Benzenesulfonic acid, 4-mono-C20-24 (even numbered)-alkyl derivs., magnesium salts

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-06-13 - 2012-11-12

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The Buehler type of sensitization test is selected at the request of the sponsor since this substance is a surfactant. Studies have shown that the Local Lymph Node Assay, which is typically used, over predicts sensitization potential of these substances. This study is a GPL-guideline study and well documented, however, as the study is used in a read across approach Klimisch 2 is assigned.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Deviations:

yes

Remarks:

Temporary deviations from the maximum level for daily mean relative humidity occurred. Evaluation: Laboratory historical data do not indicate an effect of the deviations.

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Agriculture, Forestry and Fisheries (JMAFF), 12 Nohsan, Notification No. 8147, April 2011; including the most recent partial revisions.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Food and Consumer Product Safety Autrhority (VWA)

Type of study:

Buehler test

Justification for non-LLNA method:

Study was available on a surrogate substance.

Test material

Test material information

Constituent 1

Test material form:

other: liquid

Details on test material:

- Name of test material (as cited in study report): 71786-47-5 Benzenesulfonic Acid mono and dialkylderivs magnesium salt, neutral, in diluents oil TBN = 20
- Molecular formula: UVCB
- Molecular weight: UVCB
- Substance type: organic
- Physical state: Clear brown viscous liquid
- Composition of test material, percentage of components: 100 % UVCB
- Expiration date: 2013-05-31
- Stability under test conditions: stable
- Storage condition of test material: at room temperature in the dark
- Solubility in vehicle: miscible with Kaydol White Mineral Oil
- Stability in vehicle: Stable in Kaydol White Mineral Oil

In vivo test system

Test animals

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Kisslegg, Germany
- Age at study initiation: young adult animals (approx. 5 weeks old), females were nulliparous and non-pregnant
- Housing: Group housing of maximally 5 animals per labelled Noryl cage (Tecniplast; 74 cm x 54 cm x 25 cm height) containing sterilized sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France) and shelters (CS3B02A Play tunnels (90 mm x 5 mm x 125 mm), Datesand, Manchester, UK) as cage enrichment.
- Diet (e.g. ad libitum): Complete maintenance diet for guinea pigs (SSNIFF® MS-H or MS-H Ered; SSNIFF® Spezialdiäten GmbH, Soest, Germany). Hay (TecniLab-BMI BV, Someren, The Netherlands) was provided at least twice a week.
- Water (e.g. ad libitum): Free access to tap water.
- Acclimation period: at least 5 days before the start of treatment under laboratory conditions

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24°C
- Humidity (%): 40 to 70%
- Air changes (per hr): approximately 15 room air changes/hour
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark cycle

OTHER:
Identification of the animals was guaranteed via ear tattoos.
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 skin to be treated, which was intact and free from any abnormality.

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

epicutaneous, semiocclusive

Vehicle:

other: Kaydol White Mineral Oil

Induction 2

Concentration / amount:

undiluted (pure substance) / 50 % dilution in Kaydol White Mineral Oil

Challenge

Challenge 1

Route:

epicutaneous, occlusive

Vehicle:

other: Kaydol White Mineral Oil

Challenge 2

Concentration / amount:

undiluted (pure substance) / 50 % dilution in Kaydol White Mineral Oil

No. of animals per dose:

Experimental group : 20 females.
Control group : 10 females.

Details on study design:

RANGE FINDING TESTS:
Preliminary irritation study
A preliminary irritation study was conducted in order to select test substance concentrations to be used in the Main Study. The selection of concentrations was based on the following criteria:
- The concentrations are well-tolerated by the animals.
- For the induction exposures: the highest possible concentration that produced mild irritation (grade 2).
- For challenge exposure: the maximum non-irritant concentration.

A series of test substance concentrations was tested. Practical feasibility of administration determined the highest starting-concentration (100 %). The test system, procedures and techniques were identical to those used during the main study, unless otherwise specified. The animals selected were between 4 and 9 weeks old. No body weights were determined.

