N-(4-(methoxybenzyl))-N,N-dimethylanilinium hexafluoroantimonate

Endpoint summary

Description of key information

The LLNA and the Buehler studies did not return a positive result. Therefore the test substance is not classified for skin sensitisation.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

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:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

2003

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2004

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2002

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: King Industries, Inc.
- Purity, including information on contaminants, isomers, etc.: Not indicated by the sponsor; treated as 100% pure

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: In freezer (S -15°C) in the dark
- Solubility and stability of the test material in the solvent/vehicle and the exposure medium: soluble, stability not indicated

Species:

mouse

Strain:

CBA

Remarks:

inbred, SPF-Quality

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charles River France, L'Arbresle Cedex, France
- Females nulliparous and non-pregnant: yes
- Age at study initiation: Young adult animals (approx. 10 weeks old)
- Weight at study initiation: Body weight variation was within +/- 20% of the sex mean
- Housing: Individual housing in labeled Macrolon cages (MI type; height 12.5 cm) containing sterilized sawdust as bedding material (Utalabo, S.P.P.S., Argenteuil, France)
- Diet (e.g. ad libitum): Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiaten GmbH, Soest, Germany)
- Water (e.g. ad libitum): Free access to tap water
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.0 ± 3.0°C (actual range: 21.5 - 23.4°C)
- Humidity (%): 30-70% (actual range: 41 - 76%)
- Air changes (per hr): 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours artificial fluorescent light and 12 hours darkness per day

- IN-LIFE DATES: From: June 20, 2007To: July 9, 2007

Vehicle:

dimethylformamide

Concentration:

10%, 25%, 50%.
50% is the maximum concentration that produces a solution suitable for application according to the guidelines.

No. of animals per dose:

Five

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: The vehicle was selected based on trial formulations and on test substance data supplied by the sponsor. The maximum soluble concentration that produces a solution suitable for application according to the
guidelines is 50%.
- Irritation: A preliminary irritation study was conducted in order to select the highest test substance concentration to be used in the main study. In principle, this concentration should be well tolerated systemically by the animals and may give moderate irritation (grade 2) at the highest. Two test substance concentrations were tested, 25% and 50%. Two young adult animals were selected (5-14 weeks old). Each animal was treated with one concentration on three consecutive days. Approximately 4 hours after the last exposure, the ear was cleaned of residual test substance with tap water and the irritation was assessed. Bodyweights were determined on Day 3. The animals were sacrificed after the final observation and no necropsy was performed.

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay
- Criteria used to consider a positive response: Disintegrations Per Minute (DPM) values are presented for each animal and for each dose group. A Stimulation Index (SI) is calculated for each group. The SI is the ratio of the DPM/group compared to DPM/vehicle control group. If the results indicate a SI≥ 3, the test substance may be regarded as a skin sensitizer, based on the test guideline and recommendations done by ICCVAM. The results were evaluated according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations (2003) and the EC criteria for classification and labeling of dangerous substances and preparations (Council Directive 67/548/EEC and all adaptations to technical progress and amendments of this Directive published in the Official Journal of the European Communities). Consideration was given to the EC3 value (the estimated test substance concentration that will give a SI =3).

TREATMENT PREPARATION AND ADMINISTRATION:
The test substance formulations (w/w) were prepared within 4 hours prior to each treatment. Homogeneity was obtained to visually acceptable levels.
Three groups of five animals were treated with one test substance concentration per group. The highest test substance concentration was selected from the preliminary irritation study. One group of five animals was treated with vehicle.

Induction - Days 1, 2 and 3
The dorsal surface of both ears was epidermally treated (251J1/ear) with the test substance concentration, at approximately the same time per day. The concentrations were mixed thoroughly using a vortex mixer immediately prior to dosing. The control animals were treated the same as the experimental animals, except that, instead of the test substance, the vehicle alone was administered.

