide were chosen for the main LLNA sensitisation study. These concentrations are expressed as percentage of water- and minor impurity-free test substance in the vehicle Dimethylformamide (w/w). In the main study, 20 mice (5 females / dose group) were used of which 5 served as vehicle controls. On Days 1, 2 and 3, the animals were induced by epidermal administration of the vehicle or test substance dilutions (25μL/ear/day) onto the dorsal surface of both ears. Main study animals were checked for mortality/viability twice daily and for signs of systemic toxicity once daily. In addition, bodyweights were recorded on Days 1 (prior to treatment) and 6, and irritation of the ears (erythema and edema) as well as any other local effects were recorded approximately 3-4 hours after the third treatment. 3H-methyl thymidine diluted in sterile phosphate buffered saline was injected into the tail vein on Day 6 for radioactive labelling. Five hours afterwards the draining (auricular) lymph nodes were excised and pooled per animal. On Day 7, radioactivity of precipitated DNA from the excised lymph nodes was measured by scintillation counting and automatically expressed as Disintegrations per Minute (DPM). From these data, the stimulation index (SI) reflecting the ratio of lymphocyte proliferation in treated groups to that in the vehicle control group was calculated for each group.

 

Two outlier responses, one in the vehicle control group and one in the 25% dose group, with excessively high DPM values were eliminated from the group mean DPM. The mean DPM value of the vehicle control group was 350 DPM/animal. Mean DPM values in the 10%, 25% and 50% dose groups were, 1043, 2749 and 1361, corresponding to mean stimulation index (SI) values of 3.0, 7.9 and 3.9, respectively. Hence, the SI threshold of ≥ 3.0, indicating a positive sensitisation response, was attained in all treated groups, although a dose related increase was only evident at 10% and 25% and not at 50%. From these data an EC3 value (the estimated test substance concentration that will give a SI of 3) of 10.1% was calculated. The lower response in the 50% dose group than in the 25% group may have been related to the finding of hard ears seen in all animals of the 50% group which could have impaired skin penetration in this group. This could also explain mortality/survival attained in the preliminary range-finding test, in which two of three animals of the 25% dose group died whilst both animals of the 50% group survived. There were no premature deaths and no signs of systemic toxicity in the main study. Bodyweights did not distinguish treated groups from the vehicle control group. Scoring for erythema was not possible in treated animals, because of brown discoloration of the ears by the test substance. Edema were not evident. In two animals of the 25% dose group and one animal of the 50% group auricular lymph nodes of one of the ears were enlarged, in all other main study animals their size was considered to be normal.

 

According to EU-DSD classification rules [DIRECTIVE 67/548/EEC] the results attained in this study would lead to classification and labelling as “Xi” (irritant) and “R43” (May cause sensitisation by skin contact) and according to EU-GHS classification rules [REGULATION (EC) 1272/2008] they would lead to “Category 1” (Warning: May cause an allergic skin reaction).