Epidermal application:
A series of four test substance concentrations was used, the highest concentration being the maximum concentration that could technically be applied. Two different concentrations were applied (0.5 mL each) per animal to the clipped flank using Metalline patches# (2x3 cm) mounted on Medical tape, which will be held in place with Micropore tape# and subsequently Coban elastic bandage.
After 6 hours, the dressings were removed and the skin cleaned of residual test substance with Kaydol White Mineral Oil. The resulting dermal reactions were assessed for irritation 24 and 48 hours after removal of the last dressing.

Based on the results, two additional animals were treated each with two test substance concentrations of 50 and 75% to verify the correct challenge concentration.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 3 (Days 1, 8 and 15)
- Exposure period: 6 hours (After 6 hours, the dressings were removed and skin cleaned of residual test substance using Kaydol White Mineral Oil. )
- Test groups: 0.5 mL of the undiluted test substance
- Control group: The control animals were treated as described for the experimental animals, except that, instead of the test substance, vehicle alone was administered.
- Site: The left side of the scapular region was clipped and subsequently epidermally treated with the test substance, all was held in place with Metalline patches (2x3 cm) mounted on Medical tape, which was held in place with Micropore tape and subsequently Coban elastic bandage.
- Frequency of applications: in total: trice
- Duration: 6 hours / 15 days
- Concentrations: 0.5 mL of the undiluted test substance
Evaluation: Immediately after removal of the last induction application on Day 15, the treated skin area was assessed for irritation.

B. CHALLENGE EXPOSURE
- No. of exposures: twice
- Day(s) of challenge: Day 29 and Day 36
- Exposure period: 6 hours (After 6 hours, the dressings were removed and skin cleaned of residual test substance and vehicle using Kaydol White Mineral Oil.)
- Site: The right flank of all animals was clipped and subsequently treated epidermally with the undiluted test substance and the vehicle (0.1 mL of each), using Patch Test Plasters (Curatest®, Lohmann, Almere, The Netherlands). The patches were held in place with Micropore tape and subsequently Coban elastic bandage.
- Evaluation (hr after challenge): The treated sites were assessed for challenge reactions 24 and 48 hours after removal of the dressings.

After termination, animals were sacrificed using isoflurane (Abbott B.V., Hoofddorp, The Netherlands) and an intra-peritoneal injection of Euthasol® 20% (AST Farma BV, Oudewater, The Netherlands).

Positive control substance(s):

yes

Remarks:

alpha-Hexylcinnamaldehyde

Results and discussion

In vivo (non-LLNA)

Results

Results 1

Key result

Reading:

other: first challenge

Group:

test chemical

Dose level:

50 % test substance concentration

No. with + reactions:

1

Total no. in group:

20

Clinical observations:

First challenge: Skin reactions of grade 1 were observed in one experimental animal and scaliness was observed in two experimental animals in response to the 50% test substance concentration. No skin reactions were evident in the control animals.

Remarks on result:

other: see Remark

Remarks:

Reading: other: first challenge. Group: test group. Dose level: 50 % test substance concentration. No with. + reactions: 1.0. Total no. in groups: 20.0. Clinical observations: First challenge: Skin reactions of grade 1 were observed in one experimental animal and scaliness was observed in two experimental animals in response to the 50% test substance concentration. No skin reactions were evident in the control animals..

Results 2

Key result

Reading:

other: second challenge

Group:

test chemical

Dose level:

50 % test substance concentration

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

To confirm the results of the first challenge, a second challenge was performed one week later. No skin reactions were evident after the second challenge exposure in the experimental and control animals.

Remarks on result:

other: see Remark

Remarks:

Reading: other: second challenge. Group: test group. Dose level: 50 % test substance concentration. No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: To confirm the results of the first challenge, a second challenge was performed one week later. No skin reactions were evident after the second challenge exposure in the experimental and control animals. .

Any other information on results incl. tables

PRELIMINARY IRRITATION STUDY

 

The results of the epidermal exposures for the selection of suitable test substance concentrations for the main study are described in Table 1. Based on the results, the undiluted test substance was selected for the three epidermal induction exposures. A 50 % test substance concentration was selected for the challenge phase.