Treatment - Day 6
Each animal was injected via the tail vein with 0.25 mL of sterile phosphate buffered saline (PBS) (Merck, Darmstadt, Germany) containing 20 μCi of 3H-methyl thymidine (GE Health care, Buckinghamshire, UK). After approximately five hours, all animals were killed by intraperitoneal injection with pentobarbital Euthesate® (0.2 ml/animal) (Ceva Sante Animale BV, Naaldwijk, The Netherlands). The draining (auricular) lymph node of each ear was excised. The relative size of the nodes (as compared to normal) was estimated by visual examination and abnormalities of the nodes and surrounding area were recorded. The nodes were pooled for each animal in approximately 3 mL PBS.

Radioactivity measurements - Day 7
Precipitates were recovered by centrifugation, resuspended in 1 mL TCA and transferred to 10 mL of Ultima Gold cocktail (PerkinElmer Life and Analytical Sciences, Boston, MA, US) as the scintillation fluid. Radioactive measurements were performed using a Packard scintillation counter (2800TR). Counting time was to a statistical precision of ± 0.2% or a maximum of 5 minutes whichever comes first. The scintillation counter was programmed to automatically subtract background and convert Counts Per Minute (CPM) to Disintegrations Per Minute (DPM).

Observations
Mortality/Viability: Twice daily.
Toxicity: At least once daily.
Body weights: On Days 1 (pre-treatment) and 6.
Irritation: On Day 3 (3-4 hours after treatment), the skin reactions were assessed. Skin reactions were graded according to the following numerical scoring system. Furthermore descriptions of all other (local) effects were recorded.

Grading Irritation Reactions:
Erythema and eschar formation:
No erythema 0
Slight erythema (barely perceptible) 1
Well-defined erythema 2
Severe erythema (beet redness) to slight eschar formation (injuries in depth) 3
Oedema formation:
No oedema 0
Slight oedema (barely perceptible) 1
Moderate oedema 2
Severe oedema 3

Positive control substance(s):

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

Positive control results:

The six monthly reliability check with Hexylcinnamaldehyde, indicates that the Local Lymph
Node Assay (as performed at NOTOX) is an appropriate model for testing for contact
hypersensitivity.

Parameter:

SI

Remarks:

Mean - 5 animals

Value:

1

Variability:

± 0.3 (SEM)

Test group / Remarks:

Group 1 - 0% (vehicle)

Parameter:

SI

Remarks:

Mean - 5 animals

Value:

1.1

Variability:

± 0.3 (SEM)

Test group / Remarks:

Group 2 - 10% (test substance in vehicle)

Parameter:

SI

Remarks:

Mean - 5 animals

Value:

1.3

Variability:

± 0.4 (SEM)

Test group / Remarks:

Group 3 - 25% (test substance in vehicle)

Parameter:

SI

Remarks:

Mean - 5 animals

Value:

0.9

Variability:

± 0.3 (SEM)

Test group / Remarks:

Group 4 - 50% (test substance in vehicle)

Parameter:

other: Mean DPM - 5 animals

Value:

416

Variability:

± 88 (SEM)

Test group / Remarks:

Group 1 - 0% (vehicle)

Parameter:

other: Mean DPM - 5 animals

Value:

460

Variability:

± 49 (SEM)

Test group / Remarks:

Group 2 - 10% (test substance in vehicle)

Parameter:

other: Mean DPM - 5 animals

Value:

534

Variability:

± 104 (SEM)

Test group / Remarks:

Group 3 - 25% (test substance in vehicle)

Parameter:

other: Mean DPM - 5 animals

Value:

376

Variability:

± 70 (SEM)

Test group / Remarks:

Group 4 - 50% (test substance in vehicle)

Preliminary irritation study (Table 1)

No irritation was observed in any of the animals examined.

The results of the epidermal exposures for the selection of highest test substance concentration to be tested in the main study are described in the table.

Based on the results, the highest test substance concentration selected for the main study was a 50% concentration.

 

Body weights (Table 2)

Body weights and body weight gain of experimental animals remained in the same range as controls over the study period.

 

Radioactivity measurements (Tables 3 and 4)

Mean DPM/animal values for the experimental groups treated with test substance concentrations 10, 25 and 50% were 460,534 and 376 respectively.

The mean DPM/animal value for the vehicle control group was 416.

 

Toxicity and Mortality

No mortality occurred and no symptoms of systemic toxicity were observed in the animals of the main study.

 

 

Table 1: Skin reactions after epidermal exposure and body weights
		Day 1	Day 3
			Skin reactions dorsal surface ear
			Left		Right
Animal number	% test substance	Body weight (g)	Erythema	Oedema	Erythema	Oedema	Body weight (g)
1	25	23	0	0	0	0	23
2	50	22	0	0	0	0	22

 

 

Table 2: Skin reactions, body weights and relative size auricular lymph nodes
			Day 1	Day 3				Day 6
				Skin reactions dorsal surface ear					Size nodes^4
				Left		Right			Left	Right
Group	Animal number	% test substance^1	BW (g)^3	Erythema	Oedema	Erythema	Oedema	BW (g)^3
1	1	0%	23	0	0	0	0	24	n	n
	2		25	0	0	0	0	25	n	n
	3		26	0	0	0	0	25	n	n
	4		25	0	0	0	0	25	n	n
	5		20	0	0	0	0	20	n	n
2	6	10%	24	0	0	0	0	24	n	n
	7		26	0	0	0	0	24	n	n
	8		23	0	0	0	0	23	n	n
	9		24	0	0	0	0	23	n	n
	10		21	0	0	0	0	21	n	n
3	11	25%	24	0	0	0	0	23	n	n
	12		22	0	0	0	0	21	n	n
	13		24	0	0	0	0	24	n	n
	14		25	0	0	0	0	25	n	n
	15		24	0	0	0	0	23	n	n
4	16	50%	22	0	0	0	0	23	n	n
	17		25	0	0	0	0	24	n	n
	18		23	0	0	0	0	24	n	n
	19		21	0	0	0	0	21	n	n
	20		23	0	0	0	0	22	n	n

1. Vehicle: Dimethyl formamide


2. Animal number


3. Body weight (grams)


4. Relative size auricular lymph nodes (-, -- or ---: degree of reduction, +,++ or +++: degree of enlargement, n: considered to be normal)

 

 

Table 3: Radioactivity measurements (individual animals)
Group	Animal number	% test substance	DPM / animal
1	1	0% (vehicle)	383
	2		345
	3		739
	4		208
	5		405
2	6	10%	394
	7		385
	8		620
	9		370
	10		529
3	11	25%	437
	12		565
	13		293
	14		464
	15		910
4	16	50%	415
	17		330
	18		297
	19		623
	20		214

 

 

Table 4: Disintegrations Per Minute (DPM) and Stimulation Index (S1)
Group	% test substance	Mean DPM ± SEM	Mean SI ± SEM
1	0% (vehicle)	416 ± 88	1.0 ± 0.3
2	10%	460 ± 49	1.1 ± 0.3
3	25%	534 ± 104	1.3 ± 0.4
4	50%	376 ± 70	0.9 ± 0.3

 

 

 

Interpretation of results:

GHS criteria not met

Conclusions:

The SI values calculated for the substance concentrations 10, 25 and 50% were 1.1, 1.3 and 0.9 respectively. Since there was no indication that the test substance could elicit an SI ≥ 3 when tested up to 50%, it was established that the EC3 value (if any) exceeds 50%.
Based on these results and according to the recommendations made in the test guidelines, the test substance would not be regarded as skin sensitizer.
Moreover, the test substance does not meet the GHS/CLH criteria to be classified for skin for sensitisation.

Executive summary:

The skin sensitization potential of N-(4-(methoxybenzyl))-N,N-dimethylanilinium hexafluoroantirronate in mice was assessed based on the guidelines described in:OECD, Section 4, Health Effects, No.429 (2002); EC, Council Directive 67/548/EEC, Annex V, B.42 (2004); EPA, OPPTS 870.2600 (2003) "Skin Sensitisation".

Test substance concentrations selected for the main study were based on the results of a preliminary study. In the main study, three groups of five experimental animals were treated with test substance concentrations of 10%, 25% or 50% on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with vehicle alone (Dimethyl formamide). Three days after the last exposure, all animals were injected with ³H-methyl thymidine and after five hours the draining (auricular) lymph nodes were excised. After precipitating the DNA of the lymph node cells, radioactivity measurements were performed. The activity was expressed as the number of Disintegrations Per Minute (DPM) and a stimulation index (SI) was subsequently calculated for each group.