 

MAINSTUDY

 

The skin effects observed immediately after the last (3rd) induction exposure are given in Table 2.

 

Challenge phase:

First challenge: Skin reactions of grade 1 were observed in one experimental animal and scaliness was observed in two experimental animals in response to the 50 % test substance concentration (see Table 2). No skin reactions were evident in the control animals.

Second challenge:

To confirm the results of the first challenge, a second challenge was performed one week later. No skin reactions were evident after the second challenge exposure in the experimental and control animals (see Table 3).

 

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

 

Body weights and body weight gain of experimental animals remained in the same range as controls over the study period (see Table 4).

 

Skin reactions were observed in response to a 50 % concentration of 71786-47-5 in three of the twenty experimental animals (one grade 1 skin reaction and two observations of scaliness) in the first challenge phase. These skin reactions were not confirmed at second challenge with the same concentration. No skin reactions were observed in the control animals in response to the 50 % first and second challenge exposure.

 

The six-month reliability check with Alpha-hexylcinnamicaldehyde indicates that the Buehler Test as performed at WIL Research Europe is an appropriate model for testing for contact hypersensitivity.

Table 1: preliminary irritation study
SKIN REACTIONS AFTER EPIDERMAL EXPOSURE
Animal	Conc. %	24 hours after exposure		48 hours after exposure
number		Erythema	Oedema	Erythema	Oedema
		(grade)	(grade)	(grade)	(grade)
1	100	0	1	1	0
	50	0	0	0	0
2	100	0	1	0	0
	50	0	0	0	0
3	20	0	0	0	0
	10	0	0	0	0
4	20	0	0	0	0
	10	0	0	0	0
1	75	0	0	1	0
	50	0	0	0	0
2	75	0	0	0	0
	50	0	0	0	0
Note: It was noted that the identification of the animals used in the preliminary study was not unique. The raw data of the study specifies that the animals were of separate deliveries from the supplier and treated on different occasions.

Table 2: INDUCTION AND FIRST CHALLENGE READINGS
	Induction		First Challenge
Animal	Day 15		Day 30		Day 31
			24 Hour Reading		48 Hour Reading
No	Readings		Readings		Readings
	100% #		50%	vehicle	50%	vehicle
	Er	Oe
Control
91	0	0	0	0	0	0
92	0	0	0	0	0	0
93	0	0	0	0	0	0
94	0	0	0	0	0	0
95	0	0	0	0	0	0
96	0	0	0	0	0	0
97	0	0	0	0	0	0
98	0	0	0	0	0	0
99	0p	0	0	0	0	0
100	0	0	0	0	0	0
Experimental
101	1	0	0	0	0	0
102	1p	0	0	0	0	0
103	1	0	0	0	0	0
104	1	0	0	0	0p	0
105	2	0	0	0	0	0
106	1	0	0	0	0	0
107	2p	0	0	0	1	0
108	0	0	0	0	0	0
109	1	0	0	0	0	0
110	1	0	0	0	0	0
111	1p	0	0	0	0	0
112	1	0	0	0	0	0
113	2	0	0	0	0	0
114	1p	0	0	0	0	0
115	1	0	0	0	0	0
116	1	0	0	0	0	0
117	1	0	0	0	0	0
118	1	0	0	0	0	0
119	2p	0	0	0	0	0
120	1	0	0	0	0p	0p
#. Test substance concentration (experimental animals) or vehicle (control animals).
p. Scaliness
Er: Erythema
Oe: Oedema
Vehicle: Kaydol White Mineral Oil.