No skin reactions were observed in any of the animals examined. All nodes of the experimental and control groups were considered normal in size. No macroscopic abnormalities of the surrounding area were noted. Mean DPM/animal values for the experimental groups treated with test substance concentrations 10, 25 and 50% were 460, 534 and 376 respectively. The mean DPM/animal value for the vehicle control group was 416. The SI values calculated for the substance concentrations 10, 25 and 50% were 1.1, 1.3 and 0.9 respectively. Since there was no indication that the test substance could elicit an SI ≥ 3 when tested up to 50%, it was established that the EC3 value (if any) exceeds 50%.

Based on these results and according to the recommendations made in the test guidelines, the test substance would not be regarded as skin sensitizer. The test substance does not meet the GHS/CLH criteria to be classified for skin for sensitisation.

Reference 2

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

From 19 Dec 2003 to 25 Feb 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

1998

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

1992

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

Buehler test

Justification for non-LLNA method:

The Buehler study had been completed before animal welfare rules were required.

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: MS Research Laboratories

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature and humidity


TREATMENT OF TEST MATERIAL PRIOR TO TESTING
Induction:
Challenge:

Species:

guinea pig

Strain:

Hartley

Sex:

male/female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Elm Hill Breeding Labs., Inc., Chelmsford, MA
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 3 weeks
- Weight at study initiation: 297 - 335 g for males and 298 - 342 g for females
- Housing: The animals were identified by cage notation and a uniquely numbered metal eartag and housed 1/cage in suspended wire cages. Bedding was placed beneath the cages and changed at least three times/week. The animal room reserved exclusively for guinea pigs on acute tests was temperature controlled, had a 12 hour light/dark cycle, and was kept clean and vermin free.
- Diet (e.g. ad libitum): Fresh PMI GuInea Pig Chow (Diet #5025) ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: five days
- Indication of any skin lesions: no

ENVIRONMENTAL CONDITIONS
- Temperature (°C):
- Humidity (%):
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): 12 hour light/dark cycle
- IN-LIFE DATES: From: Sep. 8, 2003 To: Oct 8, 2003

No. of animals per dose:

Group 1 (test): 10 males and 10 females
Group 2 (control) - 5 males and 5 females

Details on study design:

RANGE FINDING TESTS:

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 3
- Exposure period: 6 hours
- Test groups: Group 1 - 100%, 10 males and 10 females. 0.4 g of the test article (moistened with 0.1 ml of distilled water).
- Control group: Group 2 - naive control (untreated). 5 males and 5 females.
- Site: left shoulder area (Site 1)
- Frequency of applications: once/week on the same day each week
- Duration: three-week period, a total of 3 six-hour insults
- Concentrations: 100%


B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: 14
- Exposure period: 6 hours
- Test groups: Group 1 - 100%
- Control group: Group 2 - 100%
- Site: naive site on the left hip area (Site 3)
- Concentrations: 100% (highest non-irritating concentration)
- Evaluation (hr after challenge): 24 and 48 hours following patch removal

Challenge controls:

5 males and 5 females were dosed with 100% of the test article.

Positive control substance(s):

yes

Remarks:

Dinitrochlorobenzene (DNCB)

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

100%

No. with + reactions:

0

Total no. in group:

20

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

100%

No. with + reactions:

0

Total no. in group:

20

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

100%

No. with + reactions:

0

Total no. in group:

10

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

100%

No. with + reactions:

0

Total no. in group:

10

Dermal Observations (Table 1 & 2)

Induction 1 - Erythema was absent to very faint.

Induction 2 - Erythema was absent.

Induction 3 - Erythema was absent.

Challenge - Erythema was absent on both the induced and naive control group.

	Incidence index		Severity index
	24 h	48 h	24 h	48 h
Test article	0.00	0.00	0.00	0.00
Naive Control	0.00	0.00	0.00	0.00
Positive control	0.70	0.70	0.88	0.83
Naive Control	0.00	0.00	0.05	0.05

 

Body Weights (Table 1 & 2)

Body weight changes were normal.