Table 3: SECOND CHALLENGE READINGS
	Challenge
Animal	Day 37		Day 38
No	Readings		Readings
	50%	vehicle	50%	vehicle
Control
91	0	0	0	0
92	0	0	0	0
93	0	0	0	0
94	0	0	0	0
95	0	0	0	0
96	0	0	0	0
97	0	0	0	0
98	0	0	0	0
99	0	0	0	0
100	0	0	0	0
Experimental
101	0	0	0	0
102	0	0	0	0
103	0	0	0	0
104	0	0	0	0
105	0	0	0	0
106	0	0	0	0
107	0	0	0	0
108	0	0	0	0
109	0	0	0	0
110	0	0	0	0
111	0	0	0	0
112	0	0	0	0
113	0	0	0	0
114	0	0	0	0
115	0	0	0	0
116	0	0	0	0
117	0	0	0	0
118	0	0	0	0
119	0	0	0	0
120	0	0	0	0
#. Test substance concentration (experimental animals) or vehicle (control animals).
Er: Erythema.
Oe: Oedema.
Vehicle: Kaydol White Mineral Oil.

Table 4: BODY WEIGHTS (GRAM)
SEX/DOSE LEVEL	ANIMAL	DAY 1	DAY 38
FEMALES CONTROL
	91	309	449
	92	302	448
	93	310	525
	94	342	557
	95	323	475
	96	345	526
	97	336	509
	98	350	524
	99	319	467
	100	346	509
	MEAN	328	499
	ST.DEV.	18	37
	N	10	10
FEMALES EXPERIMENTAL
	101	337	493
	102	352	561
	103	334	510
	104	359	534
	105	306	493
	106	340	552
	107	321	478
	108	333	550
	109	323	456
	110	348	558
	111	280	519
	112	353	507
	113	333	510
	114	306	459
	115	310	497
	116	339	572
	117	334	525
	118	332	485
	119	326	543
	120	332	544
	MEAN	330	517
	ST.DEV.	19	34
	N	20	20

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: other: EU-GHS

Conclusions:

The study was performed according to the OECD Guideline 406 without deviations and according to the good laboratory practice principles, it is considered to be of high quality (reliability Klimisch 2). The criteria of validity of the test system are fulfilled. The test material did not induce a reproducible sensitisation on the intact skin of guinea pigs. The test material was considered not to be sensitising under the conditions of the test.

Executive summary:

Benzenesulfonic Acid mono and dialkylderivs magnesium salt, neutral, in diluents oil TBN = 20 (CAS 71768 -47 -5) was investigated for its sensitizing potential after repeated topical exposures in guinea pigs in a skin sensitization test according to the Buehler method (van Huygevoort, 2012, according to OECD 406, EU Method B6 and EPA OPPTS 870.2600, Klimisch 2). The Buehler type of sensitization test is selected at the request of the sponsor, since this substance is a surfactant. Studies have shown that the LLNA, which is typically used, over predicts sensitization potential of these surfactants. Test substance concentrations selected for the main study were based on the results of a preliminary study. In the main study, twenty experimental animals were epidermally treated on three occasions (once weekly, on Days 1, 8 and 15) with the undiluted test substance (100 % concentration) and ten control animals were similarly treated, but with vehicle alone (Kaydol White Mineral Oil). Two weeks after the last induction exposure (rest period), all animals were topically challenged with a 50% test substance concentration and the vehicle. A second challenge was performed approximately one week later with the same test substance concentrations and the vehicle.

Skin reactions were observed in response to a 50% concentration of the magnesium sulfonate read across substance (CAS 71786-47-5) in three of the twenty experimental animals (one grade 1 skin reaction and two observations of scaliness) in the first challenge phase.  These skin reactions were not confirmed at second challenge (one week later) with the same concentration.

So the number of animals exhibiting positive sensitization scores of 1 for the test substance at the 50% challenge and 50% rechallenge were 1/20 and 0/20, respectively (mean 24/48 hour Draize severity scores 0.00/0.05 and 0.0/0.0). No positive scores were observed in the vehicle control in the first and second challenge (mean 24/48 hour Draize severity scores 0.0/0.0). The only other observation was scaliness in two animals with scores of 0 in the first challenge with the 50% test material concentration and/or vehicle at the 48 hour reading. 

The six-month reliability check with Alpha-hexylcinnamicaldehyde indicates that the Buehler Test as performed at WIL Research Europe is an appropriate model for testing for contact hypersensitivity. Based on these results the test substance is not considered to be a skin sensitizer.