 

Systemic Observations

Wetness of the anogenital area was noted during the observation period in 1/20 animals of Group 1.

 

Table 1: Induction & Challenge Dermal Observations - Group 1 (0.4 g, 100%) - (Post Patch Removal) and Body Weights
		Body Weight (g)		Induction 1, Erythema, Site 1		Induction 2, Erythema, Site 1		Induction 3, Erythema, Site 1		Challenge, Erythema, Site 3
Animal #	Sex	Pre	Term	24 h	48 h	24 h	48 h	24 h	48 h	24 h	48 h
1	M	306	500	0	0	0	0	0	0	0	0
2	M	302	467	0	0	0	0	0	0	0	0
3	M	324	538	0.5	0	0	0	0	0	0	0
4	M	335	571	0	0	0	0	0	0	0	0
5	M	316	561	0	0	0	0	0	0	0	0
6	M	298	477	0	0	0	0	0	0	0	0
7	M	322	553	0	0	0	0	0	0	0	0
8	M	321	490	0	0	0	0	0	0	0	0
9	M	306	566	0	0	0	0	0	0	0	0
10	M	297	488	0	0	0	0	0	0	0	0
	MEAN	313	521
	SD	12.7	40.5
11	F	304	470	0	0	0	0	0	0	0	0
12	F	309	492	0	0	0	0	0	0	0	0
13	F	341	540	0	0	0	0	0	0	0	0
14	F	326	524	0	0	0	0	0	0	0	0
15	F	318	493	0	0	0	0	0	0	0	0
16	F	303	432	0	0	0	0	0	0	0	0
17	F	314	477	0	0	0	0	0	0	0	0
18	F	342	550	0	0	0	0	0	0	0	0
19	F	331	511	0	0	0	0	0	0	0	0
20	F	311	477	0	0	0	0	0	0	0	0
	MEAN	320	497
	SD	14.4	35.6

 

Table 2: Challenge Dermal Observations (Post Patch Removal) - Group 2 (0.4 g 100%)
		Body Weight (g)		Challenge, Erythema, Site 3
Animal #	Sex	Pre	Term	24 h	48 h
1	M	312	547	0	0
2	M	318	577	0	0
3	M	301	478	0	0
4	M	329	544	0	0
5	M	309	524	0	0
	MEAN	314	534
	SD	10.5	36.6
6	F	338	540	0	0
7	F	308	463	0	0
8	F	331	475	0	0
9	F	303	542	0	0
10	F	298	489	0	0
	MEAN	316	502
	SD	17.8	37

 

Interpretation of results:

GHS criteria not met

Conclusions:

N-(4-(methoxybenzyl))-N,N-dimethylanilinium hexafluoroantimonate is not a skin sensitizer.

Executive summary:

The potential of N-(4-(methoxybenzyl))-N,N-dimethylanilinium hexafluoroantimonate to promote skin sensitization reactions after repeated applications was determined using the Delayed Contact Dermal Sensitization Test - Buehler Method.

Fifteen healthy male and fifteen healthy female Hartley Albino guinea pigs were assigned to the study. Group 1 (10 males & 10 females) was induced with the moistened N-(4-(methoxybenzyl))-N,N-dimethylanilinium hexafluoroantimonate at a concentration of 100%. Group 2 (5 males & 5 females) was not induced and served as a naive control. Group 1 received three topical induction applications, one/week for three weeks. Skin reactions of the animals in Group 1 were recorded 24- and 48-hours following patch removal. Based on the results of the induction application, 100% was chosen as the highest non-irritating concentration for the challenge and ·was administered to both groups two weeks after the third induction. The skin reactions of all animals were recorded at 24- and 48-hours following patch removal. Body weights were recorded pretest and at study termination. Animals in groups 1 and 2 were observed once/day for mortality and toxicity.

Erythema was absent during the induction and challenge, the body weight changes were normal and systemic effects (wetness of the anogenital area) were observed in 1/20 animals of the test Group. The results indicate that the test substance is not a skin sensitizer and does not meet the GHS/CLH criteria for classification. 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Justification for classification or non-classification

The LLNA and the Buehler studies did not return a positive result. Therefore the test substance is not classified for skin sensitisation.